MODULE 1: Introduction to Immunohematology (part1)

MLS 18, February 23, 2021

BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
1628 William Harvey- Discovered the circulation of blood.
Legend:
The first recorded successful blood transfusion occurred in
Transcription Bullet 1665
England.
Notes/Module Packet Bullet
Jean Denis - Published in the Philosophical Transactions his
Reference Book 1667
experience in Paris with transfusing lamb blood.
Highlighted box- High yield information
Dr. James Blundell- The first to successfully transfuse
1818-
MODULE OUTLINE human blood into a patient with post-partum hemorrhage.
I. Historical Background and the current trends in Transfusion Samuel Armstrong Lane- Performs the first successful
1840
Medicine whole blood transfusion to treat hemophilia.
Joseph Lister- Uses antiseptics to control infection during
II. Blood Preservation 1867
transfusions.
III. Review of Basic Genetics US physicians transfuse milk (from cows, goats, and
IV. Review of Fundamentals of Immunology 1873-1880
humans)
V. Review of Molecular Biology Saline infusion replaces milk as a “blood substitute” due to
1884
the increased frequency of adverse reactions to milk.
LEARNING OUTCOMES  Blood was taken from 3 young men; unfortunately, all four
1. List the major developments in the history of transfusion medicine. (including the Pope) died.
o Clottin was the principal obstacle to overcome.
o Attempts to find non-toxic anticoagulant began in 1868, when
Braxton Hicks recommended sodium phosphate.
HISTORICAL BACKGROUND AND THE CURRENT TRENDS IN
o This was perhaps the first example of blood preservation research
TRANSFUSION MEDICINE
o Human blood transfusion is the process of transferring blood or BLOOD GROUPS
blood-based products from an individual into the circulatory system
of another.
1901- Edward Lindeman
o Over the past 100 years, blood transfusion has grown from the
transfusion of small amounts of fresh whole blood, to one of the o The first to succeed in designing a device for transfusion.
most common therapeutic medical practices. o Performed a vein-to-vein blood transfusion by using multiple
syringes & a special canula for transfusion from donor to
EARLY TRANSFUSION patient.

1492- Pope Innocent VII

o First blood transfusion recorded

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Karl Landsteiner- Discovered the first three human blood
1900 groups, A, B, and C. (Blood type C was later changed to O.)
Alfred Decastello and Adriano Sturli- Added AB as the
1902 fourth human blood group.
Hektoen- Suggested that the safety of transfusion might be
improved by crossmatching blood between donors and
patients to exclude incompatible mixtures. Reuben
Ottenberg performed the first blood transfusion using
1907
blood typing and crossmatching in New York. Ottenberg
also observed the mendelian inheritance of blood groups
and recognized the “universal” utility of group O donors.
MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
Audrey Smith- Reported the use of glycerol cryoprotectant
1950
for freezing red blood cells.
Gibson et al developed citrate-phosphate-dextrose
(CPD). CPD eventually replaced ACD and became
1957 commonly used preservative for storage of blood/red
cells in liquid form. Shelf-life of blood stored in CPD at
2-4 °C was 21 day.
A new anticoagulant preservative, CPDA-1, extends the
shelf life of whole blood and red blood cells to 35 days,
increasing the blood supply and facilitating resource sharing
1979
among blood banks. The addition of adenine improved the
synthesis of ATP in the stored blood, which prolonged the
storage of blood/red cells at 2-4 °C to 35 days.

1908 Moreschi- Described the antiglobulin reaction. 1941- Dr. Charles Drew
Dr. Karl Landsteiner- published the first of a series of o Developed techniques on blood transfusion and blood preservation
1990 papers demonstrating presence of the ABO blood group
during WWII
system.
o Led to the establishment of a widespread system of blood banks.
Landsteiner, Weiner, Levine and Stetson- Discovered the
1939-1940 o Director of the first American Red Cross blood bank at
Rh blood group system.
1927-1947 The MNSs and P systems were discovered Presbyterian Hospital
Coombs, Mourant, and Race- Described the use of
1945 antihuman globulin sera to detect IgG antibodies in
compatibility testing.
BLOOD STORAGE
BLOOD DERIVATIVES
1908 Alexis Carrel- Performed direct transfusion.
Edwin Cohn- Developed the cold ethanol fractionation
Long-term anticoagulants, among them sodium citrate, 1940
1914 process, called Cohn fractionation.
were developed.
Pool and Shannon- Revolutionized the treatment of
Rous and Turner- Used sodium citrate as an anticoagulant 1961
1916 hemophilia A.
with glucose.
Concentrated Rh immune globulin was introduced
1932 The first blood bank was established in a Leningrad hospital 1967
commercially.
Bernard Fantus- Established the first hospital blood bank in
1937 Dry-heated, lyophilized factor VIII and IX concentrates
the United States. 1985
became available.
Loutit and Mollison- Developed acid citrate dextrose (ACD)
1943 Genetically engineered (recombinant) factor VIII became
solution. 1993
available.
1998 Factor IX became available
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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
A test for the HIV antibody was introduced; FDA approves
BLOOD COMPONENT THERAPY 1985 enzyme-linked immunosorbent assay (ELISA), first blood-
screening test to detect HIV antibodies.
Roger Lee and Paul Dudley White- Developed the Lee- Two tests that screen for indirect evidence of hepatitis
1912
White clotting time. were developed and implemented, hepatitis B core
1987
Walter and Murphy- Introduced the use of plastic bags as a antibody (anti-HBc) and the alanine aminotransferase test
1950 (ALT).
replacement for glass bottles.
Concentrated blood platelets were recognized as useful for Testing of donated blood for human-Tlymphotropic-virus-I-
1961 1989
the treatment of thrombocytopenia. antibody (anti-HTLV-I) begins.
The first antihemophilic factor (AHF) concentrate to treat 1990 Testing for hepatitis C became routine.
1962 coagulation disorders in hemophilia patients was 2002 West Nile virus identified as transfusion transmissible.
developed through fractionation.
Plasmapheresis was introduced as a means of collecting What are the different obstacles that they need to overcome in order
1964 to have a successful blood transfusion?
plasma for fractionation.
Rh immune globulin was commercially introduced to o Problem with the clotting
1967 prevent Rh disease in the newborns of Rh-negative o Use of Anticoagulant
o Compatibility Testing
women.
o Antiseptic Techniques
Hepatitis B surface antigen (HBsAg) testing of donated
1971 o Blood Preservation
blood begins. o Component Therapy- in cases of Transfusion Overload
1972 Platelets for transfusion were collected by apheresis.

ADVERSE EFFECT OF TRANSFUSION ADVANCES IN BLOOD TRANSFUSION

Today, the blood transfusion community continues to advance its
Greenwalt et al.- Demonstrated leukocyte reduction filters transfusion systems, guided by the:
1962-
prevented febrile reactions. o ISBT (International Society of Blood Transfusion),
1970- Graw et al.- Used irradiation to prevent TA-GVHD. o AABB (American Association of Blood Banks),
Commercial testing for hepatitis B surface antigen began. o FDA (Food and Drug Administration), and
1971-
o Other federal and professional organizations.
First Acquired Immune Deficiency Syndrome (AIDS) case Researchers are establishing new surveillance systems that record
1981
was reported. data and transfusion outcomes (haemovigilance system).
1983- Transfusion-transmitted HIV was described. o They are offering more personalized treatment, limiting
Human Immunodeficiency Virus (HIV) was identified asthe transfusions based on careful assessment of need, and
1984 ultimately improving patient care.
cause of AIDS.
In addition to infectious disease risks, treating physicians must also
manage other risks, such as post-transfusion reactions.

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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
o These include:  Variant Creutzfeldt-Jakob
 Transfusion-related lung injury (TRALI);  West Nile Virus
 Transfusion associated cardiac overload (TACO); and  Malaria
 Post-transfusion iron overload.  Babesiosis
To minimize these risks, researchers studying the body’s immune  Chagas Disease
response to transfusions have found that modifying the blood prior to
transfusion can reduce reactions. STEP 3: The Abbreviated Physical Examination
o In particular, removing white blood cells or radiating blood. o The abbreviated physical examination for donors includes;
o Recently, studies found that using male plasma and platelets  Blood pressure, Pulse, and Temperature readings;
may eliminate the transmission of certain antibodies that can  Hemoglobin or Hematocrit level;
cause reactions and are found only in previously pregnant  Inspection of the arms for skin lesions
women and transfused males.
Over the last 15 years, we’ve seen a shift from whole blood–derived Currently 10 screening tests for infectious disease are performed on
platelets obtained through whole-blood collection to single-donor each unit of donated blood (See Table 1-1; Harmening, 6th Edition)
platelets obtained through apheresis.
o Blood components collected by apheresis offer several
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advantages over manually collected whole blood.
o Because apheresis separates the components, the blood
doesn’t need further processing.
Many apheresis devices allow collection of plasma and RBCs
simultaneously with platelets. Although components still need to be
tested, labeled, and stored appropriately, apheresis eliminates one
processing step. Some apheresis devices also leukoreduce RBCs and
platelets, eliminating the need to filter them in the laboratory.

DONATION PROCESS
STEP 1: Educational Materials
o Educational (ex. AABB pamphlet “An Important Message to Blood
Donors”) that contains information on the risks of infectious diseases
transmitted by blood transfusion, including the symptoms and signs of
AIDs, is given to each prospective donor to read.

STEP 2: The Donor Health History Questionnaire

o A uniform donor history questionnaire, designed to as questions that
protect the health of both the donor and the recipients, is given to
every donor.
o The health history questionnaire is used to identify donors who have
been exposed to diseases that can be transmitted in blood
 Example of blood-transmitted diseases;
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 MODULE 1: Introduction to Immunohematology (part 2)
MLS 18, February 23, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
RBC BIOLOGY
Legend: Three areas of RBC biology are crucial for normal erythrocyte survival
Transcription Bullet and function:
Notes/Module Packet Bullet 1. Normal chemical composition and structure of the RBC membrane
Reference Book 2. Hemoglobin structure and function
MODULE OUTLINE 3. RBC metabolism Defects in any or all of these areas will result in
I. Historical Background and the current trends in Transfusion Medicine RBCs surviving less than the normal 120 days in circulation.
II. Blood Preservation
Normal chemical composition and structure of the RBC membrane
A. RBC Biology
B. Blood Preservation RBC MEMBRANES
III. Review of Basic Genetics
Phospholipids
IV. Review of Fundamentals of Immunology
o The main lipid components of the membrane, are arranged in a
V. Review of Molecular Biology bilayer structure comprising the framework in which globular
proteins traverse and move.
LEARNING OUTCOMES
1. Describe the biologic properties of red blood cells (RBC) that can Integral Membrane Proteins
affect post-transfusion survival. o Proteins that extend from the outer surface and span the entire
2. Identify the metabolic pathways that are essential for normal RBC membrane to the inner cytoplasmic side of the RBC
function & survival.
3. Explain the importance of 2-3 DPG levels in transfused blood.
Peripheral Proteins
4. List the approved anticoagulant preservative solutions, explain the
o Second class of membrane proteins, found beneath the lipid
function of each component, and state the maximum storage time
bilayer
for RBCs and platelets collected in each.
o located and limited to the cytoplasmic surface of the membrane
5. Explain how additive solutions are used and list their advantages.
forming the RBC cytoskeleton
6. Explain storage requirements for platelets, including rationale.
7. Describe the indications for platelet transfusion.
8. Explain the importance of platelet corrected count increment (CCI). Integral Proteins Peripheral Proteins
Glycophorin A Spectrin
Glycophorin B Actin (band 5)
Glycophorin C Ankyrin (band 2.1)
Anion-exchange-channel protein Band 4.1 and 4.2
(band 3) Band 6
Adducin
Table 1: RBC Membrane Integral and Peripheral Proteins
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NOTE:
Red blood cells are a product of a differentiation process that starts
in the bone marrow where hematopoietic stem cells differentiate to
nucleate RBCs. After extrusion of nuclei and degradation of
endoplasmic reticulum, reticulocytes emerge in the circulation; here
they rapidly develop into mature RBCs.
Despite these features, the protein and lipid composition of the RBCs
is subject to change during its life-time. This can be particularly
observed at the level of its plasma membrane.
Changes in red blood cell morphology occurred as quickly as 22 days.
o This alteration can be harmful because red blood cells are similar in
size to the diameter of small capillaries; therefore, red blood cells
have to change shape to get through the capillaries.
Figure 1. Schematic illustration of red blood cell membrane depicting the composition
and arrangement of RBC membrane proteins.
RBC CHARACTERISTICS
The normal chemical composition and the structural arrangement
and molecular interactions of the erythrocyte membrane are crucial
to the normal length of RBC survival of 120 days in circulation. In
addition, they maintain a critical role in two important RBC
characteristics: deformability and permeability.
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
1. Deformability
To remain viable, normal RBCs must also remain flexible, deformable,
and permeable.
The loss of adenosine triphosphate (ATP) (energy) levels leads to a
decrease in the phosphorylation of spectrin and, in turn, a loss of
membrane deformability.
An accumulation or increase in deposition of membrane calcium
also results, causing an increase in membrane rigidity and loss of
pliability.
o These cells are at a marked disadvantage when they pass through
the small (3 to 5 μm in diameter) sinusoidal orifices of the
spleen, an organ that functions in extravascular sequestration and
removal of aged, damaged, or less deformable RBCs or fragments
of their membrane.
The loss of RBC membrane is exemplified by the formation of
“spherocytes” (cells with a reduced surface-to-volume ratio) and
“bite cells,” in which the removal of a portion of membrane has left a
permanent indentation in the remaining cell membrane.
o The survival of these forms is also shortened.
2. PERMEABILITY
The permeability properties of the RBC membrane and the active
RBC cation transport prevent colloid hemolysis and control the
volume of the RBC.
o Any abnormality that increases permeability or alters
cationic transport may decrease RBC survival.
The RBC membrane is freely permeable to water and anions.
Chloride (Cl–) and bicarbonate (HCO3–) can traverse the membrane
in less than a second.
The RBC membrane is relatively impermeable to cations such as
sodium (Na+) and potassium (K+).
RBC volume and water homeostasis are maintained by controlling
the intracellular concentrations of sodium and potassium.
o The erythrocyte intracellular-to-extracellular ratios for Na+
and K+ are 1:12 and 25:1, respectively.
The 300 cationic pumps, which actively transport Na+ out of the cell
and K+ into the cell, require energy in the form of ATP.
Calcium (Ca2+) is also actively pumped from the interior of the RBC
through energy-dependent calcium-ATPase pumps. Calmodulin, a
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cytoplasmic calcium-binding protein, is speculated to control these
pumps and to prevent excessive intracellular Ca2+ buildup, which
changes the shape and makes it more rigid.
When RBCs are ATP depleted, Ca2+ and Na+ are allowed to
accumulate intracellularly, and K+ and water are lost, resulting in a
dehydrated rigid cell subsequently sequestered by the spleen,
resulting in a decrease in RBC survival.
HEMOGLOBIN STRUCTURE AND FUNCTION
HEMOGLOBIN
Hemoglobin is the major protein in RBCs, and it gives to red cells the
ability to transport oxygen and carbon dioxide to and from tissues.
HEMOGLOBIN FUNCTION
Hemoglobin’s primary function is gas transport:
o Oxygen (O2) delivery from the lungs to the tissues
o Transport of Carbon dioxide (CO2) from tissues to the lungs
2,3-DPG
One of the most important controls of hemoglobin affinity for
oxygen is the RBC organic phosphate 2,3-DPG.
A carbon molecule important in erythrocyte metabolism.
o It binds to deoxygenated hemoglobin and increases oxygen off-
loading from hemoglobin into the tissues.
o As erythrocyte storage time increases, the levels of 2,3-DPG
decrease.
 Transfusion of 2,3-DPG-depleted blood may shift the
oxygen–hemoglobin dissociation curve to the left.
 As a result, red blood cells will have difficulty in unloading
oxygen from hemoglobin into the tissues.
 The unloading of oxygen by hemoglobin is accompanied by
widening of a space between chains and the binding of 2,3-
DPG on a mole-for-mole basis, with the formation of anionic
salt bridges between the chains.
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
“Tense” Form
o The resulting conformation of the deoxyhemoglobin molecule is
known as the tense (T) form, which has a lower affinity for
oxygen.
“Relaxed” Form
o When hemoglobin loads oxygen and becomes oxyhemoglobin,
the established salt bridges are broken, and chains are pulled
together, expelling 2-3-DPG. This is the relaxed (R) form of the
hemoglobin molecule, which has a higher affinity for oxygen.
HEMOGLOBIN-OXYGEN DISSOCIATION CURVE
These allosteric changes that occur as the hemoglobin loads and
unloads oxygen are referred to as the respiratory movement.
Are not directly proportional to the partial pressure of oxygen (pO2)
in its environment but instead exhibit a sigmoid-curve relationship,
known as the hemoglobin-oxygen dissociation curve.
The shape of this curve is very important physiologically because it
permits a considerable amount of oxygen to be delivered to the
tissues with a small drop in oxygen tension.
o For example, in the environment of the lungs, where the
oxygen (pO2) tension, measured in millimeters of mercury
(mmHg), is nearly 100 mm Hg, the hemoglobin molecule is
almost 100% saturated with oxygen.
As the RBCs travel to the tissues, where the (pO2) drops to an
average of 40 mm Hg (mean venous oxygen tension), the
hemoglobin saturation drops to approximately 75% saturation,
releasing about 25% of the oxygen to the tissues.
o This is the normal situation of oxygen delivery at a basal
metabolic rate.
Ligands
o The normal position of the oxygen dissociation curve depends
on three different ligands normally found within the RBC: H+
ions, CO2, and organic phosphates.
o Of these three ligands, 2,3-DPG plays the most important
physiological role. Normal hemoglobin function depends on
adequate 2, 3-DPG levels in the RBC.
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 In situations such as hypoxia, a compensatory “shift to the
right” of the hemoglobin-oxygen dissociation curve
alleviates the tissue oxygen deficit. This rightward shift of
the curve, mediated by:
 Increased levels of 2,3-DPG;
 Decreases hemoglobin’s affinity for the oxygen
molecule and;
 Increases oxygen delivery to the tissues.
 A “shift to the left” of the hemoglobin-oxygen dissociation
curve results, conversely;
 Increase in hemoglobin oxygen affinity and a decrease
in oxygen delivery to the tissues.
 With such a dissociation curve, RBCs are much less
efficient because only 12% of the oxygen can be
released to the tissues.
 Multiple transfusions of 2,3-DPG–depleted stored blood
can shift the oxygen dissociation curve to the left.
Figure 1. Diagrammatic representation of oxygen equilibrium curves of the
lug worm, man, and hemoglobin Scuba.
o The effect of hydrogen ions, 2, 3-bisphosphoglycerate, and carbon
dioxide (H++BPG +CO2) is to promote a right shift.
o If man had the hemoglobin of the lug worm (left shift), he would die
of anoxia.
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
RBC Metabolism
RBCs produce ATP anaerobically by the breakdown of glucose, thus
not using any of the oxygen for its own metabolism.
o Erythrocytes do not have mitochondria, which is the site of
aerobic respiration.
Anaerobic metabolism allows red blood cells to deliver 100% of the
oxygen to the organ sites. RBC metabolism may be divided into the
anaerobic glycolytic pathway and three ancillary pathways that
serve to maintain the structure and function of hemoglobin:
1. The Pentose Phosphate pathway
2. The Methemoglobin Reductase pathway
3. The Luebering-Rapoport shunt.
All of these processes are essential if the erythrocyte is to transport
oxygen and to maintain critical physical characteristics for its
survival.
o Glycolysis generates about 90% of the ATP needed by the RBC.
o Approximately 10% is provided by the pentose phosphate
pathway.
o The methemoglobin reductase pathway is another important
pathway of RBC metabolism, and a defect can affect RBC post-
transfusion survival and function.
RBC metabolism includes the glycolytic pathways producing both
energy (as adenosine 5′- triphosphate, or ATP) and oxidation-
reduction intermediates that support oxygen transport and
membrane flexibility. RBCs interact with their environment by
changing shape in response to pH and by secreting ATP in response
to sheer forces and nitric oxide in response to hypoxia.
At the end of their life span, RBCs undergo programmed cell death
in two ways:
o By racemization of negatively charged membrane phospholipids
in response to calcium or low concentrations of ATP, and
o By the active loss of membrane through microvesiculation.
Storage Lesion
o A blanket term used to encompass all of the “bad” things that
happen to RBCs during storage. These “bad” things include:
 Decreased concentrations of ATP and 2,3-
diphosphoglycerate (2,3-DPG)
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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
 Increased concentrations of extracellular potassium, o Two molecules of lactic acid contain exactly the same number of
changes in cell shape carbons, hydrogens, and oxygens as one molecule of glucose;
 Loss of RBC viability  there is sufficient free energy available from the cleavage
 Hemolysis and rearrangement of the glucose molecule to produce 2
moles of ATP per mole of glucose converted into lactate.
CLINICAL SIGNIFICANCE The RBC uses most of this ATP to maintain electrochemical and ion
1. Elevated extracellular potassium concentrations in stored RBC units gradients across its plasma membrane.
Not usually of clinical significance One mole of glucose is converted to 2 moles of lactate during
However, can be fatal if RBC concentrates are infused in large anaerobic glycolysis.
volumes through central vascular catheters No oxygen is consumed, nor is CO2 produced in this pathway. There
o As in neonatal exchange transfusions or when priming is a net yield of 2 mole of ATP per mole of glucose converted to
cardiopulmonary bypass machines. lactate.

2. Hemolysis LUEBERING-RAPAPORT PATHWAY

Reduction of the relative number of stored RBCs that survive
An offshoot of the Embden-Meyerhof pathway is the Luebering-
when returned to circulation
Rapaport bypass.
Release of harmful breakdown products.
This pathway permits the accumulation of an important RBC organic
phosphate, 2,3-diphosphoglycerate (2,3- DPG).
3. Free hemoglobin
o Has a significant effect on the affinity of hemoglobin for oxygen
Vasoconstriction
and therefore affects how well RBCs function post-transfusion.
Endothelial cell activation
Renal tubular damage. The 2,3- DPG alternate route does not yield ATP, but does modulate
levels of 2,3-DPG.
4. Free membrane phospholipids Erythrocyte 2,3-DPG concentration is especially sensitive to pH
Are procoagulant because the enzymes catalyzing its production are inhibited or
Clinical consequences of the early forms of RBC shape change or stimulated by hydrogen ions.
reduced 2,3-DPG concentrations are much less clear. Defects in the Luebering-Rapaport bypass can affect the levels of
2,3-DPG available to erythrocytes. Red blood cell 2,3-DPG regulates
5. Loss of viability during storage oxygen release depending on the needs of tissues.
Reduces the effective transfused RBC dose, but the Whenever the peripheral tissues have an increased amount of
consequences of administering intact but nonviable cells are not deoxygenated blood (deoxyhemoglobin), glycolysis is stimulated
known. and 2,3-DPG levels rise.
o Again, pH changes within the cell probably contribute to this
ANAEROBIC GLYCOLYTIC PATHWAY process.
o The result is that 2,3- DPG attaches to deoxyhemoglobin and
In RBCs, which lack mitochondria and oxidative metabolism,
causes hemoglobin to resist binding to oxygen. This decrease in
pyruvate is reduced to lactic acid, a three-carbon hydroxyacid, the
oxygen affinity by hemoglobin increases oxygen release to
product of anaerobic glycolysis.
tissues.
Each mole of glucose yields 2 moles of lactate, which are then
excreted into blood.
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HEXOSE MONOPHOSPHATE SHUNT/ PATHWAY
Red blood cells are capable of limited aerobic glycolysis
Also called the phosphogluconate pathway or the pentose
phosphate shunt.
The major role of the hexose monophosphate shunt, is the
generation of reduced nicotinamide adenine dinucleotide
phosphate (NADPH).
o Erythrocyte NADPH converts oxidized glutathione to reduced
glutathione, the major red blood cell antioxidant.
o Red blood cell enzymes, and especially hemoglobin, are
protected from oxidant damage through the action of
glutathione, which maintains hemoglobin in a reduced, active
form.
Although oxidants are damaging to cells, cells commonly produce
them.
o Macrophages, for example, produce them in response to
infection.
o Erythrocytes even produce them when certain drugs are present.
If the level of reduced glutathione is not sufficient to neutralize
intracellular red blood cell oxidants, globin will denature and
precipitate as Heinz bodies, ultimately producing membrane
damage. The levels of nicotinamide adenine dinucleotide phosphate
(NADP)/NADPH regulate the amount of glucose metabolized by the
hexose monophosphate shunt.
As NADPH generates reduced glutathione, NADP is produced, which
stimulates glucose metabolism in the hexose monophosphate
shunt. This mechanism arms the red blood cell with more reducing
capability during an oxidative challenge.
METHEMOGLOBIN REDUCTASE PATHWAY
An important auxiliary process of erythrocyte metabolism is the
methemoglobin reductase pathway.
This pathway maintains heme iron in the reduced, or active state
(ferrous).
It requires reduced pyridine nucleotides generated from the
Embden-Meyerhof pathway.
Unlike the hexose monophosphate shunt, which provides a
mechanism for preventing the denaturation of the globin
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
component of hemoglobin, the methemoglobin reductase pathway
ensures that the iron (heme) in the hemoglobin molecule does not
become oxidized. Methemoglobin with iron in the ferric state is
useless as an oxygen carrier. This pathway uses the enzyme
methemoglobin reductase and NAD to maintain hemoglobin in a
reduced state.
THE IMPORTANCE OF 2-3 DPG LEVELS IN TRANSFUSED BLOOD
The two main regulators of oxygen uptake and delivery are the pH
of tissues and the content of 2,3-diphosphoglycerate (2,3-DPG) in
red cells.
The pH of blood is kept relatively constant at the slightly alkaline
level of about 7.4 (pH less than 7 indicates acidity, more than 7
alkalinity). The effect of pH on the ability of hemoglobin to bind
oxygen is called the Bohr effect:
o When pH is low, hemoglobin binds oxygen less strongly, and
when pH is high (as in the lungs), hemoglobin binds more tightly
to oxygen.
o The Bohr effect is due to changes in the shape of the hemoglobin
molecule as the pH of its environment changes. The oxygen
affinity of hemoglobin is also regulated by 2,3-DPG, a simple
molecule produced by the red cell when it metabolizes glucose.
Effect of 2,3-DPG is to reduce the oxygen affinity of hemoglobin.
When the availability of oxygen to tissues is reduced, the red cell
responds by synthesizing more 2,3-DPG, a process that occurs over
a period of hours to days. 2,3-DPG is an intermediary metabolite in
the Embden–Meyerhof glycolytic pathway in the red cells, which
affects haemoglobin affinity for oxygen.
↑ Concentration of 2,3-DPG ↓ Concentration of 2,3-DPG
o decreases the affinity and thus o Has the opposite effect.
increases the fraction of o Caused by acidosis.
haemoglobin-bound oxygen
available to the tissues
o found in response to hypoxia or
anemia
Table 2: Effects of 2,3-DPG
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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
BLOOD PRESERVAT ION ANTICOAGULANT PRESERVATIVE SOLUTIONS IN BLOOD BAGS
The goal of blood preservation is to provide viable and functional Prevent blood from clotting
blood components for patients requiring blood transfusion. Also used to preserve the life and survival of RBCs so as to have the
maximum post transfusion survival.
RBC Viability Citrate-based anticoagulants are used for the collection of blood
A measure of in vivo RBC survival following transfusion. for transfusion.
Because blood must be stored from the time of donation until the o Citrate binds calcium, preventing the activation of plasma
time of transfusion, the viability of RBCs must be maintained during coagulation factors.
the storage time as well. EDTA, which binds calcium more strongly compared to citrate, is not
To maintain optimum viability, blood is stored in the liquid state used for the collection of blood transfusion products.
between 1°C and 6°C for a specific number of days, as determined Heparin is rarely used for the collection of blood transfusion
by the preservative solution(s) used. components as its effective anticoagulant half-life (approximately
2,3-DPG is re-formed in stored RBCs after in vivo circulation an hour) is limited.
Whole blood is collected into a primary blood bag containing a
Factors affecting the rate of 2,3-DPG restoration citrate based anticoagulant (CPD, CPDA-1 or CP2D) and adequate
o Recipient’s Acid-Base status mixing is ensured. The anticoagulant solution may contain nutrients
o Phosphorus Metabolism such as glucose and adenine.
o Degree of Anemia Additional cell specific nutrients are supplied to the stored red cells
o Overall severity of the disorder and platelets by the additive solutions in the respective red cell and
RBC clearance occurs within the first hour after transfusion platelet satellite bags.
Approximately 220 to 250 mg of iron in one RBC unit Citrate is the most common anticoagulant used in apheresis blood
Rapid RBC clearance of a single unit of blood delivers a massive load collection. Sodium citrate (4%) is used for plasma apheresis and
of iron to the monocyte and macrophage system ACD-A is used for platelet apheresis.
Incorporation of Adenine & its effects on glycolysis and ATP levels
Food and Drug Administration requirements o Adenine incorporated into the CPD solution (CPDA-1) increases
1. Average 24-hour post transfusion RBC survival of more than 75% ADP Levels, thereby driving glycolysis toward the synthesis of
ATP.
2. Free Hemoglobin less then 1% of total hemoglobin
NAME ABBREVIATION STORAGE
Determination of Post-Transfusion RBC survival TIME (DAYS)
 Taking RBCs from healthy subjects Acid -citrate-dextrose ACD 21
 Storage of RBCs labeled with radioisotopes Citrate-Phosphate-Dextrose CPD 21
 Reinfusion back to the original donor Citrate-Phosphate-Dextrose-Adenine CPDA-1 35
 Measurement 24 hours after transfusion Citrate-Phosphate-Double-Dextrose CP2D 21
Table 3: Additive Solutions in use in North America

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Pathophysiologic effects of the transfusion of RBCs with low 2,3-
DPG levels & increased affinity for Oxygen:
o ↑ Cardiac Output
o ↓Mixed venous (pO2) tension
o ↑Cardiac output & ↓pO2 tension
Plastic containers used for storage of blood affects its viability
di (ethylhexyl)-phthalate (DEHP) in PVC bags
o Plasticizer
o Leach from the plastic into the lipids of the plasma medium and
RBC membranes of the blood during storage
o Break at low temperature
Added to the RBCs after removal of the plasma
Without ADSOL:
o Decreased nutrients necessary to maintain RBCs during
storage
o Decreased in viability, particularly in the last 2 weeks of
storage.
ACID CITRATE DEXTROSE (ACD):
The original ACD solution had a pH of 5;
Was made with citric acid, dextrose, and sodium citrate; and stably
survived being autoclaved.
It was used primarily for glass bottle storage of whole blood and
allowed for 72 % RBC survival after 21 days with little hemolysis.
CITRATE PHOSPHATE DEXTROSE (CPD)
(CPD) Citrate–phosphate–dextrose (CPD), is the mainstay of blood
preservation.
Citrate works as an anticoagulant by binding to and inhibiting the
function of calcium (factor IV).
Phosphate stabilizes pH which maintains proper levels of 2,3-DPG.
The dextrose component is necessary for red blood cell ATP
production.
If adenine, a purine nucleotide, is added to CPD (CPD-A), storage
time jumps from 21 days to 35 days.
o Adenine assists in the production of ATP.
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CITRATE PHOSPHATE DEXTROSE ADENINE (CPDA-1)
CPD to which adenine (A) is added, becomes CPDA-1 (the‘1’
signifies the formula used) and improves the synthesis of ATP.
CPDA-1 is usually used when the collected donation is to be stored
as whole blood.
NOTE:
The volume of anticoagulant required to prevent clotting and
preserve red cells is dependent on the volume of blood taken
from the donor.
Some collection bags are designed for the collection of 500 mL
blood and contain 70 mL anticoagulant; others are designed for
450 mL collections and contain 63 mL anticoagulant.
If smaller quantities of blood are to be drawn, then the volume
of anticoagulant is reduced proportionately. 15 ml of ACD, 14ml
of CPD or CPDA is used for preserving 100ml of blood.
ACTION OF INGREDIENTS OF ANTICOAGULANT SOLUTION
CITRATE Acts by chelating Calcium.
DEXTROSE Necessary for the metabolism of stored RBCs. It passes
from plasma into the red cells and is utilized for energy
production. The principal pathway being Anaerobic
glycolysis.
CITRIC ACID Prevents carmalization of glucose in citrate dextrose
solution during autoclaving.
ADENINE Improves the viability of red cells.
CPDA – 2 Here, the amount of Adenine is increased to 0.55g and
that of dextrose to 44.6g.
o This is a better anticoagulant preservative
solution than CPDA–1.
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STORAGE TEMPERATURES
Collection of blood into anticoagulant-preservatives maintains
component function and viability only if storage is within the
correct temperature range.
Low temperature storage slows glycolytic activity and allows
the shelf life of the component to be extended.
Low temperatures also retard bacterial proliferation, should
bacteria have gained access during the time of donation;
either from the venipuncture site, the donor’s circulation or
other source.
Additive Solution
Additive solutions are preserving solutions that are added to the
RBCs after removal of the plasma with/without platelets. Additive
solutions replace nutrients lost when the plasma is removed from
red blood cells. When additive solutions are added, red blood cell’s
storage time can be increased from 35 days to 42 days.
Two of the solutions (Adsol, Optisol) contain adenine, glucose,
saline, and mannitol.
o Reason for their development: removal of the plasma
component during the preparation of RBC concentrates removed
much of the nutrients needed to maintain RBCs during storage.
Also overcome the problem of high viscosity of RBC
concentrates.
With CPD anticoagulant in the primary bag, the additive solution used is
SAGM (saline, adenine, glucose, mannitol).
ADVANTAGES
Extends the storage of RBCs and lowers the viscosity of packed
red cells for ease of transfusion. Maximum amount of fresh
plasma is harvested – platelets and cryoprecipitate. Red cell
concentrates that are prepared from whole blood donations
collected into CPD are suspended in additive solution for
improved storage and shelf life.
When plasma is removed after the centrifugation of whole blood
donations, most of the anticoagulant and nutrients in CPD are
removed along with it. At this stage, blood has been effectively
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
anticoagulated so the presence of CPD is no longer required by
the red cell concentrate remaining in the primary collection bag.
However, the red cells need nutrients to survive, and should also
be suspended in sufficient fluid to allow for normal flow
characteristics. This is achieved by the use of additive solutions.
Additive solutions (AS) vary in composition depending on the
supplier. They are sometimes referred to by their brand names or
simply as SAGM (saline, adenine, glucose and mannitol) or AS-1, AS-
3, AS-5 and so on. The typical composition of an additive solution is
as follows:
SALINE the fluid in which the red cells are
suspended to provide the desired flow
rate conditions.
GLUCOSE (OR DEXTROSE) Provides the basic nutrients for glycolysis.
ADENINE AND MANNITOL assist in the process of ATP generation.
When an additive system is used then the blood donation is
collected in CPD, which has no adenine.
o The unit is processed within 24 hours of collection and the
adenine is added with the red cell additive solution.
o The volume of additive solution required to preserve red cells
during storage varies according to the volume of the whole blood
donation.
o Red cells from a donation of 500 mL require about 111 mL of
additive solution, whereas 450 mL donations need 100 mL.
Red cell concentrates and platelet concentrates are stored as fresh
products ready for transfusion, although in special circumstances
can be frozen using specific cryopreservation procedures.
o Red cell concentrates are stored refrigerated (2– 6 °C) for up to
42 days (maximum duration may vary depending on the type of
additive solution used and/or local regulatory criteria).
o Platelet concentrates are stored at 20–24 °C for up to 5 days,
with continuous gentle agitation to maintain maximum biological
function.
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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
During storage, red cells and platelets continue to metabolize and This interprets as a shelf life of 21 days for ACD, 28 days for CPD, 35
undergo a range of physiochemical changes, collectively referred to as days for CPDA-1 and 42 days for CPD replaced with a suitable additive
the 'storage lesion’. solution.
o The storage lesion ultimately affects the in vivo function and
survival of transfused red cells and platelets and thus limits their For Platelet Concentrates
shelf life. Shelf life at +22°C ± 2°C is determined by its efficacy when transfused.
This may be related to platelet viability and function during storage in
Currently approved AdSol: the correct conditions of temperature and motion, and is considered to
1. Adsol (AS-1) – Baxter Healthcare be up to 7 days.
2. Nutricel (AS-3) – Pall Corporation Most blood services allocate a 5-day shelf life to limit the increased risk
3. Optisol (AS- 5) – Teruo Corporation of bacterial growth resulting from the room temperature storage
requirement.
AS-1 AS-3 AS-5
Storage period (days) 42 42 42 For Plasma
pH (measured at 37°C) 6.6 6.5 6.5 The levels of stable clotting factors (FII, FVII, FIX, FX and fibrinogen) are
24-hour survival (%) 83 85.1 80 quite well maintained at +4°C ± 2°C.
ATP (% initial) 68 67 68.5 Labile clotting factors (FV, FVIII) deteriorate to levels that are not useful
2,3-DPG (% initial) 6 6 5 if not frozen within 24 hours of collection.
Hemolysis 0.5 0.7 0.6 Shelf life of labile coagulation factors is up to 3 years if frozen (colder
Table 4: Red Cell Additives: Biochemical Characteristics than –25°C).
However, a storage temperature that does not reach –25°C but is colder
SHELF LIFE OF WHOLE BLOOD COMPONENTS than –18°C reduces shelf life to 3 months.
SHELF LIFE is the maximum allowable storage time that a blood
product may be stored, provided that the requirements of RBC FREEZING
temperature, preservative solutions and physical environment are For Autologous Units & storage of rare blood types
met. Involves the addition of a cryoprotective agent to RBCs that are less
Because of the great diversity in collection containers and than 6 days old
anticoagulant preservatives, as well as storage capabilities within 20% or 40% Glycerol
different blood services, the shelf life of different components o cryoprotectant
varies considerably. Shelf-life specifications must comply with local Rapid freezing and stored in a freezer
standards. Transfusion of frozen cells must be followed by deglycerolization
Cryoprotectant us systematically replaced with decreasing
For Red Cells concentrations of saline
Shelf life varies at +4°C ± 2°C according to anticoagulant/preservative Current Trend: FDA licenses frozen RBCs for a period of 10 years
and additive solution used. from the date of freezing
The requirement that determines shelf life is that at least 75% of red
cells transfused at the end of the proposed storage period must still be
in circulation 24 hours after transfusion.

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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
RBC REJUVENATION pH 6.2: current standard for maintaining satisfactory platelet
Process of enhancing and restoring ATP & 2,3-DPG levels viability
RBCs (liquid state) can be rejuvenated at outdate or up to 3 days Second generation storage containers
after outdate o Increased gas transport properties
o Ex. Rejuvesol
Incubation of RBC unit with 50 mL of the rejuvenating solution for 1 PLATELET STORAGE AND BACTERIAL CONTAMINATION
hour at 37°C Storage of platelets at 20-24°C may cause bacterial growth
Following Rejuvenation, the RBCs can be washed with Saline & o Room temperature storage and the presence of Oxygen can
Transfused within 24 hours encourage bacterial proliferation
o Sepsis due to contaminated platelets: most common infectious
PLATELET STORAGE complication of transfusion
o 10-40% of patients transfused with a bacterially contaminated
MAJOR CHALLENGE IN PLATELET STORAGE
platelet unit develop life-threatening sepsis
5-day shelf life (in the US)
Bacterial contamination at incubation of 22°
A varying degree of platelet activation/ aggregation
INDICATIONS FOR BLOOD COMPONENTS
Release of intracellular granules RED BLOOD CELL TRANSFUSION
Suppression in ATP &ADP levels Are used to treat hemorrhage and to improve oxygen delivery to
tissues.
CLINICAL USE OF PLATELET It should be based on the patient's clinical condition.
Treatment of bleeding associated with thrombocytopenia Indications for transfusion include:
Transfused to hematology-oncology thrombocytopenic patients to o Symptomatic Anemia (causing shortness of breath, dizziness,
prevent bleeding congestive heart failure, and decreased exercise tolerance)
Current Trend: Platelets are prepared as concentrates from whole o Acute Sickle Cell Crisis
blood & increasingly by apheresis o Acute Blood Loss of more than 30 percent of blood volume.
Fresh frozen plasma infusion can be used for reversal of
CURRENT CONDITION OF PLATELET PRESERVATION anticoagulant effects.

PLATELET CONCENTRATES
Prepared from whole blood and apheresis components
PLATELET TRANSFUSION
Storage: 20-24°C with continuous agitation
Expiration time (FDA): midnight of Day 5
Should contain a minimum of 5.5 x 10^10 platelets in a volume of
between 45 & 65 mL
RATIONALE AND ADVANE CONCEPT FOR PRESERVATION
Maintenance of pH
o Key parameter for retaining platelet viability in vivo when
platelets were stored at 20-24°C
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Indicated to prevent hemorrhage in patients with
thrombocytopenia or platelet function defects.
Cryoprecipitate is used in cases of hypofibrinogenemia, which most
often occurs in the setting of massive hemorrhage or consumptive
coagulopathy.
Transfusion-related infections are less common than non-infectious
complications.
o All noninfectious complications of transfusion are classified as
noninfectious serious hazards of transfusion.
o Acute complications occur within minutes to 24 hours of the
transfusion
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o Delayed complications may develop days, months, or even years
later.
Platelet transfusion may be indicated to prevent hemorrhage in
patients with thrombocytopenia or platelet function defects.
Contraindications to platelet transfusion include thrombotic
thrombocytopenic purpura and heparin-induced
thrombocytopenia.
o Should not be transfused in patients unless a life-threatening
hemorrhage has occurred.
o Transfusion of platelets in these conditions can result in further
thrombosis.
o One unit of apheresis platelets should increase the platelet count
in adults by 30 to 60 × 103 per μL (30 to 60 × 109 per L).
o In neonates, transfusing 5 to 10 mL per kg of platelets should
increase the platelet count by 50 to 100 × 103 per μL (50 to 100 ×
109 per L).
o One apheresis platelet collection is equivalent to six pooled
random donor platelet concentrates.
DISEASES IMPORTANT IN BLOOD BANKING
o Immunodeficiency
o Hypersensitivity
o Monoclonal and polyclonal gammopathies
o Autoimmune disease
o Hemolytic Disease of Newborn (HDN)
Further discuss in Module 1- Lesson 4
IMPORTANCE OF PLATELET CORRECTED COUNT INCREMENT (CCI)
Refractory is defined as failure to achieve acceptable increase in
platelet count following platelet transfusion on at least two
occasions and no alternate cause for refractoriness such as fever,
sepsis, DIC, bleeding, splenomegaly, or drug interaction (e.g.
amphotericin B).
Platelet count must be measured within one hour after transfusion.
Response to platelet transfusion is typically measured within one
hour. However, a sample collected 10 minutes after transfusion
yields similar information and may be easier to obtain routinely. A
post-transfusion platelet count can alternatively be taken at 20
hours.
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
Rule of thumb:
A unit apheresis platelet (or a pool of 6 platelet concentrates)
should achieve an increment of 30,000 to 50,000/μL in an
average adult. Alternative way to determine whether a
patient is refractory is by calculating the corrected count
increment or CCI.
Corrected count increment (CCI) and percent platelet recovery
(PPR) are measures of response to platelet transfusion that
"correct" the count increment for blood volume and number of
platelets transfused.
Corrected count increment (CCI) is a measure of the expected
increase in platelets following a platelet transfusion.
o The “count increment” refers to the increase in platelets
following a transfusion.
o The “correction” is based on the patient’s size and the number of
platelets transfused.
o It is used because CCI is a more accurate measure of
refractoriness, as it adjusts for the number of platelets
transfused and the patient's blood volume.
o It can guide the decision to pursue platelets with improved
compatibility (i.e., HLA-matched platelets).
FORMULA:
CCI = (count increment, per μL) × body surface area (m2) / unit
content
Units (m2/μL) are usually omitted when reporting the result.
Example:
For an adult patient with a BSA of 2.0 m2 whose platelet count rose
from 5 × 109/L to 25 × 109/L after a platelet transfusion containing
4.0 × 1011 platelets:
CCI for above patient = (20 × 109/L × 2.0 m2) / 4.0 = 10,000
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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
APPENDIX

SUPPLEMENTARY NOTES:
1. Glycolysis Reaction:
https://www.youtube.com/watch?v=uWOURkrxpH4

2. Luebering-Rapaport Pathway:
https://www.youtube.com/watch?v=ZB2yaxi4zV0&feature=youtu.be

-END OF TRANSCRIPTION-
Sana’y mahaba din pasensya mo this sem. Kasi paubos na yung sakin.
Charr : ))

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 MODULE 1: Introduction to Immunohematology (part 3)
MLS 18, February 23, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
o The pairing that occurs is specific: adenine is partnered with
Legend: guanine and thymine is partnered with cytosine. Through the
Transcription Bullet process of binding, the strands twist and form a double helix.
Notes/Module Packet Bullet The double helix DNA forms an actual unit of inheritance, a gene.
MODULE OUTLINE
I. Historical Background and the current trends in Transfusion Medicine GENES
II. Blood Preservation Genetics is the study of inheritance (transmission of characteristics)
from parent to offspring
III. Review of Basic Genetics
o It is based on the biochemical structure of chromatin
A. Molecular Genetics o Chromatin is composed of nucleic acids, structural proteins and
B. Population Genetics enzymes.
C. Cellular Genetics The gene is an area of DNA that controls a trait or characteristic.
IV. Review of Fundamentals of Immunology The product of the gene is usually a protein or RNA.
V. Review of Molecular Biology
CENTRAL DOGMA OF MOLECULAR BIOLOGY
LEARNING OUTCOMES First described by James Watson and Francis Crick
1. Explain Mendel’s laws of independent segregation and random Framework for understanding the transfer of sequence
assortment. information between Information-carrying Biopolymers in living
2. Correlate the concepts of dominance and recessive traits with organisms
examples of the inheritance of blood group antigens.
3. Describe the process of mitosis and meiosis. DNA
4. Differentiate some of the ways in which Genetics can be used in Dictate the synthesis of RNA.
modern transfusion laboratory. Produce new DNA by replication
Produce RNA by transcription

MOLECULAR GENETICS: BASIC GENETIC COMPONENTS RNA

DNA Dictate the synthesis of Proteins
Produces proteins by translation
Deoxyribonucleic acid, the main building block of genetic material
Composed of four building blocks: adenine, guanine, cytosine, and
PROTEINS
thymine. These building blocks are bases.
Dictate the function of the cell
o Each base attaches to a sugar molecule and a phosphate molecule.
These building blocks form strings. Following the “stringing” of a
The location of a specific gene on the chromosome is known as its
single strand, there is a “pairing” process where the partner for each
genetic locus.
building block attaches and forms a double strand.
The alternate gene forms for a specific locus are known as alleles.
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o For example, eye color might be determined by allele: blue, brown,
hazel, or green.
A single allele for eye color would be inherited from each parent. This
simple pattern of inheritance of a single allele from each parent will be
applicable for blood group antigen inheritance.
When multiple alleles exist at a single locus, this is known as
polymorphism.
o Some systems are more polymorphic than others.
o A trait that has ten possible alleles at a single locus will be more
polymorphic than a trait with four possible alleles.
o Example of a polymorphic gene is the ABO blood group
Antithetical genes are a pair (or more than 2) of genes that are loated in
different alleles
o Opposite form of gene
POLYMORPHISM of a locus determines the likelihood that two individuals
will be found with an identical allelic composition.
Hundreds of genes comprise each chromosome.
Humans have 23 pairs of chromosomes.
o Each individual inherits one half of his or her chromosomes from
each parent.
o The pairs consist of 22 pairs of autosomal chromosomes and one
pair of sex chromosomes.
o With rare exception, genes that code for blood group antigens and
the production of blood group antigens are found on the autosomal
chromosomes.
GENE EXPRESSION
With inherited physical traits, there are certain patterns of expression.
Gene expression of simple physical traits typically follows one of three
patterns.
A gene is dominant if it is always expressed even when found in
combination with a second gene. In this case, the second gene is not
expressed.
The “silent” recessive gene can only be expressed if two identical genes
are present.
A third level of gene expression is codominant.
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
CODOMINANCE is the expression of two different genes that are
inherited at the same loci on a pair of chromosomes.
With rare exceptions, blood group systems are expressed as codominant
characteristics. For example, when an individual inherits an A gene from
one parent and a B gene from the other parent, the genotype is AB. The
expressed blood type, or phenotype, will be AB. In this case, neither the A
nor B gene is expressed in a dominant manner.
AMORPH are genes that do not code for the production of any
detectable product. These genes appear to be recessive.
When the amorphic gene is inherited in conjunction with an allele that
does produce a detectable product, the detectable product of that allele
is expressed. This allele is not dominant over the amorph nor is the
amorph recessive to the expressed allele. A common example of an
amorph is the gene that codes for the O blood group. When inherited in
a homozygous state, two O genes, there is no detectable product.
HARDY-WEINBERG PRINCIPLE
Mathematical equation to predict allele distribution and frequencies in
a population
Allows for the study of Mendellian inheritance in great detail
o Predictions are not always accurate
Formula:
p+q=1
o p = percentage/ gene frequency for the dominant allele
o q = percentage/ gene frequency for the recessive allele
Also expressed by
p2+2pq+q2=1
o p2 = Homozygous dominant allele (AA)
o q2 = Homozygous recessive allele (aa)
o 2pq = Heterozygous allele (Aa)
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ZYGOSITY
ZYGOSITY describes the similarity or dissimilarity of genes at an allelic
position on two homologous chromosomes.
o When the genes are identical, they are said to be homozygous.
Conversely, when the genes are different, they are said to be
heterozygous.
The concept of zygosity can be related to antigen strength.
o There are some gene products that exhibit dosage.
o When genes are inherited as homozygous (i.e. two identical alleles
coding for the same product), the individual is said to have a
“double dose” of this product. The expression of the trait or
product is stronger when present in the homozygous state.
o In comparison, an individual that inherits a heterozygous set of
genes (i.e. two different alleles coding for two different products) is
said to have a “single dose” of each gene since only one gene for
each product has been inherited. In this case, the expression of each
trait or product is weaker than when two identical genes are
inherited.
o Dosage is exhibited with some blood group systems. Red cells that
are heterozygous for a specific antigen will demonstrate a weaker
reaction than the homozygous cells when they react with the
specific antisera.
This is an important concept in blood group testing.
o Interactions may occur between two genes.
o Interactions are dependent on location of the inherited genes. The
likelihood of an interaction occurring is determined by the
proximity of the genes on the autosomal chromosomes.
Cis-
Relationship of two genes that are inherited on the same
chromosomes
Gene expression is stronger compared to Trans genes
Trans-
Genes that are inherited on different/ seperate chromosomes.
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The steric arrangement of two genes may create a weakened
expression of one of the gene products.
o This is a position effect or steric hindrance of that particular gene
product.
An example of gene interaction exists within the Rh blood group system.
o D and C are specific genes in this system.
o All genes in the Rh system are inherited on the same chromosome.
o When C and D genes are inherited trans to each other (C on one
chromosome and D on the opposite homologous chromosome), the
steric effect will weaken the expression of the D antigen.
o When the genetic relationship is cis (D and C on the same
homologous chromosome), there is no effect on the antigen
expression. For each genetic characteristic, an individual receives a
gene from each of his or her parents. The sum total of both genes is
known as the individual’s genotype.
For example, the inheritance of a “blue” gene from the mother and a
“blue” gene from the father produces a genotype of “blue/blue.” This
individual is homozygous for the “blue” allele.
An individual that inherits a P gene from his or her mother and a Q gene
from his or her father has a genotype of P/Q and is heterozygous for the
P/Q alleles.
NOTE:
The frequency of each genotype is reflective of the degree of
polymorphism within that system.
Systems that display a greater degree of polymorphism (i.e. more
alleles at each locus) will have a lower frequency for each allele than
those that are less polymorphic.
PHENOTYPE
The phenotype is a function of gene expression.
o The product of a recessive gene will not be expressed in a
phenotype.
The dominant gene will produce a detectable product, whether in the
homozygous or heterozygous state.
If the alleles are codominant, both will be expressed in the phenotype.
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o In a system where the alleles are codominant, an individual with a
genotype of Z/Y will have a phenotype of ZY while an individual with
the genotype Z/Z will have a phenotype of Z.
POPULATION GENETICS: MENDEL’S LAWS
Gregor Mendel is recognized as the father of genetics.
o The results of his genetic research can be directly applied to the
inheritance of blood group antigens.
o Mendel was an Austrian monk and mathematician who used sweet
pea plants growing in a monastery garden to study physical traits in
organisms and how they are inherited.
He first described the law known as independent segregation which
refers to the transmission of a trait between generations in a
predictable fashion.
He determined the physical traits to be due to factors he called
elementen (eventually known as genes) within the cell.
He studied the inheritance of several readily observable pea plant (a
good model organism) characteristics
o notably flower color, seed color, and seed shape—and based his
first law of inheritance, the law of independent or random
segregation, on these results.
MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
o The F2 generation has a ratio of three red-flowered plants to one
white flowered plant.
o This is because the plants that have the R gene, either RR
homozygous or Rr heterozygous, will have red flowers because the
red gene is dominant.
o Only when the red gene is absent and the white gene occurs in
duplicate, as in the rr homozygous white-flowered plant, will the
recessive white gene expression be visible as a phenotype.
This illustrates Mendel’s first law, the law of independent segregation.
o Therefore, each gene is passed on to the next generation on its
own.
o Specifically, Mendel’s first law shows that alleles of genes have no
permanent effect on one another when present in the same plant
but segregate unchanged by passing into different gametes.

MENDEL’S 1ST LAW: INDEPENDENT SEGREGATION

The first generation in the study, called the parental, pure, or P1
generation, consisted of all red or all white flowers that bred true for
many generations.
The plants were either homozygous for red flowers (RR, a dominant
trait; dominant traits are usually written with uppercase letters). or
homozygous for white flowers (rr, a recessive trait; recessive traits are
usually written with lowercase letters).
o When these plants were crossbred, the second generation, called
first-filial, or F1, had flowers that were all red.
o Thus the dominant trait was the only trait observed.
When plants from the F1 generation were crossbred to each other, the
second-filial, or F2, generation, of plants had flowers that were red and
white in the ratio of 3:1.
o All the plants from the F1 generation are heterozygous (or hybrid)
for flower color (Rr).
An intermediate situation can also occur when alleles exhibit partial
dominance.
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o This is observed when the phenotype of a heterozygous organism is
a mixture of both homozygous phenotypes seen in the P1
generation.
o An example of this is plants with red and white flowers that have
offspring with pink flowers or flowers that have red and white
sections.
o It is important to remember that although the phenotype does not
show dominance or recessive traits, the F1 generation has the
heterozygous genotype of Rr. It is essential to understand how a
genotype can influence a phenotype, and using flower color is a
good basic model system to study this.
Unlike the flower color of many types of plants, most blood group genes
are inherited in a codominant manner.
o In codominance, both alleles are expressed, and their gene
products are seen at the phenotypic level.
o In this case, one gene is not dominant over its allele, and the protein
products of both genes are seen at the phenotypic level.
o An example of this is the MNSs blood group system, in which a
heterozygous MN individual would type as both M and N antigen
positive.
o Blood group antigens are inherited in this fashion with only rare
variations. The outcome of this type of inheritance can be predicted
by a Punnett Square.
PUNNETT SQUARES
Prediction of possible genotypes and phenotypes in an offspring can
be performed using a Punnett Square.
In order to use the Punnett Square as a predictive tool, the user must
know the exact genotype or inferred genotype of both parents.
Using this information, a Punnett Square can be set up to predict the
likelihood of the genotype and phenotype of the offspring.
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
PEDIGREE CHARTS
A method for tracking family history and inheritance patterns is a
pedigree chart.
It is a visual representation of the parents and the possible genotypes
and phenotypes for the offspring.
This chart illustrates the inheritance patterns of all the family
members and can be used for visualization of inherited traits,
including blood group systems.
o The pedigree chart is useful since it is more detailed than the
Punnett Square.
MENDEL’S 2ND LAW: INDEPENDENT ASSORTMENT
The second concept of Mendelian genetics/ Mendel’s second law
States that genes for different traits are inherited separately from
each other.
o This allows for all possible combinations of genes to occur in
the offspring.
o Specifically, if a homozygote that is dominant for two
different characteristics is crossed with a homozygote that is
recessive for both characteristics, the F1 generation consists
of plants whose phenotype is the same as that of the
dominant parent.
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o However, when the F1 generation is crossed in the F2
generation, two general classes of offspring are found.
One is the parental type; the other is a new phenotype called a
reciprocal type and represents plants with the dominant feature of
one plant and the recessive feature of another plant.
o Recombinant types occur in both possible combinations.
Mendel formulated this law by doing studies with different types of
seeds produced by peas and noted that they can be colored green or
yellow and textured smooth or wrinkled in any combination.
Genes located on different chromosomes are inherited separately
and expressed discreetly from one another.
o In most cases, this applies to the blood group antigens.
Even blood groups that are inherited on the same chromosome, such
as Rh and Duffy (Chromosome #1), are inherited as separate entities.
o One is not dependent on the other for inheritance or
expression.
o Separate genes account for the inheritance of blood group
system antigens.
LINKED GENES
Genes are in very close proximity, that are inherited as a unit rather
than as separate entities.
Independent assortment does not occur with antigens that are
linked.
For example, the two genes that code for M/N and S/s antigen pairs
are very close to one another. They are inherited as a “package”
from each parent.
Two genes are linked if the products appear with greater
frequency than expected if inherited independently
o This deviance from anticipated frequencies is termed linkage
disequilibrium.
LINKED GENES
Set of genes inherited via one of the two parental gamates
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
Linkage can be determined by examining the frequency of the antigen
or product in the general population.
A more complex example of linkage disequilibrium occurs within the
HLA antigen system.
o The HLA-A and HLA-B antigens are more closely linked than
the M/N and S/s genes.
o An individual’s haplotype is the set of HLA antigens inherited
from one parent.
o For example, the mother of an offspring may be typed as HLA-
A3, A69; B7, B45. This mother may pass along to herprogeny
the haplotype A3, B7, or A69, B45 but neverA3, B45, or A69,
B7.
NOTE:
Aside from Mendel’s work other major areas of population genetics are
due to:
 The pioneering work of Linnaeus and Darwin
 Hardy-Weinberg Principle
 Inheritance patterns
CONCEPTS OF DOMINANCE AND RECESSIVE TRAITS
A variation on incomplete dominance is wherein both alleles for the
same characteristic are simultaneously expressed in the
heterozygote.
An example of codominance occurs in the ABO blood groups of
humans. The A and B alleles are expressed in the form of A or B
molecules present on the surface of red blood cells.
Homozygotes (IAIA and IBIB) express either the A or the B phenotype,
and heterozygotes (IAIB) express both phenotypes equally.
o The IAIB individual has blood type AB.
In a self-cross between heterozygotes expressing a codominant trait,
the three possible offspring genotypes are phenotypically distinct.
o However, the 1:2:1 genotypic ratio characteristic of a
Mendelian monohybrid cross still applies.
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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
MULTIPLE ALLELES
Although individual humans (and all diploid organisms) can only have
two alleles for a given gene, multiple alleles may exist at the
population level, such that many combinations of two alleles are
observed.
o Mendel implied that only two alleles, one dominant and one
recessive, could exist for a given gene, which is an
oversimplification.
Note that when many alleles exist for the same gene, the convention is
to denote the most common phenotype or genotype in the natural
population as the wild type (often abbreviated “+”).
All other phenotypes or genotypes are considered variants (mutants) of
this typical form, meaning they deviate from the wild type.
o The variant may be recessive or dominant to the wild-type allele.
An example of multiple alleles is the ABO blood-type system in humans.
o In this case, there are three alleles circulating in the population. The
IA allele codes for A molecules on the red blood cells, the IB allele Notice that instead of three genotypes, there are six different
codes for B molecules on the surface of red blood cells, and the i genotypes when there are three alleles.
allele codes for no molecules on the red blood cells. o The number of possible phenotypes depends on the dominance
o In this case, the IA and IB alleles are codominant with each other relationships between the three alleles.
and are both dominant over the i allele.
o Although there are three alleles present in a population, each
individual only gets two of the alleles from their parents.
o This produces the genotypes and phenotypes shown in the table GENOME
below. It takes two gametes to make a fertilized egg with the correct (2N)
number of chromosomes in the nucleus of a cell.
DOSAGE EFFECT o Therefore, each parent contributes only half (1N) of the inherited
Excess unbound immunoglobulin that leads to Prozone effect genetic information, or genes, to each child.
An antibody gives a stronger reaction with RBC double-dosed for the In order to be completely healthy, each child must have the correct
target antigen number of genes and chromosomes (2N), without major mutations
affecting necessary biochemical systems.
A homozygous recessive trait may be expressed more strongly than a
o At the smallest level, genes are composed of discrete units of DNA
heterozygous trait
arranged in a linear fashion, similar to a strand of pearls, with
Affected by inheritance of genotype
structural proteins wrapped around the DNA at specific intervals to
pack it into tightly wound bundles.
PROZONE EFFECT
effectiveness of antibodies to form immune complexes is sometimes
impaired when concentrations of an antibody or an antigen are very
high

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CHROMOSOMES are DNA that is organized at a higher level
o Each chromosome being one incredibly long strand of duplex
(double-stranded) DNA.
A GENE is a section, often very large, of DNA along the chromosome. It
controls a trait or characteristic
o The specific sequence of nucleotides and the location on the
chromosome determines a gene.
o In addition, each gene has specific and general sequences that
occur upstream (before the start site) and downstream (after the
termination signals) that contribute to how the gene functions.
RNA is the product of a gene, that codes for proteins
A LOCUS (plural = loci) is the specific location of a gene on a
chromosome
ALLELES are specific locus formed by an alternate gene. They one or
several different forms of the same gene at each locus
GENOTYPE is the sequence of DNA that is inherited.
PHENOTYPE is anything that is physically manifested; the ratio of
muscle fibers; the level of hormones produced; and such obvious traits
as eye, skin, and hair color.
KEEP IN MIND that more than one gene can affect a particular trait (part
of a phenotype), such as the height of an individual; all relevant genes can
be considered as part of the genotype for that trait. Depending on the
alleles inherited, an organism can be either homozygous or heterozygous
for a specific trait. The presence of two identical alleles results in a
homozygous genotype (i.e., AA), and the phenotype is group A blood. On
the other hand, the inheritance of different alleles from each parent gives
a heterozygous genotype.
Another important concept is that of the “silent” gene, or amorph, and
the term hemizygous.
An AMORPH is a gene that does not produce any obvious, easily
detectable traits and is seen only at the phenotypic level when the
individual is homozygous for the trait.
HEMIZYGOUS refers to the condition when one chromosome has a copy
of the gene and the other chromosome has that gene deleted or absent.
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
CELLULAR GENETICS: CELL DIVISION
In eukaryotic cells such as human cells, the cell cycle is divided into four
distinct stages and is represented by a clock or circular scheme
indicating that it can repeat itself or can be stopped at any one point in
the cycle.
G0 / resting stage The first step, the state of cells not actively
dividing.
G1 pre-replication stage
S Phase The step at which DNA is synthesized
G2 Postreplication stage
M phase mitosis occurs
Chromosomes are in the interphase stage of mitosis in the span from
G0 to the end of the G2 phase.
Cells that are completely mature and no longer need to divide to
increase their numbers, such as nerve cells, can remain in the G0 stage
for a very long time.
It is a hallmark of cancer cells, such as the transformed cells seen in the
various leukemias and solid tumors, that they can go through the stages
of cell division much faster than non-transformed, normal cells and
therefore outgrow them.
o In this way, they take up the bulk of nutrients needed by the
nontransformed cells and crowd them out of existence and
potentially overgrow the adult organism in which they occur.
MITOSIS
The process by which cells divide to create identical daughter cells is
called mitosis
During cell division, the chromosomes are reproduced in such a way
that all daughter cells are genetically identical to the parent cell.
o Without maintaining the same number and type of chromosomes,
the daughter cells would not be viable.
The chromosomes are duplicated, and one of each pair is passed to the
daughter cells.
During the process of mitosis, quantitatively and qualitatively identical
DNA is delivered to daughter cells formed by cell division.
The complex process of mitosis is usually divided into a series of stages,
characterized by the appearance and movement of the chromosomes.
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DNA is in the form of chromatin and is dispersed
throughout the nucleus. This is the stage of the DNA
INTERPHASE
when cells are not actively dividing. New DNA is
synthesized by a process called replication.
The chromatin condenses to form chromosomes. In
PROPHASE
prophase, the nuclear envelope starts to break down.
The chromosomes are lined up along the middle of
the nucleus and paired with the corresponding
METAPHASE
chromosome. In this stage, chromosome preparations
are made for chromosome analysis in cytogenetics.
The cellular spindle apparatus is formed and the
chromosomes are pulled to opposite ends of the cell.
ANAPHASE
The cell becomes pinched in the middle, and cell
division starts to take place
the cell is pulled apart, division is complete, and
the chromosomes and cytoplasm are separated
TELOPHASE
into two new daughter cells.
MEIOSIS
A different process is used to produce the gametes or sex cells.
The process results in four unique, rather than two identical, daughter
cells.
o The uniqueness of the daughter cells generated with meiosis allows
for great genetic diversity in organisms and controls the number of
chromosomes within dividing cells.
If cells with 2N chromosomes were paired, the resulting daughter cells
would have 4N chromosomes, which would not be viable. Therefore,
gametes carry a haploid number of chromosomes, 1N, so that when
they combine, the resulting cell has a 2N configuration.
MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
MEIOSIS I The first stages of meiosis are nearly identical to
those in mitosis, in which chromatin is
condensed, homologous chromosomes are
paired in prophase, and chromosomes are
aligned along the center of the cell.
However, there is no centromere division, and at
anaphase and telophase, the cell divides and
enters interphase once again, in which there is
no replication of DNA.
PROPHASE II Chromosomes are condensed
METAPHASE II centromeres divide and chromosomes line up along
the center again
ANAPHASE II Chromosomes are pulled to opposite ends of the cell
TELOPHASE II The two cells divide, giving rise to four 1N daughter
cells.
In addition, during meiosis, crossing over and recombination can
happen between maternal- and paternal-derived chromosomes.
o This allows for the creation of new DNA sequences that are
different from the parent strains. Combined with random
segregation, it is possible to have very large numbers of new DNA
sequences.
o In humans with 23 pairs of chromosomes, the total possible number
is several million.

Meiosis only occurs in the germinal tissues and is important for

reproduction.
o Without the complicated process of meiosis, there would be no
change from generation to generation, and evolution would not
occur or happen too slowly for organisms to adapt to environmental
changes.

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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
PATTERNS OF INHERITANCE
AUTOSOMAL DOMINANT
Genes expressed with equal frequency in males and females
Non-sex chromosome
One parent can transmit a trait to a son or daughter

SEX-LINKED DOMINANT
Trait carried on the X chromosome
Father to daughter transmission only
o No father to son transmission

SEX-LINKED RECESSIVE
Trait carried on the X chromosome
Carrier mother to son transmission
Traits are exhibited most commonly in males
o Example: Hemophilia A

-END OF TRANSCRIPTION-
….for now (0_0)

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 MODULE 1: Introduction to Immunohematology (part 4)
MLS 18, February 23, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
Cellular Immunity
Legend: Mediated by various IS cells, such as macrophage, T cells, and
Transcription Bullet dendritic cells.
Notes/Module Packet Bullet Lymphokines- are other effector molecules that play critical roles in
MODULE OUTLINE the cellular system by activating and deactivating different cells,
I. Historical Background and the current trends in Transfusion Medicine which allows cells to communicate throughout the host body. It
II. Blood Preservation includes cytokines and chemokines.
III. Review of Basic Genetics
Humoral Immunity
IV. Review of Fundamentals of Immunology
Consists of the fluid parts of the IS, such as antibodies and
V. Review of Molecular Biology complement components found in plasma, saliva, and other
secretions.
LEARNING OUTCOMES Antibody- also known as immunoglobulins (immune because of
At the end of this module, the student shall be able to: their function and globulin because they are a type of globular
1. Outline the cellular and humoral components of the immune soluble protein). Its function is to bind to foreign molecules called
system. antigens.
2. Describe the characteristics of immunoglobulins in relation to its o Antigen-antibody reactions are specific. Only one antibody
significance in transfusion medicine. reacts with one antigen or one part (an epitope/antigenic
3. List the methods used in the blood bank to detect antibodies and determinant) of a complex antigen.
complement bound to red blood cells.
4. Identify the various factors that affect agglutination reactions. Innate Immunity
5. Describe some of the common diseases that can affect blood bank
It is the immediate line of immune defense.
testing.
First line of defense, not specific.
It doesn’t need modification to function to the same antigen
WHAT IS IMMUNITY? Physical barriers that includes are:
Refers to the process by which a host organism protects itself from o skin
attacks by external and internal agents. o mucous membrane
Confers protection from non-self and abnormal self-elements which o cilia lining
are controlled at different levels. o cough mechanism
The response and elimination of organisms and unwanted cells is
accomplished through cellular and/or humoral mechanisms. Biochemical barriers
o bactericidal enzyme (lysozymes, RNase, fatty acid, sweat,
digestive enzyme, stomach acid, and vaginal low ph)

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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
Acquired/ Adaptive Immunity Characteristics of Immunoglobulins
Acquired refers to the fact that the immunity is acquired via specific A complex protein produced by plasma cells, with specificity to
contact with a pathogen or aberrant cell. antigens that stimulate their production.
Adaptive refers to the ability to adapt to and destroy new complex 20% Immunoglobulins present to an individual
pathogens, although it must first react to them through complex Antibodies bind antigen, fix complement, facilitate phagocytosis and
recognitions processes. neutralize toxic substances in the circulation.
They are classified according to the molecular structure of their
heavy chains. Five classifications:
CELLS AND ORGANS OF THE IMMUNE SYSTEM
Different types of immune system cells can be distinguished by the IgG Most concentrated in serum (80%) of total
membrane markers they possess known as clusters of differentiation (gamma heavy chain) serum Ig.
(CD) markers and are detected by immunotyping methods. secreted by the plasma in the blood
acts in long term immune system
Types of Cells: ability to cross the placenta
1. Lymphocytes (B cell/T cell) IgA Is next with 13% (although majority it is
2. Plasma cell (alpha heavy chain) major found in body secretions)
3. Natural Killer cells found in mucous, saliva, tears
present in secretions
B cells IgM 6%
Antibodies are secreted by mature B cells called plasma cells and bind (mu heavy chain) responsible for the early stages of
to antigens in specific manner. immunity
When the receptor on the B cell reacts with a specific antigen and 1gD 1%
recognizes it, the B cell is activated to divide. The cells produced from (delta heavy chain) receptor in B cells
this rapid division mature into plasma cells and memory B cells. triggers or activates basophils or mast
Memory B cells have antibody on their surfaces that is of the same cells
conformation as that of the B cell from which they were derived. found in the membrane of immature B
cell
helps in the activation of plasma cell
T cells
IgE Least common, <1%
(epsilon heavy chain) present against allergic reaction
They require help in the form of cell membrane proteins known as
protect against parasitic worms
major histocompability complex (MHC) molecules
MHC 2 classes
Immunoglobulin Structure
o Class 1: found in all nucleated cells
o Class 2: found in APC (Antigen Presenting Cell) Basic Ig structural unit is composed of four polypeptide chains; two
The MHC genes determine the human leukocyte antigens (HLA) identical light chains (MW=approximately 22,500 daltons) and two
present on leukocytes and other cells; they have been known for identical heavy chains (MW= app. 50,000 to 75,000 daltons).
many years to cause rejection of tissue grafts. Covalent disulfide bonding holds the light and heavy chains
T cell mediated immunity involve response against fungal and viral together. The covalent disulfide linkages in Ig molecules provide
infection, intracellular parasite, tissue grafts, and tumor.
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greater structural strength than hydrogen bonding and van der
Waals forces. However, they limit the flexibility of the Ig molecule.
The heavy chains are also interconnected by disulfide linkages in
the hinge region of the molecule.
Two types of light chains: kappa and lambda (both are present in
all classes of immunoglobulins, regardless of the heavy chain
classification).
Ig molecules are protein and therefore have two terminal regions:
the amino (-NH2) terminal and the carboxyl (-COOH) terminal.
Enzyme papain splits the antibody molecule at the hinge to give
three fragments: one crystallizable Fc fragment and two antigen
binding fragments, Fab.
Domains of the immunoglobulins are the regions of the light and
heavy chains that are folded into compact globular loop structures.
IgG
Immunoglobulins Significant for Blood Bank
All immunoglobulins can be significant for transfusion medicine;
however, IgG, IgM and IgA have the most significance.
Most clinically significant antibodies that react at body
temperature (37⁰C) are IgG isotype and are capable of destroying
transfused antigen-positive RBCs, causing anemia and transfusion
reactions of various severities.
IgM
IgM antibodies are most commonly encountered as naturally
occurring antibodies in the ABO system and are believed to be
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
produced in response to commonly occurring antigens like intestinal
flora and pollen grains.
o Other blood groups such as Lewis, Ii, P and MNS may also
produce IgM antibodies, which usually react best at ambient
temperature (22⁰C to 24⁰C)
The primary testing problem encountered with IgM antibodies is
that they can interfere with the detection of clinically significant IgG
antibodies by masking their reactivity. Unlike IgG, IgM exist both
monomeric and polymeric forms (as pentamers) containing a J
(joining) chain.
The pentameric form can be dissociated through cleavage of
covalent bonds interconnecting the monomeric subunits and the J
chain by chemical treatment with sulfhydryl reducing reagents such
as ß-2-mercaptoethanol (2-ME) or dithiothreitol (DTT). These
reagents can distinguish a mixture of IgM and IgG antibodies
because only IgM is removed by the use of such compounds;
therefore, the removal allows unexpected IgG to be detected.
IgG antibodies are significant in transfusion medicine because they
are the class of immunoglobulins that are made in response to
transfusion with non-self and therefore are incompatible RBCs and
other blood products.
IgG antibodies are important in hemolytic disease of the newborn
(HDN) because antibodies can be formed in response against
alloantigens on fetal RBCs that enter the mother’s circulation,
usually during delivery.
IgG has the greatest number of subclasses: IgG1, IgG2 , IgG3 , and
IgG4 , and all four are easily separated by electrophoresis.
o The small differences in the chemical structure within the
constant regions of the gamma heavy chains designate the
various subclasses, and the number of disulfide bonds
between the two heavy chains in the hinge region of the
molecule constitutes one of the main differences between
subclasses.
Functional differences between the subclasses include the ability to
fix complement and cross the placenta.
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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
IgA IgD
Like IgM, IgA exists in two main forms, a monomer and polymer IgD, present as less than 1 percent of serum immunoglobulins,
form, as dimers or trimers composed of two or three identical appears to have functions that deal primarily with maturation of B
monomers, respectively, joined by a J chain. cells into plasma cells. IgD is usually found bound to the membrane
IgA is located in different parts of the IS depending on subclass. of immature B cells. Therefore, IgD may be necessary for regulatory
o Serum IgA is found in both monomeric and polymeric forms; roles during B-cell differentiation and antibody production but is
Serum IgA can cause problems when transfused in probably the least significant for blood banking.
plasma product to patient having deficient IgA can cause
fatal anaphylaxis and can induce IgG rbc hemolysis CHARACTERISTICS IgG1 IgG2 IgG3 IgG4
o Secretory IgA is usually found in the mucosal tissues of the Proportion of 65-70 23-28 4-7 3-4
body. total serum IgG
• Its polymer form acquires a glycoprotein secretory (%)
component as it passes through epithelial cell walls of Complement ++ + +++ 0
mucosal tissues and appears in nearly all body fluids, fixation (classic
including saliva, tears, bronchial secretions, prostatic pathway)
fluid, vaginal secretions, and the mucous secretions of Binding to +++ ++ +++ +
the small intestine. macrophage FC
Also, anti-IgA antibodies can cause severe problems if transfused in receptors
plasma products to patients who are deficient in IgA, as potentially
Ability to cross + + + +
fatal anaphylaxis can result. Another reason for the importance of
placenta
IgA is that IgA can increase the effect of IgG-induced RBC hemolysis.
Dominant antibody activities:
Anti-Rh ++ 0 + +
IgE Anti-factor VII 0 0 0 +
IgE is normally found only in monomeric form in trace
Anti-dextran 0 + 0 0
concentrations in serum, about 0.004% of total immunoglobulins,
Anti-Kell + 0 0 0
and is important in allergic reactions.
Anti-Duffy + 0 0 0
The Fc portion of the IgE molecule attaches to basophils and mast
cells and facilitates histamine release when an allergen binds to the Anti-platelet 0 0 + 0
Fab portion of the molecule and cross- links with a second molecule Biological half-life 21 21 7-8 21
on the cell surface. (days)
Histamine is critical for bringing about an allergic reaction.
o Although hemolytic transfusion reactions are not caused by Immunoglobulin variations
IgE, urticaria may occur because of the presence of IgE Isotype (class variation)
antibodies. Refers to variants present in all members of a species, including the
o Because IgE causes transfusion reactions by release of different heavy and light chains and the different subclasses.
histamines, patients who have several allergic reactions to
blood products can be pretreated with antihistamines to
Allotypic
counteract the response when receiving blood products.
Present primarily in the constant region not all variants occur in all
members of a species.
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Idiopathic
which determines the antigen-binding specificity regions and is
specific for each antibody molecule.
Immunoglobulin Fc Receptors
Macrophages and monocytes have receptors for the attachment
and can bind CH3 domain of the Fc portion.
Only the IgG1 and IgG3 subclasses are capable of attachment to
phagocytic receptors. This is one way that incompatible RBCs coated
with IgG antibody are removed by phagocytosis.
The other phagocytic cells with Fc receptors include neutrophils, NK
cells and mature B cells.
Complement system
Is a complex group of over 20 circulating and cell membrane
proteins that have a multitude of functions within the immune
response
Primary roles include direct lysis of cells, bacteria, and enveloped
viruses as well as assisting with opsonization to facilitate
phagocytosis.
Another role is production of peptide fragment split products, which
play roles in inflammatory responses such as increased vascular
permeability, smooth muscle contraction, chemotaxis, migration
and adherence.
The complement proteins are activated in a cascade of events
through three main pathways: the classical, alternative and lectin
pathways.
The three pathways converge at the activation of the complement
C3:
Classical Pathway
The classical pathway is activated by binding of an antigen with an
IgM, IgG1 or IgG3 antibody.
Alternative Pathways
Alternative pathway is activated by high molecular weight
molecules with repeating units found on the surfaces of target cells.
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Lectin Pathway
The lectin pathway is activated by attachment of plasma mannose-
binding lectin(MBL) to microbes. MBL in turn activates proteins of
the classical pathway.
Characteristics of Antigens
The immune response is initiated by the presentation of an antigen
(can initiate formation of and react with an antibody) or
immunogen (can initiate an immune response) to the IS and the IS
determining that the antigen is non-self.
The immune reaction to any immunogen, including antigens, is
determined by the host response as well as by several biochemical
and physical characteristics of the immunogen.
Properties such as size, complexity, conformation, charge,
accessibility, solubility, digestibility, and biochemical composition
influence the amount and type of immune response.
Molecules that are too small cannot stimulate antibody production.
Immunogens having a molecular weight (MW) less than 10,000
daltons (D), for example, are called haptens and usually do not elicit
an immune response on their own. A hapten coupled with a carrier
protein having a MW greater than 10,000 D, however, can produce
a reaction.
The biochemical composition of the stimulus plays a role in immune
stimulation.
Remember that RBC antigens are very diverse in structure and
composition and may be proteins (such as the Rh, M, and N blood
group substances) or glycolipids (such as the ABH, Lewis, Ii, and P
blood group substances).
Human leukocyte antigens (HLAs) are glycoproteins. Because of
these differences in structure, conformation, and molecular nature,
not all blood group substances are equally immunogenic in-vivo.
Characteristics of Blood Group
Polyclonal and Monoclonal Antibodies
Polyclonal or serum antibodies and are produced in response to a
single antigen with more than one epitope.
Monoclonal antibodies- are produced by isolating individual B cells
from a polyclonal population and propagating them in cell culture
with hybridoma technology
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The supernatant from the cell culture contains antibody from a single
type of B cell, clonally expanded, and therefore with the same
variable region and having a single epitope specificity.
This results in a monoclonal antibody suspension. Monoclonal
antibodies are preferred in testing because they are highly specific,
well characterized, and uniformly reactive. Most reagents used today
are monoclonal in nature.
Naturally Occurring and Immune Antibodies
RBC antibodies are considered naturally occurring when they are
found in the serum of individuals who have never been previously
exposed to RBC antigens by transfusion, injection, or pregnancy.
These antibodies are probably produced in response to substances
in the environment that are highly similar to RBC antigens such as
pollen grains and parts of bacteria membranes.
Most naturally occurring antibodies are IgM cold agglutinins, which
react best at room temperature or lower; activate complement, and
when active at 37⁰C may be hemolytic. In blood banking, the
common naturally occurring antibodies react with antigens of the
ABH, Hh, Ii, Lewis, MN, and P blood group systems.
RBC antibodies are considered immune when found in the serum of
individuals who have been transfused or pregnant. These antigens
are not generally found in nature, and their molecular makeup is
unique to human RBCs.
Most immune RBC antibodies are IgG antibodies that react best at
37⁰C and require the use of antihuman globulin sera (Coombs sera)
for detection. The most common immune antibodies encountered
in testing include those that react with the Rh, Kell, Duffy, Kidd, and
Ss blood group systems.
Unexpected Antibodies
All other antibodies directed against RBC antigens are considered
unexpected and must be detected and identified before blood can
be safely transfused, even if antibodies react at room temperature
or only with Coombs sera.
Also, autoreactive antibodies must be investigated. The reactivity of
unexpected antibodies is highly varied and unpredictable as they
may be either isotype IgM or IgG; rarely, both may be present in the
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
same sample. These antibodies may be able to hemolyze,
agglutinate, or sensitize RBCs.
Some antibodies require special reagents to enhance their reactivity
and detection. Due to the enormous polymorphism of the human
population, a diversity of RBC antigens exists, requiring a variety of
standardized immunologic techniques and reagents for their
detection and identification.
Alloantibodies and Autoantibodies
Alloantibodies
Alloantibodies are produced after exposure to genetically different,
or non-self, antigens of the same species, such as a different RBC
antigen after transfusion.
Transfused components may elicit the formation of alloantibodies
against antigens (red cell, white cell and platelets) not present in the
recipient.
Autoantibodies
Autoantibodies are produced in response to self-antigens. They can
cause reactions in the recipient if they have a specificity that is
common to the transfused blood.
Some autoantibodies do not have a detectable specificity and are
referred to as pan- or polyagglutinins.
Autoantibodies can react at different temperatures, and cold or
warm autoantibodies may both be present.
Characteristics of Antigen-Antibody Reactions
The antigen-binding site of the antibody molecule is uniquely
designed to recognize a corresponding antigen; this antibody amino
acid sequence cannot be changed without altering its specificity.
The extent of the reciprocal relationship, also called the fit between
the antigen and its binding site on the antibody, is often referred to
as a lock and key mechanism.
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Detection of RBC Antigen-Antibody Reactions
Various factors influence detection of RBC antigen-antibody
reactions. These include having a correct sample (a sample that is
stored under the right conditions) and the proper reagents
performing the correct test and understanding how the test should
be done.
Traditional Laboratory Methods
Commonly used techniques including hemagglutination (a special
type of agglutination), precipitation, agglutination inhibition, and
hemolysis. Other techniques such as radioimmunoassay (RIA),
enzyme-linked immunosorbent assay (ELISA) or enzyme
immunoassay (EIA), and immunofluorescence (IF), which quantifies
antigen or antibody with the use of a radioisotope, enzyme, or
fluorescent label, respectively, may be used in automated or
semiautomated blood banking instrumentation.
Hemagglutination
Are methods for the analysis of blood group antigen-antibody
responses and typing for ABO, Rh, and other blood group antigens is
accomplished by red cell agglutination reactions.
o Agglutination is a straightforward process and can be shown to
develop in two stages. In the first stage, called sensitization,
antigen binding to the antibody occurs.
o Epitopes on the surfaces of RBC membranes combine with the
antigen combining sites (Fab region) on the variable regions of
the immunoglobulin heavy and light.
o Antigen and antibody are held together by various non-covalent
bonds, and no visible agglutination is seen at this stage. In the
second stage, a lattice-type structure composed of multiple
antigen-antibody bridges between RBC antigens and antibodies
is formed. A network of these bridges forms, and visible
agglutination is present during this stage.
Precipitation reaction
The development of an insoluble antigen-antibody complex, resulting
from the mixing of equivalent amounts of soluble antigen and
antibody.
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Agglutination inhibition
A method in which a positive reaction is the opposite of what is
normally observed in agglutination. Agglutination is inhibited when
an antigen-antibody reaction has previously occurred in a test system
and prevents agglutination.
The antigen and antibody cannot bind because another substrate has
been added to the reaction mixture and blocks the formation of
antigen-antibody agglutinates
RIA, ELISA and IF Techniques
radioimmunoassay (RIA), enzyme-linked immunosorbent assay
(ELISA) or enzyme immunoassay (EIA), and immunofluorescence (IF)
techniques are immunologic methods based on quantization of
antigen or antibody by the use of a radioisotope, enzyme, or
fluorescent label, respectively.
Factors That Influence Agglutination Reactions
Typical of most biochemical reaction systems, agglutination reactions
are influenced by the concentration of the reactants (antigen and
antibody) as well as by factors such as pH, temperature, and ionic
strength. The surface charge, antibody isotype, RBC antigen dosage,
and the use of various enhancement media, antihuman globulin
reagents, and enzymes are all important in antigen-antibody
reactions.
Centrifugation
It decreases reaction time by increasing the gravitational forces on
the reactants as well as bringing reactants closer together. Under the
right centrifugation conditions, sensitized RBCs overcome their
natural repulsive effect (zeta potential) for each other and
agglutinate more efficiently
Antigen-Antibody Ratio
Antigen and antibody have optimal concentrations; in ideal reactive
conditions, an equivalent amount of antigen and antibody binds. Any
deviation from this decreases the efficiency of the reaction and a loss
of the zone of equivalence between antigen and antibody ratio that
is necessary for agglutination reactions to occur.
Page 7 of 10

o An excess of unbound immunoglobulin leads to a prozone
effect, and a surplus of antigen (antigen-binding sites) leads
to a postzone effect.
o Another reason antigen amount may be altered is due to
weak expression of antigen on RBCs (dosage effect). Weak
expression occurs as a result of the inheritance of genotypes
that give rise to heterozygous expression of RBC antigens
and resultant weaker phenotypes.
Effect of pH
The ideal pH of a test system for antigen-antibody reactions is a
range between 6.5 and 7.5, which is similar to the pH of normal
plasma or serum. Exceptions include some anti-M and some Pr(Sp1)
group antibodies that show stronger reactivity below pH 6.5.
Temperature
Different isotypes of antibodies may exhibit optimal reactivity at
different temperatures. IgM antibodies usually react optimally at
ambient temperatures or below 22⁰C, whereas IgG antibodies usually
require 37⁰C.
Ig type
Examples of IgM antibodies that have importance in blood banking
include those against the ABH, Ii, MN, Lewis (Lea , Leb ), Lutheran
(Lua ), and P blood group antigens.
Important IgG antibodies are those directed against Ss, Kell (Kk, Jsa ,
Jsb , Kpa , Kpb ), Rh (DCEce), Lutheran (Lub ), Duffy (Fya , Fyb ), and
Kidd (Jka , Jkb ) antigen.
Enhancement media
Agglutination reactions for IgM antibodies and their corresponding
RBC antigens are easily accomplished in saline medium as these
antibodies usually do not need enhancement or modifications to
react strongly with antigens.
Many of the commercially available enhancement media accomplish
this by reducing the zeta potential of RBC membranes. The net
negative charge surrounding RBCs (and most other human cells) in a
cationic media is part of the force that repels RBCs from each other
and is due to sialic acid molecules on the surface of RBCs.
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MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
Factors that influence Agglutination Reactions: ENHANCEMENT MEDIA
Protein Media
Colloidal substances, or colloids are a type of clear solution that
contains particles permanently suspended in solution. Colloidal
particles are usually large moieties like proteins as compared with
the more familiar crystalloids, which usually have small, highly
soluble molecules, such as glucose, that are easily dialyzed.
The colloidal solutes can be charged or neutral and go into solution
because of their microscopic size. Colloids include albumin,
polyethylene glycol (PEG), polybrene, polyvinylpyrrolidone (PVP),
and protamine. These substances work by increasing the dielectric
constant (a measure of electrical conductivity), which then reduces
the zeta potential of the RBC.
LISS Media
Low Ionic Strength Solution (LISS) Media (LISS), or low salt media,
generally contain 0.2 percent sodium chloride.
They decrease the ionic strength of a reaction medium, which
reduces the zeta potential and therefore allows antibodies to react
more efficiently with RBC membrane antigens.
LISS media are often used because they result in an increased rate of
antibody uptake during sensitization and a decreased reaction
incubation time (from 30 to 60 minutes to 5 to 15 minutes as
compared with protein potentiators such as albumin).
However, they can result in false-positive reactions and may cause
wasted time if reactions have to be repeated with albumin.
Polyethylene Glycol (PEG)
Polyethylene Glycol (PEG) and Polybrene are macromolecule
additives used with LISS to bring sensitized RBCs closer to each other
to facilitate antibody cross-linking and agglutination reactions. T
they are often used in place of albumin and have some advantages
and possible drawbacks. Polybrene can detect ABO incompatibility as
well as clinically significant IgG alloantibodies, whereas PEG produces
very specific reactions with reduction in false positive or nonspecific
reactions.
PEG is considered to be more effective than albumin, LISS, or
polybrene for detection of weak antibodies.
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Proteolytic Enzymes
Are protein molecules that function by altering reaction conditions
and bring about changes in other molecules without being changed
themselves.
Enzymes used in the detection and identification of blood group
antibodies include ficin (isolated from fig plants), papain (from
papaya), trypsin (from pig stomach), and bromelin (from
pineapple).
The use of enzymes provides enhanced antibody reactivity to Rh,
Kidd, P1, Lewis, and I antigens and destroys or decreases reactivity to
Fya , Fyb, M, N, and S antigens
Antihuman Globulin (AHG) Reagents
When RBCs become coated with antibody or complement or both
but do not agglutinate in regular testing, special reagents are needed
to produce agglutination.
The direct antihuman globulin (AHG) test is designed to determine if
RBCs are coated with antibody or complement or both.
o Polyspecific AHG can determine if RBCs have been sensitized
with IgG antibody or complement (components C3b or C3d) or
both.
o Monospecific AHG reagents react only with RBCs sensitized with
IgG or complement.
Chemical Reduction of IgG and IgM Molecules
The reagents generally act on covalent sulfhydryl bonds and facilitate
antibody identification by removal of either IgG or IgM antibodies.\
Dithiothreitol (DTT) and ß-2-mercaptoethanol (2-ME) are thiol
reducing agents that break the disulfide bonds of the J (joining) chain
of the IgM molecule but leave the IgG molecule intact
Another reagent, ZZAP, which consists of a thiol reagent plus a
proteolytic enzyme, causes the dissociation of IgG molecules from
the surface of sensitized RBCs and alters the surface antigens of the
RBC.
Chemical reduction of the disulfide bond of the IgG molecule is also
used to produce chemically modified reagents that react with RBCs in
saline. Sulfhydryl compounds reduce the strong but less flexible
covalent disulfide bonds in the hinge region of the IgG molecule,
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allowing the Fab portions more flexibility in facilitating agglutination
reactions.
New and Nontraditional Laboratory Methods
Flow Cytometry
Solid-Phase Adherence
Gel Test
RBC Affinity Column Test
DISEASES IMPORTANT IN BLOOD BANK SEROLOGIC TESTING
Immunodeficiency diseases
Can result from various defects in the IS at many different levels of
immune function and may be congenital or acquired. They can
result from defects in either innate or adaptive immunity or both;
the cause is often not known.
Hypersensitivity (or allergy)
An inflammatory response to a foreign antigen and can be cell- or
antibody-mediated or both. There are four different types of
hypersensitive reactions, and the symptoms and treatment required
for each are different. All four types can be caused by blood product
transfusions and may be the first sign of a transfusion reaction.
Type I Reactions
Also called anaphylaxis or immediate hypersensitivity, involves
histamine release by mast cells or basophils with surface IgE
antibody.
It can occur in IgA-deficient individuals who receive plasma products
containing IgA. Urticarial reactions (skin rashes) may also result
from transfusion of certain food allergens or drugs in plasma
products.
Type II Reactions
Reaction can involve IgG or IgM antibody with complement,
phagocytes, and proteolytic enzymes.
HDN or transfusion reactions caused by blood group antibodies as
well as autoimmune hemolytic reactions are all type II reactions.
Page 9 of 10

Type III Reactions
Involve phagocytes and IgG and IgM and complement. Type III
reactions result in tissue damage from the formation of immune
complexes of antigen-antibody aggregates, complement, and
phagocytes and are therefore very serious.
Penicillin and other drug induced antibodies can lead to hemolytic
reactions through type III hypersensitivity.
Type IV Reactions
The type IV reaction involves only T cell–mediated responses and
their cytokines and can be fatal if untreated. The most important
type IV reaction is graft versus-host, of which there is also more
than one type.
MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
Hemolytic Disease of the Newborn
(HDN) can result when the maternal IS produces an antibody
directed at an antigen present on fetal cells but absent from
maternal cells.
The mother is exposed to fetal RBCs as a result of feto-maternal
transfer of cells during pregnancy or childbirth.
o Maternal memory cells can cause a stronger response during a
second pregnancy if the fetus is positive for the sensitizing
antigens. IgG1, IgG3, and IgG4 are capable of crossing the
placenta and attaching to fetal RBCs whereas IgG2 and IgM are
not.
o Severe HDN is most often associated with IgG1 antibodies and
may require exchange transfusion

Monoclonal and Polyclonal Gammopathies -END OF TRANSCRIPTION-

Plasma cell neoplasms result in proliferation of abnormal
immunoglobulin (or gamma globulin) from either a single B cell clone
(monoclonal gammopathies) or multiple clones (in polyclonal
gammopathies) and may be a specific isotype or only light or heavy
chain molecules.
Increased serum viscosity is a result of these diseases and can
interfere with testing. The increased concentrations of serum
proteins can cause nonspecific aggregation (as opposed to
agglutination) of erythrocytes called rouleaux, which is seen as a
stacking of RBCs, like a stacking of coins. It often occurs in multiple
myeloma patients.
If rouleaux is suspected, the saline replacement technique may be
needed to distinguish true cell agglutination from nonspecific
aggregation.

Autoimmune Disease
Autoantibodies are produced against the host’s own cells and
tissues.
It is unknown why this loss of tolerance to self-antigens occurs, but
there are many possible explanations such as aberrant antigen
presentation, failure to obtain clonal deletion, anti-idiotypic network
breakdown, and cross reactivity between self and non self -antigens.
Autoimmune hemolytic anemias are an important problem in
testing and transfusion
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 MODULE 1: Introduction to Immunohematology (part 5)
MLS 18, February 23, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
(S) strain, which has a smooth polysaccharide capsule, kills mice through
Legend: pneumonia.
Notes/Module Packet Bullet o The nonvirulent rough (R) strain lacks the outer capsule and is
Reference Book nonlethal to mice.

MODULE OUTLINE 1944, Oswald T. Avery and his group

I. Historical Background and the current trends in Transfusion Medicine o at Rockefeller Institute were able to reproduce this transformation
II. Blood Preservation in vitro.
III. Review of Basic Genetics
IV. Review of Fundamentals of Immunology Max Delbrück of Vanderbilt University, Salvador Luria of Indiana
University, and Alfred Hershey of Carnegie’s Department of Genetics
V. Review of Molecular Biology
at Cold Spring Harbor
A. DNA: The Genetic Material
o Confirmation that DNA was the genetic material came from the
B. Nucleic Acid Testing
“phage group”.
C. Central Dogma
o Coincided in their interest to research bacteriophages, viruses that
1. Replication
infect bacteria.
2. Transcription
o Bacteriophages are very small viruses that consist only of a DNA
3. Translation
core enclosed in a protein capsule. It was known that, during
bacterial infection, the phage particles reproduce inside the cell and
LEARNING OUTCOMES
lyse the bacteria to release a new generation of viruses.
At the end of this module, the student shall be able to:
1. Explain how DNA was proven to be the carrier of genetic
Alfred Hershey and Martha Chase
information.
o In 1952, they reported an experiment that convinced the scientific
2. Explain the central dogma of molecular biology.
community that DNA was indeed the genetic material.
3. Describe the basic mechanism replication, transcription, and
translation.
DNA STRUCTURE
4. Explain how nucleic acid testing (NAT) in blood donor improves the
process of screening for infectious disease. By 1950, the chemical composition of nucleic acids was known.
o Nucleic acids were known to be long molecules composed of three
distinct chemical subunits:
DNA: THE GENETIC MAT ERIAL  Five-carbon sugar
1928, Fred Griffith  Acidic phosphate
o A British microbiologist that presented a model system that was key  Four types of nitrogen-rich bases.
to demonstrating that DNA is the genetic material. o Two forms of nucleic acids were differentiated by their sugar
o He used two naturally occurring strains of pneumococcus composition: RNA contained ribose and DNA 2-deoxyribose.
bacterium that differed in their infectivity of mice. The virulent smooth

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o Both possessed adenine, guanine, and cytosine. RNA contained
uracil, and DNA contained thymine.
1950, Erwin Chargaff of Columbia University
o He reported a consistent one-to-one ratio of adenine to
thymine and guanine to cytosine in DNA samples from
different organisms.
1951, Maurice Wilkins and Rosalind Franklin
o He produced x-ray diffraction photographs of DNA, which
suggested a helical molecule with repeats of 34 angstroms
(Å) and a width of 20 Å.
1953, James Watson (trained in the phage group) and Francis
Crick (a physicist trained in x-ray crystallography, published a
paper in the journal Nature in which they assembled the
puzzle pieces of DNA structure.)
o They proposed that the DNA molecule was a helix. DNA was
a helical ladder, the rails of which were built from alternating
units of deoxyribose and phosphate.
o Each rung of the ladder was composed of a pair of
nucleotides (a base pair) held together by hydrogen bonds.
The double helix consisted of two strands of nucleotides that
ran in opposite directions (antiparallel).
o Consistent with the 34-Å repeat from x-ray diffraction,
10 base pairs were stacked on top of each other at
each turn of a helix.
o In agreement with Chargaff’s observation, adenine always
paired with thymine, and guanine always paired with
cytosine. Thus the nucleotide alphabet of one half of the
DNA helix determined the alphabet of the other half.
DNA FUNCTION
Genes must be replicated and passed down to each new cell and each
new generation.
Genes must also provide the information for protein synthesis.
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Expression of Genetic Information
Genes act by determining the sequence of amino acids and therefore
the structure of proteins.
Knowledge of the structure of DNA revealed that the genetic
information must be specified by the order of the four bases (A, C, G,
and T).
Proteins are polymers of 20 amino acids, the sequence of which
determines their structure and function.
Because DNA was in the nucleus, separated from the cytoplasm by the
nuclear membrane, an intermediary molecule had to be responsible
for conveying the genetic information from the nucleus to the
cytoplasm.
RNA was a good candidate for this role. Its structure suggested that
RNA could be produced from a DNA template and that RNA was
located mainly in the cytoplasm, where protein synthesis occurred.
THE CENTRAL DOGMA OF MOLECULAR BIOLOGY
The Central Dogma of Molecular Biology states that: DNA makes RNA
makes proteins.
Deciphering the genetic code confirmed the central dogma of
molecular biology first formulated by Francis Crick.
The genetic material is DNA.
DNA is self-replicating and is transcribed into mRNA, which in turn
serves as a template for the synthesis of proteins.
Although Crick’s central dogma remains true, the knowledge acquired
in the following
years has refined and enlarged it, a process that continues now and
into the future.
Scientists have added further qualifications to the central dogma:
1. Genes are not “fixed.”
2. DNA sequences and protein amino acid sequences are not entirely
collinear.
3. Some RNA molecules display catalytic activity; by “RNA
interference,” small RNA molecules help to regulate gene
expression.
Page 2 of 5

DNA REPLICATION
The Watson-Crick structure revealed how DNA could replicate.
The DNA molecule is made of two antiparallel complementary strands.
Cytosine always pairs with guanine, and thymine always pairs with
adenine.
The information in one DNA strand predicts the information in the
other strand. During replication, the hydrogen bonds break, the
strands separate, and each one functions as template for the synthesis
of another complementary half molecule.
Two identical DNA molecules are generated, each containing an
original strand and a new complementary strand, each to be passed to
a daughter cell
Because each daughter double helix contains an “old” strand and a
newly synthesized strand, this model of replication is called
semiconservative.
DNA TRANSCRIPTION
Transcription is the process by which DNA is copied (transcribed) to
mRNA, which carries the information needed for protein synthesis.
Transcription takes place in two broad steps.
o First, pre-messenger RNA is formed, with the involvement of
RNA polymerase enzymes. The process relies on Watson-Crick
base pairing, and the resultant single strand of RNA is the
reverse-complement of the original DNA sequence.
o The pre-messenger RNA is then "edited" to produce the desired
mRNA molecule in a process called RNA splicing.
Reverse transcription
o RNA is "reverse transcribed" into DNA
o catalyzed by reverse transcriptase enzymes, allows retroviruses,
including the human immunodeficiency virus (HIV), to use RNA
as their genetic material
o Reverse transcriptase enzymes have also found applications in
biotechnology, allowing scientists to convert RNA to DNA for
techniques such as PCR.
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DNA TRANSLATION
The mRNA formed in transcription is transported out of the nucleus,
into the cytoplasm, to the ribosome (the cell's protein synthesis
factory). Here, it directs protein synthesis.
Messenger RNA is not directly involved in protein synthesis − transfer
RNA (tRNA) is required for this.
The process by which mRNA directs protein synthesis with the
assistance of tRNA is called translation.
Recombinant DNA
The big breakthrough came with the development of recombinant
DNA technology.
o DNA from one organism, humans for example, can be
“cut and pasted” into a carrier DNA molecule or vector.
o The new DNA molecule, which is a “recombinant” of the
original DNA with the vector DNA, can be introduced into
another, usually simpler, host organism. Because the
genetic code is almost universal, the host organism treats
the gene as its own. This technique is called molecular
cloning.
o Cloning is the reproduction of daughter cells from one
single cell by fission or mitotic division, giving rise to a
population of genetically identical clones.
o In DNA cloning, the DNA fragment of interest
carried by the vector is introduced into a host
cell. Successive divisions of the host cell create a
population of clones containing the DNA
fragment of interest.
DETECTION OF NUCLEIC ACIDS AND PROTEINS
Nucleic Acid Hybridization
Base pairing between complementary strands of DNA or RNA allows
the specific detection of nucleic acid sequences.
Double-stranded nucleic acids denature at high temperature (90⁰ to
100⁰C) and renature when cooled to form double-stranded molecules
as dictated by complementary base pairing.
Nucleic acid hybrids can be formed between two strands of DNA, two
strands of RNA, or one strand of RNA and one of DNA. DNA or RNA
Page 3 of 5

sequences complementary to any purified DNA fragment can be
detected by nucleic acid hybridization.
o Southern Blotting
 In this technique developed by E. M. Southern, the DNA to
be analyzed is digested with one or more restriction
endonucleases and the fragments separated by agarose gel
electrophoresis.
 The gel is then placed over a nitrocellulose or nylon
membrane and overlaid with transfer buffer. The DNA
fragments are transferred or “blotted” when vacuum is
applied by the flow of transfer buffer.
 The membrane-bound fragments have the same relative
positions as the fragments separated by size on the gel.
o Northern Blotting
 This technique is a variation of the Southern blotting and is
used for detection of RNA instead of DNA.
 Total cellular RNAs are extracted and fractioned by size
through gel electrophoresis.
 The RNAs are then blotted onto a filter and detected by
hybridization with a labeled probe. This technique is used
for studying gene expression.
o DNA microarrays
 also called gene chips, allow tens of thousands of genes to
be analyzed simultaneously.
 A gene chip consists of a glass slide or a membrane filter
onto which oligonucleotides or fragments of cDNA are
printed by a robot in small spots at high density.
 Because a chip can contain more than 10,000 unique DNA
sequences, scientists can produce DNA microarrays
containing sequences representing all the genes in a
cellular genome.
 A common application of this technique is the study of
differential gene expression; for example, the comparison
of the genes expressed by a normal cell as opposed to
those of a tumor cell.
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o Fluorescent in situ Hybridization (FISH)
 In this technique, scientists use fluorescent probes to
detect homologous DNA or RNA sequences in
chromosomes or intact cells.
 In this case, the hybridization of the probe to specific cells
or sub-cellular structures is determined by direct
microscopic examination.
PCR-Based Techniques
Amplification of DNA by PCR allows the detection of even single
copies of DNA molecules.
The specificity of the PCR amplification depends on the primers that
hybridize to complementary sequences of the template molecule
spanning the target DNA fragment.
With carefully chosen primers, PCR can be used to selectively
amplify DNA molecules from complex mixtures, such as genomic
DNA from cell extracts.
o Reverse Transcription-PCR (RT-PCR)
 By adding a step of cDNA synthesis by reverse
transcription (RT) prior to the PCR amplification, single
copies of RNA can be detected. By RT-PCR,
 RNA molecules can be specifically amplified from total
RNA obtained from cell extracts or tissue sections.
o Real-Time PCR in real-time PCR (also called RT-PCR)
 the product formed in each cycle of amplification is
detected by fluorescence at the same time that it is
produced.
 Combined with reverse transcription, this method allows
the quantification of specific mRNA in complex mixtures
and is extremely useful in the study of gene expression.
 the high sensitivity of PCR-based DNA and RNA detection
techniques makes them valuable for early detection of
transfusion-transmitted viruses, such as HIV and hepatitis
B and C.
 Nucleic acid testing (NAT) is rapidly becoming a standard
method in blood banks. It allows the detection of
Page 4 of 5
 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
pathogens before the appearance of a testable immune
response, such as screening of antibodies.
 Reducing the “window” period (during which donors can
be infected but do not yet test positive) helps to enhance
the safety of blood products.

Antibodies as Probes for Proteins

Gene expression at the protein level can be studied by using labeled
antibodies as probes. In particular, monoclonal antibodies are
widely used in the technique called immunoblotting.

Recombinant DNA Libraries

A way of isolating single genes is by the production of recombinant
DNA libraries. Instead of trying to fish one gene out of a mass of
genomic DNA or cDNA, each gene is physically separated and
introduced into a vector.
The target gene is selected by screening each of the individual
pieces with a specific probe. Recombinant DNA libraries are
collections of clones that contain all genomic or mRNA sequences of
a particular cell type.
Clones containing a specific gene are identified by hybridization with
a labeled probe, such as a cDNA or genomic clone or a PCR product.

Red Cell Genotyping

In cases where serology is not possible or not sensitive enough or
where discrepancies occur, genotyping provides results that can be
used to obtain a blood type on a donor or patient.

Applications for genotyping:

1. Fetal DNA typing
2. Blood group typing of donors for alloimmunized patients
3. Screening blood donors to locate rare blood group phenotypes
4. Screening blood inventory for antigen negative units
5. Determining the frequency of blood group polymorphism
6. Determining zygosity for the fathers of fetuses at risk for HDFN
7. Blood group typing of patients with autoimmune hemolytic anemia
and other disease.

-END OF TRANSCRIPTION-
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 MODULE 2&3: Rh Naming
MLS 18, March 13, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`

SUMMARY OF BLOOD GROUP SYSTEMS Note: “p” refers to short arm of chromosome; “q” refers to long arm of
ISBT # BLOOD GROUP CHROMOSOME NUMBER
For Rh Blood group
001 ABO 9q Chr1: RHD gene: D polypeptide; RHCE gene: RHCe,RHcE,RHce,RHCE
002 MNS 4q polypeptides Chr 6: RHAG: Rh associated glycoprotein that forms complexes
003 P 22q with Rh polypeptides
004 Rh 1p
005 Lutheran 19q Rh Nomenclatures
006 Kell 7q 1. Fisher-Race: genetics and serology
007 Lewis 19p 2. Wiener: shorthand
008 Duffy 1q 3. Rosenfield: presence or absence of a given antigen
009 Kidd 18q 4. ISBT: catalogues each antigen within a blood group
system
010 Diego 17q
011 Cartwright 7q Fisher-Race Terminology
012 Xga Xp o Based on closely linked alleles D, C/c, and E/e
013 Scianna 1p o d is an amorph and does not produce a phenotypic product
014 Dombrock 12p o d= absence of D antigen
015 Colton 7p
016 Landsteiner-Weiner 19p Wiener terminology
017 Chido-Rogers 6p • Wiener is the “shorthand” version of Fisher-Race
018 H 19q o R= presence of D
019 Kx Xp o r= d, or absence of D antigen
o 1 or single prime= presence of C
020 Gerbich 2q
o 2 or doubleprime= presence of E
021 Cromer 1q
022 Knops 1q Antigen Wiener
023 Indian 11p D R
024 Ok 19p D r
025 Raph 11p C 1 or ‘
026 John-Milton Hagen 15q E 2 or “
027 I 6p
028 Globoside 3q
029 GIL 9p
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 MODULE 2-3- RH NAMING
Fisher-Race Wiener Sample Question:
Rh positive Dce R0
Question 1
DCe R1 • A patient’s red blood cells are tested for the following Rh antigens:
DcE R2 Anti-D : +
DCE Rz Anti-C : +
Rh negative dce r Anti-E: 0
dCe r’ anti-c: +
dcE r” anti-e: +
dCE y
Antigens present: D, C, c, e
Rosenfield Most likely genotype: DCe/dce R1r
o This system simply describes the presence or absence of the antigen Other possibilities: Dce/dCe R0r’
on the RBC. There is no genetic basis. DCe/Dce R1R0
o D=1, C=2, E=3, c=4, e=5 Question 2
o Example: R1r (DCe/dce): Rh:1,2,-3,4,5 • A patient’s red blood cells are tested for the following Rh antigens:
• E is number 3; E antigen is not present and is therefore designated with Anti-D: 0
-3 Anti-C: 0
ISBT Anti-E: 0
o International Society of Blood Transfusion Numeric Terminology. anti-c: +
o Rh blood group is assigned the prefix 004 anti-e: +
o Each antigen assigned to the Rh blood group is given a unique
number to complete the six-digit number. Antigens present: c, e
o Example: E antigen 004003 Most likely genotype: dce/dce rr
o Advantage over Rosenfield is that it is a purely numeric system, Other possibilities: None
which is easier for data processing.
Question 3
• A patient’s red blood cells are tested for the following Rh antigens:
Anti-D: +
Anti-C: +
Anti-E: 0
anti-c: 0
anti-e: +

Antigens present: D, C, e
Most likely genotype: DCe/DCe R1R1
Other possibilities: DCe/dCe R1r’

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 MODULE 2 Part 1: The Anti-globulin Test
MLS 18, March 5, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
ANTI-GLOBULIN TEST
Legend: Antiglobulin test (also called Coombs’ test)
Transcription Bullet Based on the principle that: anti-human globulins (AHGs) obtained
Notes/Module Packet Bullet from immunized nonhuman species bind to human globulins such as
IgG or complement, either free in serum or attached to antigens on red
MODULE OUTLINE blood cells (RBCs).
I. Anti- Globulin Test There are two major types of blood group antibodies, IgM and IgG.
A. History o Because of their large pentamer structure, IgM antibodies bind to
B. AHG Reagents corresponding antigen and directly agglutinate RBCs suspended in
1. Polyspecific AHG saline.
2. Monospecific AHG o IgG antibodies are termed non-agglutinating because their
C. Preparation of AHG monomer structure is too small to agglutinate sensitized RBCs
D. Antibodies required in AHG directly.
E. Principle of the Anti-Globulin Test The addition of AHG containing anti-IgG to RBCs sensitized with IgG
F. Direct and Indirect Anti-Globulin Test antibodies allows for hemagglutination of these sensitized cells. Some
G. Factors Affecting the Anti-Globulin Test blood group antibodies have the ability to bind complement to the RBC
H. Modified and Automated Antiglobulin Test Techniques membrane. Antiglobulin tests detect IgG and/or complement-sensitized
I. Use of Polyspecific Versus Monospecific AHG in the IAT RBCs.

LEARNING OUTCOMES HISTORY

1. State the principle of antiglobulin test.
2. Differentiate monoclonal from polyclonal and monospecific from
polyspecific antihuman globulin (AHG) reagents.
3. Compare and contrast the indirect antiglobulin test (IAT) and the
direct antiglobulin test (DAT).
4. Identify the factors that affect the antiglobulin test.
5. Formulate the steps necessary to resolve errors associated with
performance of the antiglobulin test.
Before the discovery of the antiglobulin test, only IgM antibodies had
been detected.
The introduction of the antiglobulin test permitted the detection of non-
agglutinating IgG antibodies and led to the discovery and
characterization of many new blood group systems.
In 1945, Coombs and associates
o Described the use of the antiglobulin test for the detection of weak
and nonagglutinating Rh antibodies in serum.

In 1946, Coombs and coworkers

o Described the use of AHG to detect in-vivo sensitization of the RBCs
of babies suffering from hemolytic disease of the newborn (HDN).

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o The first of the Kell blood group system antibodies and the
associated antigen were reported only weeks after Coombs had
described the test.
In 1908, Moreschi
o Described the Principle of Coombs test.
o Moreschi’s studies involved the use of rabbit antigoat serum to
agglutinate rabbit RBCs that were sensitized with low non-
agglutinating doses of goat anti-rabbit RBC serum. The antiglobulin
test can be used to detect RBCs sensitized with IgG alloantibodies,
IgG autoantibodies, and complement components.
How does the AHG reagent was produced?
o It involves the injection of Human Serum into the Rabbit to
produce an Anti-human Serum.
o After the adsorption, to remove the heterospecific antibodies and
the dilution to avoid the Prozone Effect
 Prozone Effect- excessive Antibodies
o AHG Serum still remains sufficient activity to permit cross linking of
the adjacent RBCs synthesize with IgG Antibodies.
 This can later on produce the heme agglutination.
 AHG able to detect in vivo and in vitro sensitization of RBCs
o In vivo: Direct Antiglobulin Test (DAT)
 One-stage procedure
o In vitro: Indirect Antiglobulin Test (IAT)
 Two-stage procedure
AHG REAGENTS
POLYSPECIFIC AHG
Contains antibody to human IgG and to the C3d component of human
complement.
Other anticomplement antibodies, such as anti-C3b, anti-C4b, and anti-
C4d, may also be present.
Commercially prepared polyspecific AHG contains little, if any, activity
against IgA and IgM heavy chains.
However, the polyspecific mixture may contain antibody activity to
kappa and lambda light chains common to all immunoglobulin classes,
thus reacting with IgA or IgM molecules.
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MONOSPECIFIC AHG
Contain only one antibody specificity: either anti-IgG or antibodies to
specific complement components such as C3b or C3d.
Licensed monospecific AHG reagents in common use are anti-IgG and
anti-C3bC3d.
Anti-IgG Reagent
Reagents labeled anti-IgG contain no anticomplement activity.
Anti-IgG reagents contain antibodies specific for the Fc fragment of
the gamma heavy chain of the IgG molecule.
If not labeled “gamma heavy chain–specific,” anti-IgG may contain
anti–light chain specificity and therefore react with cells sensitized
with IgM and IgA as well as with IgG.
Anti-Complement
Anti-complement reagents, such as anti-C3b-C3d reagents, are
reactive against the designated complement components only and
contain no activity against human immunoglobulins.
PREPARATION OF AHG
POLYCLONAL ANTIBODIES are a mixture of antibodies from different
plasma cell clones. The resulting polyclonal antibodies recognize
different antigenic determinants (epitopes), or the same portion of the
antigen but with different affinities. Hybridoma technology can be used
to produce monoclonal antiglobulin serum.
MONOCLONAL ANTIBODIES are derived from one clone of plasma cells
and recognize a single epitope.
PREPARATION OF POLYSPECIFIC AHG
POLYCLONAL AHG PRODUCTION
Polyclonal AHG is usually prepared in rabbits, although when large
volumes of antibody are required, sheep or goats may be used. In
contrast with the early production methods, in which a crude globulin
fraction of serum was used as the immunogen, modern production
commences with the purification of the immunogen from a large pool of
normal sera.
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MONOCLONAL AHG PRODUCTION
The monoclonal antibody technique devised by Kohler and Milstein has
been used to produce AHG and has proved particularly useful in
producing high-titer antibodies with well-defined specificities to IgG and
to the fragments of C3.
Monoclonal antibody production begins with the immunization of
laboratory animals, usually mice, with purified human globulin.
After a suitable immune response, mouse spleen cells containing
antibody-secreting lymphocytes are fused with myeloma cells.
The resulting “hybridomas” are screened for antibodies with the
required specificity and affinity. The antibody-secreting clones may then
be propagated in tissue culture or by inoculation into mice, in which
case the antibody is collected as ascites.
PREPARATION OF MONOSPECIFIC AHG
It is prepared by a production process similar to that described for
polyspecific AHG; however, it contains only one antibody specificity.
Monospecific anti-IgG is usually of polyclonal origin; however,
monoclonal anti-IgG has been prepared effectively by hybridoma
technology.
Monospecific anticomplement reagents are often a blend of monoclonal
anti-C3b and monoclonal anti-C3d.
ANTIBODIES REQUIRED IN AHG
Anti-IgG
AHG must contain antibody activity to non-agglutinating blood group
antibodies. The majority of these antibodies are a mixture of IgG1 and
IgG3 subclass.
Rarely, non-agglutinating IgM antibodies may be found; however, they
have always been shown to fix complement and may be detected by
anticomplement.
IgA antibodies with Rh specificity have been reported; however, IgG
antibody activity has always been present as well.
The only RBC alloantibodies that have been reported as being solely IgA
have been examples of anti-Pr,12 and those antibodies were
agglutinating.
IgA autoantibodies have been reported, although very rarely.
o Therefore, anti-IgG activity must be present in the AHG reagent.
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Anti-Complement
Some antibodies “fix” complement components to the RBC membrane
after complexing of the antibody with its corresponding antigen.
These membrane-bound complement components can be detected by
the anticomplement activity in AHG.
Use of Polyspecific Versus Monospecific AHG in the IAT
Petz and coworkers
Compared monospecific anti-IgG with polyspecific AHG.
They also compared the albumin technique with low ionic strength
solutions (LISS)-suspended RBCs.
Four Jka antibodies were detected with polyspecific but not with
monospecific anti-IgG using albumin or LISS-suspended RBCs.
An additional anti-Jka was detected only with polyspecific AHG when
using LISS but not with albumin.
Also, five antibodies of antiKell, anti-Jka, and Fya specificities were
detected when using LISS, but not albumin, with both polyspecific AHG
and antiIgG.
o Their results concluded that some clinically significant antibodies
are detected with the anticomplement component of AHG but not
with anti-IgG.
Also determined the number of false-positive reactions obtained when
using polyspecific AHG versus anti-IgG with LISS and albumin.
o False-positive reactions were defined as those caused by antibodies
with no definable specificity or by antibodies considered to be
clinically insignificant because of optimum reactivity at cold
temperatures (anti-I, anti-H, anti-P1, anti-M).
o Of the unwanted positive reactions, 93% were shown to be caused
by C3 on the cells.
Engelfriet and others
have also shown that degradation of C3b to C3d can occur in vitro,
providing that the incubation period is greater than 1 hour.
In 1976, Garratty and Petz confirmed the need for anti-C3d activity in
AHG for use in the DAT. They also confirmed Engelfriet’s observation
that, given sufficient time, cell-bound C3b could be degraded to C3d in
vitro.
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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
NOTE: Positive results are monitored by a DAT panel using monospecific anti-
The detection of C3d on the RBC membrane is important in the IgG and anti-C3d to determine the specific type of protein sensitizing
investigation of both warm and cold autoimmune hemolytic anemia the cell.
(AIHA). Many cases of warm AIHA are associated with both IgG and The saline control serves to detect spontaneous agglutination of cells
C3d coating the RBCs. In cold AIHA, C3d may be the only globulin or reactions occurring without the addition of AHG reagents.
detectable on the RBC. In warm AIHA, including drug-induced hemolytic anemia, the RBCs
In the investigation of AIHA, a DAT is performed initially with may be coated with IgG or C3d, or both.
polyspecific AHG. If globulins are detected on the RBC membrane,
follow-up testing with monospecific AHG (antiIgG, anti-C3d) is
performed to identify the coating proteins. Although the RBCs of
most patients with AIHA are coated with IgG, the cells of some
patients will exhibit both IgG and complement coating or
complement alone. The presence of complement alone may support
the diagnosis of AIHA, rendering the finding significant.

PRINCIPLES OF THE ANTIGLOBULIN TEST

1. Antibody molecules and complement components are globulins.
2. Injecting an animal with human globulin stimulates the animal to
produce antibody to the foreign protein (i.e., AHG). Serologic tests
employ a variety of AHG reagents reactive with various human
globulins, including anti-IgG, antibody to the C3d component of
For the case of HDN:
human complement, and polyspecific reagents that contain both anti-
o The RBC of the Fetus contains antigens to its surface. This will bind
IgG and anti-C3d activity.
to the Antibodies formed by the mother
3. AHG reacts with human globulin molecules, either bound to RBCs or
o After the Antigen-Antibody binding, the Anti-IgG found in Coomb’s
free in serum.
Reagent will bind itself to the Antibody of the mother.
4. Washed RBCs coated with human globulin are agglutinated by AHG.
o This will lead to the formation of agglutination.

DAT (Direct Antiglobulin Test)

Evaluation of Positive DAT
Detects in-vivo sensitization of RBCs with IgG and/or complement
 The American Association of Blood Banks Technical Manual states that:
components.
“The results of serological tests are not diagnostic; their significance can
Clinical conditions that can result in in-vivo coating of RBCs with
only be assessed in relationship to the patient’s clinical condition.
antibody and/or complement are:
o Hemolytic disease of the newborn (HDN)
o Hemolytic transfusion reaction (HTR) IAT (Indirect Antiglobulin Test)
o Autoimmune and drug-induced AIHA. The IAT is performed to determine in-vitro sensitization of RBCs
Initial DATs include testing one drop of a 3 to 5 percent suspension of Used in the following situations:
washed RBCs with polyspecific (anti-IgG, anti-C3d) reagent. o Detection of incomplete (non-agglutinating) antibodies to
potential donor RBCs (compatibility testing) or to screening
cells (antibody screen) in serum
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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
o Determination of RBC phenotype using known antisera (e.g., MODIFIED AND AUTOMATED ANTIGLOBULIN TEST TECHNIQUES
Kell typing, weak D testing)
Low Ionic Polybrene Technique
o Titration of incomplete antibodies
In 1980, Lalezari and Jiang reported on the adaptation of the automated
low ionic polybrene (LIP) technique for use as a manual procedure.
The technique relies on low ionic conditions to rapidly sensitize cells
with antibody.
Polybrene, a potent rouleaux-forming reagent, is added to allow the
sensitized cells to approach each other to permit cross-linking by the
attached antibody.
A high ionic strength solution is then added to reverse the rouleaux;
however, if agglutination is present, it will remain. The test can be
carried through to an AHG technique if required.
If this is performed, a monospecific anti-IgG reagent must be used
because the low ionic conditions cause considerable amounts of C4 and
C3 to coat the cells and would give false-positive reactions if a
polyspecific reagent were used.

Enzyme-Linked Antiglobulin Test (ELAT)

In IAT: There are Two Stages involved: An RBC suspension is added to a microtiter well and washed with saline.
o Stage 1 AHG, which has been labeled with an enzyme, is added.
 We have the serum of the patient, it contains free Antibody, The enzyme labeled AHG will bind to IgG-sensitized RBCs. Excess
this will be collected and allowed to be mixed with the blood antibody is removed, and enzyme substrate is added.
of the donor. The amount of color produced is measured spectrophotometrically and
 The Antigen present at the surface of the donor’s blood will is proportional to the amount of antibody present.
bind to the Antibody of the patient. The optical density is usually measured at 405 nm. The number of IgG
o Stage 2 molecules per RBC can also be determined from this procedure.
 After the Antibody-Antigen binding, the Coomb’s Reagent
containing Anti-IgG will now bind to the Antibody present in Solid Phase
the patient.
Solid-phase technology may be used for the performance of antiglobulin
FACTORS AFFECTING THE ANTIGLOBULIN TEST tests. Several different techniques have been reported using either test
1. Ratio of serum to cells. tubes or microplates.
2. Reaction medium. With the availability of microplate readers, this modification lends itself
3. Temperature. to the introduction of semi-automation.
4. Incubation time Direct and indirect tests can be performed using solid-phase
5. Washing of RBC methodology.
6. Addition of AHG o In the former, antibody is attached to a microplate well, and RBCs
7. Centrifugation for reading are added. If antibody is specific for antigen on RBCs, the bottom of
-Refer to reference book for in depth explanation

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 MODULE 1- INTRODUCTION TOIMMUNOHEMATOLOGY
the well will be covered with suspension; if no such specificity
occurs, RBCs will settle to the bottom of the well.
o In the latter, known RBCs are bound to a well that has been treated
with glutaraldehyde or poly L-lysine. Test serum is added to RBC-
coated wells, and if antibody in serum is specific for antigen on fixed
RBCs, a positive reaction occurs.

Anti-Complement
The gel test is a process to detect RBC antigen-antibody reactions by
means of using a chamber filled with polyacrylamide gel. The gel acts as
a trap; free unagglutinated RBCs form pellets in the bottom of the tube,
whereas agglutinated RBCs are trapped in the tube for hours.
Therefore, negative reactions appear as pellets in the bottom of the
microtube, and positive reactions are fixed in the gel.
There are three different types of gel tests: neutral, specific, and
antiglobulin.
o A neutral gel does not contain any specific reagent and acts only by
its property of trapping agglutinates. The main applications of
neutral gel tests are antibody screening and identification with
enzyme-treated or untreated RBCs and reverse ABO typing.
o Specific gel tests use a specific reagent incorporated into the gel
and are useful for antigen determination.

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 MODULE 2 Part 2: ABO BLOOD GROUP SYSTEM
MLS 18, March 5, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo` 11. Interpret the results from an ABO typing and resolve any
discrepancies, if present.
Legend:
Transcription Bullet
Notes/Module Packet Bullet BLOOD GROUP
MODULE OUTLINE Classification of blood based on inherited differences (polymorphisms)
I. Blood Group in antigens on the surfaces of the red blood cells
II. ABO Antigens Inherited differences of white blood cells (leukocytes), platelets
III. ABO Antibodies (thrombocytes), and plasma proteins also constitute blood groups.
IV. Inheritance of the ABO Blood Groups The human ABO blood groups were discovered by Austrian-born
V. ABO Subgroups American biologist Karl Landsteiner in 1901.
VI. Bombay Phenotype o Landsteiner found that there are substances in the blood, antigens
VII. Effects of Disease on the Expression of ABH Antigens and Antibodies and antibodies that induce clumping of red cells when red cells of
VIII. Laboratory tests to detect and identify ABO Antigens and Antibodies one type are added to those of a second type.
IX. ABO Discrepancies o He recognized three groups—A, B, and O—based on their reactions
to each other. He was inadvertently the first individual to perform
LEARNING OUTCOMES forward and reverse grouping.
1. Describe the reciprocal relationships between ABO antigens and
antibodies for blood groups O, A, B, and AB. Forward grouping (front type)
2. Explain the effects of age on the production of isoagglutinins. Defined as using known sources of commercial antisera (anti-A, anti-
3. Describe the immunoglobulin classes of ABO antibodies in group O, A, B) to detect antigens on an individual’s RBCs.
and B individuals. Reverse grouping (back type)
4. Predict the ABO phenotypes and genotypes of offsprings from various Defined as detecting ABO antibodies in the patient’s serum by using
ABO mating. known reagent RBCs, namely A1 and B cells.
5. Explain the formation of H, A, and B antigens on the red blood cells
from precursor substance to immunodominant sugars. AB Group
6. Describe the qualitative and quantitative differences between A1 and Was identified a year later by another research team
A2 phenotypes. Red cells of the A group clump with donor blood of the B group; those
7. Describe the reactivity of Ulexeuropaeus with the various ABO of the B group clump with blood of the A group; those of the AB
groups. group clump with those of the A or the B group because AB cells
8. Describe the characteristics of the weak subgroups of A (A3, Ax, Aend, contain both A and B antigens
Am, Ay, Ael) and of the Bombay phenotypes. Those of the O group do not generally clump with any group, because
9. Explain the effects of disease on the expression of ABH antigens and they do not contain either A or B antigens.
antibodies.
10. Perform laboratory tests to detect and identify ABO antigens and
antibodies.

ABO forward and reverse grouping tests must be performed on all
donors and patients. ABO grouping is the most frequently performed
test in the blood bank.
There is always an inverse reciprocal relationship between the
forward and reverse type; thus, one serves as a check on the other.
IMPORTANCE OF ANTIGENS AND ANTIBODIES
The red cells of an individual contain antigens on their surfaces that
correspond to their blood group and antibodies in the serum that
identify and combine with the antigen sites on the surfaces of red cells
of another type.
Agglutination
The reaction between red cells and corresponding antibodies that
cause the clumping of RBC
Agglutinogens
Antigens on the surfaces of red cells responsible for agglutination
Antibodies are part of the circulating plasma proteins known as
immunoglobulins, which are classified by molecular size and weight and
by several other biochemical properties.
Most blood group antibodies are found either on immunoglobulin G
(IgG) or immunoglobulin M (IgM) molecules,
o Occasionally the immunoglobulin A (IgA) class may exhibit blood
group specificity.
Naturally occurring antibodies are the result of immunization by
substances in nature that have structures similar to human blood
groups.
o Present in an individual despite the fact that there has been no
previous exposure to the corresponding red cell antigens
o Example: anti-A in the plasma of people of blood group B and anti-B
in the plasma of people of blood group A.
Immune antibodies are evoked by exposure to the corresponding red
cell antigen.
Immunization (i.e., the production of antibodies in response to antigen)
against blood group antigens in humans can occur as a result of
pregnancy, blood transfusion, or deliberate immunization.
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o The combination of pregnancy and transfusion is a particularly
potent stimulus.
Individual blood group antigens vary in their antigenic potential
o For example, some of the antigens belonging to the Rh and ABO
systems are strongly immunogenic (i.e., capable of inducing
antibody formation), whereas the antigens of the Kidd and Duffy
blood group systems are much weaker immunogens.
o The blood group antigens are not restricted solely to red cells or
even to hematopoietic tissues.
The antigens of the ABO system are widely distributed throughout the
tissues and have been unequivocally identified on platelets and white
cells (both lymphocytes and polymorphonuclear leukocytes) and in skin,
the epithelial (lining) cells of the gastrointestinal tract, the kidney, the
urinary tract, and the lining of the blood vessels.
Evidence for the presence of the antigens of other blood group systems
on cells other than red cells is less well substantiated.
Among the red cell antigens, only those of the ABO system are
regarded as tissue antigens
o Therefore, need to be considered in organ transplantation.
ABO ANTIGENS
Located on the surface of the red blood cell.
Also present on lymphocytes, thrombocytes, organs, endothelial cells,
and epithelial cells.
First detected by Landsteiner as he performed his mixing tests
The biochemistry and structure of ABO antigens are well-established.
Antigens of the ABO system are well-developed in adults.
o They are detectable at 5 to 6 weeks of gestation.
Newborns demonstrate weaker antigens, but ABO antigens are fully
developed by two to four years of age.
One factor contributing to the difference in ABO antigen strength
between newborns and adults is the number of branched
oligosaccharides.
o Adults demonstrate greater numbers of branched chains compared
to newborns, who have more linear chains.
o Adults have more branched chains and, hence, the ability to add on
more terminal sugars and produce more antigens.
o Newborns and infants have fewer antigen sites on their red cells.
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The branched chains permit attachment of more molecules to
determine H antigen specificity.
Following H antigen development, the A and/or B specific molecule may
be attached.
INHERITANCE OF ABH ANTIGENS
As Bernstein discovered, ABO antigens are inherited in a simple
Mendelian fashion from an individual’s parents.
o Each individual possesses a pair of genes.
o Each gene occupies an identical locus on chromosome 9.
o There are three possible genes that can be inherited: A, B, and O.
A and B genes produce a detectable product while the O gene is an
amorph that does not produce a detectable product.
o The expression of the A and B genes is codominant.
The H antigen is required to produce A and/or B antigens.
o The H gene is also inherited in Mendelian fashion and occupies a
locus on chromosome 19.
o Each parent contributes one gene, either H or h.
o The possible genetic combinations are HH, Hh, or hh.
Individuals who are genetically either HH or Hh will produce the H
antigen, and it can be detected on their red cells.
The frequency of occurrence of the H antigen in the Caucasian
population is greater than 99.99%.
Individuals inheriting an hh genotype do not produce the H antigen and
have the Bombay Phenotype, Oh.
Anti-H – Frequently demonstrated on the plasma of an individual with a
Bombay phenotype
BIOCHEMICAL AND STRUCTURAL DEVELOPMENT OF ABH ANTIGENS
Expression of A, B, and H genes does not result in the direct production
of antigens.
o Rather, each gene codes for the production of an enzyme known as
a transferase.
Each transferase catalyzes the transfer of a carbohydrate molecule to an
oligosaccharide chain. The attached carbohydrate provides antigenic
specificity.
The O gene codes for an enzymatically inactive protein and, hence, no
antigen is produced.
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DEVELOPMENT OF H ANTIGEN
The H allele codes for the transferase, L-fucosyltransferase.
o This enzyme catalyzes the formation of the H antigen by transfer of
L-fucose to either type one or type two oligosaccharide chains.
o The L-fucose is the immunodominant sugar for the H antigen. It is
the sugar that confers antigenic specificity to the H antigen.
The H antigen serves as a precursor for A and B antigens.
The h allele is an amorph and does not produce a detectable product.
DEVELOPMENT OF A AND B ANTIGENS
The H antigen oligosaccharide chain serves as a precursor for both the
A and B antigens.
The A and B alleles each code for a transferase that attaches a sugar
molecule to the terminal end of the H antigen oligosaccharide chain,
which forms either the A or B antigen.
o The A allele codes for N-acetylgalactosamine transferase.
o This transferase attaches N-acetyl-D-galactosamine to the H antigen
forming the A antigen.
o The B allele codes for D-galactosyltransferase.
o This transferase attaches D-galactose to the H antigen forming the B
antigen.
The product of the O allele is an enzymatically inactive protein.
o Hence, this allele produces no detectable antigen (amorph)
Conversely, group O cells contain the most H antigen. This results from
no conversion of H antigen to A and/or B antigens.
o In comparison, group A1 B cells have the least amount of H antigen
since quantitatively the most H is converted to A1 and B antigens.
ABO ANTIBODIES
“Antibodies directed against ABO antigens are the most important
antibodies in transfusion medicine.”
The ABO blood group presents a unique situation in
Immunohematology.
It is the only example of a blood group where each individual
produces antibodies to antigens not present on the red cells.
These ABO antibodies were originally thought to be natural antibodies
formed with no apparent antigenic stimulus.
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Since the antibodies are not stimulated by exposure to red cells, they
may also be considered non-red cell stimulated antibodies.
o However, some form of an antigenic stimulus must exist.
o The proposed mechanism is environmental.
These “naturally occurring” substances resemble A and B antigens
and stimulate the production of complementary antibodies to the
antigens that are not present on the red cell surface.
NOTE:
Newborns have no ABO antibodies. When newborns are tested, only
a forward group is performed. Newborns may exhibit passive ABO
antibodies that have crossed the placental barrier. Reverse grouping
of a newborn or umbilical cord serum indicates the blood group of
the mother. The child will begin antibody production, and have a
detectable titer, at three to six months of age. ABO antibody
production peaks at age five to ten years of age and continues in
immunocompetent individuals throughout life. Titers begin to wane
in the elderly.
THE IMMUNOGLOBULIN CLASSES OF ABO ANTIBODIES IN GROUP ABO
IMMUNOGLOBULIN CLASS
ABO antibodies are typically isoagglutinins.
o They are saline agglutinins with optimal reactivity at 4°C.
These naturally occurring antibodies are mostly IgM isotype
o IgG and IgA classes of ABO antibodies have been detected.
The development of IgG antibodies occurs without apparent antigen
exposure via transfusion of incompatible red cells or fetal maternal
incompatibility.
Anti-A,B
Group O individuals do not have A or B antigens on their cells.
o Consequently, they produce anti-A, anti-B, and anti-A,B.
Anti-A,B is an antibody that has cross- reactivity with A and B cells.
This cross-reactive antibody detects a common molecular structure in
both antigens.
o Although the antibody reacts with both antigens, it cannot be
divided into individual components (i.e. anti-A plus anti-B).
o Anti-A,B is used as a third antisera in forward grouping.
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MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
o Anti-A,B is not required in forward grouping.
Since it is a valuable reagent for determining subgroups of A and B, anti-
A,B is often included as a routine part of forward grouping.
Monoclonal anti-A,B have replaced the use of human anti-A, B in
forward grouping.
Anti-A1
As per Landsteiner’s Law, group B and O individuals produce anti-A.
This anti-A can be separated by absorption procedures.
These absorption procedures can produce two components of the
antibody found in group B and O individuals.
o These components are anti-A and anti-A1.
o The anti-A1 antibody reacts specifically with A1 cells and not with
A2 cells or cells from other subgroups of A.
Like other ABO antibodies, this antibody reacts optimally at room
temperature or colder.
Anti-A1 is not considered clinically significant as it relates to transfusion.
o It is, however, significant when it causes incompatible crossmatches
at the immediate spin phase.
Antibodies to other A subgroups, such as A2, are not produced. These
subgroups have the A antigen but in reduced amounts.
o Therefore, transfusion of A1 individuals with A2 cells will not
stimulate the production of anti-A2 since both A1 and A2 individuals
have the A antigen in common.
CLINICAL SIGNIFICANCE OF ABO ANTIBODIES
ABO antibodies are capable of causing both Hemolytic Disease of the
Fetus and Newborn (HDFN) and Hemolytic Transfusion Reactions
(HTR).
These issues explain the clinical significance of “naturally occurring”
antibodies.
HDFN usually presents itself with a maternal antibody of an IgG
isotype that corresponds to an antigen on the surface of the baby’s
red cells.
The most common scenario is a group O mother and a group A baby.
o ABO hemolytic disease may affect a woman’s first pregnancy.
This is in contrast to Rh HDFN where the antigenic stimulation
usually occurs in the first pregnancy and subsequent antigen-
positive newborns are affected.
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Hemolytic transfusion reaction occurs when a recipient is transfused
with red cells that are an ABO group incompatible with the antibodies
in his or her serum. Because of the complement-binding ability of the
ABO antibodies, this is always a life-threatening situation.
As the recipient antibodies react with the incompatible red cells,
complement is activated and in vivo hemolysis, agglutination, and
red blood cell destruction occurs.
ABO compatibility is also significant in solid organ transplantation.
o For most organs, an ideal scenario for transplant is an ABO
compatible solid organ.
o Post-transfusion antibody titer, and pheresis to reduce the
titer of the incompatible antibody, will assist in achieving a
positive outcome when an ABO incompatible organ is
transplanted.
EFFECTS OF AGE ON THE PRODUCTION OF ISOAGGLUTININS
ISOAGGLUTININS
Isoagglutinins are antibodies produced by an individual that cause
agglutination of RBCs in other individuals.
People possess isoagglutinins directed toward the A or B antigen
absent from their own RBCs.
For example, type B or O individuals will usually possess anti-A. The
anti-A is formed in response to exposure to A-like antigenic structures
found in ubiquitous non-red cell biologic entities (eg, bacteria).
Isoagglutinins present in the newborn are passively acquired from
maternal circulation.
o Such passively acquired isoagglutinins will gradually
disappear, and the infant will begin to produce
isoagglutinins at 3 to 6 months of age.
Isoagglutinins production may vary in patients with certain
pathologic conditions.
o Decreased levels of isoagglutinins may be seen in patients
with acquired and congenital hypogammaglobulinemia and
agammaglobulinemia.
o Some individuals with roundworm infections will have
elevated levels of anti-A.
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MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
ANTI-A AND ANTI-B
Anti-A and anti-B are found in the sera of individuals who lack the
corresponding antigens.
They are produced in response to environmental stimulants, such as
bacteria, and have therefore been termed natural antibodies.
Antibody production begins after birth, reaching a peak at age 5 to
10 and declining with increasing age.
The antibodies formed to carbohydrate antigens are mostly
immunoglobulin M (IgM).
o IgM antibodies activate complement, which, in conjunction
with the high density of ABO antigen sites on RBCs,
o is responsible for the severe, life-threatening transfusion
reactions that may be caused by ABO-incompatible
transfusions.
Hemolytic disease of the Newborn
Hemolytic disease of the newborn caused by ABO antibodies is
usually mild, for the following reasons:
o placental transfer is limited to the fraction of IgG anti-A and
anti-B found in maternal serum
o fetal ABO antigens are not fully developed
o ABO tissue antigens provide additional targets for the
antibodies.
ABO-HDN is most often seen in non-group O infants of group O
mothers, because anti-A, anti-B, and anti-A,B of group O mothers
often has a significant IgG component.
Bombay Phenotype
Potent anti-H (along with anti-A and anti-B) found in Oh (Bombay)
or para-Bombay nonsecretors destroys transfused RBCs of any ABO
group, so these individuals must be transfused only with blood of
the Bombay phenotype.
In contrast, anti-H in non-Bombay individuals is usually IgM and
clinically insignificant.
Anti-IH is not uncommonly found in patient sera and is usually IgM;
compatible blood is easily found among donors of identical ABO
type.
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 MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
INHERITANCE OF THE ABO BLOOD GROUPS o The specific combination of these four components
The theory for the inheritance of the ABO blood groups was first determines an individual's type in most cases.
described by Bernstein in 1924. The table below shows the possible permutations of antigens and
o He demonstrated that an individual inherits one ABO gene antibodies with the corresponding ABO type ("yes" indicates the
from each parent and that these two genes determine presence of a component and "no" indicates its absence in the
which ABO antigens are present on the RBC membrane. blood of an individual).
The inheritance of ABO genes, therefore, follows simple Mendelian
genetics. ABO Blood Antigen A Antigen B Antibody Antibody
o ABO, like most other blood group systems, is codominant in Type anti-A anti-B
expression. A YES NO NO YES
o One position, or locus, on each chromosome 9 is occupied B NO YES YES NO
by an A, B, or O gene. O NO NO YES YES
The O gene is considered an amorph, as no detectable antigen is AB YES YES NO NO
produced in response to the inheritance of this gene.
o Therefore, the group O phenotype is an autosomal For example;
recessive trait with the inheritance of two O genes that are People with type A blood will have the A antigen on the surface of
nonfunctional. their red cells (as shown in the table below).
o The designations group A and B refer to phenotypes, o As a result, anti-A antibodies will not be produced by them
whereas AA, BO, and OO denote genotypes. because they would cause the destruction of their own
o In the case of an O individual, both phenotype and blood.
genotype are the same, because that individual would have However, if B type blood is injected into their systems, anti-B
to be homozygous for the O gene. antibodies in their plasma will recognize it as alien and burst or
o An individual who has the phenotype A (or B) can have the agglutinate the introduced red cells in order to cleanse the blood of
genotype AA or AO (or BB or BO). alien protein.

THE ABO GENOTYPES AND PHENOTYPES. ABO Blood Antigen A Antigen B Antibody Antibody
Serologically, it is not possible to determine the genotype from the Type anti-A anti-B
phenotype of an A or B individual. A YES NO NO YES
o Family studies or molecular assays would have to be B NO YES YES NO
performed to determine the exact genotype. O NO NO YES YES
o The phenotype and genotype are the same in an AB AB YES YES NO NO
individual because of the inheritance of both the A and B
gene. Individuals with type O blood do not produce ABO antigens.
All humans and many other primates can be typed for the ABO Therefore, their blood normally will not be rejected when it is given
blood group. to others with different ABO types.
o There are four principal types: A, B, AB, and O. o As a result, type O people are universal donors for
o There are two antigens and two antibodies that are mostly transfusions, but they can receive only type O blood
responsible for the ABO types. themselves.

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 MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
Those who have type AB blood do not make any ABO antibodies. H AND SE ANTIGEN
Their blood does not discriminate against any other ABO type. The H antigen is actually the precursor structure on which A and B
o Consequently, they are universal receivers for transfusions, antigens are made.
but their blood will be agglutinated when given to people o Inheritance of the H gene results in the formation of the H
with every other type because they produce both kinds of antigen.
antigens. o The H and Se genes are closely linked and located on
chromosome 19, in contrast to ABO genes, which are
ABO Blood Antigen A Antigen B Antibody Antibody located on chromosome 9.
Type anti-A anti-B o The H and Se genes are not part of the ABO system;
A YES NO NO YES however, their inheritance does influence A and B antigen
B NO YES YES NO expression.
O NO NO YES YES The H gene must be inherited to form the ABO antigens on the
AB YES YES NO NO RBCs, and the Se gene must be inherited to form the ABO antigens
in secretions.
It is easy and inexpensive to determine an individual's ABO type from a
few drops of blood. PRECURSOR SUBSTANCE
o A serum containing anti-A antibodies is mixed with some of the The precursor substance on erythrocytes is referred to as type 2.
blood. o This means that the terminal galactose on the precursor
o Another serum with anti-B antibodies is mixed with the remaining substance is attached to the N-acetylglucosamine in a beta
sample. 1 → 4 linkage .
Whether or not agglutination occurs in either sample indicates the ABO
A type 1 precursor substance refers to a beta 1 → 3 linkage
type.
It is a simple process of elimination of the possibilities. between galactose and N-acetylglucosamine.
ABH antigens on the RBC are constructed on oligosaccharide chains
For instance, if an individual's blood sample is agglutinated by the anti-A
of a type 2 precursor substance.
antibody, but not the anti-B antibody, it means that the A antigen is
present but not the B antigen. Therefore, the blood type is A.
ABH ANTIGEN DEVELOPMENT
FORMATION OF ABH ANTIGENS ON THE RBCS FROM PRECURSOR The ABH antigens develop early in fetal life but do not increase
much in strength during the gestational period.
SUBSTANCE TO IMMUNODOMINANT SUGARS
The RBCs of the newborn have been estimated to carry anywhere
The formation of ABH antigens results from the interaction of genes from 25% to 50% of the number of antigenic sites found on the
at three separate loci (ABO, Hh, and Se). adult RBC.
o These genes do not actually code for the production of As a result, reactions of newborn RBCs with ABO reagent antisera
antigens but rather produce specific glycosyltransferases are frequently weaker than reactions with adult cells.
that add sugars to a basic precursor substance. The expression of A and B antigens on the RBCs is fully developed by
A, B, and H antigens are formed from the same basic precursor 2 to 4 years of age and remains constant throughout life.
material (called a paragloboside or glycan) to which sugars are In addition to age, the phenotypic expression of ABH antigens may
attached in response to specific enzyme transferases elicited by an vary with race, genetic interaction, and disease states.
inherited gene.

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INTERACTION OF Hh AND ABO GENES
Blood Group O
Individuals who are blood group O inherit at least one H gene
(genotype HH or Hh) and two O genes.
The H gene elicits the production of an enzyme called α-2-L-
fucosyltransferase, which transfers the sugar L-fucose to an
oligosaccharide chain on the terminal galactose of type 2 chains.
o The sugars that occupy the terminal positions of this
precursor chain and confer blood group specificity are called
the immunodominant sugars.
o Therefore, L-fucose is the sugar responsible for H specificity
(blood group O).
The O gene at the ABO locus, which is sometimes referred to as an
amorph, does not elicit the production of a catalytically active
polypeptide transferase; therefore, the H substance remains
unmodified.
Consequently, the O blood group has the highest concentration of
H antigen.
o The H substance (L-fucose) must be formed for the other
sugars to be attached in response to an inherited A and/or B
gene.
The H gene is present in more than 99.99% of the random
population. Its allele, “h,” is quite rare, and the genotype hh is
extremely rare.
The term Bombay has been used to refer to the phenotype that
lacks normal expression of the ABH antigens because of the
inheritance of the hh genotype.
o The hh genotype does not elicit the production of α-2-L-
fucosyltransferase.
o Therefore, L-fucose is not added to the type 2 chain, and H
substance is not expressed on the RBC.
o Even though Bombay (hh) individuals may inherit ABO
genes, normal expression, as reflected in the formation of A,
B, or H antigens, does not occur.
#MLSWATER2022
MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
BLOOD GROUP A
In the formation of blood group A, the A gene (AA or AO) codes for
the production of α-3-N-acetylgalac - tosaminyltransferase, which
transfers an N-acetyl-D-galac - tosamine(GalNAc) sugar to the H
substance.
o This sugar is responsible for A specificity (blood group A).
o The A-specific immunodominant sugar is linked to a type 2
precursor substance that now contains H substance through
the action of the H gene.
The A gene tends to elicit higher concentrations of transferase than
the B gene. This leads to the conversion of practically all of the H
antigen on the RBC to A antigen sites.
o As many as 810,000 to 1,170,000 antigen sites exist on an
A1 adult RBC in response to inherited genes.
BLOOD GROUP B
Individuals who are blood group B inherit a B gene (BB or BO) that
codes for the production of α-3-D-galactosyltransferase, which
attaches D-galactose (Gal) sugar to the H substance previously
placed on the type 2 precursor substance through the action of the
H gene.
o This sugar is responsible for B specificity (blood group B).
o Anywhere from 610,000 to 830,000 B antigen sites exist on
a B adult RBC in response to the conversion of the H antigen
by the α-3-Dgalactosyltransferase produced by the B gene.
When both A and B genes are inherited, the B enzyme (α-3-D-
galactosyltransferase) seems to compete more efficiently for the H
substance than the A enzyme (α-3-
Nacetylgalactosaminyltransferase).
o Therefore, the average number of A antigens on an AB adult
cell is approximately 600,000 sites, compared with an
average of 720,000 B antigen sites.
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REACTIVITY OF ULEX EUROPAEUS WITH THE VARIOUS ABO GROUPS
The H antigen is a basic blood group antigen present in human beings.
There is considerable variation in the H antigen content in different
individuals of the same ABO group but the general pattern indicates
their strength as O>A2>A2B>B>A1>A1B.
Water soluble H substance can also be demonstrated in saliva or body
fluids of individuals who are secretors.
Human red blood cells that do not agglutinate with Anti-H lectin are
classified as Bombay Phenotype (Oh).
o The Bombay Phenotype is more common in India than other parts
of the world and the estimated gene frequency of Oh phenotype in
Bombay is 0.0066%.
REAGENT
Anti-H lectin is a ready to use purified extract of Ulex europaeus seeds.
It contains a phytohaemagglutinin, which is virtually specific for the H
antigen on human red blood cells.
Anti-H lectin is used for recognition of the H antigen on human red
blood cells.
It is useful, especially for assessing the H secretor status of group 'O'
individuals and also in differential grouping of Aint subgroup along with
Anti-A1 lectin.
PRINCIPLE
Human red blood cells possessing the H antigen will agglutinate in
the presence of seed extract (lectins) containing
phytohaemagglutinin specifically directed towards it.
Water soluble H substance present in saliva neutralises Anti-H
lectin.
Agglutination of red blood cells / Neutralization of Anti-H lectin by
saliva is a positive test result and indicates the presence of
H substance on/in the red cell / saliva respectively.
No agglutination / Neutralization of Anti-H lectin is a negative test
result and indicates the absence of H substance on / in the red cell /
saliva respectively.
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MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
NOTE
1. In vitro diagnostic reagent for laboratory and professional use only.
 Not for medicinal use.
2. The reagent contains sodium azide 0.1% as preservative. Avoid
contact with skin and mucosa.
 On disposal flush with large quantities of water.
3. Extreme turbidity may indicate microbial contamination / reagent
deterioration. Such reagents should be discarded.
ABO SUBGROUPS
A AND B SUBGROUPS
A1 AND A2 SUBGROUPS
Group A antigens can be differentiated into multiple subgroups. The
two major subgroups are
o A1, 80% of Group A individuals, and
o A2, 20% of group A individuals
Persons typing as AB can be divided into the same percentages of A
antigen presentation.
o A1B make up approximately 80% and
o A2B are 20% of all AB individuals.
The remaining group A individuals fall into one of many minor
subgroups.
A1 and A2 antigens have qualitative and quantitative differences.
Qualitative differences of A1 and A2 antigens
For qualitative test, a subgroup phenotype are distinguished from A1
phenotype by a glycolipid antigen expression because of the
structural differences in the branching of the oligosaccharide chain
The structural difference explain why the A subgroup individuals
often make the anti-A1
The reagent dolichos biflorus lectin will distinguish A1 from A2
When red cells are qualitatively tested for antigens, A1 and A2 red
cells have differing amounts of antigens on the cell surface.
o The A1 gene produces a transferase that has a greater ability to
convert H antigen to A antigen than the A2 gene.
o This quantitative difference results from the kinetics of the
reaction catalyzed by each of the transferases.
There are also differences in the quantity of transferase produced.
Page 9 of 16

o Individuals exhibiting the A1 phenotype have five to ten times
more transferase than those with an A2 phenotype.
Two mutations have been detected that produce this A2 phenotype:
o a Pro156Leu substitution
o a single nucleotide deletion (nucleotide 1060).
These substitutions are responsible for the decreased enzyme activity
that differentiates A2 from A1 cells. -Refer to Module packet regarding Quali & Quanti differences of A1 &A 2 antigens
(pp. 11-12)
The antigens also differ qualitatively.
ADDITIONAL A SUBGROUPS
Quantitative differences of A1 and A2 antigens
For quantitative test, produces so little antigen because the number
of A antigen is reduced in A2.
There is a little amount of antigen in A2 than in A1
A2 antigens are composed mainly of linear oligosaccharide chains
A1 cells have a greater number of branched chains
o In routine testing, this qualitative difference is not detectable but
can be determined biochemically.
Typing of A1 and A2 cells is unremarkable with routine antisera.
Both A1 and A2 cells will react equally with anti-A and anti-A,B.
The lectin, Dolichos biflorus, is used to obtain an extract with anti-A1
specificity.
o Dolichos biflorus will react specifically with A1 cells and will be
negative with A2 cells.
A2 individuals can develop antibodies to the A1 antigens.
The typical reaction pattern of reverse grouping in a group A individual
is no agglutination with the A cells (no anti-A) and agglutination with B SUBGROUPS OF B
cells (anti-B present).
o In A2 persons with an anti-A1, the A cells will also be agglutinated in
the reverse grouping.
o This discrepancy should be confirmed by testing the red cells with
the Dolichos biflorus lectin.
MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
QUANTITATIVE QUALITATIVE
↓ Number of Antigen Sites Differences in the precursor
oligosaccharide chains
↓ Amount of Transferase Enzyme Subtle differences in transferase
enzymes
↓ Amount of Branching Formation of anti-A1, in a
percentage of some subgroups
Occurring subgroups of A exist less frequently.
o These subgroups are also genetically controlled.
Subgroups of A include Aintr, A3, Ax , Am, Aend, Ael,and Abantu.
o These subgroups follow the patterns of A1 and A2 with regard to
quantitative and qualitative antigenic differences.
o The cells of these subgroups exhibit fewer antigen sites on their
surface while many demonstrate an anti-A1 in the plasma.
Adsorption and elution techniques may be necessary for detection of
antigens on the surface of red cells.
The classification of subgroups is based on reactions of the patient’s
red cells with anti-A, anti-B, anti-A,B, and anti-A1 antisera as well as A1,
A2, and B reverse grouping cells.
o While testing for subgroups of A, a mixed field agglutination
reaction may be noted.
o A3 cells will demonstrate this pattern of agglutination with anti-A
and anti-A,B.
Very rare and encountered less frequently than subgroups of A.
Methods of detection and classification are similar to those used for the
subgroups of A.
WEAK SUBGROUPS OF A
The prevalence of A subgroups of A weaker than A1 and A2 is less than
1%.
Fewer antigen sites on the RBC means weaker reactions with antisera.
It is possible for an Ax donor to be mistyped as O.

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 MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
This unit could then be transfused into an O recipient, who has anti- THE BOMBAY PHENOTYPES (OH)
A,B. The Bombay phenotype was first reported by Bhende in 1952 in
The anti-A,B antibody in the recipient could agglutinate and lyse the Bombay, India.
donor Ax RBCs and cause intravascular hemolysis. It represents the inheritance of a double dose of the h gene,
producing the very rare genotype hh.
Subgroup Laboratory Results Number of A o As a result, the ABO genes cannot be expressed, and ABH
Antigens sites antigens cannot be formed, since there is no H antigen
A3 Mixed field reaction with anti-A and 35,000 per made in the Bombay phenotype.
most anti-A,B reagents RBC More than 130 Bombay phenotypes have been reported in various
Ax Characteristically not agglutinated 4000 parts of the world. These RBCs are devoid of normal ABH antigens
with anti-A but do agglutinate with most and, therefore, fail to react with anti-A, anti-B, and anti-H.
examples of anti-A,B In RBC testing using anti-A and anti-B, the Bombay would
Aend Mixed field reaction with anti-A and 3500 phenotype as an O blood group.
anti-A,B. Aend is inherited as an allele at Using conventional or non-potent anti-A and anti-B sera
the ABO locus. Anti-A1 is found in some would result in the Bombay phenotype to be mistaken as
sera. Only H is found in secretions. O-blood group.
Am Characteristically not agglutinated, or very 200-1900 To differentiate group O from Bombay, use anti-A, anti-B,
weakly agglutinated by all anti-A and anti- and anti-H since group O (with H-antigens) would always
A,B have an agglutination with anti-H
reagents. Usually do NOT produce anti-A1 o However, the RBCs of the Bombay phenotype (Oh) do not
in react with the anti-H lectin (Ulex europaeus), unlike those of
sera. the normal group O individual, which react strongly with
anti-H lectin.
WEAK B SUBGROUPS Bombay serum contains antiA, anti-B, anti-A,B, and anti-H.
With the addition of the anti-A and anti-B reagent to the cell, there Unlike the anti-H found occasionally in the serum of A1 and A1B
can be two reactions: weak agglutination or no agglutination at individuals, the Bombay anti-H can often be potent and reacts
all. strongly at 37°C.
It is an IgM antibody that can bind complement and cause RBC
Weak
lysis.
B(3) – mix field reaction with anti-A and anti-B
Transfusing normal group O blood (with the highest concentration
B(x)– weak agglutination with anti-A and anti-AB only
of H antigen) to a Bombay recipient (anti-H in the serum) would
cause immediate cell lysis.
No agglutination o Therefore, only blood from another Bombay individual will
B(m) – demonstrates the presence of A-substance be compatible and can be transfused to a Bombay recipient.
B(el) – shows the presence of H-substance ABH substance is also absent in saliva.
The (Oh) Bombay phenotype is inherited as an autosomal recessive
-Refer to reference book for further information. trait.
The underlying molecular defect is most commonly a mutation in
the gene FUT1 (H gene), which produces a silenced gene that is

#MLSWATER2022 Page 11 of 16

incapable of coding for the enzyme, α(1,2)fucosyltransferase (H
transferase).
This enzyme catalyzes the transfer of fucose in an α-1,2 linkage to
the terminal galactose of the precursor molecule on RBCs forming
the H antigen.
This mutation underlying the Bombay phenotype is also associated
with a silenced FUT2 gene (Se gene), which normally encodes a very
similar α(1,2)fucosyltransferase and normally transfers a fucose to
form H antigens in secretions when active.
When family studies demonstrate which ABO genes are inherited in
the Bombay phenotype, the genes are written as superscripts (OhA,
OhB, OhAB).
EFFECTS OF DISEASE ON THE EXPRESSION OF ABH ANTIGENS
AND ANTIBODIES
ABH ANTIGENS AND ANTIBODIES IN DISEASE
Associations between ABH antigens and practically any disorder
known to man can be found throughout medical literature.
Even more profound are the associations of blood group specificity
with such things as a more pronounced:
o “hangover” in A blood groups
o “criminality” in group B blood groups
o “good teeth” in group O individuals.
There are also several papers correlating blood groups with
personality traits. It is no surprise that many scientists refer to
these associations as a part of blood group mythology.
However, more relevant associations between blood groups and
disease are important to the blood banker in terms of blood group
serology.
Various disease states seem to alter red cell antigens and result in
progressively weaker reactions or additional acquired
pseudoantigens, which can be seen during forward grouping.
Leukemia, chromosome 9 translocations, and any hemolytic
disease that induces stress hematopoiesis (e.g., thalassemia) have
been shown to depress antigen strength.
o Often the cells will appear to show a mixed-field
agglutination (tiny agglutinates in a sea of unagglutinated
cells).
#MLSWATER2022
MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
Hodgkin’s disease also has been reported to weaken or depress
ABH red cell antigens, resulting in variable reactions during forward
grouping similar to those found in leukemia.
o The weakening of the antigen tends to follow the course of
the disease.
o The antigen strength will increase again as the patient
enters remission.
The isoagglutinins (anti-A, anti-B, or anti-A,B) also may be weak or
absent in those leukemias demonstrating
hypogammaglobulinemia, such as chronic lymphocytic leukemia
(CLL).
Various lymphomas, such as the malignant (non-Hodgkin’s) variety,
may yield weak isoagglutinins, owing to moderate decreases in the
gamma globulin fraction.
Also, immunodeficiency diseases, such as congenital
agammaglobulinemia, will also yield weak or absent isoagglutinins.
o If this problem is suspected, a simple serum protein
electrophoresis will confirm or rule out this condition.
Individuals with intestinal obstruction, carcinoma of the colon or
rectum, or other disorders of the lower intestinal tract may have
increased permeability of the intestinal wall, which allows passage
of the bacterial polysaccharides from Escherichia coli serotype O86
into the patient’s circulation.
o This results in the acquired B phenomenon in group A1
individuals.
o The patient’s group A red cells absorb the B-like
polysaccharide, which reacts with human-source anti-B.
A lack of detectable ABO antigens can occur in patients with
carcinoma of the stomach or pancreas.
o The patient’s red cell antigens have not been changed, but
the serum contains excessive amounts of blood group–
specific soluble substances (BGSS) that may neutralize the
antisera utilized in the forward grouping.
All these disease states previously mentioned may result in
discrepancies between the forward and reverse groupings,
indicating that the patient’s red cell group is not what it seems.
All ABO discrepancies must be resolved before blood for transfusion
is released for that patient.
o In some cases, secretor or molecular studies may help
confirm the patient’s true ABO group.
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LABORATORY TESTS TO DETECT AND IDENTIFY ABO
ANTIGENS AND ANTIBODIES
ENZYME-CONVERTED O CELLS (ECO)
Blood group O is considered the universal donor because it can be
transfused to patients of all ABO types. Therefore, enzymes that
remove terminal carbohydrates from the nonreducing end of
carbohydrate chains could be used to remove terminal A and B
sugars to convert the blood supply to all universal group O units.
An enzyme from coffee beans, α-galactosidase, has been the most
successful at removing galactose to convert blood group B to group
O.
RBCs treated in this manner have normal survival when transfused
to group B, A, or O recipients.
Removal of N-acetylgalactosamine to convert group A to group O
has been much more problematic, owing to the inaccessibility of the
carbohydrates on internal branching chains, especially those found
on A1 cells.
The procedures required to convert B to O, which include exposure
to low pH followed by numerous washings, make them impractical
for general use. This is an active area of research and alternative
enzymes and improved methodologies are under investigation.
BLOOD TYPING
Blood typing is a method to tell what type of blood you have.
Is done so you can safely donate your blood or receive a blood
transfusion.
It is also done to see if you have a substance called Rh factor on the
surface of your red blood cells.
Your blood type is based on whether or not certain proteins are on
your red blood cells.
o These proteins are called antigens. Your blood type (or
blood group) depends on what types your parents passed
down to you.
FORWARD AND REVERSE GROUPING
ABO FORWARD GROUPING
As previously described, ABO antigens are present on the surface of
red cells, while the antibodies are found in plasma or serum.
#MLSWATER2022
MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
Routine testing for antigens and antibodies is performed as a
forward and reverse grouping, respectively.
The forward grouping is a test performed for antigens using known
antisera with patient’s cells that may contain unknown antigens.
Test methods for forward grouping include:
o Tube typing
o Gel Technology
o Automation
o Solid Phase Technology
ABO forward grouping with tube typing uses a saline suspension of
3 to 5% washed patient red cells. These cells are combined in a 1:1
ratio with commercial antisera.
When utilizing gel technology, the cell suspension consists of a
0.8% suspension of washed patient cells in the manufacturer’s
recommended diluent.
These cells are applied to the anti-A and anti-B tubes of the gel card.
In both methods, centrifugation is applied and results interpreted.
ANTISERA
When performing tube typing, three antiseras are available for ABO
forward grouping.
Forward grouping may be performed using all three, or in the case
of patients or transfusion recipients anti-A and anti-B are used.
Antisera are combined in a 1:1 ratio with the patient’s cell
suspension.
When evaluating reaction patterns, the antigens on the cells are
reacting with the specific antibodies in the antisera.
o Group A individual has the A antigen and reacts with both
anti-A and anti-A,B.
o A group O will react with no antisera since these cells have
neither A nor B antigens.
The inclusion of anti-A,B antisera in forward grouping is significant.
o It is not a mixture of anti-A and anti-B, but rather a separate
antibody that will react with both the A and B antigens.
It is included in forward grouping and serves two purposes:
1. To confirm the results of the anti-A and anti-B.
2. To detect weak subgroups of A and B.
These subgroups may demonstrate a positive reaction with anti-A,B
but not with anti-A and anti-B.
Page 13 of 16
 MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
SAMPLE PROCEDURE 1: REACTION PATTERNS FOR ABO GROUP
ABO FORWARD GROUPING: TUBE TYPING METHOD BLOOD GROUP ANTI-A ANTI-B ANTI-A,B
Procedure: A POSITIVE NEGATIVE POSITIVE
1. Prepare a 3 to 5% suspension of patient’s red cells. B NEGATIVE POSITIVE POSITIVE
2. Label three small test tubes with the patient’s name and AB POSITIVE POSITIVE POSITIVE
identification number. O NEGATIVE NEGATIVE NEGATIVE
3. Each of these tubes should then be labeled as follows: First tube: In Forwarding Typing the Antigens are used as Antisera.
“Anti-A” Second tube: “Anti-B” Third tube: “Anti-A, B” o Blood Type O has no agglutination
NOTE: Labeling should be done with care since clerical errors are the o Blood Type A only has the reaction seen in Anti-A since Blood type A
most frequent errors in the blood bank. contains Antigen A
4. Check clarity and expiration date on antisera; record information. o Blood Type B only has the reaction seen in Anti-B since Blood type B
5. To each of these tubes, add one drop of the corresponding antisera. contains Antigen B
NOTE: Use a free-floating drop. Do not touch the dropper to the side o Blood Type AB has both the Antigen AB therefore it is present in
of the tube. Always add antisera before cells. both reactions.
6. Using a transfer pipet, add one drop of the well-mixed 5% cell
suspension to each of these three tubes. Molecular Testing
NOTE: Use a free-floating drop. Do not touch the pipet to the side of
As molecular diagnostic testing continues to develop, applications
the tube.
to ABO forward grouping may become commonplace in the
7. Gently mix all tubes.
laboratory.
8. Serofuge all three test tubes for 15 seconds.
Molecular testing has the potential to solve typing discrepancy in
NOTE: Time may vary with each serofuge. Check the calibration
recently or chronically transfused patients.
information for each individual serofuge.
These patients present unique challenges to the blood bank, since
9. Remove each tube and examine for hemolysis.
typing through traditional methods frequently produces discrepant
10. Using an agglutination viewer, gently resuspend each cell button,
or erroneous results.
and examine for agglutination.
11. Grade each reaction and record the results. POLYMERASE CHAIN REACTION (PCR) METHODS
Have been proven to be more reliable than traditional serological
methods in resolution of typing discrepancies in recipients that have
SAMPLE PROCEDURE 2:
been transfused within the last 30 days.
1. Prepare a 0.8% suspension of test cells in the appropriate diluent.
As PCR test results are available within several hours, this testing
2. Choose a gel card for ABO forward grouping and label with patient
method presents a promising future for resolution of discrepancies
identification.
that were previously major compatibility challenges.
3. Add 10μl of cell suspension of test cells to each microtube on the
card.
4. Centrifuge the card. Reverse Grouping
5. Evaluate each microtube for agglutination. Record and interpret
ABO reverse grouping uses patient plasma combined in a 2:1 ratio
results.
with commercially prepared cells.
The cells are packaged in sets of two (A1 and B) or three (A1, A2,
and B). The cells are used to detect unknown antibodies in the
plasma.

#MLSWATER2022 Page 14 of 16
 MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
The result is evaluated by examining the tubes for hemolysis and INTERPRETATION OF REVERSE GROUPING TEST RESULTS
agglutination. BLOOD GROUP A1 CELLS B CELLS
NOTE: A NEGATIVE POSITIVE
Recall that antibodies present in the test plasma correspond to B POSITIVE NEGATIVE
antigens missing on the red cell surface. O POSITIVE POSITIVE
For example, group A has the A antigen and the B antigen is not AB NEGATIVE NEGATIVE
present. For the reverse typing, this is the detection of antibodies in our serum
The corresponding B antibody is demonstrated in the group A o Both positive reaction for Blood type O since it contains both Anti
individual’s plasma. A,B
When the plasma reacts with the reagent red blood cells, the B o In Blood type A, only B cells has the reaction since Blood type A
antibody reacts with specific antigens on the B cells, but not individual has the Anti B
antigens on the A cells. o In Blood type B, only A cells has the reaction since Blood type B has
Therefore, a positive reaction will be seen in the B tube, but not in the Anti- A
the A tube. o In Blood type AB, there is no reaction for the reverse typing since
Blood type AB contains no any antibodies in their serum
SAMPLE PROCEDURE:
1. Label two 10 × 75 test tubes with the patient’s name and
identification number. ABO DISCREPANCIES
2. Label one of the tubes A and one of them B. ABO discrepancies occur when unexpected reactions occur in the
NOTE: Labeling is a crucial step in the blood typing procedure. Fatal forward and reverse grouping.
errors are made when clerical errors occur. These can be due to:
3. Add two drops of serum to each tube. o Problems with the patient’s serum (reverse grouping)
4. To the appropriate tube, add one drop of well-mixed reagent red o Problems with the patient’s red cells (forward grouping)
cells. o Problems with both the serum and cells.
NOTE: Before adding reagent red cells, be certain they are well- The unexpected reaction can be due to an extra positive reaction or
mixed and that all of the cells are resuspended from the bottom of a weak or missing reaction in the forward and reverse grouping.
the vial. All ABO discrepancies must be resolved prior to reporting a patient
5. Gently mix the two tubes. or donor ABO group.
6. Serofuge for 15 seconds.
NOTE: The time for centrifugation may vary with each serofuge. Technical Errors
Check the calibration on the serofuge being used for testing. Technical errors can also cause ABO discrepancies.
7. Remove each tube and examine for hemolysis. This includes errors in labeling the blood sample at the patient’s
8. Using an agglutination viewer, gently resuspend each cell button and bedside or in the laboratory; therefore, patient and sample
examine for agglutination. identification are essential!
9. Grade and record each reaction. Other errors include the failure to add reagents or the addition of
incorrect reagents or sample. Therefore, it is recommended that
serum and antiserum be added first, then the patient or reagent red
cells.

#MLSWATER2022 Page 15 of 16
 MODULE 2 PART 2- ABO BLOOD GROUP SYSTEM
It is also recommended that results be recorded immediately to
Forward vs. Reverse Typing
avoid transcription errors.
In addition, contaminated reagents can cause errors in testing. Forward Typing Reverse Typing
Therefore, looking at all the reagent vials when performing ABO Other term Direct or Front Typing Indirect or Back Typing
testing and during quality control testing is extremely important. Detection A & B antigen within Anti-A and Anti B in
patient’s RBCs patient’s serum
Resolution Specimen 1 drop of whole blood or 2- 2 drops of patient’s serum
5% red cell suspension
If the initial test was performed using RBCs suspended in serum or
Reagent 2 drops of Anti-A (blue)and 1 drop of A1 red cells and B
plasma, repeat testing the same sample using a saline suspension of
Anti-B (yellow) red cells derived from
RBCs can usually resolve the ABO discrepancy.
human source, 4-5% RBC
It is important to make sure that any and all technical factors that
suspension is used as
may have given rise to the ABO discrepancy are reviewed and
reagents
corrected.
It is also essential to acquire information regarding the patient’s Blood Forward Typing Antigens Reverse Typing Antibody
age, diagnosis, transfusion history, medications, and history of Group Px’s RBC with in RBCs Px’s Serum with in Serum
pregnancy. Anti-A Anti-B A1 Cells B Cells
If the discrepancy persists and appears to be due to an error in
O 0 0 No A/ B + + Anti-A &
specimen collection or identification, a new sample should be
Anti-B
drawn from the patient and the RBC and serum testing repeated.
A + 0 A Ag 0 + Anti-B
When a discrepancy is encountered, results must be recorded, but
B 0 + B Ag + 0 Anti-A
interpretation of the ABO type must be delayed until the
AB + + A&B 0 0 none
discrepancy is resolved.
Px: Patient; 0: Negative/ No reaction; +: Positive
If blood is from a potential transfusion recipient, it may be
necessary to administer group O–compatible RBCs before the
discrepancy is resolved. Investigation of Weak A Subgroup: https://drive.google.com/file/d/1z-
In general, when investigating ABO discrepancies, it should be 4kYqsCKm7Yz2oMklMuV9FiixmEylrN/view?usp=sharing
noted that RBC and serum grouping reactions are very strong (3+ to
4+); therefore, the weaker reactions usually represent the Investigation of Weak B Subgroup:
discrepancy. https://drive.google.com/file/d/16C6F2j8rxVVBdKVw8NkPSiT6QHJ34Tn6/vi
ew?usp=sharing
Categories of ABO Discrepancies
-END OF TRANSCRIPTION -
ABO discrepancies may be arbitrarily divided into four major Bloody hell isn’t it? But imagine the lives that you will
categories: group I, group II, group III, and group IV discrepancies. save in the future!
Be Positive (B+) Dreamer!
-Refer to ABO Discrepancies Assignment for the complete description of each
Category. -quote by no one.

APPENDICES
#MLSWATER2022 Page 16 of 16
 MODULE 2 Part 3: Rh-Hr Blood Group System
MLS 18, March 10, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
HISTORY OF THE RH SYSTEM
Legend: Before 1939, the only significant blood group antigens recognized were
Notes/Module Packet Bullet those of the ABO system.
MODULE OUTLINE Transfusion medicine was thus based on matching ABO groups.
I. History of the Rh System
II. Nomenclatures of the Rh System Levine and Stetson
A. Fisher Race: DCE Terminology Described a hemolytic transfusion reaction in an obstetrical patient.
B. Wiener: Rh-Hr Terminology o After delivery of a stillborn infant, a woman required transfusions.
C. Rosenfield and Coworkers: Alphanumeric Terminology Her husband, who had the same ABO type, was selected as her
D. ISBT Committee: Numeric Terminology donor.
III. Proposed Genetic Pathways o After transfusion, the recipient demonstrated the classic symptoms
A. Mechanisms of Antigen Production of an acute hemolytic transfusion reaction.
B. Biochemistry of the Rh Antigens Subsequently, an antibody was isolated from the mother’s serum that
IV. Weak D: Variations of the Rho (D) Antigen Expression reacted both at 37C and at 20C with the father’s RBCs.
V. Detection of Rh Antibodies and Antigens It was postulated that the fetus and the father possessed a common
VI. Clinical Consideration factor that the mother lacked.
VII. Rh Deficiency Syndrome: Rhnull and Rhmod o While the mother carried the fetus, she was exposed to this factor
VIII. Unusual Phenotypes and Rare Alleles and subsequently built an antibody that reacted against the
IX. The LW Antigen transfused RBCs from the father, which resulted in the hemolytic
transfusion reaction.

LEARNING OUTCOMES Landsteiner and Wiener

1. Explain the derivation of the term Rh. Reported on an antibody made by guinea pigs and rabbits when they
2. Differentiate Rh from LW blood group systems were transfused with rhesus monkey RBCs a year after Levine and
3. Compare and contrast the Fisher-Race and Weiner theories of Rh Stetson’s discovery.
inheritance.
This antibody, which agglutinated 85 percent of human RBCs, was
4. Differentiate the mechanisms that result in the weakened
named Rh.
expression of D on the red blood cells.
Demonstrated that the agglutinin that had caused the hemolytic
5. List some instances in which the weak-D status of an individual
transfusion reaction and the antibody described by Landsteiner and
must be determined.
Wiener appeared to define the same blood group.
6. Describe the characteristics of Rh antigens (excluding dCcEe).
o Many years later it was recognized that the two antibodies were
7. Describe the characteristics of Rh antibodies.
different.
8. Compare & contrast Rhnull&Rhmod
o However, the name Rh was retained for the human-produced
9. Describe the role of RhAG in Rh antigen expression.
antibody, and the anti-rhesus antibody formed by the animals was

#MLSWATER2022 Page 1 of 9

renamed anti-LW in honor of those first reporting it (Landsteiner
and Wiener).
Further research resulted in defining Rh as a primary cause of hemolytic
disease of the newborn (HDN, also called erythroblastosis fetalis) and a
significant cause of hemolytic transfusion reactions.
By the mid-1940s, five antigens made up the Rh system. Today the Rh
blood group system is made up of nearly 50 different specificities.
NOMENCLATURES OF THE RH SYSTEM
The terminologies used to describe the Rh system are derived from four
sets of investigators:
o Two of the terminologies are based on the postulated genetic
mechanisms of the Rh system.
o The third terminology describes only the presence or absence of a
given antigen.
o The fourth is the result of the combined efforts of the International
Society of Blood Transfusion (ISBT) Working Party on Terminology
for Red Cell Surface Antigens.
Fisher-Race: The DCE Terminology
In the early 1940s, Fisher and Race were investigating the antigens
found on human RBCs, including the newly defined Rh antigen.
o They postulated that the antigens of the system were produced by
three closely linked sets of alleles.
Each gene was responsible for producing a product (or antigen) on the
RBC surface. Each antigen and corresponding gene were given the same
letter designation (when referring to the gene, the letter is italicized).
Fisher and Race named the antigens of the system D, d, C, c, and E and
e.
o Now it is known that “d” represents the absence of D antigen.
o The phenotype (blood type observed during testing) of a given RBC
is defined by the presence of D, C, c, E, and e expression.
According to the Fisher-Race proposal, each person inherits a set of Rh
genes from each parent (i.e., one D or d, one C or c, and one E or e).
o Because Rh genes are codominant, each inherited gene expresses
its corresponding antigen on the RBC.
o The combination of maternal and paternal haplotypes determines
one’s genotype (the Rh genes inherited from each parent) and
#MLSWATER2022
MODULE 2 Part 3: Rh-Hr Blood Group System
dictates one’s phenotype (the antigens expressed on the RBC that
can be detected serologically).
An individual’s Rh phenotype is reported as DCE rather than CDE
because Fisher postulated that the C/c locus lies between D/d and E/e
loci. This information is based on frequencies of the various gene
combinations.
Rh-positive person exhibiting a deletion phenotype such as these is
written -De or -DE, CD- or cD-, or -D-, respectively.
o The last is sometimes referred to as a double deletion.
o The person expressing no Rh antigens on the RBC is said to be
Rhnull, and the phenotype may be written as –––/–––.
Weakened expression of all Rh antigens of an individual has also been
reported.
o These individuals are said to have the Rhmod phenotype, and there
is no unique way of indicating this using the Fisher-Race terminology
Wiener: The Rh-Hr Terminology
Wiener believed that the gene responsible for defining Rh actually
produced an agglutinogen that contained a series of blood factors.
According to Rh-Hr terminology, this Rh gene produces at least three
factors within an agglutinogen.
o The agglutinogen may be considered the phenotypic expression of
the haplotype. Each factor is an antigen recognized by an antibody.
Antibodies can recognize single or multiple factors (antigens).
Page 2 of 9

When describing an agglutinogen, the uppercase R denotes the
presence of the original factor, the D antigen.
The lowercase r indicates the absence of the D antigen.
The presence of uppercase C is indicated by a one (1) or a single
prime (′).
Lowercase c is implied when there is no 1 or ′ indicated.
When referring to the Rh antigens (or factors) in Wiener nomenclature,
o the single prime (′) refers to either C or c,
o The double prime (′′) to either E or e.
o If the r precedes the h (i.e., rh′ or rh′′), this refers to the C or E
antigens, respectively.
o When the h precedes the r, this refers to either the c (hr′) or e (hr′′)
antigen.
Rh0 is equivalent to D. In the Wiener nomenclature, there is no
designation for the absence of D antigen.
#MLSWATER2022
MODULE 2 Part 3: Rh-Hr Blood Group System
Rosenfield and Coworkers: Alpha/ Numeric Terminology
As the Rh blood group system expanded, it became more difficult to
assign names to new antigens using existing terminologies.
In the early 1960s Rosenfield and associates proposed a system that
assigns a number to each antigen of the Rh system in order of its
discovery or recognized relationship to the Rh system.
o This system has no genetic basis, but simply demonstrates the
presence or absence of the antigen on the RBC.
o A minus sign preceding a number designates absence of the
antigen.
For the five major antigens, D is assigned Rh1, C is Rh2, E is Rh3, c is Rh4,
and e is Rh5.
For RBCs that type D C E c negative, e negative, the Rosenfield
designation is Rh: 1, 2, 3, 4, 5. If the sample was not tested for e, the
designation would be Rh: 1, 2, 3, 4. All Rh system antigens have been
assigned a number.
International Society of Blood Transfusion: Numeric Terminology
The ISBT adopted a six-digit number for each authenticated blood
group specificity.
o The first three numbers represent the system and the remaining
three the antigenic specificity.
o The number 004 was assigned to the Rh blood group system, and
then each antigen assigned to the Rh system was given a unique
number to complete the six-digit computer number.
o 001 was assigned to -------
o Therefore, D is RH1, C is RH2, and so forth.
The phenotype designation includes the alphabetical symbol that
denotes the blood group, followed by a colon and then the specificity
numbers of the antigens defined.
A minus sign preceding the number indicates that the antigen was
tested for but was not present.
o The phenotype D CE c e or DccEe or R2 r would be written RH:1,-
2,3,4,5.
When referring to a gene, an allele, or a haplotype, the symbols are
italicized, followed by a space or asterisk, and then the numbers of the
specificities are separated by commas.
o R1 or DCe would be RH 1,2,5.
Page 3 of 9

PROPOSED GENETIC PATHWAYS
MECHANISMS OF ANTIGEN PRODUCTION
Two theories of Rh genetic control were initially postulated.
o Wiener postulated that a single gene produces a single product that
contains separately recognizable factors.
o In contrast, Fisher and Race proposed that the Rh locus contains
three distinct genes that control the production of their respective
antigens.
It is currently accepted that only two closely linked genes located on
chromosome 1 control the expression of Rh; one gene (RHD) codes for
the presence or absence of the D polypeptides and the second gene
(RHCE) for either RHCe, RHcE, RHce, or RHCE polypeptides.
Another gene (RHAG), which resides on chromosome 6, produces a Rh-
associated glycoprotein that is very similar in structure to the Rh
proteins and within the RBC membrane forms complexes with the Rh
polypeptides. RHAG is called a coexpressor and must be present for the
successful expression of the Rh antigens but by itself does not express
any of the Rh antigens.
The Rh genes are inherited as codominant alleles. Offspring inherit one
Rh haplotype from each parent. In rare instances, individuals express no
Rh antigens on their RBCs. These individuals are said to have the Rhnull
phenotype.
BIOCHEMISTRY OF THE RH ANTIGENS
Rh antigens are nonglycosylated protein.
o This means that there are no carbohydrates attached to the protein.
The Rh antigens are transmembrane polypeptides and are an integral
part of the RBC membrane.
The gene products of RHD and RHCE are remarkably similar in that both
encode for proteins composed of 417 amino acids that traverse the cell
membrane 12 times and that their sequence differs by only 44 base
pairs.
#MLSWATER2022
MODULE 2 Part 3: Rh-Hr Blood Group System
WEAK D: VARIATIONS O F THE RH0 (D) ANTIGEN EXPRESSION
RBCs carrying the weaker D antigen have historically been referred to as
having the Du type.
o Now they are referred to as expressing weak D and are considered
Rh-positive.
o Three different mechanisms have been described that can explain
the weakened expression of the D antigen:
1. Genetic Weak D
Inheritance of D genes that code for a weakened expression of the D
antigen.
The D antigens expressed appear to be complete but few in number. On
a molecular level, these quantitative differences in D expression are
attributed to mutations of the Rh polypeptide.
Inheritance of these genes can be tracked vertically from one
generation to the next and are seen most frequently in blacks. The
genetic weak D is rare and seldom found in whites.
2. C Trans
Position effect or gene interaction effect
In individuals showing the gene interaction weak D, the allele carrying D
is trans (or in the opposite haplotype) to the allele carrying C
o for example, Dce/dCe.
The Rh antigen on the RBC is normal, but the steric arrangement of the
C antigen in relationship to the D antigen appears to interfere with the
expression of the D antigen.
3. Partial D (D Mosaic)
One or more of the D epitopes within the entire D protein is either
missing and/or is altered.
Cells with a partial-D antigen usually type weaker than expected or may
not react at all when routine procedures are used with most commercial
anti-D reagents.
Page 4 of 9

DETERMINATION OF D STATUS
Is required when testing donor bloods
Blood for transfusion is considered Rhpositive if either the D or the
weak-D test is positive.
Any donor blood sample that is typed Rh0(D)-negative by the slide or
rapid tube method must be tested further by an indirect antihuman
globulin technique.
NOTE:
If both tests are negative, the donor sample is considered Rh-
negative.
If the donor sample tests positive in any phase of Rh0(D) testing, the
sample is considered Rh-positive.
Determining the Rh0(D) status (including weak-D status) of obstetric
patients is critical.
All Rh-negative, weak D–negative obstetric patients are candidates for
Rh immune globulin (RhIg) (a drug injected to prevent Rh-negative
individuals who are exposed to Rh-positive RBCs from developing
antiD).
o Likewise, when the mother is Rh-negative and the newborn is typed
Rh-negative, the weak-D status of the newborn must be determined
to assess the likelihood of maternal sensitization and the need for
Rh immune globulin prophylaxis for the mother.
o If a newborn’s cells are coated with maternal IgG anti-D in utero,
very few D antigen sites are available to react with reagent anti-D
(termed “blocking phenomena”).
Elution of the sensitizing antibody (removing the antibody) and
identifying it as anti-D will verify that the infant’s RBCs are D-positive.
DETECTION OF RH ANTIBODIES AND ANTIGENS
Rh Antibodies
Although the Rh system was first recognized by saline tests used to
detect IgM antibodies, most Rh antibodies are IgG immunoglobulins and
react optimally at 37 degrees C or after antiglobulin testing.
Rh antibodies are usually produced following exposure of the
individual’s immune system to foreign RBCs, through either transfusion
#MLSWATER2022
MODULE 2 Part 3: Rh-Hr Blood Group System
or pregnancy. Rh antigens are highly immunogenic; the D antigen is the
most potent.
Exposure to less than 0.1 mL of Rh-positive RBCs can stimulate antibody
production in an Rh-negative person.
IgG1, IgG2, IgG3, and IgG4 subclasses of Rh antibodies have been
reported. IgG1 and IgG3 are of the greatest clinical significance because
the reticuloendothelial system rapidly clears RBCs coated with IgG1 and
IgG3 from the circulation.
As with most blood group antigen sensitization, IgM Rh antibodies are
formed initially, followed by a transition to IgG.
Rh antibodies do not bind complement. For complement to be fixed (or
the complement cascade activated), two IgG immunoglobulins must
attach to an RBC in close proximity. Rh antigens (to which the antibody
would attach) are not situated on the RBC surface this closely.
Therefore, when an Rh antibody coats the RBCs, intravascular,
complement-mediated hemolysis does not occur.
RBC destruction resulting from Rh antibodies is primarily extravascular.
Because Rh antibodies are primarily IgG and can traverse the placenta
and because Rh antigens are well developed early in fetal life, Rh
antibodies formed by Rh-negative pregnant women do cross the
placenta and may coat fetal RBCs that carry the corresponding antigen.
This results in the fetal cells testing positive by the direct antiglobulin
test and in HDN, the coated fetal cells are removed prematurely from
the fetal. Until the discovery of Rh immune globulin, anti-D was the
most frequent cause of HDN.
Rh Antigen Typing Reagents
The reagents used to type for D and for the other Rh antigens may be
derived from a variety of sources.
o The reagents may be high-protein–based or low-protein–based,
saline-based, chemically modified, monoclonal, or blends of
monoclonals.
Saline reactive reagents, which contain IgM immunoglobulin, were the
first typing reagents available to test for the D antigen.
o Saline anti-D has the advantage of being lowprotein–based and can
be used to test cells that are coated with IgG antibody. The primary
disadvantages of saline typing reagents are their limited availability,
Page 5 of 9

cost of production, and lengthy incubation time. Because saline
anti-D is an IgM immunoglobulin, it cannot be used for weak-D
typing.
In the 1940s, high-protein anti-D reagents were developed.
o Human plasma containing high-titer D-specific antibody is used as
the raw material.
o Potentiators of bovine albumin and macromolecular additives such
as dextran or polyvinylpyrrolidone are added to the source material
to optimize reactivity in the standard slide and rapid tube tests.
o The presence of potentiators and the higher protein concentration,
however, increase the likelihood of false positive reactions.
To assess the validity of the high-protein Rh typing results, a control
reagent was manufactured and had to be tested in parallel with each Rh
test. If the control reacted, the test result was invalid and had to be
repeated using a different technique or reagent anti-D. The major
advantages of high-protein anti-D reagents are reduced incubation time
and the ability to perform weak-D testing and slide typing with the same
reagent.
In the late 1970s, scientists chemically modified the IgG anti-D
molecule by breaking the disulfide bonds that maintain the antibody’s
rigid shape.
Monoclonal antibody reagents have become available more recently.
These reagents are derived from single clones of antibody-producing
cells.
CLINICAL CONSIDERATIONS
Transfusion Reactions
Rh antigens are highly immunogenic. The D antigen is the most
immunogenic antigen outside the ABO system.
Circulating antibody appears within 120 days of a primary exposure and
within 2 to 7 days after a secondary exposure.
Rh-mediated hemolytic transfusion reactions, whether caused by
primary sensitization or secondary immunization, usually result in
extravascular destruction of immunoglobulin-coated RBCs.
The transfusion recipient may have an unexplained fever, a mild
bilirubin elevation, and a decrease in hemoglobin and haptoglobin.
The direct antihuman globulin test is usually positive, and the antibody
screen may or may not demonstrate circulating antibody.
#MLSWATER2022
MODULE 2 Part 3: Rh-Hr Blood Group System
Hemolytic Disease of the Newborn (HDN)
HDN caused by Rh antibodies is often severe because the Rh antigens
are well developed on fetal cells, and Rh antibodies are primarily IgG,
which readily cross the placenta.
After years of research, a method was developed to prevent susceptible
(Rh0 D-negative) mothers from forming antiD, thus preventing Rh0(D)
HDN.
Rh-immune globulin, a purified preparation of IgG anti-D, is given to a D-
negative woman during pregnancy and following delivery of a D positive
fetus. Rh-immune globulin is effective only in preventing anti-D HDN.
RH DEFICIENCY SYNDROME: RHNULL AND RHMOD
It is the rare individual who fails to express any Rh antigens on the RBC
surface or exhibits a severely reduced expression of all Rh antigens. The
individuals who lack all Rh antigens on their RBCs are said to have the
Rhnull syndrome, which can be produced by two different genetic
mechanisms:
o In the regulator-type Rhnull syndrome, there is a mutation in the
RHAG gene. This results in no Rh polypeptides or RHAG antigen
expression on the RBCs, even though these individuals usually have
a normal complement of RHD and RHCE genes. These individuals
can pass the normal RHD and RHCE genes to their children.
o In the second type of Rhnull syndrome (the amorphic type), there is
a mutation in each of the RHCE genes and a deletion of the RHD
gene. The RHAG gene is normal.
Individuals with the Rhnull syndrome demonstrate a mild compensated
haemolytic anemia, reticulocytosis, stomatocytosis, a slight-to-
moderate decrease in haemoglobin and hematocrit levels, an increase
in hemoglobin F, a decrease in serum haptoglobin, and possibly an
elevated bilirubin level.
When transfusion of individuals with Rhnull syndrome is necessary, only
Rhnull blood can be given.
NOTE:
Individuals of the Rhmod phenotype have a partial suppression of Rh
gene expression and exhibit features similar to those with the
Rhnull syndrome
Page 6 of 9

However, the clinical symptoms are usually less severe and rarely
clinically remarkable.
33 RBCs classified as Rhmod do not completely lack Rh or LW
antigens.
Rhnull and Rhmod RBCs exhibit other blood group antigens; however,
S, s, and U antigen expression may be depressed.
34 Rhnull RBCs are negative for FY5.
UNUSUAL PHENOTYPES AND RARE ALLELES
Cw
Originally considered an allele at the C/c locus.
Later studies showed that it can be expressed in combination with both
C and c and in the absence of either allele.
Now known that the relationship between C/c and Cw is only
phenotypic and that
Antithetical to the high-incidence antigen MAR.
Found in about 2 percent of whites and is very rare in blacks.
Anti-Cw has been identified in individuals without known exposure to
foreign RBCs as well as after transfusion or pregnancy.
o Anti-Cw may show dosage (i.e., reacting more strongly with cells
from individuals who are homozygous for Cw). Because of the low
incidence of Cw, Cw antigen–negative blood is readily available.
f (ce)
The f antigen is expressed on the RBC when both c and e are present on
the same haplotype (i.e., cis position); it has been called a compound
antigen.
Phenotypically, the following samples appear the same when tested
with the five major Rh antisera: Dce/DCE and DcE/DCe. However, when
tested with anti-f, only the former reacts.
Anti-f has been reported to cause HDN and transfusion reactions.
rhi (Ce)
Similar to f, rhi is present when C and e are in the cis configuration, has
been called a compound antigen, and is a single entity.
#MLSWATER2022
MODULE 2 Part 3: Rh-Hr Blood Group System
A sample with the phenotype D C E c e can be either DcE/DCe or
Dce/DCE. Anti-rhi reacts only with Dce/DCe RBCs.
G
G is an antigen that is present on most D-positive and all Cpositive RBCs.
In the test tube, anti-G reacts as though it were a combination of anti-C
plus anti-D.
Was originally described in an rr person who received Dccee RBCs.
Subsequently, the recipient produced an antibody that appeared to be
anti-D plus anti-C, which should be impossible because the C antigen
was not on the transfused RBCs.
Rh:13, Rh:14, Rh:15, Rh:16 Rh:13, Rh:14, Rh:15, and Rh:16
Define four different parts of the D mosaic, as it was originally
described.
o Although these parts are included in the partial-D categories II to VII
as defined by Tippett and Sanger, 25, 27 they are not directly
comparable.
Hr0 Hr0
antigen present on all RBCs with the “common” Rh phenotypes (e.g.,
R1R1 , R2R2 , rr).40 When RBCs phenotype as D--, the most potent
antibody they make is often one directed against Hr0.
Rh:23, Rh:30, Rh:40
all low-frequency antigens associated with a specific category of partial-
D. Rh:23 (also known as Wiel and Dw) is an antigenic marker for
category Va partialD.41 Rh:30 (also known as Goa or Dcor) is a marker
for category Iva partial-D.42 Rh:40 (also known as Tar or Targett) is a
marker for category VII.
Rh:33
The low-incidence antigen Rh:33 is associated with a rare variant of the
R0 (Dce) gene called R0 Har. 44 R0 Har gene codes for normal amounts
of c, reduced amounts of e, reduced f, reduced Hr0, and reduced
amounts of D antigen.
The D reactions are frequently so weak that the cells are frequently
mistakenly typed as Rh negative.
Page 7 of 9

To denote the weakened expression of an antigen in Fisher-Race
nomenclature the letter is placed in parentheses. The R0 Har gene
expresses (D)c(e) and has been found in whites.
Rh:32
Rh:32 is a low-frequency antigen associated with a variant of the R1
[D(C)(e)] gene that is called RN.
The C antigen and e antigen are expressed weakly.
The D antigen expression is exaggerated or exalted. This gene has been
found primarily in blacks.
e Variants
It appears, especially in the black population, that the e antigen may
exhibit the same mosaic quality described for D. Because of these
variations, e typings can be unreliable.
Among the variants at the e locus are hrs, hrB, and VS(es ), with a
variant R0 or r gene making e plus one or the other of these pieces.
Such variants are usually recognized when they make antibodies that
behave as anti-e, even though their RBCs type as e-positive with routine
Rh typing reagents.
V, VS
The V(ceS) antigen is found in about 30 percent of randomly selected
American blacks.
In selected individuals, it appears to be the serologic counterpart of f
because it is present when c is cis with eS.
(The VS(eS) antigen is also relatively common in blacks with the VS
antibody, reacting with all V-positive RBCs and additionally with r′s
RBCs.48) Although the relationship of V to VS remains somewhat less
than clear, both are markers associated with the black population.
Deletions
There are very uncommon phenotypes that demonstrate no Cc and/or
Ee reactivity.
Many examples lacking all Cc or Ee often have an unusually strong D
antigen expression, frequently called exalted D.
The deletion phenotype is indicated by the use of a dash (–), as in the
following examples:
#MLSWATER2022
MODULE 2 Part 3: Rh-Hr Blood Group System
o DC–, Dc–, D–E, D--. The antibody made by D-- people is called anti–
Rh 17 or anti-Hr0.
o A variation has been recognized within the deletion D--, called D••.
The D antigen in the D•• is stronger than that in DC–, D–E, Dc–, or D–e
samples but weaker than that of D-- samples.
A low-incidence antigen called Evans (Rh:37) accompanies the Rh
structure of D•• cells.
Transfusion of individuals with a Rh deletion or D•• phenotype is
difficult if multiple antibodies are present; blood of a similar phenotype
would be required.
THE LW ANTIGEN
LW systems.
o Anti-LW reacts strongly with most D-positive RBCs, weakly
(sometimes not at all) with Rh-negative RBCs, and never with Rhnull
cells.
The independent segregation of LW from the Rh blood group genes was
established by a family study on a D-positive LW-negative woman; other
family studies support this point.
There are three alleles at the LW locus: LWa , LWb , and LW (a silent
allele). Persons lacking LW antigen altogether are LW/LW and express
no LW on the RBCs.
LWa is very common, and LWb is rare. When the Rhnull is present due
to the suppression mechanism, the Rh and LW genes are not expressed
on the RBC; however, the Rh and LW genes are normal and, when
passed to offspring, can be expressed normally.
The amorphic Rhnull individual inherits the rr genes, which do not
express Rh antigens; therefore, LW genes cannot be expressed.
Anti-LW usually reacts more strongly with D-positive RBCs than with D-
negative adult RBCs.
A weak anti-LW may react only with D-positive RBCs, and enhancement
techniques may be required to demonstrate its reactivity with
Dnegative cells.
Anti-LW reacts equally well with cord cells regardless of their D type.
This is an important characteristic to remember when trying to
differentiate anti-LW from anti-D.
Also, anti-LW more frequently appears as an autoantibody, which does
not present clinical problems.
Page 8 of 9
 MODULE 2 Part 3: Rh-Hr Blood Group System
APPENDICES CLASS ACTIVITY
1. Patient is D+C+E+c and e was not tested
Wiener Fisher-Race Wiener Fisher-Race o Convert to Rosenfield: Rh: 1, 2, 3, -4
Ro Dce r dce How about when D is positive, C is negative, E is positive, c is positive
R1 DCe r’ dCe and e is negative?
R2 DcE r” dcE o Convert to Rosenfield: Rh: 1, -2, 3, 4, -5
Rz DCE R^y dCE
2. Convert to Fisher Race
ISBT FISHER-RACE WIENER ROSENFIELD o R1r= DCe/dce
004001 D Rho Rh1 o R1R1= DCe/DCe
o r1r1= dCe/dcE
004002 C rh’ Rh2
o R2ry= DcE/dCe
004003 E rh” Rh3
004004 c hr’ Rh4
3. Rh: 1, -2, 3, 4 ,5
004005 e hr” Rh5
o What is the Phenotype? D+C+E+c+e or DcE/ce
o Convert to Weiner: R2r
COMMON Rh TYPES BY THREE NOMENCLATURES
COMMON GENOTYPE 4. Wiener to Rosenthal
WIENER FISHER-RACE ROSENFIELD FREQUENCY (%) o R1r= Rh: 1, 2, -3, 4, 5
(approx., White) o R2R0= Rh: 1, -2, 3, 4, 5
R1r DCe/dce Rh: 1,2, -3, 4, 5 33 o RzR1= Rh: 1, 2, 3, -4, 5
R1R1 DCe/DCe Rh: 1, 2, -3, -4, 5, 18 o ryr= Rh: -1, 2, 3, 4, 5
rr dce/dce Rh: -1, -2, -3, 4, 5, 15
R1R2 DCe/DcE Rh: 1, 2, 3, 4, 5, 11 5. An Individual’s red blood cell give the following reactions with
R2r DcE/dce Rh: 1, -2, 3, 4, 5, 9 antisera:
R2R2 DcE/DcE Rh: 1, -2, 3, 4, -5, 2
RARER GENOTYPES Anti-D 4+
r’r dCe/dce Rh: -1, 2, -3, 4, 5, 1 Anti-C 3+
r’r’ dCe/dCe Rh: -1, 2, -3, -4, 5, 0.01 Anti-E 0
r”r dcE/dCe Rh: -1, -2, 3, 4, 5, 1 Anti-c 3+
r”r” dcE/dcE Rh: -1, -2, 3, 4, -5, 0.03 Anti-e 3+
R0r Dce/dce Rh: 1, -2, -3, 4, 5, 2 Rh control 0
R0R0 Dce/Dce Rh: 1, -2, -3, 4, 5, 0.1
ryr dCE/dce Rh: -1, 2, 3, 4, 5, rare The Individual’s most probable genotype is: Dce/dce
Rosenthal: 1, 2, -3, 4, 5

-END OF TRANSCRIPTION -
Kung na kaya mo si Complement System, makaya mo man si Rh Typing.
Kung indi, basta kaya mo na ah.

#MLSWATER2022 Page 9 of 9
 MODULE 3: MINOR BLOOD SYSTEMS
MLS 18, March 13, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`

Legend: LEWIS SYSTEM

Transcription Bullet The gene for the Lewis system is linked to the third component of
Notes/Module Packet Bullet complement and is located on chromosome 19.
❖ PPT
The Lewis system differs from most other blood group systems in that
MODULE OUTLINE the Lewis genes must interact with Hh and secretor genes in order for
I. Systems that produce Cold-reacting Antibodies Lewis antigens to be produced
A. Lewis System
B. I Blood Group Collection ❖ Among all the blood group system, Lewis blood group is not
C. P blood Group manufactured by the RBC.
D. MNSs Blood Group System ❖ It is manufactured by tissue cells
II. Systems that Produce Warm-reacting Antibodies ❖ Type 1 glycosphingolipid that are possibly absorbed into the RBC
A. Kell System membrane
B. Kidd Blood System
C. Duffy blood System LEWIS ANTIGENS
III. Other Blood Group Antigens Lewis antigens are probably synthesized in the intestinal epithelium,
A. Lutheran Blood Group System are soluble in body fluids, and the antigens present in plasma are
IV. Clinical Significance of Antibodies of Minor Blood Groups adsorbed onto red cells.
LEARNING OUTCOMES The Lewis antigenic specificity is determined by the carbohydrate
1. Describe the characteristics of the antigens of the different minor fucose.
blood groups systems. The exact Lewis antigen produced is determined by the presence or
2. Describe the characteristics antibodies of the different minor blood absence of the secretor gene (Se).
groups systems and their clinical significance. Lewis antigens are easily and conveniently detected in human saliva by
3. Perform laboratory tests for the detection and identification of hemagglutination inhibition.
antigens &/or antibodies of the minor blood group systems Lewis antigens have also been detected in, and isolated from, human
milk, gastrointestinal juices, urine, seminal fluid, ovarian cyst fluid, and
amniotic fluid.
Leᵃ and Leᵇ cannot usually be detected on cord red cell samples by
SYSTEMS THAT PRODUCE COLD -REACTING ANTIBODIES direct agglutination.
A total of 30 blood group systems are recognized by the International Lewis antigens start to appear on red cells soon after birth.
Society of Blood Transfusion (ISBT) Lewis antigens in the saliva of neonates with an Le gene are the same
Nine blood group systems (ABO, Rhesus, Kell, Kidd, Duffy, MNS, P, as those detected in adult salivas
Lewis, and Lutheran) are considered to be clinically significant as Agglutinability of red cells with anti-Leᵃ or anti-Leᵇ may be reduced
these are known to cause hemolytic transfusion reactions (HTR) and during pregnancy and pregnant women with a transient Le(a–b–)
hemolytic disease of fetus and newborn (HDFN). phenotype may produce Lewis antibodies
#MLSWATER2022 Page 1 of 18

LEWIS ANTIBODIES
❖ Lewis antigenic specificity
o Determined by the carbohydrate fucose.
o Lewis gene will code for fucosyltranferase enzyme
• Fucosyltranferase enzyme can catalyse the addition of
fucose to the 4th carbon of N-acatylgalactosamine of the
type 1 precursor substance
❖ Antigen in Lewis Blood Group System
o Do not develop as an integral part of the RBC membrane
MODULE 3: MINOR BLOOD SYSTEMS
Lewis antibodies are common, usually react below body temperature,
are mainly or entirely IgM, and are not clinically significant
Lewis antibodies that are only active in vitro at temperatures below
37°C do not cause increased clearance of antigen-positive transfused
red cells in vivo
Lewis antibodies do not cause serious hemolytic disease of the
newborn, presumably because Lewis antigens are present in fetal
secretions, but generally not on fetal red cells.

❖ Expression in Lewis Blood Group System 1. Anti-Leᵃ

o The amount of antigen present is variable in partially dependent on 2 Types:
individuals ABO, phenotype and pH.
• Because Lewis antigen expression is affected by H gene, Human anti-Leᵃ
secretor gene and Lewis secretor gene o Leᵃ antibodies are usually ‘naturally occurring’ and predominantly
o May decrease dramatically during pregnancy, most especially after IgM
delivery o Agglutination of red cells with anti-Leᵃ is generally strongest at low
temperatures, often not occurring at all at 37°C.
4 Common Phenotype o Anti-Leᵃ usually fix complement
Lewis A+B- Lewis A, presence of A positive o A two-stage complement addition antiglobulin test is often a
Are mostly non secretors of the ABH successful way of detecting anti-Leᵃ
Lewis A- B- Lewis B, presence of B positive
Secretors of ABH It can best react in room temperature but can also react in 37°C
Although most of Lewis antibodies are considered to be clinically
Lewis A+ B+ Lewis AB, presence of A,B positive
insignificant but they can have the ability to bind with the
Frequently seen in Africans
complement therefore it can trigger in-vitro hemolysis
Lewis A- B- Lewis null, it does not contain any antigen
Anti-Leᵃ are not associated with hemolytic disease of the newborn
Frequently seen in Asians
since there is no presence of anti Le a in the fetal RBC
The antibodies that would react with 37°C, that can trigger the
❖ Most neonates/newborns/infant are Lewis A-B- regardless of which
complement and capable also in triggering in-vitro hemolysis can be
Lewis gene is inherited
associated with the hemolytic transfusion reaction
❖ When the neonates/newborns/infant contain Lewis gene and secretor
anti-Le a can trigger hemolytic transfusion reaction by triggering in-
gene, the A- will become A+ in 2 weeks – 6 months
vitro hemolysis
❖ A+ with the presence of secretor gene can change to B+
Anti-Leᵃ can be enhanced by enzyme treatment
❖ Non secretor will have A+B- but if secretor gene is present A-B+ is the
Lewis gene
Monoclonal anti-Leᵃ

#MLSWATER2022 Page 2 of 18

2. Anti-Leᵇ
Human anti-Leᵇ
Like anti-Leᵃ, anti-Leᵇ are found most often in people with Le (a– b–)
red cells; unlike anti-Leᵃ, these people are generally non secretors of
ABH
Anti-Leᵇ are generally IgM and would react best in room temp.
❖ They can bind oppositely with Anti-Leᵃ
It binds with complement poorly
Activities are enhance by enzyme treatment
Not associated with hemolytic disease of the newborn
can cause hemolytic transfusion reaction very rarely
may appear transiently during pregnancy in Lewis A-B- women, but
disappears after delivery
Animal anti-Leᵇ
Polyclonal and monoclonal Anti-ALeᵇ
Anti-A1Leᵇ, an antibody that reacts only with the red cells of A1 Lewis-
positive secretors [A1 Le(a–b+)], was first found in the serum of a
group A1B Le(a–b–) man
I BLOOD GROUP COLLECTION
Cold agglutination involves the occurrence and reaction of
autoantibodies, reacting optimally in the cold (0°C) with red blood
cells. These autoantibodies are termed cold agglutinins.
Low titer cold agglutinins are present in the sera of all adults. The most
prevalent of these autoantibodies is a heterogeneous assembly of
specificities called anti-I.
Anti-I are generally weak, but potent examples may be found as
autoantibodies in patients with cold haemagglutinin disease (CHAD) or
following Mycoplasma pneumoniae infection
The i antigen has a reciprocal relationship with I
I blood group antigens (I and i) are structurally-related with the ABO
antigens; found in the RBC membrane, in the plasma in the milk, and
even in the amniotic fluid
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
Ii ANTIGENS
Clausen and Hakomori refer to I and i as histoblood group antigens
because, like ABH, Ii antigens are not restricted to red blood cells, but
are usually found on the surface of most human cells and on soluble
glycoproteins in various body fluids.
I and i antigens have been detected in amniotic fluid, urine, and
ovarian cyst fluid
I and i are present on lymphocytes from cord and adult blood. Anti-I
and -i are potent cold lymphocytotoxins, effective at killing B and T
lymphocytes
The i antigen is a characteristic of dividing cells present on a variety of
cell types including lymphoblasts, fibroblasts, erythroblasts, and
thymocytes
Ii antigens represent developmental antigens on red cells and in many
other tissues
1. I antigen (I1)
I antigen can be detected in saliva by hemagglutination inhibition
I is present in human milk in greater quantities than in saliva and
most anti-I are inhibited to some extent by milk
I antigen is subdivided into:
o Iᴰ (developed)
• Is the I antigen on most adult cells, but not cord or adult i
cells;
“D” for developed; usually seen in adults
poorly developed at birth, I antigenic strength increases = i
antigenic strength decreases
o Iᶠ (fetal)
• Is the I antigen expressed on all human red cells including
cord and adult i cells. Iᴰ therefore has a reciprocal
relationship with i.
• “f” for fetal; usually seen with newborns
• Antigenic strength would only be seen with neonates or
infants
Treatment of red cells with proteases or with sialidase generally
enhances expression of Ii antigens.
Page 3 of 18

2. i Antigen (I2)
3. Iᵀ antigen
Iᵀ is the name given by Booth et al. to an antigen detected by cold
agglutinins present in the sera of a high proportion of Melanesians
and expressed strongly on cord cells, weakly on normal adult cells,
and weaker still on adult i cells.
The ‘T’ stands for ‘transition’ as Iᵀ was assumed to represent a
developing I antigen at the transition from I to i
Ii ANTIBODIES
1. Anti-I
3 Types:
Autoanti-I
o These antibodies directly agglutinate I-positive red cells at 4°C with
varying thermal amplitude, but are generally inactive above 30°C
Naturally-occurring, “reacting IgM antibodies”, fail to react w cord
RBCs, some react in a broader temp range, and can cause cold
agglutinin disease, cannot bind or be associated with HDN;
related/associated w mycoplasma pneumoniae which causes
mycoplasma pneumonia (primary atypical pneumonia)
Alloanti-I
o It does not react with autologous cells.
o These antibodies are almost invariably IgM and usually only active
at low temperatures.
Anti-I lectin
o Lectin prepared from the gonads of Aplysia depilans, a marine
mollusc (sea slug), behaved serologically as anti-I
2. Anti-i
Anti-i are heterogeneous in specificity
Anti-i is often found in the serum of patients with infectious
mononucleosis and occasionally causes hemolysis
The presence of anti-i is associated with immunodeficiency
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
associated with infectious mononucleosis, may also be associated with
hemolytic anemia (disappears as the infection is resolved), can be
associated w HDN, hemolytic anemia, & infectious mononucleosis
3. Anti-Iᵀ
Anti-Iᵀ defines an antigen expressed strongly on cord cells, weakly on
most adult cells, and very weakly on adult i cells
4. Anti-j
These antibodies reacted with protease- and sialidase-treated red
cells, but not with cells treated with endo-b-galactosidase
They resembled most pathogenic anti-I and -i by expressing the 9G4
idiotype, a characteristic of antibodies encoded by a gene utilizing a
V4- 34 sequence
P BLOOD GROUP
P antigenic determinants on red cells reside in the carbohydrate
residues of glycosphingolipids, oligonucleotide chains attached to
ceramide
P antigens are structurally-related to ABO antigens; exist as glycolipid
or glycoprotein
Common Phenotypes:
P1 • One of the most (out of two) common phenotypes
involved; approx. 75% of the population have this
o P1 individuals have both P and P1 antigens
o P antigen is well developed at birth while P1
antigen is poorly developed
• The P expression in adult is wildly variable
P2 • The other most common phenotype, individuals with
this phenotype have only P antigen in their RBC.
P1k • Is very rare, individuals have both P1 and PK antigen
in their rbc
P2k- • Also rare, have both Pk and P2 antigens in their rbc
P • Extremely rare, rbc with this phenotype, negative for
P, P1, and Pk antigen.
Page 4 of 18
 MODULE 3: MINOR BLOOD SYSTEMS
P ANTIGENS Monoclonal anti-P1
1. P1 antigen
P1 on red cells is a glycosphingolipid 2. Anti-P
Anti-P is found in the serum of all Pᴷ individuals and can be separated
2. Pᴷ antigen from serum of p individuals by adsorption with P1ᴷ or P2ᴷ cells, or by
Pᴷ was present on the fibroblasts of all P1 and P2 individuals, and only inhibition with HCF
absent from those of p people 3 Types:
Pk (also known as CD77) has been detected on lymphocytes,
granulocytes,monocytes, platelets, smooth muscle of the digestive Alloanti-P
track and urogenital system, and in other tissues. Are produced by individuals with PK1 and PK2 phenotypes. Can
Pk is also expressed on malignant cells and cell lines derived from them trigger severe hemolytic transfusion reaction.
and is a useful marker for Burkitt’s lymphoma Also associated with Donath–Landsteiner (DL) antibodies causes
cold reacting antibody associating with paroxysmal cold
3. P antigen (globoside) hemoglobinuria
Globoside is the most abundant red cell membrane glycolipid
P antigen, the glycolipid globoside , is found on all red cells except Donath–Landsteiner (DL) antibodies
those of the rare phenotypes p and Pk o Generally, have P specificity
P antigen is found on malignant cells and cell lines derived from them. o Paroxysmal cold hemoglobinuria (PCH) is a rare form of
P has also been detected on fetal liver, fetal heart, and on placenta autoimmune hemolytic anemia (AIHA), occurring predominantly in
young children following viral infections.
4. LKE antigen o Sera from patients with PCH usually give a positive Donath–
Landsteiner test; i.e. the antibody binds in the presence of
complement at 0°C and hemolyses the cells when subsequently
warmed
P antibodies
Anti-P1 Monoclonal anti-P
Are naturally occurring antibodies, are called reacting IGM, often
seen with individuals with P2 phenotype.
3. Anti-Pᴷ
They rarely react at high room temperature but bind with
3 Types:
compliment. Do not cause Htn but rarely associated with hemolytic
o Alloanti-Pᴷ
transfusion reaction. Can be neutralized using hyatid cyst fluid from
o Autoanti-Pᴷ
echinococcus granulosus infection.
o Monoclonal anti-Pᴷ
3 Types:
• A monoclonal antibody was shown to define the neutral
glycolipid
Alloanti-P1
o Alloanti-P1 is a common specificity, usually as a weak agglutinin
active only at low temperature.

Animal anti-P1
#MLSWATER2022 Page 5 of 18

MNSs BLOOD GROUP SYSTEM
The second blood group system discovered, is probably second only to
Rh in its complexity
M and N determinants are carried on glycophorin A (GPA), the major
red cell sialic acid-rich glycoprotein (sialoglycoprotein, SGP).
M differs from N in the amino acid composition of the extracellular tip
of GPA:
M has serine at position 1 and glycine at position 5;
N has leucine at position 1 and glutamic acid at position 5
S and s are carried on another red cell SGP called glycophorin B (GPB).
GPB, another sialic acid-rich red cell membrane glycoprotein, is closely
related in structure to GPA
The S/s distinction arises from a single amino acid substitution at
position 29 of GPB:
S has methionine at position 29
s has threonine at position 29
Anti-M & Anti-N antibodies
IgM saline reaction
Dosage effect
Possible HDN & HTR if reaction at AHG
M and N antigens
The amino acid sequence of GPA demonstrates polymorphic variation
at positions 1 and 5, represented serologically as the MN blood groups.
GPA isolated from M+N– individuals has serine at position 1 and
glycine at position 5
GPA from M–N+ individuals has leucine at position 1 and glutamic acid
at position 5.
Both forms of GPA can be isolated from M+N+ individuals.
The terminal serine of M-active GPA is not glycosylated; amino acid
residues 2, 3, and 4 of GPAM and GPAN are O-glycosylated.
Various proteolytic enzymes have proved very useful in the serological
identification, analysis, and definition of antigens belonging to the MNS
system
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
1. Mᵍ
a very rare antigen first described by Allen et al. in 1958, is encoded by
a gene that produces virtually no M or N antigen.
Like M and N, Mᵍ is denatured by treatment of the cells with trypsin,
but not chymotrypsin; unlike most anti-M and -N, anti-Mᵍ generally
react with sialidase-treated Mᵍ + cells
Anti-Mg is easily inherited by the glycosylated N terminal octapeptide
cleaved from GPAMg, but not by that from GPAN
Mᵍ is extremely rare, yet anti-Mᵍ is possibly the most common MNS
antibody
2. Mᶜ
Cannot strictly be regarded as a blood group antigen as no anti-Mᶜ
exists.
Mᶜ is often considered to represent an intermediate between M and N
Rare allele of M and N found by Dunsford et al. in an English family,
produces a determinant that reacts with the majority of anti-M (as
demonstrated by the red cells of the N/Mᶜ member of the family) and
with the minority of rabbit anti-N (as demonstrated by the two M/Mᶜ
members).
subsequently been defined by a pattern of reactions with known anti-
M and -N reagents
NOTE:
Trypsin catalyzes the hydrolysis of peptide bonds on the carboxyl
side of lysine and arginine residues.
Chymotrypsin (a-chymotrypsin) normally hydrolyses the peptide
bond on the carboxyl side of the aromatic amino acids
phenylalanine, tryptophan, and tyrosine, as well as leucine,
methionine, asparagine, and glutamine
Papain, ficin, and bromelin are enzymes, and have a rather broad
specificity and the preparations available are often crude compared
with trypsin and chymotrypsin. Most GPA- and GPB borne antigens
are destroyed by treatment of red cells with these enzymes, only
those situated close to the red cell membrane survive.
GPA and GPB carry about 15 and 11 O-linked oligosaccharides,
respectively, most of which contain two molecules of sialic acid. In
addition, GPA has one N-glycan, which may also be sialylated.
Page 6 of 18

Sialidase (neu-raminidase) treatment of red cells removes at least
some of these sialic acid residues, altering the charge and possibly
the shape of the molecules.
Most monoclonal anti-M and -N do not react, or react comparatively
weakly, with desialylated red cells or isolated glycophorins
Unusual MNS phenotypes caused by two very rare gene deletions:
En(-a)
deletion of the coding region of GYPA, causes a deficiency of GPA,
but not GPB.
En(a–) cells have normal or enhanced expression of S and/or s
Anti-Ena represents an umbrella term, which describes antibodies
to determinants on various parts of GPA.
The En(a–) phenotype can arise in a number of ways. Typically,
En(a–) represents homozygosity for a rare gene deletion (called En
for convenience) at the GYPA locus, resulting in no production of
GPA, but normal production of GPB.
Mᴷ
a deletion of the coding regions of GYPA and GYPB is responsible
for deficiency of GPA and GPB
The name Mᴷ was coined by Metaxas and Metaxas-Bühler for a
new allele of M and N that appeared to produce neither M nor N
Mᴷ produces neither GPA nor GPB; red cells from Mᴷ/Mᴷ
homozygotes are devoid of GPA and GPB . Red cells of people
heterozygous for Mᴷhave about half the normal quantity of GPA
and GPB
S AND S ANTIGENS
The S/s polymorphism is represented by a single amino acid
substitution in GPB at position 29; S-active GPB has Met29 and the s-
active molecule has Thr29
S/s genotyping on genomic DNA can be carried out by PCR-based
techniques with an allele-specific primer
S, s, and most other MNS system antibodies are not sialic acid-
dependent
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
U–red cells are almost always S–s–, but S–s– cells are often U+.
S–s–U+ is often referred to as S–s–U+var.
Strength of U antigen expression on S–s–U+ red cells is variable;
adsorption–elution tests or sensitive agglutination tests with a
particularly potent antibody may be required for its detection
anti-U is unclear, but the term is traditionally used to describe
antibodies produced by S–s– individuals to high-frequency
determinants on GPB
S–s–U– and S–s–U+var cells usually lack the trypsin-resistant N antigen
carried on GPB (‘N’)
U antigen is generally resistant to denaturation by sialidase, trypsin,
chymotrypsin, papain, and ficin. However, unusual examples of anti-U
do not react with papain-treated cells
S–s–U– red cells do not show most of the unusual serological reactions
associated with reduced sialic acid that are characteristic of red cells
deficient in GPA , though Glycine soja lectin may agglutinate U-
deficient cells
S–s–U– red cells are deficient in GPB. This has been demonstrated by
failure to inhibit anti-S, -s, or -U with sialoglycoproteins isolated from
S– s–U– cells, by SDS PAGE of red cell membranes or isolated
sialoglycoproteins , and by immunoblotting with antibodies and lectins
directed at determinants on GPB
MNSsU ANTIBODIES
1. Human anti-M
Most anti-M are only reactive at temperatures below 37°C, with an
optimum temperature of 4°C, but occasional examples will agglutinate
red cells at body temperature
Most human anti-M contain an IgM component, although 78% were
found to be at least partially IgG and these IgG antibodies could
agglutinate saline suspensions of M+ red cells.
Naturally occurring, may be both IgM and IgG, do not bind w a
complement, react at room temp or lower, rarely associated w HDR &
HTR, some are pH-dependent (best at 6.5) and glucose-dependent
2. Human anti-N
Red cells of individuals with the rare M+N–S–s–(U– or U+var)
phenotypes lack ‘N’ and may produce a potent alloanti-N, which will
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agglutinate all cells carrying an N determinant, whether on GPA or
GPB.
These antibodies have been referred to as anti‘N’, -N‘N’, or -NU
Most anti-N are ‘naturally occurring’, IgM, and inactive above 25°C
Rare, weak, naturally-occurring IgM antibodies, react at room temp or
below, usually not associated w HDN & HTR
MNSs antibodies follow the dosage effect; homozygous = stronger
agglutination compared to heterozygous
3. Anti-S
S, s, and U antibodies usually react at 37°C, but most are optimally
reactive at temperatures between 10 and 22°C by manual antiglobulin
tests under normal ionic conditions
Anti-S reagents are notorious for containing antibodies to private
antigens.
Anti-S has been implicated in haemolytic transfusion reactions and has
caused severe and fatal HDN
Autoanti-S has been responsible for AIHA
Autoantibodies that are probably detecting nonpolymorphic
determinants on GPB may ‘mimic’ anti- S because of the greater
quantity of GPB molecules on S+ cells than on S– cells
4. Anti-s
Anti-s are usually optimally reactive at 22°C or below
Anti-s has been responsible for severe and fatal HDN and for a delayed
haemolytic transfusion reaction
5. Anti-U
Anti-U are generally non-complement-binding IgG antibodies
containing an IgG1 component
no ‘naturally occurring’ anti-U has been reported.
Like anti-S and -s, U antibodies may have greater reactivity at
temperatures below 22°C than at body temperature
“U” = universal; usually reactive in the anti-globulin phase, rare and
occurs in S-s-U-people, Black origin (African black)
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
SYSTEMS THAT PRODUCE WARM-REACTING ANTIBODIES
KELL SYSTEM
Kell antigen
None of the Kell antigens is expressed on cells of the Kell-null
phenotype, Ko, which arises from homozygosity for a silent gene at the
KEL locus.
Ku antigen is present on all cells save those of the Ko phenotype
The Kell antigens are located on CD238, a red cell transmembrane
glycoprotein of apparent Mr 93000, a metalloendopeptidase
The molecular bases for most of the Kell antigens are known and all the
Kell system polymorphisms result from single amino acid substitutions.
KEL is situated on chromosome 7q32–q36
McLeod syndrome is a form of neuroancanthocytosis, which includes
an abnormal Kell red cell phenotype.
McLeod phenotype red cells have depressed Kell antigens and lack the
high frequency antigen Kx.
The inheritance of Kx is controlled by an X-borne gene, XK, and
represents a blood group system (the Kx system) independent of Kell.
The Kx protein and Kell glycoprotein are linked by a disulphide bond
Kell antigens are well developed at birth. K was found in fetuses of 10–
11 weeks’ and k at 6–7 weeks’ gestation
K18 is the only Kell antigen not shown to be inherited.
TOU (KEL26) is an antigen of high frequency absent from Ko cells and
shown to be located on the Kell glycoprotein by a MAIEA analysis
Kell system antigens are destroyed by disulphide-bond reducing agents
and so must be dependent on the native conformation of the molecule
Compromised of 21 high and low incidence antigen. The most
significant of which are the K and k antigen.
KO phenotype - lack all K antigen and the antigens associated with Kell
system.
Kx antigen – located in the x chromosome, rbc that lacks Kx antigen
also have greatly weaken expression of other kell antigen. Rbc are
morphologically acanthocytic they have decrease survival and less
permeable to water and this syncdrome is called Mcleod syndrome.
Mcleod Syndrome – characterized by splenomegaly with
reticulocytosis and occasional associated with chronic granulomatous
disease.
Kell antigens are inactivated by sulfhydryl reagents.
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The Kx system
The Kx system consists of one antigen, Kx (XK1 or 019001),
encoded by an X-linked gene, XK.
Absence of Kx from red cells results in severe reduction in
expression of Kell antigens, the McLeod phenotype.
KELL ANTIBODIES
1. Anti-K
Anti-K is a relatively common cause of severe hemolytic disease of the
newborn (HDN)
Anti-K can be responsible for severe hemolytic transfusion reactions,
including reactions caused by incompatibility between donations given
to the same patient
2. Alloanti-K
Anti-K is the most common immune red cell antibody outside of the
ABO and Rh systems
Anti-K is often found in sera containing antibodies to high incidence
Kell system antigens.
Anti-K, like other Kell system antibodies, are generally IgG, and
predominantly IgG1
3. Anti-k
Most anti-k are IgG (often IgG1 ) and work best by the antiglobulin test,
but cold agglutinating IgM anti-k are known
Anti-k has been responsible for haemolytic transfusion reactions,
including severe intravascular haemolysis, and for HDN
Considered to only be second to D antigen in autoimmunity. Usually
react best in 37 degree Celsius. May occasionally bind with the
compliment and may cause hemolytic disease to newborn and
haemolytic transfusion reaction
4. Anti-Kpᵃ
Many examples of anti-Kpa are known. The first (Penney) appeared to
be ‘naturally occurring’ but, as with most anti-Kpa, reacted best by the
antiglobulin test.
Anti-Kpa very rarely causes HDN severe enough to require transfusion
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
5. Anti-Kpᵇ
The first anti-Kpᵇ(Rautenberg) was found during routine
crossmatching ; the serum also contained antiK
Anti-Kpᵇ has been responsible for a delayed haemolytic transfusion
reaction
6. Anti-Kpc
The first anti-Kpc , called anti-Levay for 34 years, was made by a
patient with lupus erythematosus diffusus in response to transfusion
7. Jsa and Jsb (KEL6 and KEL7)
The Jsa /Jsb polymorphism is associated with two nucleotide changes
in exon 17 of KEL, one encoding an amino acid substitution: Jsa , C1910
Pro597, G2019 Leu633; Jsb, T1910 Leu597, A2019 Leu633
8. Anti-Jsᵃ
Anti-Jsa generally react best by the antiglobulin test and are red cell
immune in origin
9. Anti-Jsᵇ
All examples of anti-Jsᵇhave been found in black people.
They generally work best by the antiglobulin test.
Anti-Jsᵇhas caused severe HDN , resulting in fatal hydrops fetalis
10. Anti-Ku (-KEL5)
Anti-Ku is the typical antibody of immunized Ko individuals and detects
an antigen present on all red cells apart from those of the Ko
phenotype.
It appears to be a single specificity and cannot be separated, by
adsorption and elution, into components of other Kell specificity
The original anti-Ku caused both HDN and a haemolytic transfusion
reaction.
Anti-Ku was also responsible for a severe transfusion reaction resulting
in jaundice, renal failure, and anuria
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 MODULE 3: MINOR BLOOD SYSTEMS
KIDD BLOOD GROUP SYSTEM Anti - Jkᵃ has been responsible for severe and fatal immediate HTRs
Jkᵃ and Jkᵇ of the Kidd system are the products of alleles and are and is regularly associated with delayed HTRs, which may be severe,
polymorphic in all populations tested. leading to oliguria, renal failure, and even death.
Both jka and jkb may exhibit dosage effect. Reactivity may also be Anti - Jkᵇ has also been incriminated in severe delayed HTRs
enhanced with enzyme treatment. Poorly immunogenic and can cause A major reason why Kidd antibodies are such a common cause of
severe haemolytic transfusion reaction. Both are noted for causing delayed HTRs is their tendency to fall rapidly to low or undetectable
delayed haemolytic transfusion reaction. Would react best in 37 levels in the plasma
degrees Celsius.
Most of Kidd Antibodies are Igg1 and Igg3 that’s why they can bind 2. Anti-Jkᵃ and -Jkᵇ Autoantibodies
complement efficiently and they would cross the placenta and cause
mild to severe hemolytic transfusion reaction. Very hard to detect, 3. Anti-Jkᵃ and -Jkᵇ Monoclonal antibodies
enzymes are used (LISS, PEG) IgM human monoclonal anti - Jkᵃ and - Jkᵇ have been produced by
Kidd antibodies are often difficult to work with. They are potentially Epstein - Barr virus - transformation of lymphocytes from immunised
dangerous, as they are a common cause of delayed haemolytic donors and fusion with mouse myeloma cells to form
transfusion reactions. heterohybridomas
A rare null phenotype, Jk(a–b–), is generally inherited recessively and
is most commonly found in Polynesians.
4. Anti - Jk3 (Jk3 Antibodies)
o Jk(a–b–) cells lack the high incidence antigen Jk3.
react optimally by an antiglobulin test, the reaction being enhanced by
o Jk(a – b – ) red cells lack the Kidd glycoprotein
enzyme treatment of the cells.
The Kidd glycoprotein functions as a urea transporter
Enzyme -treated cells may be hemolyzed by anti - Jk3 in the presence
The JK (SLC14A1) locus is on chromosome 18 at 18q11–q12
of fresh serum
Jkᵃand Jkᵇ are inherited as codominant alleles.
Anti - Jk3 are usually IgG
Jka, Jkb, and Jk3 are papain-, ficin-, trypsin-, chymotrypsin-, and
Anti - Jk3 may decline rapidly in vivo
pronase-resistant
Anti - Jk3 has been responsible for severe immediate and delayed
HTRs.
Kidd antibodies Most babies of mothers with anti - Jk3 are clinically unaffected,
1. Anti-Jkᵃ and -Jkᵇ Alloantibodies although the baby’s red cells may give a positive DAT, and in a few
Generally, an antiglobulin test is required to detect Kidd antibodies. cases phototherapy was administered
Kidd antibodies are usually IgG or a mixture of IgG and IgM; they are
rarely pure IgM
Anti - Jkᵃ detectable only by the manual Polybrene test was responsible
for an HTR, emphasizing the importance of detecting weak Kidd
antibodies.
Many anti - Jkᵃ react more strongly with Jk(a + b – ) than with Jk(a + b
+ ) cells, and some anti - Jkᵃ can only be detected with Jk(a + b – ) cells
Kidd antibodies, which are often difficult to detect, are a hazard in
blood transfusion.

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The Kidd glycoprotein is the red cell urea transporter, UT -B
The Kidd glycoprotein is present on endothelial cells of the vasa
recta, the vascular supply of the renal medulla, but is not present in
renal tubules.
Urea transporters in the kidney play an important role in
concentrating urea in the renal medulla, whilst conserving water, in
order to produce concentrated urine
UT - B in human colon epithelium could participate in the transport
of urea across the colon mucosa and assist in the maintenance of a
normal colonic bacterial population
2 Main Functions:
transporting urea rapidly in and out of the cells to prevent
shrinkage as they pass through the high urea concentration of the
renal medulla, and to prevent swelling as they leave;
to prevent the red cells from carrying urea away from the renal
medulla, which would decrease the urea concentrating efficacy of
the kidney
-Please refer to reference book for the missing information (On Jk antigens &
antibodies)
DUFFY BLOOD GROUP
Has 4 alleles that are important for the major antigen and are resulting
from the type.
Duffy A and B are produced by codominant alleles
FYx is a weaken form of Duffy b
FY are produces no gene factor
Fy (A-B-) would have resistance from the infection of Plasmodium
vivax.
Duffy Antigens
1. Fyᵃ and Fyᵇ
Fy a and Fyb result from a Gly42Asp substitution within the Duffy
glycoprotein.
Fyᵃ and Fyᵇ are very sensitive to most proteolytic enzymes:
They are completely destroyed by treatment of the red cells with
papain, ficin, bromelin, pronase, or chymotrypsin, but trypsin does not
abolish Fyᵃ and Fyᵇ activity.
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
Fyᵃ and Fyᵇ are fully developed at birth and have been detected on red
cells from embryos as early as 6 – 7 weeks gestation
The expression of Fyᵃ and Fyᵇ is as strong on red cells of very young
fetuses as on those of adults, and remains unmodified throughout fetal
life
In addition to its presence on red cells, DARC, detected with anti-Fy6, is
abundant on endothelial cells lining post - capillary venules throughout
the body, except for the liver , and on Purkinje neurons of the
cerebellum
Fyᵃ and Fyᵇ are not present on lymphocytes, monocytes, granulocytes,
or platelets
Use of impure preparations of trypsin containing chymotrypsin
probably accounts for some early reports that Duffy antigens are
trypsin - sensitive.
Sialidase treatment of red cells does not affect the activity of Duffy
antigens.
2. FYx
The presence of Fyˣ may be confirmed serologically by adsorption and
elution of anti - Fyᵇ
Fyˣ behaves as a weak Fyᵇ antigen; there is no anti – Fyˣ.
3. Fy3
Fy3 is present on all red cells apart from those of the Fy(a – b – )
phenotype.
Fy3 is a public antigen in people of European and Asian origin,
polymorphic in many black populations, and a private antigen in some
parts of Africa.
In contrast to Fyᵃ and Fyᵇ, Fy3 is resistant to the treatment of red cells
with proteases
Red cells of some primates have Fy3, but not Fyᵃ or Fyᵇ
4. Fy5
Fy5 closely resembles Fy3
it differs by its absence from Fy3 - positive Rh null cells (amorph and
regulator type), weak expression on red cells of D – – phenotype, and
presence on Fy(a – b – ) cells from people of non - African origin.
Like Fy3, Fy5 is a protease – resistant antigen.
Fy5 is expressed equally strongly on red cells of adults and newborns
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DARC appears to be part of a protein complex that contains the Rh
proteins, so Fy5 expression could be dependent on an interaction
between those proteins.
Duffy Antibodies
1. Anti – Fyᵃ
Anti - Fyᵃ is estimated to be three times less frequent than anti – K
Duffy antibodies are not commonly detected in the first 6 months
following transfusion, but, relative to other antibodies, are more
common after 6 months and even more so after 5 years
Anti – Fyᵃ are usually IgG, mostly IgG1
Anti-Fyᵃ generally react best by an antiglobulin test, but rarely anti -
Fyᵃ may be directly agglutinating.
Anti – Fyᵃ in donor blood was responsible for a transfusion reaction
in a Fy(a + b + ) patient
Anti - Fyᵇ
Anti - Fyᵇ is a relatively rare antibody usually found only in mixtures
of red cell antibodies.
o It has been stimulated by pregnancy and transfusion, and by
intrauterine transfusion in the mother
o naturally occurring anti - Fyᵇ has been found
o Often consisting entirely of IgG1
o Fyᵇ antibodies generally react best by an antiglobulin test, but
directly agglutinating examples are known
o Some anti - Fyᵇ bind complement
o Anti - Fyᵇ has been responsible for immediate and delayed HTRs,
two of which were reported to have been fatal
Anti - Fy3
Anti - Fy3 is potentially hemolytic and has been responsible for
immediate and delayed HTRs, including intravascular hemolysis in
an acute reaction and hyper hemolysis in a patient with sickle cell
disease
Fy(a−b−) red cells should be selected for transfusion to patients with
anti - Fy3.
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
Duffy Glycoprotein
Binds a variety of pro- inflammatory chemokines and is often known
as the Duffy Antigen Receptor for Chemokines (DARC)
Duffy antigens are located on a glycoprotein of apparent MW 35 –
50 kDa, a chemokine receptor of the G protein - coupled family.
Duffy glycoprotein is present on endothelial cells of post - capillary
venules and on other cells throughout the body
Individuals with the Fy(a – b – ) phenotype are resistant to infection
by the malarial parasite Plasmodium vivax and Fy(a – b – ) red cells
are refractory to invasion by P. vivax in vitro
Interaction between the Duffy glycoprotein and receptors on P.
vivax merozoites are essential, but not sufficient, for red cell
invasion.
The Duffy (FY or DARC) locus is on chromosome 1q21 - q22.
o DARC is N - glycosylated, but has no, or very little, O -
glycosylation.
Treatment of red cells with the N – glycanase endo F prior to
solubilization and immunoblotting with anti - Fyᵃ resulted in a
dramatic reduction in apparent MW and sharpening of the band on
the blot
Similar results were obtained by treatment of purified DARC or of
tryptic peptide derived from it
Variation in the degree of N - glycosylation may account for the
range of MW
Duffy Chemokines
Chemokines are chemotactic cytokines that are involved in many
cellular processes, especially the recruitment, activation, and
directional movement of leucocytes
There are two main classes of chemokines, called CXC and CC based
on the position of two highly conserved cysteine residues at their N
- termini, plus two minor classes, C and CXXXC.
o Most chemokine receptors belong to a very large family of
integral cell - membrane glycoproteins, G protein - coupled
receptors, which traverse the membrane seven times and
have an extracellular N - terminal domain
o Most chemokine receptors are specific for one or more
chemokines of a single class, but DARC, a promiscuous
receptor, binds with high affinity to 60% of inflammatory
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 MODULE 3: MINOR BLOOD SYSTEMS
chemokines from both CX and CC classes, but not with OTHER BLOOD GROUP ANTIGENS
homeostatic chemokines LUTHERAN BLOOD GROUP SYSTEM
o Unlike almost all other G protein - coupled receptors, DARC Two most common antigen (A and B)
lacks the Asp - Arg – Tyr (DRY) motif in the second The Lutheran system consists of 20 antigens: LU1 to LU22 in the
cytoplasmic loop and does not appear to be coupled to a numerical notation, with two declared obsolete
guanosine triphosphate - binding protein (G - protein) o Four pairs of these antigens have allelic relationships and
o Duffy is a chemokine - binding protein with no signalling represent SNPs in the Lutheran gene, LU :
function and has been referred to as a ‘ silent receptor ’ or • Luᵃ (LU1) and Luᵇ (LU2);
interceptor (internalising receptor)
• Lu6 and Lu9;
o DARC is present on the endothelial cells of post -capillary
• Lu8 and Lu14; and
venules throughout the body, in both Duffy -positive people
• Auᵃ (LU18) and Auᵇ(LU19)
and Fy(a – b – ) Africans
The null phenotype, Lu null or Lu(a – b – ), in which the red cells
Fy(a –b–) human red cells are refractory to invasion by P.
lack all Lutheran system antigens, results from homozygosity for
knowlesi and P. vivax merozoites, in vitro , whereas Fy(a+b+)
inactivating in the Lutheran gene
cells are invaded
o Individuals with the Lu null phenotype may make an
o Despite being Fy(b + ) and Fy:3, red cells of the Old World
antibody to the Lutheran glycoproteins (Lu - gps), anti - Lu3
rhesus monkey are Fy: – 6 and are not invaded by P. vivax ,
o Nucleotide changes in LU are associated with loss of eight
but are invaded by P. knowlesi
other antigens of high frequency, which are also absent
o New World capuchin monkey cells are Fy(a – b – ) Fy:3, – 6
from Lu null cells
and are not invaded by P. knowlesi or P. vivax merozoites
o The molecular bases for two other antigens of high
o These data suggest that the Fy6 epitope is important forthe
frequency absent for Lu null cells are unknown.
invasion of P. vivax
Lutheran antigens are located on two red cell membrane
o The Duffy gene is subject to opposing selection pressures:
glycoproteins (CD239) of apparent MW 78 and 85 kDa, which
the need to maintain effective chemokine binding versus
belong to the immunoglobulin superfamily of receptors and
the benefits of reducing P. vivax invasion.
adhesion molecules.
The Lu - gps are ligands for the extracellular matrix glycoprotein,
Disease associations laminin LU or BCAM is situated on chromosome 19q12 - q13 and
HIV - 1 attaches to red cells via DARC, effecting infection of target consists of 15 exons, with alternative splicing accounting for the two
lymphocytes isoforms of the Lu – gps
Red cell Duffy expression is strongly associated with severity of
sickle cell disease (SCD) and especially with organ damage.
The Lutheran glycoproteinsis Lu –gps
o Twice as many Duffy - negative patients had evidence of
Components of apparent MW 85 and 78 kDa were revealed by
organ damage compared with Duffy -positive patients;
immunoblotting of red cell membranes with monoclonal anti - Lu b
o Duffy - negative patients were nearly four times more likely
or with alloanti - Luᵃ , - Luᵇ, - Lu3, - Lu4, - Lu6, - Lu8, - Lu12, - Auᵃ, or
to have proteinuria
- Auᵇ
o Duffy - positive SCD patients have higher plasma levels of
chemokines IL - 8 and RANTES than Duffy - negative patient
The Lu-glycoproteins belong to the immunoglobulin superfamily (IgSF)
The immunoglobulin superfamily (IgSF) is a large collection of
glycoproteins, abundant on leucocytes, but also present on other
#MLSWATER2022 Page 13 of 18

cells, which contain repeating extracellular domains with sequence
homology to immunoglobulin variable (V), constant (C1 or C2), or
intermediate (I) domains.
o Each IgSF domain consists of approximately 100 amino acids
and is structured into two β – sheets stabilised by a
conserved disulphide bond
o IgSF glycoproteins mostly function as receptors and
adhesion molecules, and may be involved in signal
transduction
Luᵃ and Luᵇ (Lu1 ad Lu2)
The first Lutheran antibody, anti - Luᵃ, was described in 1945 by
Callender et al.
Variation in Antigenic Strength
The Lutheran antigens are very variable in strength
o Luᵃ on red cells from different families may vary
quantitatively, but the antigenic strength remains roughly
constant within the family.
o Occasionally adsorption and elution tests are required to
detect weak Luᵇ on Lu(a + b + ) cells
o There is also heterogeneity of Lutheran antigen strength
between individual red cells within a person, which
accounts for the characteristic mixed - field agglutination
patterns often seen with Lutheran antisera, especially anti -
Luᵃ, and the wide range of survival times of Lu(b + ) cells
introduced into an Lu(a + b – ) person with anti - Luᵇ
o The abundance of Luᵇ antigens on red cells, as determined
by Scatchard analysis with purified monoclonal anti - Luᵇ, is
relatively low and shows wide variation
Red cells from cord samples and from infants in the first year of life
have markedly weakened expression of Luᵃ and Luᵇ compared with
those from adults
o Adult levels of Luᵃ and Luᵇ antigenic expression are reached
by the age of 15
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
Lutheran Antibodies
Anti- Luᵃ
Luᵃ antibodies are usually IgM, but, like other Lutheran - system
antibodies, often have IgG and IgA components
Anti - Luᵃ often agglutinate Lu(a + ) red cells directly, with a thermal
optimum well below 37 °с
o Some also react in an antiglobulin test, and a few,
predominantly IgG examples, are reactive only by an
antiglobulin test
o A single - chain variable - fragment (scFv) with Luᵃ specificity
has been produced by phage display and recombinant DNA
technology, and a monoclonal anti - Luᵃ constructed
Anti-Luᵇ
Anti-Luᵇ is relatively rare, often found as a single antibody
o It has been stimulated by transfusion and by pregnancy
o Anti - Luᵇ are often optimally active in the antiglobulin test,
but directly agglutinating anti - Luᵇ have been described,
many with a temperature optimum of about 20°с.
o Most anti - Luᵇ are mixtures of IgG and IgM, although IgA
may also be present
o IgG anti - Luᵇ may be predominantly IgG1, although IgG2
and IgG4 may be present
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Clinical Significance of anti - Luᵃ and - Luᵇ
No case of HDFN caused by anti - Luᵃ or - Luᵇ and requiring any
treatment other than phototherapy is reported, although raised
bilirubin or a positive DAT may be detected
No case of HDFN caused by anti - Luᵃ or - Luᵇ and requiring any
treatment other than phototherapy is reported, although raised
bilirubin or a positive DAT may be detected:
o Babies of mothers with high - titre IgG1 anti - Luᵇ or -
Lu6 had no sign of HDFN, their red cells gave negative
DATs, and Lutheran antibody could not be detected in
their sera.
o Maternal IgG1 usually becomes concentrated in the
fetal circulation by active placental transfer.
o As Lu-gp is present on placental tissue, it is possible that
Lutheran antibodies are adsorbed by placental cells,
preventing their transfer to the fetus
Other Lutheran antigens and antibodies
All Lutheran antigens are absent from Lu null cells and absent from
or expressed very weakly on In(Lu) cells
LU11
Lu11 has not been shown to be inherited and has not been shown
to be located on the Lu-gps or encoded by the LU gene, and so
should be referred to as a para -Lutheran antigen
LU6 and LU9
Lu6 and Lu9, Lutheran antigens of high and low frequency,
respectively, have an antithetical relationship, and represent a SNP
and Cfo I restriction polymorphism in LU encoding an amino acid
substitution in the third IgSF domain
LU8 and LU14
Lu8 and Lu14, Lutheran antigens of high and low frequency,
respectively, have an antithetical relationship, and represent a SNP
and Fat I and Nla III restriction polymorphisms in LU , encoding an
amino acid substitution in the second IgSF domain
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
Recombinant Lutheran Antigens
Lutheran antigens have been used as models for the application of
recombinant proteins in antibody identification
Recombinant proteins containing all or some of the IgSF domains of
the Lutheran protein have been expressed in eukaryote or
prokaryote cells
The purified protein was then used in agglutination inhibition tests,
attached to polystyrene plates for detection by an ELISA procedure,
or coupled to superparamagnetic particles for detection in a particle
gel immunoassay.
Alloanti-Luᵃ or Alloanti-Luᵇ were detected with high sensitivity and
specificity
Effects of Enzymes and Reducing Agents on Lutheran Antigens
Lutheran antigens are destroyed by treatment of red cells with
trypsin or α - chymotrypsin; papain has little effect
Sulphydryl reducing agents, such as AET and DTT, break inter- and
intra - polypeptide chain disulphide bonds resulting in the unfolding
of the protein
Red cells treated with 6% AET or 200mM DTT at pH8.0 did not react
with most Lutheran antibodies tested, including many examples of
anti - Luᵃ and - Luᵇ
Lu null and Anti-Lu3 (LU3)
Lu null phenotype is extremely rare and has a recessive mode of
inheritance
o Lu null cells lack all Lutheran system antigens
o Individuals with the Lu null phenotype may make an
antibody, anti - Lu3, which reacts with all red cells apart
from those with the Lu null phenotype
o Lu null red cells have normal expression of those antigens,
such as AnWj, that are expressed very weakly on In(Lu) red
cells
Anti-Lu3 (-LU3)
All Lu null propositi have been found following the detection of an
antibody to a high frequency antigen, anti-Lu3
o Anti - Lu3 has a single specificity and reacts equally strongly
with Lu(a+b–), Lu(a + b + ), and Lu(a – b + ) cells
Page 15 of 18

o Adsorption with Lu(a + b – ) cells will remove the activity for
Lu(a – b + ) cells and vice versa [90,92]
o Lu3 is present on all red cells that express any Lutheran
antigen
Luᵇ was not detected on lymphocytes, granulocytes, monocytes,
platelets, or the erythroleukaemic cell lines K562 and HEL
Disease associations
The IgSF gps expressing Lutheran and LW blood group activity are
overexpressed on SS red cells in sickle cell disease: SS red cells
express about 67% more Lu-gp than normal cells and bind increased
quantities of laminin
For more information regarding Lutheran Blood Group and probably
everything that confuses you. Refer to: Daniels, Human Blood Group, 3rd
Edition https://books.google.com.ph/books?id=CHT_-
c45qQcC&printsec=frontcover
CLINICAL SIGNIFICANCE OF ANTIBODIES OF MINOR BLOOD
GROUPS
Lewis Blood Group
Although some cases of hemolytic transfusion reactions caused by
anti-Lea have been reported and there have been cases of in vivo
RBC destruction due to anti-Leb, Lewis antibodies are generally
considered insignificant in blood transfusion practices. This is
because:
1. Lewis antibodies can be neutralized by the Lewis substances
present in the plasma and can thereby be decreased in quantity.
2. The Lewis antigens dissociate from the RBCs as readily as they
bind to the RBCs. In other words, the Lewis-positive donor RBCs
can become Lewis-negative RBCs following transfusion into an
individual with a Lewis-negative phenotype. These antigens
released into the plasma can further neutralize any Lewis
antibodies present in the recipient plasma.
3. Lewis antibodies are generally IgM and therefore cannot cross
the placenta and cause HDN. In addition, Lewis antigens are not
fully developed at birth.
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
For these reasons, the presence of Lewis antibodies in a patient’s
serum does not require transfusions of Lewis negative RBCs, as long
as pre-transfusion tests performed at 37⁰C and Coombs phase are
compatible and there is no evidence of in vitro hemolysis.
The Lewis antibodies reactive at 37⁰C and antihuman globulin
phase, however, should not be ignored because these antibodies
can cause in vivo RBC destruction. In the presence of multiple
antibodies, Lewis antibodies often complicate antibody
identification, but they can be easily inhibited with saliva from
secretors or with commercially available Lewis substance.
Factors Contributing to Clinical Insignificance of Lewis Antibodies
o Neutralization of Lewis antibodies by Lewis substances present in
the plasma
o Loss of red cell Lewis antigen(s) into the plasma
o Lack of reactivity at 37⁰C and antihuman globulin phase
o Generally, lgM in nature and incapable of crossing placenta
o Lewis antigens poorly developed in newborn infants
The MNS (002) Blood Group System
Disease Association
GPAM may serve as the receptor by which certain
pyelonephritogenic strains of Escherichia coli gain entry to the
urinary tract.
The malaria parasite Plasmodium falciparum appears to use
alternative receptors, including GPA, GPB, and GPC23 for cell
invasion; some of these receptors also involve NeuNAc.
In an attempt to identify the receptor, the invasion rate into cells
with normal and rare phenotypes was studied. Reduced invasion is
seen with En(a), U, MkMk, Tn and Cad RBCs (which have altered
oligosaccharides on glycophorins), Ge, and normal RBCs treated
with neuraminidase and trypsin
Page 16 of 18

The P Blood Group: P (003) and Globoside (028) Blood Group Systems
and Related Antigens
Disease Associations
Several pathologic conditions associated with the P blood group
antigens have been described: parasitic infections are associated
with anti-P1 , early abortions with anti-PP1 Pk or anti-P, and PCH
with autoanti-P. The P system antigens may also be associated with
urinary tract infections.
Some pyelonephritogenic strains of E. coli ascend the urinary tract
in ladderlike fashion by adhering to P1 and/or Pk glycolipids on
uroepithelial cells. The fimbriae or pili of such organisms have
receptor sites for structures involving Gal(ß1-4)Gal( 1-4), the
terminal sugars for P1 and Pk.
Other globoside associations have been identified with infection.
Streptococcus suis, which occasionally causes meningitis and
septicemia in humans, binds exclusively to Pk antigen. A class of
toxins secreted by Shigella dysenteriae, Vibrio cholerae, Vibrio
parahaemolyticus, and some pathogenic strains of E. coli also have
binding specificity for a Gal(ß1-4)Gal( 1-4) moiety. In addition,
globoside is the receptor of human parvovirus B19.
The I (027) Blood Group System and i Antigen
Disease Associations
Anti-I and cold agglutinin syndrome and M. pneumoniae
Anti-i and infectious mononucleosis
Anti-IT and Hodgkin’s lymphoma.
Cold autoantibodies have also been reported in influenza infections,
but other associations are rare.
Diseases can also alter the expression of I and i antigens on RBCs.
Conditions associated with increased i antigen on RBCs include
those with shortened marrow maturation time or dyserythropoiesis:
acute leukemia, hypoplastic anemia, megaloblastic anemia,
sideroblastic anemia, thalassemia, sickle cell disease, paroxysmal
nocturnal hemoglobinuria (PNH), and chronic hemolytic anemia.
Except in some cases of leukemia, the increase in i on RBCs is not
usually associated with a decrease in I antigen; the expression of I
antigen can appear normal or sometimes enhanced.
Reactive lymphocytes in infectious mononucleosis are reported to
have increased i antigen. Those from patients with chronic
#MLSWATER2022
MODULE 3: MINOR BLOOD SYSTEMS
lymphocytic leukemia have less lymphocyte i antigen than normal
control subjects.
Chronic dyserythropoietic anemia type II or hereditary
erythroblastic multinuclearity with a positive acidified serum test
(HEMPAS) is associated with much greater i activity on RBCs than
control cord RBCs.
HEMPAS RBCs are very susceptible to lysis with both anti-i and anti-
I, and lysis by anti-I appears to be the result of increased antibody
uptake and increased sensitivity to complement.
I antigen may be involved in binding immune complexes consisting
of drug and drug antibodies. Immune complexes involving rifampin,
nitrofurantoin, dexchlorphenyramine, and thiopental have been
associated with binding to I antigens on RBCs and subsequent
complement activation and hemolysis.
In Asians, the adult i phenotype has been associated with congenital
cataracts. Mutations at the I locus have been identified in three
Taiwanese families with the adult i phenotype, which suggests a
molecular genetic mechanism for this condition in this population.
The Kell (006) and Kx (019) Blood Group Systems
Antibodies of the Kell system should be considered potentially
clinically significant, both from the point of view of causing severe
hemolytic disease of the fetus and newborn (HDFN) and hemolytic
transfusion reactions (HTRs).
Patients with Kell-system antibodies should be transfused with
antigen-negative blood whenever possible.
Kell-system antibodies are usually IgG and predominantly IgG1.
HDFN caused by anti-K differs from that resulting from anti-D.
Anti-K HDFN is associated with lower concentrations of amniotic
fluid bilirubin than anti-D HDFN of comparable severity. Postnatal
hyperbilirubinemia is not prominent in babies with anemia caused
by anti-K. There is also reduced reticulocytosis and erythroblastosis
in the anti-K disease, compared with anti-D HDFN
The infrequent cases of HDN caused by Kell immunization tend to
result in severe fetal anemia because maternal anti-Kell target fetal
red blood cell (RBC) precursors, suppressing the fetal production of
RBCs.
Page 17 of 18
 MODULE 3: MINOR BLOOD SYSTEMS
The Duffy (008) Blood Group System
The Duffy-Malaria Association
Since 1955, Miller and colleagues confirmed that malaria
merozoites invaded only RBCs carrying normal Fya or Fyb antigen.
When antigen sites were blocked by antibody or denatured with
certain enzymes, the RBCs became resistant to invasion. Because
the resistance factors for P. knowlesi and P. vivax were so parallel in
West African populations, it was suggested that Fya and Fyb might
also be the invasion receptor for P. vivax.
Close evaluation of the invasion process of P. knowlesi suggests that
two receptor sites are involved: one for attachment and one for
invasion.Initial attachment of the merozoite to the RBC occurs
regardless of Duffy type, but the junction and invasion are Duffy
antigen–dependent.
Data from human and old and new world monkey RBCs and their
susceptibility to invasion by P. vivax and P. knowlesi indicate that
Fy6 is important for invasion for P. vivax. The monoclonal anti-Fy6
has been shown to block invasion of RBCs by P. vivax

The Kidd (009) Blood Group System

Disease Associations
Although Jka and Jkb are thought to be human RBC antigens, three
organisms have been associated with Jkb-like specificity. Two,
Enterococcus faecium and Micrococcus, were able to convert Jk(b)
cells to Jk(b), and one, Proteus mirabilis, may have been the
stimulus for an autoanti-Jk

-END OF TRANSCRIPTION -

#MLSWATER2022 Page 18 of 18
 MODULE 4: PRE-TRANSFUSION TESTING
MLS 18, March 17, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
The crossmatch is an actual mixing of the recipient and donor’s blood to
Legend: insure in vitro compatibility.
Transcription Bullet Only a part of compatibility testing
Notes/Module Packet Bullet Check for any untoward reaction between the donor and recipient’s
 PPT blood
MODULE OUTLINE
I. Compatibility Testing SEROLOGIC CROSSMATCH
II. Antibody Screening Two main functions of the serologic crossmatch test:
III. Selection of Appropriate Donor Units o It is a final check of ABO compatibility between donor and patient.
IV. Routine Blood Bank Laboratory o It may detect the presence of an antibody in the patient’s serum
that will react with antigens on the donor RBCs but that was not
LEARNING OUTCOMES detected in antibody screening because the corresponding antigen
1. Define compatibility test. was lacking from the screening cells.
2. Identify tests included in compatibility test. Consists of mixing the patient’s serum with the donor’s RBC
3. Describe appropriate methods for proper patient identification in
sample collection.
Major Crossmatch
4. Outline the procedure for testing donor and patient specimens.
An in-vitro determination that combines recipient’s plasma with a
5. Compare and contrast major and minor crossmatch procedures.
5% cell suspension of the donor’s cells.
6. Resolve incompatibilities in the crossmatch.
This test is performed at different phases in the same manner as the
7. State the limitations of compatibility testing procedures.
antibody screen and antibody identification.
8. Discuss future issues of compatibility testing.
The immediate spin crossmatch is the first phase of the major
crossmatch procedure.
o This procedure provides a determination of compatibility
COMPATIBILITY TESTING
that encompasses ABO incompatibility.
Series of procedures designed to ensure the safety of blood for
The final phase is the anti-human globulin (AHG) test.
transfusion.
o Antibodies present in the recipient that are reactive at the
Vital part of the blood bank laboratory. AHG phase are most likely to cause an in vivo reaction
o The determination of compatibility of a unit of blood for transfusion
is performed each time a unit of red blood cells is to be transfused.
Minor Crossmatch
Included in the compatibility test is a crossmatch.
Performed by mixing donor plasma with cells from the recipient.
The minor crossmatch is no longer performed.
CROSSMATCHING/CROSSMATCH TESTING The majority of cellular products transfused are red blood cells
The crossmatch test is the testing of the patient’s serum with the donor rather than whole blood.
RBCs, including an antiglobulin phase or simply an immediate spin phase
to confirm ABO compatibility.
 The volume of donor plasma transfused with red cell products is
very small and not significant enough to induce a reaction with
recipient cells
Compatibility test results:
Compatibility test procedures are performed and the results
assessed.
Compatible units of red cells will have no agglutination or hemolysis
at any phase of testing.
Tests that have either hemagglutination or hemolysis noted are
incompatible and should not be transfused without further
evaluation.
The cause of the incompatibility should be determined and
documented.
Objective of testing: to select donor units that can provide maximal
benefit to the patient, which should be kept in mind when developing
the test protocol.
Conventional Crossmatching
Three Phases: Immediate Spin Crossmatch, 37°C Incubation with
the use of enhancement media, Antiglobulin Crossmatch
Immediate Spin Crossmatch
This is accomplished by mixing the recipient’s serum with donor
RBCs and centrifuging immediately.
Absence of hemolysis or agglutination indicates ABO compatibility.
The immediate spin does not detect all ABO incompatibilities
False reactions may be seen in:
o The presence of other immediate spin-reactive antibodies
(e.g., autoanti-I)
o Patients with hyperimmune ABO antibodies
o May be seen when the procedure is not performed correctly
(e.g., delay in centrifugation or reading)
o When rouleaux is observed
o When infants’ specimens are tested.
#MLSWATER2022
Adding ethylenediaminetetraacetic to the test system reportedly
eliminates some of the false-positive reactions, thus improving the
sensitivity of the immediate spin crossmatch.
2-3 drops of serum sample with a drop of a wash of 2-5% RBC
suspension; observe presence of hemolysis in the centrifuge
accomplished by mixing the recipient’s serum with the donor’s RBC
and centrifuging immediately (no incubation)
Type-And-Screen Procedure
Involves testing the patient’s blood sample for ABO, Rh, and
clinically significant unexpected antibodies.
The patient sample is then stored in the blood bank refrigerator for
future crossmatch if blood is needed for transfusion.
The type-and-screen procedure has application for patients
undergoing many elective procedures who may need blood;
because the process may not always require transfusion, the
crossmatch is not performed until necessary.
37°C Incubation with the use of enhancement media:
Enhancement Media – there is the option to use 22% albumin, low
ionic saline solution, or polyethylene glycol
Antiglobulin Crossmatch
The antiglobulin crossmatch procedure begins in the same manner
as the immediate spin crossmatch, continues to a 37°C incubation,
and finishes with an antiglobulin test.
Several enhancement media may be applied to boost antigen-
antibody reactions
o These may include albumin, low ionic strength solution
(LISS), polyethylene glycol, and polybrene
For greatest sensitivity, an antihuman globulin (AHG) reagent
containing both anti-IgG and anticomplement may be selected for
the final phase of this crossmatch method.
An autocontrol, consisting of the patient’s own cells and serum, may
be tested in parallel with the crossmatch test.
Used to detect (unagglutinated IgG) antibodies that were not
detected with 37°C Incubation and Immediate spin crossmatch
Page 2 of 16
 Computer Crossmatch
Uses computer technology that is developed and increases
sensitivity of tests
As computer technology has developed and the sensitivity of
antibody detection has increased, a process for computer or
electronic crossmatch has been developed.
The electronic crossmatch is a viable option when there is no
current or previous history of clinically significant antibodies.
One of the primary advantages is maximum utilization of blood
supply.
When the electronic crossmatch is used with appropriate patients,
wastage of units by holding units for specific patients is significantly
reduced.
Many believe that the computer crossmatch is safer than the
immediate spin because of the integrity of the computer software
to detect ABO incompatibility between the sample submitted for
pretransfusion testing and the donor unit.
The computer crossmatch compares recent ABO serologic results
and interpretations on file for both the donor and the recipient
being matched and determines compatibility based on this
comparison.
Advantages of the Electronic Crossmatch:
a. More efficient management of blood inventory
b. Less wastage of blood components due to outdate
c. More efficient use of technician time
d. Increase in staffing flexibility
e. Smaller recipient sample required
f. Potential for a centralized transfusion service data bank
accessible by all interested parties
g. annual savings
h. Reduced sample requirements
Reduced expenses
i. Reduced handling of biological materials
j. Elimination of false reactions associated with the immediate
spin crossmatch.
#MLSWATER2022
Blood Sample Collection and Labelling
Acceptable collection tubes for pre-transfusion testing are:
 Plain Red Tube (No Anticoagulant; No Serum Separator)
 Yellow Top Tube (Acid Citrate Dextrose or ACD, Formula B)
 Pink or Purple Top Tube (EDTA)
 Blue Top Tube (Sodium Citrate)
Historically, the use of anticoagulant for crossmatching was
discouraged, since the binding of calcium inhibited the activation of
complement in the compatibility test.
o The clinical significance of these complement-binding
antibodies has been determined insignificant. Hence, the
use of anticoagulated samples has become acceptable.
Anticoagulated samples are readily available for crossmatching in an
emergency situation since the time required for a sample to clot
prior to processing may delay testing and treatment.
o In addition, the formation of fibrin that may interfere with
testing is less likely when using an anticoagulated specimen.
The sample should not be contaminated with IV solutions
o A sample may be collected from an infusion line only if the
line is flushed with saline and a volume of blood equal to
twice the test volume is withdrawn and discarded prior to
obtaining the sample.
The collection process should employ a needle of sufficient size to
avoid hemolyzing the red cells in the sample.
o The presence of hemolysis in plasma of the original sample
may mask hemolysis resulting from an antigen-antibody
interaction in the testing process.
Lipemic serum may also provide an environment that is difficult for
evaluation of serological reactions.
o At times it may be necessary to accept test samples with
some amount of hemolysis or lipemia.
o Each institution should establish criteria and procedures for
acceptable samples
Individual institutions should establish the maximum age for pre-
transfusion testing samples.
Page 3 of 16
 Patient specimen free transfusion testing begins with properly
labelled, properly collected and identified patient specimen; patient
identification is first in compatibility testing
Proper identification of the patient is imperative; patient must have
a wristband with the identification information, tube should be
labelled on the bedside immediately after the sample was drawn
Label should include the date, time of collection, name of the
person collected, and sample should not be collected into a
previously labeled tube.
During collection serum is a preferred specimen for compatibility
testing, hemolysis should be avoided, and blood should not be
drawn from intravenous site.
Stop the infusion for 5-10 minutes before collecting blood and then
the lye should be flush with normal saline and first 5-10ml blood lye
should be discarded.
o Red top for serum sample
o Yellow, pink and purple top used for collecting from donor
Fresh sample (not older than 72 hours) should be the best option
used for compatibility testing if patient has previously undergone
transfusion or transfusion history is unknown.
o Also, the same for pregnant women
For sample storage, EED standards states that patient sample
should be stored between 1° C and 6° Celsius for at least 7 days
following transfusion.
Lipemic samples or lipemia can interfere with the testing
Hemolyzed sample should also be avoided
AABB Standards for Blood Bank and Transfusion Services, 25th Edition,
states that a recipient sample must be:
a. Collected within three (3) days of the scheduled transfusion if
the patient has been transfused or pregnant in the preceding
three (3) months or if the history is uncertain or unavailable.
The day of collection is labeled as day zero (0).
b. These patient samples, as well as a segment from any red cell
containing component, are all refrigerated and retained for at
least seven (7) days after transfusion.
The AABB designates that the request for blood and blood products
contain sufficient information to uniquely identify the patient. This
includes a minimum of two independent identifiers:
#MLSWATER2022
These two independent identifiers should be confirmed at the
time of collection and the blood sample labeled with this unique
information:
the date of collection
The phlebotomist’s signature.
This labeling must take place at the bedside.
Most institutions use hospital identification bands to identify
patients
o Past records should be compared to current test results.
The requirement further states that the current ABO and Rh
type must be compared to results obtained within the past
12 months.
o Discrepancies are investigated and resolved before any
units are issued for transfusion.
The AABB Standards require that all donor blood have the ABO and
Rh confirmed from an attached segment prior to transfusion of any
whole blood or red blood cell component.
o The standards do not require that the institution issuing the
blood for transfusion perform a test for the confirmation of
the weak D antigen
According to the AABB, patient samples must be tested for ABO, Rh,
and unexpected antibodies to red cell antigens prior to transfusion
of red cells.
o The weak D test is not required, but the antibody screen
must include “incubation at 37°C preceding an antiglobulin
test using reagent red cells that are not pooled.”
The compatibility test procedure is only applicable for red cell
products that are to be transfused
o Plasma based products such as cryoprecipitate, platelet
concentrates, and frozen plasma contain virtually no red
cells and require no compatibility testing.
o These components have been screened for antibodies and
infectious diseases. Hence, they are transfused as ABO-
compatible.
o Platelet-pheresis products and granulocyte concentrates
may contain some red cells, but require crossmatching only
if more than 2 ml of red cells are present.
Page 4 of 16
 PROBLEM SOLVING INCOMPATIBLE CROSSMATCHES o The procedure must include a system to ascertain that the
autologous units are administered before any allogeneic units
PROBLEM are issued.
POSSIBLE CAUSE RESOLUTION o The administration of allogeneic units before the stored
ENCOUNTERED
ABO typing  Patient identification  Repeat the ABO autologous units presents a liability risk for the administering
error group of the institution.
 Sample identification recipient
error  Recheck the label COMPATIBILITY TESTING OF NEONATES UNDER 4 MONTHS OF AGE
 Choice of unit of of the selected unit Neonates require special consideration
incompatible ABO  Recollect a sample Antibodies present have originated from the mother since the
group from the recipient neonate is unable to produce their own antibodies.
Unexpected  Cold alloantibody  Test with panel Neonates actually have the antibody of their mother
antibodies when  Cold autoantibody cells and Initial pretransfusion testing uses the neonate’s red cells for ABO
antibody screen is  anti-A1 in an A2 determine clinical and Rh typing.
negative at AHG individual significance of Reverse grouping is not performed on neonates due to the lack of
antibody antibody production.
 Prewarm Antibody screen may be performed using either the infant or
compatibility maternal plasma.
testing If the initial antibody screen is negative, there is no need to perform
 Test plasma of additional antibody screens or crossmatches on the neonate during
recipient with A2 this admission, so long as ABO-compatible and Rh identical red cell
cells products are administered.
Positive AHG phase  If the antibody screen is positive, the antibody must be identified
Positive DAT in donor  Perform DAT test
and antigen negative units administered for assured compatibility.
of compatibility  Antibody to low on donor
Additionally, an antiglobulin crossmatch should be performed and
test incidence antigen in  Choose alternate
only compatible units released.
recipient with antigen unit for
Crossmatching of all units needs to be continued until the maternal
positive cells in donor compatibility test
antibody is no longer detectable in the infant’s plasma.
Units of blood are crossmatched and divided since only small
COMPATIBILITY TESTING FOR AUTOLOGOUS TRANSFUSION quantities are administered to neonates.
Autologous donation is the process of collecting units of blood and Blood bags with multiple satellite bags or “pedi-packs” are
storing them for transfusion to the original donor commercially available for this purpose.
o This procedure is most often used for patients anticipating o This further reduces the possibility of incompatibility or
elective surgery. disease transmission
o Procedures to ensure that the collected units are transfused CMV negative units for use specifically in neonates may be obtained
to the intended recipient are established by the transfusing from the component source
institution.
o This procedure may be computerized with all records retained
electronically.

#MLSWATER2022 Page 5 of 16
 CAUSES OF POSITIVE RESULTS IN THE SEROLOGIC CROSSMATCH
Incorrect ABO grouping of the patient or donor.
An alloantibody in the patient’s serum reacting with the
corresponding antigen on donor RBCs.
An autoantibody in the patient’s serum reacting with the
corresponding antigen on donor RBCs.
Prior coating of the donor RBCs with protein, resulting in a positive
antihuman globulin test.
Abnormalities in the patient’s serum.
Contaminants in the test system
ANTIBODY SCREEN
The recipient’s serum or plasma must be tested for clinically
significant unexpected antibodies.
Usually done or is part of the antibody testing
The object of the antibody screening test is to detect as many
clinically significant unexpected antibodies as possible.
In general, “clinically significant unexpected antibody” refers to
antibodies that are reactive at 37°C or in the antihuman globulin
test and are known to have caused a transfusion reaction or
unacceptably short survival of transfused RBCs
Detection of unexpected antibodies is important for the selection of
donor RBCs that will have the best survival rate in the patient’s
circulation and reduce the risk of hemolytic transfusion reaction.
Antibody screening or antibody panels are used
Antibody screening tests should demonstrate the presence of all
potentially clinically significant alloantibodies in the recipient’s
serum or plasma and indicate the need for further studies.
All antibodies encountered in the screening test must be identified
to determine potential clinical significance and to decide whether
there is a need to select antigen-negative units for transfusion.
Multiple antibodies are more commonly found in patients older
than 60 years old or have undergone transfusion multiple times
are usually the ones detected with unexpected reactions
In antibody identification we make use of screening cells (cells that
have no antigen present)
3 Phases Involved (Immediate screen, 37°C, Antihuman globulin
phase)
#MLSWATER2022
Goal of Antibody Screening
o To detect as many clinically significant antibodies as
possible
o To detect as few insignificant
o To detect the red cell antibody other than anti a and anti b
SELECTION OF APPROPRIATE DONOR UNITS
The first choice for transfusion is blood and blood components of
the patient’s own ABO and Rh group. This is defined as ABO group–
specific.
When blood and blood components of the patient’s ABO blood
group are not available or some other reason precludes their use,
units selected must lack any antigen against which the recipient has
a clinically significant antibody.
When a recipient must be given blood of a different ABO group,
only packed RBCs can be given.
Whole blood cannot be administered in these situations because
incompatible, preformed ABO antibodies are present in the whole-
blood plasma.
o Ex. Group A whole blood cannot be transfused into a group
AB recipient, because the plasma of the group A whole
blood has anti-B antibodies present.
Group O packed RBCs can be safely used for all patients; however,
conservation of a limited supply of group O blood should dictate its
use for recipients of other ABO types only in special circumstances.
If ABO group–specific blood is not available or is in low supply,
alternative blood groups are chosen
Rh-negative blood can be given to Rh-positive patients; however,
good inventory management should conserve this limited resource
for use in Rh-negative recipients.
o But if the Rh-negative unit is near expiration, the unit
should be given rather than wasted.
Rh-positive blood should not be given to Rh-negative female
patients of childbearing age.
Transfusion of Rh-negative male patients and female patients
beyond menopause with Rh-positive blood is acceptable as long as
no preformed anti-D is demonstrable in their sera.
Page 6 of 16
 BLOOD BANK LABORATORY
A facility involved in the collection, storage, processing, and
distribution of human blood and blood products for transfusion
BLOOD BANK AREA AND FUNCTION
AREA FUNCTIONS
Component Preparation  Separation of whole blood into packed
And Storage RBCs, plasma, platelets and
cryoprecipitate
 Storage of blood products at
appropriate temperatures
 Apheresis procedure
Donor Processing Donor units tested for:
 ABO and Rh
 Antibody Screen
 Serologic Test for Syphilis
 Transfusion-Transmitted viruses
Product Labelling  RBCs and any other component are
labeled
 Products stored at their proper
temperatures
Main Laboratory Patient Samples tested for:
o ABO and Rh
o Antibody Screen
o Crossmatch
o DAT
o Prenatal Evaluation
o Postpartum Evaluation
o Cord blood studies
o Issue blood products
Reference Laboratory Resolution of:
o ABO and Rh discrepancy
o Antibody identification
o Positive DAT
o Warm autoantibodies
o Cold autoantibodies
o Transfusion reaction
#MLSWATER2022
Donor Processing
 Before unit blood can be placed in general inventory, testing must
be performed
 Blood Processing centers utilize the testing methods that will
provide the safest blood products for patients
Product Labelling
Appropriate ABO and Rh type
Expiration date
Stored at proper temperature
Issue of Blood Products
The individual in the blood bank who will issue the blood product
inspects the unit for any abnormal appearance and verifies that all
required transfusion forms and labels are complete and that they
adequately identify the transfusion recipient.
Personnel Requirements
Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88)
establishes personnel qualifications for laboratories performing
certain types of testing:
o include gel technologies
o Solid phase RBC adherence.
Standard Operating Procedures
These manuals, usually located at the workbench and accessible to
all personnel, contain information outlining the operations of the
laboratory; details on how, when and why particular activities are
done; and procedures for all tests performed.
SOP manuals are integral components of any blood bank
laboratory’s quality assurance program.
They are reviewed at least annually and updated on a regular basis
to reflect changes in operations and implementation of new
regulations.
Page 7 of 16
 Equipment o In addition to patient information, the label should also
 Blood Storage Refrigerator include the date and time of collection and the name of the
 Donor Couches person who collected the sample
 Dielectric Tube Sealer 2. Collection
 Blood Mixer & Collector o Serum is the preferred specimen for compatibility testing.
 Platelet Incubator o Hemolysis should be avoided.
 Platelet Agitator o Blood should not be drawn from an intravenous site unless
 Plasma Expressor absolutely necessary. In such a case, the infusion should be
 ELISA Reader with washer stopped, the line should be flushed with normal saline, and
 Refrigerated Centrifuge the first 5 to 10 mL of blood should be discarded before the
 Binocular Microscope CH 21 specimen is collected.
 Universal Hot air oven 3. Age of specimen
 Bacteriological Incubation o The freshest sample possible should be used for compatibility
 Centrifuge Machine testing.
 Rh view box o If the patient has previously undergone transfusion or if the
transfusion history is unknown, the sample should be no older
than 72 hours.
SUPPLEMENTARY NOTES: PRE TRANSFUSION TEST ING o Pregnant patients should also be tested with samples not
CROSSMATCHING more than 72 hours old
4. Sample storage
Only a part of compatibility testing.
o The AABB requires that patient samples must be stored
Type and Screen procedure has emerged as an acceptable
between 1◦C and 6◦C for at least 7 days following
alternative to crossmatching blood
transfusion.
Major Crossmatch
COMPATIBILITY TESTING:
o DONOR RBCS + PATIENT SERUM
Minor Crossmatch for homologous transfusion (ALLOGENIC):
o DONOR SERUM + PATIENTS RBCS ABO and Rh on donor units
Autocontrol ABO and Rh on recipient
o PATIENT RBCS + PATIENT SERUM Antibody screening of recipient
Antibody identification
Autocontrol
SPECIMEN COLLECTION Crossmatch
Pretransfusion testing begins with a properly collected and
identified patient specimen for autologous transfusion:
ABO and Rh on autologous units
1. Patient identification ABO and Rh on recipient
o The tube should be labelled at bedside immediately after the Antibody screening and major crossmatch
sample is drawn o not required, although an immediate spin major crossmatch
is often performed
#MLSWATER2022 Page 8 of 16
for neonatal transfusion: FDA Licensed Screening Cells
ABO and Rh on the infant unspooled reagent red cells
Antibody screen on the infant or the mother 2-3 vials (R1R1, R2R2,rr)
o If the antibody screen is negative, a crossmatch is not detects clinically significant Ab
necessary reagents red cell express the ff: D,C,E, c,e
o If the donor cells are not group O, the infant must be tested M,N,S,s,P1,Lea,Leb,K,k,Fya,Fyb,Jka,Jkb
for anti-A and anti-B antibodies. If either is present, ABO- Autocontol not required
compatible RBCs should be used. A crossmatch is not DAT-not required
necessary
NOTE: Group O cells are used so that anti-A and anti-B will not interfere
ANTIBODY DETECTION in the detection of antibodies to other blood group systems.
Antibody detection/ antibody screening is testing the patient’s
serum against 2 or 3 reagent group O screening cells. ANTIGRAM/ WORKSHEET
Screening cells are commercially prepared cells suspension from It is a listing of the antigen make up of each screening cells that is
individual donor that is phenotyped for most common antigens. provided with every lot of screening cells.
In test tube testing: 3 phases Can be used as a worksheet during antibody detection process.
o Immediate spin o Caution: Lot number must match against the screen cells in
o 37C incubation use
o AHG Sample (link to worksheet):
Testing can also be: gel technique or solid phase method https://drive.google.com/drive/folders/1d_Lz_7fXfjYItsjN_yUhbbk7dStDOo
Performed as part of: Hi?usp=sharing
o testing to provide compatible red cell transfusion
o prenatal evaluation
o evaluation of HDFN ANTIBODY IDENTIFICAT ION
o resolution of transfusion reactions ANTIBODY PANEL
o processing of donor units
Just an expanded antibody screen
Uses group O reagent RBCs
Uses Antibody Panels - used to identify an unexpected antibody
RBCs from 8-20 donors
detected by the antibody screening
Patient serum or plasma
Expanded version of antibody screening
Autocontrol
antibody screening uses screening cells 2-3 reagents of group O
IS / 37 C / AHG if tubes
screening cells
AHG only if gel or solid phase
Antibody panel uses 8-20 screening cells, these panels usually
Reactions documented on a sheet that outlines every RBC’s
contain 10-15 or 8-20 vials of group O cells each of which yield a
phenotype
different antigen reaction pattern

Each of the panel cells has been antigen typed (shown on antigram)
o + refers to the presence of the antigen
o 0 refers to the absence of the antigen
#MLSWATER2022 Page 9 of 16

All cells are negative at AHG, so add “Check” Cells
Check cells are added to all negative tubes.
Check cells are cells coated with IgG and should react positively with
the AHG in the tube.
o If check cells are negative, the procedure was not
performed correctly and should be repeated.
#MLSWATER2022
INTERPRETING ANTIBODY PANELS
1. Check History
Will give you clue what possible Ab is involved.
Check racial profile, recent transfusion, pregnancy and recent
bacterial or viral infection.
Notes the previous condition of the patient.
o Ex. pregnant: Rh blood group system is involved
Important also to note the racial profile of the patient since African-
American lacks Duffy antigen and Whites has high frequency
antigen
2. Check Autocontrol
Negative - alloantibody
Positive – autoantibody or DTR (i.e., alloantibodies)
Check if the reaction if zero or plus or positive
If the reaction is positive that would indicate that there is presence
of autoantibody
If the reaction is negative there might be the presence of
alloantibody
o Another thing for negative control, it would show a uniform
reaction that means all cells are reactive. It would suggest there
might be presence of multiple alloantibodies and single
alloantibodies related with high frequency antigen
Antibodies will only react with cells that have the corresponding
antigen; antibodies will not react with cells that do not have the
antigen
3. Check the Reaction Phase
IS – cold reacting (IgM); clinically insignificant
37° - cold (some have higher thermal range) or warm reacting (IgG)
AHG – warm (IgG) and significant Ab. It detects Ab that have been
coated/sensitized RBCs but were not visible as agglutinates
4. Look at the general pattern (Reaction strength)
1 consistent strength (Uniform)– one antibody
Different strengths(Mixed reaction)s – multiple antibodies or
dosage
Page 10 of 16
 NOTE:
5. Ruling Out Strength of reaction may be due to “dosage”
Look at the first completely negative cells ( panel cells that has 0 or If panel cells are homozygous, a strong reaction may be seen
no reaction) If panel cells are heterozygous, reaction may be weak or even
Go blood group by blood group or paired antigen non-reactive
Panel cells that are heterozygous should not be crossed out
Example case: because antibody may be too weak to react

6. Look what IS there (Look at the usual Ab reactivity)

Cell # 3 and 4 shows double dosage (homozygous Lea) Lea with

(+) reaction should be eliminated since the IS results should be
negative. Leb cannot be rule out since it has the same reaction
with IS results.
Cell # 5 and 7 shows single dose (heterozygous) cannot be ruled
out.
Cell #9 and 10 shows double dosage (homozygous) Leb with (+)
reaction can be ruled out.

7. Look for Matching Pattern

Single antibodies usually shows a pattern that matches one of the
antigens.
Multiple antibodies are more difficult to match because they often
show mixed reaction strengths.

#MLSWATER2022 Page 11 of 16
 RULE OF THREE: FOR EXAMPLE:
Patient Serum must be: Let’s say you ran a panel and identified 3 different antibodies:
o Positive with 3 cells with the antigen anti-S, anti-Jka, and anti-P1
o Negative with 3 cells without the antigen o Selected cells could help…
o If there are not enough cells in the panel to fulfill the rule,
then additional cells from another panel could be used. Selected cells S Jka P1 IS LISS AHG
37
8. Use Special Techniques if Necessary #1 + 0 0 0 0 2+
#5 0 + 0 0 0 3+
9. Ensure statistical significance #8 0 0 + 0 0 0 (check cell +)

SPECIAL TECHNIQUES These results show that instead of 3 antibodies, there are actually
2: anti-S and anti-Jka
PHENOTYPING
In addition to the rule of three, antigen typing the patient red cells
can also confirm an antibody
NEUTRALIZATION/ INHIBITION
Only perform this if the patient has NOT been recently transfused
Other substances in the body and in nature have antigenic
(donor cells could react)
structures similar to RBC Antigens which can neutralize antibodies
If reagent antisera (of the suspected antibody) is added to the
present in serum, allowing separation
patient RBCs, a negative reaction should result.

NOTE: Individuals DO NOT make allo-antibodies against antigens SUBSTANCE ANTIBODY NEUTRALIZED
they have – Landsteiner Rule Anti P1
Hydatid cyst fluid, pigeon droppings,
turtledove’s egg whites
MULTIPLE ANTIBODIES
Selected cells are chosen from other panel or screening cells to Plasma/serum;secretor saliva Anti-Le
confirm or eliminate the antibody
Plasma or serum from Ch/Rg(+) individuals Anti-Chido; Anti-Rodgers
The cells are “selected” from other panels because of their
characteristics Anti-Sda
Urine, guinea pig urine
The number of selected cells needed depends on how may
antibodies are identified Human breast milk Anti0I

Secretor saliva ABH antibodies

#MLSWATER2022 Page 12 of 16
ENZYMES (PROTEOLYTIC) AUTOANTIBODIES
Can be used to enhance or destroy certain blood group antigens; Autoantibodies can be cold or warm reacting
Enzymes remove the sialic acid from the RBC membrane, thus A positive autocontrol or DAT may indicate that an auto-antibody is
“destroying” it and allowing other antigens to be “enhanced” present
Several enzymes exist: Sometimes the autocontrol may be positive, but the antibody
o Ficin (figs) screening may be negative, meaning something is coating the RBC
o Bromelin (pineapple)
o Papain (papaya) WHAT CAN THE DAT TELL US?
Although not always performed in routine pretransfusion testing,
DESTROYED ENHANCED NO EFFECT a positive DAT can offer valuable information
If the patient has been transfused, the patient may have an
MN, Duffy, Xg, JMH, alloantibody coating the transfused cells
ABO, Rh, Lewis, P, Ii,
Ss, Chido/Rodgers, Pr, Kell, Diego, Colton ,U If the patient has NOT been transfused, the patient may have an
Kidd
Lutheran autoantibody coating their own cells

One-stage: Enzyme is added directly to the serum/cell mixture Note: Auto-antibodies can sometimes “mask” clinically significant allo-
Two-stage: Panel cells are pre-treated with enzyme, incubated and antibodies, so it’s important to differentiate between auto- and allo-
washed. Patient serum is added to panel cells and tested antibodies

SULFHYDRYL REAGENTS
Cleave the disulfide bonds of IgM molecules and help differentiate COLD AUTOANTIBODIES
between IgM and IgG antibodies React at room temperature with most (if not all) of the panel cells
Good to use when you have both IgG and IgM antibodies and give a positive autocontrol
(warm/cold) The DAT is usually positive with anti-C3 AHG (detects complement)
Dithiothreitol (DTT) is a thiol and will denature Kell antigens Could be due to Mycoplasma pneumoniae, infectious mono, or cold
2-mercaptoethanol (2-ME) agglutinin disease
A combination of proteolytic enzymes and DTT
Denatures Kell, M, N, S, Duffy and other less frequent blood group
antigens
Does not denature the Kx antigen
Good for adsorption techniques
“frees” autoantibody off patient’s cell, so that autoantibody can
then be adsorbed onto another RBC

#MLSWATER2022 Page 13 of 16
AVOIDING REACTIVITY
Cold autoantibodies can be a nuisance at times. Here are a few ways to
avoid a reaction:
Use anti-IgG AHG instead of polyspecific. Most cold antibodies
react with polyspecific AHG and anti-C AHG because they fix
complement
Skipping the IS phase avoids the attachment of cold
autoantibodies to the red cells
Use 22% BSA instead of LISS
If the antibodies remain, then prewarmed techniques can be
performed:
Red cells, serum, and saline are incubated at 37° before being Types of Elution:
combined
Autoadsorption is another technique in which the autoantibody is
removed from the patients serum using their own red cells
The serum can be used to identify any underlying alloantibodies
WARM AUTOANTIBODIES
More common that cold autoantibodies
Positive DAT due to IgG antibodies coating the red cell
Again, the majority of panel or screening cells will be positive ADSORPTION
The Rh system (e antigen) seems to be the main target although
others occur
Cause warm autoimmune hemolytic anemia (WAIHA)
Sources of Warm autoantibodies:
o Idiopathic
o Known disorder (SLE, RA, leukemias, UC, pregnancy,
infectious diseases, etc) Two Types:
o Medications
#MLSWATER2022
ELUTION (WHENEVER DAT IS POSITIVE)
Elution techniques “free” antibodies from the sensitized red cells so
that the antibodies can be identified.
Testing the eluate is useful in investigations of positive DATs
o HDN
o Transfusion reactions
o Autoimmune disease
The red cells can also be used after elution for RBC phenotyping if
needed
When tested with panel cells, the eluate usually remains reactive
with all cells if a warm autoantibody is present
Acid elutions (glycine acid)
o Most common
o Lowers pH, causing antibody to dissociate
Organic solvents (ether, chloroform)
o Dissolve bilipid layer of RBC
Heat (conformational change)
Freeze-Thaw (lyses cells)
Adsorption procedures can be used to investigate underlying
alloantibodies
ZZAP or chloroquine diphosphate can be used to dissociate IgG
antibodies from the RBC (may take several repeats)
After the patient RBCs are incubated, the adsorbed serum is tested
with panel cells to ID the alloantibody (if present)
Autoadsorption
o No recent transfusion
o Autoantibodies are removed using patient RBCs, so
alloantibodies can be identified
Allogenic (Differential) adsorption
o If recently transfused
o Uses other cells with the patients serum
Page 14 of 16
 ANTIBODY PANEL VS. ANTIBODY SCREEN
CHLOROQUINE DIPHOSPHATE
Quinilone derivative often used as an antimalarial
May not remove autoantibody completely from DAT positive cells
Partial removal may be enough to antigen type the cells or to be
used for autoadsorption of warm autoantibodies
ADD-ONS FROM MA’AM JOANA’S DISCUSSION
When do we need to identify the antibody?
ANTIGRAM/WORKSHEET
COMMON REASONS TO IDENTIFY AN ANTIBODY PANEL TESTING
o Uses Antibody Panels - are used to identify an unexpected antibody
detected by the antibody screening
o This is usually done following a positive antibody screening when
testing would suggest a presence of new antibody and confirm a
previously identified antibody
Why do we need to identify the antibody present in a reaction?
o it is needed for transfusion processes
o it is essential component for the compatibility testing
o identification of any unexpected antibodies in the patient’s serum
In antibody panel testing, it is basic to know what is alloantibody and
autoantibody
o Alloantibodies – are antibodies against RBC agent not present on
patient’s own RBC
o Autoantibodies- are antibodies against the RBC antigen present on
patient’s own RBC
IMMEDIATE SPIN PHASE
RBC screening
o uses 2-3 screening cells to detect if any antibodies are present in the
serum
o it follows the concept about when detecting/identifying the
antibodies, test the patient’s serum (contains the unknown), weigh
the reagents RBC (contains the known)
#MLSWATER2022
o Group O reagents but with antibody panel it uses 10-15 or 8-20 vials
or donors blood compared into 2-3 vials in antibody screening.
o Patient serum is also used just like in antibody screening.
o It also involves 3 phases: immediate spin, 37° Celsius, and
antihuman globulin
o We use 10 panel cells, the number 11 is usually the auto control
o Group O cells are used so that anti-A and anti-B will not interfere in
the detection of antibody to the other blood group system
o Each of the panel cells have been antigen type
o The table is called the antigram
o antigram or worksheet is a listing of the antigen make up of each of
the screening cells or panel cells that is provided with a lot of
screening cells. This can be used as a worksheet during antibody
detection process
o (+) positive or plus sign symbol: refers to presence of antigen
o (0) zero: refers to the absence of antigen
o Auto controls are located below, it is used to determine the
antibody present is an autoantibody or alloantibody
 There is the presence of autoantibody if the auto control with
the patient’s RBC will result a (+) positive reaction
 If (0) zero or no agglutination that would occur during the auto
control testing would mean that the antibody present is
alloantibody
o It is also needed to check in the antigram the reaction in different
phases: immediate spin, 37° Celsius, and antihuman globulin
o 10 panel: a drop in each panel cells and 2 drops of patient’s serum
o Detect clinically insignificant cold antibodies usually IgM in nature
and it involves immediate centrifugation of the mixture at room
temperature for 20 seconds using the centrifuge
 There are some laboratories that will not require immediate
spin phase they directly undergo LISS phase and anti-human
globulin
Page 15 of 16
37° CELSIUS WITH ENHANCEMENT MEDIUM DIFFERENT GRADING FOR AGGLUTINATION
o it detects the warm reactive IgG antibodies 1 CLUMP 4+
 Enhancement Media are potentiators that added to the cells IF DISTANCED 3+
serum mixture before the 37° Celsius or incubation to increase SCATTERED 2+
the sensitivity of the test system HAS AGGLUTINATION BUT VERY 1+
MINIMAL
DIFFERENT ENHANCEMENT MEDIA NO AGGLUTINATION REPORTED AS NEGATIVE
1. 20% Albumin
DO NOT FORGET TO ADD THE CHECK CELLS TO ANY NEGATIVE AHG
o Incubation period 30-60 minutes, works in decreasing zeta
o Example: Encounter a reaction that can be only seen in Immediate
potential Spin Phase
o Low Ionic Strength Solution/ Saline Solution: Incubation time 10- o No reaction was recorded for the LISS and no reaction was also seen in
15 minutes and it contains .2% of Sodium Chloride. Meaning it anti-human globulin.
has low ionic content compared to the normal saline solution o To confirm that the procedure is performed correctly, it doesn’t need
that contains glycine, dextrose or glucose in addition to saline to repeat and check cells must be performed. Since check cells are added
reduce the zeta potential in all negative tubes
o increases the amount of the antibody taken up by the red cell  Check cells – are cells coated with IgG and should react
during the sensitization process positively with anti-human globulin reagent within the tube.
o Sensitization – is the first page in the agglutination process If the check cells are negative, procedure was not
performed correctly and should be repeated
2. Polyethylene Glycol
-END OF TRANSCRIPTION -
o Works in removing water from the test system thereby
ඞ
concentrating any antibodies that are present in the reaction.
More sensitive compared to LISS and 22% albumin
 Disadvantage: don’t usually used in high motile patients
having high protein levels just like in the cases of multiple
myeloma since it can interfere in the testing procedure

3. Antiglobulin phase
o Detects non-agglutinating warm reactive antibodies that are
possibly not detected with 37° Celsius incubation period.
o It would also detect antibodies that has been coated or
sensitized, RBC that did not show any visible agglutination.

#MLSWATER2022 Page 16 of 16
 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 1)
MLS 18, April 6, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
Has regulatory authority over drugs, biologics and devices as
Legend: mandated by the Federal Food, Drug and Cosmetic (FDC) Act of
Transcription Bullet 1938 and the Public Health Services (PHS) Act of 1944.
Notes/Module Packet Bullet Blood establishments in the US are therefore:
 PPT • Regulated by the FDA
• Required to follow federal regulations provided by the FDA in
MODULE OUTLINE
the composite form of the CFR (Code of Federal Regulations)
I. Organizations that Regulate or Accredit the Immunohematology
• Inspected at regular intervals by agents of the FDA
Laboratory
Blood establishments that ship products across state lines must be
II. Blood Donor Selection
licensed by the FDA, while blood banks and hospital transfusion
III. Types of Donation
services that operate within state boundaries must be registered
IV. Blood Component
with the FDA
V. Safe Storage of Blood
Hospital transfusion services that do not perform any
VI. Packing Blood Components for Transportation
manufacturing processes as defined by the FDA are not required to
be licensed or registered, though they must be in compliance with
LEARNING OUTCOMES applicable portions of the CFR and state regulations.
At the end of this module, the student shall be able to:
1. Identify the organizations that regulate or accredit the A government agency that regulates and establishes blood products
immunohematology laboratory. and its guidelines, transportation temperature, storage temperature
2. State the minimum acceptable levels for the following tests in and the products used in blood bank laboratory
allogenic & autologous donation Established CBER (Center for Biologics Evaluation and Research) is
3. Identify the medical history information that would cause responsible for the regulation of collection of the blood and blood
permanent deferral or temporary deferral and state the length of components that is being used in the transfusion and for the
deferral period. manufacture of the pharmaceutical derived from the blood and
4. Explain the reasons for permanent or temporary deferral given the blood components. It developed and enforces quality standard and
various medical conditions. inspection in blood establishments and monitor reports about
errors, accidents and adverse clinical events that occurs in the blood
transfusion practice. In hospital transfusion services it will not
ORGANIZATIONS THAT REGULATE OR ACCREDIT THE
perform any manufacturing processes defined by the FDA are not
IMMUNOHEMATOLOGY LABORATORY
required to licensed or registered.
FDA
Food and Drug Administration AABB (American Association of Blood Banks)
An organizational arm of the Department of Health and Human A professional organization which serves a number of key functions
Services (HHS) often referred to as the Agency. for the blood industry, including education, supporting science and
research and its dissemination, and public advocacy.

#MLSWATER2022 Page 1 of 16
 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
• The National Blood Foundation (NBF), a subsidiary of AABB,
was created with the sole mission of providing seed funding Donor Requirements
and financial support for scientific inquiry related to 1. Identification card
transfusion medicine Includes photographic identification of the donor

College of American Pathologists (CAP) 2. Donor registration information

Participates in laboratory accreditation and proficiency testing. Includes full name, date and time of donation, address,
The CAP program uses a system of peer review, and participating telephone/cell phone number, sex, age and date of birth
institutions must provide inspectors for future accreditation visits.
3. Consent Form
BLOOD DONOR SELECTIO N
Two questions that must be answered before proceeding with the 4. Additional Information
blood donation: The name of the patient whom the blood is intended
1. Will a donation of approximately 450 mL of whole blood at this time Race of the donor for the rare phenotype
be harmful to the donor? CMV status
2. Could blood drawn from this time potentially transmit a disease to
the recipient Calculations for the Blood Volume:
 Calculate adjusted blood volume and anticoagulant:
If “yes”: Defer donor: temporarily or permanently In athletes, pulse rate of <50 bpm is considered normal and not a factor
for deferral
If “No”: Proceed to Blood Donation
Volume to Collect:
General Requirements for Donation
𝑑𝑜𝑛𝑜𝑟 ′ 𝑠 𝑤𝑒𝑖𝑔ℎ𝑡 𝑖𝑛 𝑘𝑔
= 𝑥 450 𝑚𝐿
General Appearance: Observe the prospective donor for the 50 𝑘𝑔
presence of excessive anxiety, drug or alcohol
influence, or nervousness.
Age At least 17-18 years old Reduced Volume of Anticoagulant:
Oral Temperature < 37.5 °C or 99.5 °F
Blood Pressure Less than or equal 180/100 mmHg 𝑉𝑜𝑙𝑢𝑚𝑒 𝑡𝑜 𝐶𝑜𝑙𝑙𝑒𝑐𝑡
= 𝑥 63 𝑚𝐿
450 𝑘𝑔
Hemoglobin ≥12.5%
Hematocrit ≥ 38%
Pulse 50-100beats/minute Volume of Solution to be removed:
Weight ≥ 110 lbs or ≥ 50 kg
= 63 𝑚𝐿 − 𝑟𝑒𝑑𝑢𝑐𝑒𝑑 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐴𝑛𝑡𝑖𝑐𝑜𝑎𝑔𝑢𝑙𝑎𝑛𝑡
Volume Maximum of 525 mL can be collected
WB Donation Maximum of 10.5 mL of blood /kg of donor See Appendices for Sample Problem
weight

#MLSWATER2022 Page 2 of 16
 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
Blood Collection Process
1. Donor Registration
2. Educational material is distributed to the donor.
 If the prospective donor shows symptoms of an infectious disease,
the donor is excluded from the donation.
Explain to the donor what might happen to his blood since it will
undergo different blood test
They must also sign the consent form allowing for the blood
collection as well as other blood tests
Medical technologists must further explain to the donor the
other blood tests to be conducted to his blood
3. Actual Donor selection/ Identification
No fasting is required for blood donation, but donor must eat
Donors who have not eaten food for 4hrs or more are required
to eat before blood collection
Position needle at 10-20° angle
16-gauge needle usually used for blood donation; instruct
donor to open-and-close their fists every 10-20secs
Mix the unit periodically 1-2 times per minute.
o Blood Mixer – automatically mixes the blood with
anticoagulant
o If there is no blood mixer, the medical technologist will do
the mixing manually.
Amount of blood collected: 450mL ± 10%
Duration of collection: 7-10 minutes
o If the correct duration time is not observed, the blood
collected would not be suitable for platelet
concentration/concentrate preparation, FFP, or
cryoprecipitate (>15mins of blood collection would make
the blood not viable, especially in cryoprecipitate).
Color Coding for Blood Bags

4. Blood Collection FDA (1985) *RA 1517

 Use Aseptic Technique: Blood Type Color Label Blood Type Color Label
o PVP Iodine compound or chlorhexidine gluconate & A Yellow A Blue
Isopropyl alcohol B Pink B Yellow
o Scrub site at least 4 cm in all directions for 20 seconds AB White AB Pink
o Apply tourniquet or blood pressure cuffs: 40-60 mmHg O Blue O White
o Position the needle at 10-20 ° angle *Blood Bank Law; the standard/ what used
 Mix the unit periodically 1-2 times per minute
 Amount of blood collected: 450 mL ± 10% Donor Deferral
 Duration of collection: 7-10 mins
o Component must be prepared after collection TEMPORARY DEFERRAL
 EXAMPLE: Donor has received a blood transfusion; defer for 12
Blood collection facilities confirms the identity of the donor months from date of transfusion
through the photographic identification such as the use of  Tooth Extraction
showing their driver’s license for confirmation  Persons who had experienced convulsions
The antecubital fossa should be free from lesions, scars and
track marks INDEFINITE DEFERRAL
During blood collection 2 blood bank personnel, 1 Medical  These donors may be eligible to donate autologous blood
Technologist and 1 Head of Blood Bank is required to be The donor is unable to donate their blood for some reason and
present. They do interview and look for multiple needle marks, there is no specified period of time due to regulatory requirements;
unexplained weight loss or if the patient is immunosuppressed, but donors are still eligible to donate autologous blood
oral thrush or Kaposi sarcoma
#MLSWATER2022 Page 3 of 16
 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
PERMANENT DEFERRAL
TEMPORARY DEFERRAL
 EXAMPLE: Donor states that he or she has hepatitis C; defer Deferral if the donor received the following vaccine:
permanently 2-week deferral Smallpox, Polio, Measles, Mumps, Influenza
The donor will never be eligible to donate blood for someone else; 4-week deferral German Measles, Chicken-pox
donor is only eligible to donate autologous blood
12-month deferral Rabies vaccine if given after the bite of a
o Definite disease or habits strongly associated with bloodborne
rabid animal
pathogens
May donate if afebrile Diphtheria, Pertussis, Typhoid, Tetanus,
o Narcotic addiction
Cholera, Influenza
o Alcohol addiction
1 Year  Skin penetration w/ instruments
o Received pituitary growth hormones of human origin
(Possible exposure to contaminated w/blood (surgery)
o History of chaga’s disease, malaria, or babesiosis; cancer,
Hepatitis, HIV or Malaria)  Closed contact with persons with viral
leukemia, lymphoma
hepatitis
o Persons w severe thrombocytopenia
 (+) STS- 12 months from completion of
therapy
PERMANENT DEFERRAL  Traveled to endemic area for malaria w/
Definite disease or Viral Disease or w/o antimalarial drugs
habits strongly  Hepatitis after the age of 11
 ≥72 hours in correctional institution
associated with  (+) confirmatory test for HbsAg
bloodborne pathogens  Repeatedly (+) test for anti-Hbc  Female donor who had sex with bisexual
 Gave only unit to recipient who men
developed post transfusion hepatitis and  Anyone who had given someone money
HIV or drugs in payment for sexual contact
 Present and Past infection with Hepa C 3 Years  Visit/ immigrant from area endemic for
and HIV (Possible exposure to malaria
 Narcotic addiction malaria)  Had malaria, but presently asymptomatic
 Alcohol addiction
Other diseases: **plasma preparation w/o red cells are
 Received pituitary growth hormone of exempted from these restriction
human origin No deferral if afebrile  Diphtheria, pertussis, typhoid, tetanus,
 History of Chaga’s disease, babesiosis or cholera, influenza, previous history of tb
malaria that has been successfully treated and is
 History of cancer, leukemia, or lymphoma no longer active
 Person’s with severe thrombocytopenia

#MLSWATER2022 Page 4 of 16
 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
Intake of Medication injections given over
a 6-month period
Medication Indication Deferral Period Unlicensed Vaccine Research Proposal 12 months unless
Proscar (Finasteride) Prostate Gland 2 months otherwise
Avodart (Dutasteride) enlargement 6 months indicated by
Propecia Baldness 2 months medical director
Accutane (Amnesteem, Severe Acne 2 months
Claravis, Sotret, How it affects donation?
Isotretinoin)  HBIG does not prevent Hepatitis B in every case
Soriatane (Acitretin) Severe Psoriasis 3 years
Tegison (Etretinate) Permanent Contraceptive Pills
 May donate anytime
How it affects donation?
 These medications can cause birth defects. Donated blood with high Highly Allergenic Drugs like Penicillin, Aspirin, and others
enough levels to damage the unborn baby if transfused to a  May donate anytime provided the blood collected will not be used
pregnant woman. to prepare platelets
 Once medications are cleared from the body, one can donate again  Donor may donate only after 24 hours of taking the medications.

Medication Indication Deferral Period Piroxicam

Growth Hormone from Delayed impaired Permanent  Deferred until TB is completely cured
Human Pituitary Glands growth (used only until
1985) TYPES OF DONATION
Insulin from cows Indefinite
ALLOGENIC DONATION
(Bovine or beef insulin)
Genetically different individual but same specie
Blood is taken from an individual of the same specie as the recipient
How it affects donation?
 Can lead to development of rare nervous system disorder called CJD
(Creutzfeldt-Jakob Disease) REQUIREMENTS FOR ALLOGENIC DONATION
 If Insulin was imported from countries infected in which Mad Cow
Disease had been found, it could contain material from infected Philippine Standard AABB Standards
cattle Age 16-65 years old At least 17 years old
 MCD can be transmitted through blood transfusion Temperature ≤37.5 °C or ≤99.5 °F ≤37.5 °C or ≤99.5 °F
Pulse Rate 60-100 bpm 50-100 bpm
Medication Indication Deferral Period Hemoglobin ≥ 12.5 g/dL ≥ 12.5 g/dL
HBIg  Following exposure 12 months after
Blood Pressure
to hepatitis B receipt of vaccine
 Systolic 90- 160 mmHg ≤ 180mm Hg
 Different for
 Diastolic 60-100 mmHg ≤ 100 mm Hg
Hepatitis B vaccine,
which is a series of 3

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
AUTOLOGOUS DONATION 3. INTRAOPERATIVE COLLECTION
Autologous means “self”  Involved collecting shed blood from the surgical site, a device that
Blood is given to the recipient came from the recipient himself utilizes vacuum is used to collect shed blood.
REQUIREMENTS FOR AUTOLOGOUS DONATION  Blood is the washed with saline and concentrated to reach
hematocrit of 50-60%, then reinfusing those cells immediately.
Collection and Reinfusion of lost blood during a surgery
Age No age requirement
Hemoglobin 11 g/dL
Hematocrit 33% 4. POSTOPERATIVE COLLECTION
 General Condition: Patient should have no condition predisposing to  Collected from a drainage tube placed at the surgical site.
bacteremia or any form of severe cardiovascular/ pulmonary  It is reinfused with or without processing, via a microaggregate filter
condition. to screen out any debris
 Single unit is removed at a time, with at least 3 days intervals
 Final phlebotomy must be at least 72hrs before surgery DIRECT DONATION
 A direct donation is a unit collected under the same requirements as
those for allogenic donors, except that the unit collected is directed
TYPES OF AUTOLOGOUS DONATION towards a specific patient.
The patient selects his/her own donor for an anticipated non-
1. PREOPERATIVE COLLECTION emergency transfusion
 Removal of blood 5-6 weeks immediately preceding a scheduled, Donor is typically a friend or relative of the patient
elective surgical procedure
The storage of blood and its components before an elective surgery; APHERESIS DONATION
blood is used during or after surgery  Aphaeresis= “taking away”
o Possible Problems: presurgical anemia or hypovolemia can  An effective mechanism for collecting a specific blood component
occur, clinical identification errors, outdating of liquid-stored while returning the remaining whole blood components back to the
blood, homologous blood transfusion instead of autologous patient
 Amount of time for a particular procedure can range from 45-120
2. ACUTE NORMOVOLEMIC HEMODILUTION minutes
 Collection of whole blood with the concurrent infusion of crystalloid  ACD: Most common and Primary Anticoagulant in Apheresis
or colloid solutions procedure
 The idea is that the patient bleeds more dilute blood during the It is the withdrawal of blood from a donor removing selected
procedure, and the patient’s heart may pump more effectively due components and reinfusion of the remaining components back to
to decreased blood viscosity the donor
Isovolemic Hemodilution
o Multiple units of blood are collected; the blood is reinfused Methods of Centrifugation
near-end the surgery
1. INTERMITTENT FLOW CENTRIFUGATION
 The last unit collected must be the first unit to go back
 Blood is drawn and reinfused through the same needle
 Once the desired component is separated, the remaining
components are reinfused to the donor

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The blood is processed in batches or cycle and requires only 1
venipuncture.
Spins, process in the machine and gives back the unused part to the
donor. The blood is return to the same site where it was extracted.
To stop the blood from coagulating, anticoagulant is automatically
mixed with the blood as it pumps the blood into the apheresis
machine.
2. CONTINUOUS FLOW CENTRIFUGATION (CFC)
 Involve withdrawal and processing and reinfusing of blood to the
individual simultaneously
MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
 Donor’s RBCs should be ABO- and Rh-compatible, relatively fresh
(leukoreduced, negative for hemoglobin S and partially phenotype-
matched for the Rh (C, c, E, e) and K1 antigens
WBC PHERESIS (LEUKAPHERESIS)
The granulocyte concentrate from leukapheresis are needed for
patients who have conditions such as severe neutropenia or
conditions that are unresponsive to antibiotic.
Storage time is 24 hours at 20-24°Celsius.
Compatibility testing is typically performed for WBC pheresis.
PLASMAPHERESIS (PLASMA EXCHANGE)
 Equivalent of at least two whole-blood derived plasma units
o Infrequent plasmapheresis
o Frequent or Serial
 FDA recommends 12 L (14.4 L for donors weighing more than 175
pounds) as the maximum allowable plasma volume donated per
year
Maximum of 2 donation in a 7 day period with 2 days gap period is
allowed.

THERAPEUTIC PROCEDURES
 The rationale of Therapeutic Apheresis (TA) is based on the
following:
o A pathologic substance exists in the blood that contributes
Types of Apheresis to a disease process or its symptoms
PLATELETPHERESIS (THROMBOCYTOAPHERESIS) o The substance can be more effectively removed by
 Donor’s platelet count: at least 150,000/ µL apheresis than by the body’s own homeostatic mechanisms
 Donor should have not taken aspirin 3 days before donation
 Interval of at least 2 days 1. Therapeutic Plasma Exchange (TPE)
 pH ≥ 6.2  Removal and retention of the plasma, with return of all cellular
components to the patient.
 To remove the agent in the plasma, such as an antibody, toxin, or
RBC PHERESIS (ERYTHROCYTAPHERESIS) abnormal protein, that is causing the clinical symptoms.

REQUIREMENTS FOR ERYTHROCYTAPHERESIS Factors Removed by Therapeutic Plasmapheresis

Height Weight Hct Immune Complexes SLE
Male 5’11” 130 lbs 40% Alloantibodies Antibody
Female 5’5” 150 lbs 40% Autoantibody Gullain-Barré syndrome
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Immunoglobulin causing Waldenstrōm’s macroglobulinemia
hypersensitivity
Protein-bound toxins or drugs Amanita mushroom poisoning,
barbiturate poisoning
LPPs Familial hypercholesterolemia,
hypertriglyceridemia
Phytanic Acid Refsum’s disease
MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
NAUSEA OR VOMITING
 Management:
1. Instruct the donor to breathe slowly
2. Apply cold compresses to the forehead
3. Turn the donor’s head to one side and provide an appropriate
receptacle
4. The donor may be given water after vomiting has ceased

LOSS OF CONSCIOUSNESS
2. Therapeutic Platelet Pheresis  Management:
 Thrombocytosis (at least 500,000/ µL) can occur in myeloproliferative 1. Check vital signs frequently
disorders (ET, PV, CML) or as a reactive process in response to 2. Administer 95% O2 and 5% CO2
splenectomy, infection, chronic inflammation, or malignancy
SEVERE REACTIONS
3. Therapeutic Leukapheresis
CONVULSIONS
 Elevated level of WBCs placed the patient at risk for complication
 Management:
associated with leukostasis, including organ dysfunction due to the
1. Call for help immediately; notify blood bank physician
formation of microthrombi in the pulmonary and cerebral
2. Try and restrain the donor to prevent injury to self or others
microvasculature
3. Ensure an adequate airway

STEM CELL PHERESIS

CARDIAC OR RESPIRATORY
 Management:
DONOR REACTIONS
1. Perform CPR until medical help arrives
MILD REACTION
HEMATOMA
SYNCOPE  Management:
 Management:
1. Remove the tourniquet and withdraw needle
2. Place cold compression on the donor’s forehead POST DONOR CARE
3. Raise the donor’s legs above the level of the head  Raise arm and apply pressure on the puncture site after collection.
4. Loosen tight clothing and secure airway  Rest after blood collection, reclining for few minutes, then sit upright
5. Monitor vital signs and allow to talk.
 Instruct the donor to drink a lot of water, refrain from smoking and
TWITCHING OR MUSCLE SPASMS avoid strenuous work or driving.
 Management:
1. Disengage the hyperventilation sequence by conversing with
the donor and having the donor breathe into a paper bag.

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NOTES FROM MODULE PACKET:
Blood Donor Selection
The primary responsibility of a blood transfusion service is to
provide a safe, sufficient and timely supply of blood and blood
products
The purpose of blood donor selection is to:
o Protect donor health and safety by collecting blood only
from healthy individuals
o Ensure patient safety by collecting blood only from donors
whose donations, when transfused, will be safe for the
recipients
o Identify any factors that might make an individual
unsuitable as a donor, either temporarily or permanently
o Reduce the unnecessary deferral of safe and healthy donors
o Ensure the quality of blood products derived from whole
blood and apheresis donations
o Minimize the wastage of resources resulting from the
collection of unsuitable donations.
Blood donors have a responsibility to self-defer if they are aware of
having been exposed to any risk of an infection or a known health
condition or treatment that could influence their suitability to
donate blood.
Blood donors also have the right to withdraw at any stage of the
donation process.
A donor questionnaire is the key tool in donor selection for
assessing donor health and safety and for reducing the risk of
transmission of infection, in particular for infections for which no
Donor registration
suitable screening tests are available.
o The use of a donor questionnaire prompts donor selection
staff to ask important questions and carefully assess the
donor’s health.
o By presenting all relevant information in a standard format,
a donor questionnaire facilitates decisions on the
acceptance or deferral of the donor.
Effective public information and donor education are the first steps
in the process of donor selection.
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MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
Key principles of blood donor selection:
The health and safety of the donor as well as the recipient must
be safeguarded
Only individuals in good health should be accepted as donors of
whole blood and blood components
The selection of blood donors should be based on regularly
reviewed selection criteria, without discrimination of any kind
including gender, race, nationality or religion
A prospective donor’s health status and medical history should be
evaluated for each donation, on the day of donation prior to
blood collection
The blood donor selection (BTS) should provide appropriate
donor information and a simple donor questionnaire for health
and risk assessment and obtain the donor’s informed consent to
blood donation
Staff should be suitably qualified and trained in the donor
selection process
Good communication should be established between the BTS
staff and the donor, and donor confidentiality should be assured
The BTS has a duty of care to provide counselling to all deferred
donors and referral for their further management.
Steps Involved in The Donor Selection Process
Compliance with all donor selection criteria is crucial to ensure a
safe blood donation process and outcomes.
All prospective donors who meet the general criteria for blood
donation such as age and good health should be registered when
they attend a blood donation session, even if they are subsequently
not accepted for donation.
Essential donor registration information includes the individual’s full
name, date of birth, gender and contact details.
During donor registration, prospective donors should be provided
with donor information and education materials and the donor
questionnaire, which should be completed on each occasion of
donation.
Page 9 of 16

Pre-donation information
The process of donor selection begins even before donors come to
give blood through public awareness campaigns and donor
education.
At the donation session, pre-donation information should be
provided either orally or through printed, graphic, audio-visual or
online materials, presented in a simple and clear format and in
appropriate languages.
Pre-donation information provides an opportunity for the
prospective donors to know about health conditions or high-risk
behaviour that would make them unsuitable to donate blood.
This information assists the donors in deciding whether to self-
defer;
• It may also assist in donor return if they understand the
reason why they should not donate blood on this occasion
Objectives of Pre-donation Information:
Increase donor awareness of the donor selection criteria, the
process of blood donation and the tests that will be performed on
donors’ blood
Encourage prospective donors to inform the BTS of any medical
conditions or TTI-related risks that may affect their suitability to
donate blood
Encourage individuals to self-defer from blood donation if they
recognize that they are not suitable to donate blood due to
general health or medical conditions or risk for TTI.
Pre-donation Information Coverage:
Nature and use of blood and its components; the need for
voluntary nonremunerated blood donors; and the importance of
maintaining healthy lifestyles
The blood donation process, including the donor questionnaire,
donor medical history, health and risk assessment, venipuncture,
blood collection as whole blood or apheresis procedure, post-
donation care and the screening tests performed on donated
blood
Rationale for the donor questionnaire and pre-donation health
assessment and the importance of donor compliance in the
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MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
donor selection process; and donor’s duties, responsibilities and
rights
Options for the donor to decide about blood donation prior to
proceeding further, to withdraw or self-defer at any time during
or after the donation process, without any undue embarrassment
or questioning
Transfusion-transmissible infections, including HIV, HBV, HCV and
syphilis, routes of their transmission, natural history and
prevention; types of screening tests performed; and window
period of infection and alternative testing sites for individuals
seeking to ascertain their infection status
Possible consequences for donors and the donated blood in the
case of abnormal TTI test results; the mechanism for notification
about abnormal test results and post-donation counselling,
assurance of confidentiality and if necessary, referral for further
testing, treatment and care
The possibility of adverse donor reactions.
Completion of donor questionnaire
Each prospective blood donor should complete a donor
questionnaire to provide information in relation to the donor
selection criteria defined in the national guidelines.
In most situations, the donor questionnaire is given to donors at the
time of registration for completion before the donor interview and
assessment.
Alternatively, the donor questionnaire may be sent to the donor’s
residence to be completed before donation.
• This has the advantage of allowing donors time to think about
the answers and saves time at a blood donation session.
• However, donors may misunderstand some of the questions
and self-defer for the wrong reasons.
The donor questionnaire may also be administered electronically as
a computer-based questionnaire.
It is essential that donors are aware of the importance of the
questionnaire, the significance of the questions and the need for
providing accurate information
The information provided by the donor can then be further
elaborated on during the interview.
Page 10 of 16

Donor interview and pre-donation counselling
The completed donor questionnaire should be reviewed prior to
donation in a one-to-one confidential interview between the donor
and a donor selection staff member so that an assessment can be
made of the donor’s general health, medical history and any TTI
risks.
It also provides an opportunity to check whether the donor has
understood the questions and has answered them correctly.
Assurance about the confidentiality of the donor’s medical history is
essential.
Pre-donation counselling is an integral part of the donor interview.
It enables donor selection staff to:
• Check that the donor has understood all questions and
responded accurately to the questionnaire
• Answer the donor’s questions and provide reassurance in case
of anxiety
• Explain reasons for any deferral and give advice about further
medical care, if needed
• Ensure that the donor is able to give informed consent to
donate and recognizes that his/her signature is an affirmation
that responses provided to the questionnaire are accurate.
Donor health and risk assessment
The assessment of donor suitability and deferral, where
appropriate, aims to exclude donations from individuals at risk of
TTI, particularly from those with recently acquired infection that
cannot or may not be detected by routine screening tests or with
infections for which no effective blood screening tests are available.
The donor assessment not only enables the review of the donor’s
medical history and medications, but also provides an opportunity
for a basic health check to assess whether the donor is in general
good health.
Any signs of debility, under-nutrition, pallor, jaundice, cyanosis,
dyspnea or intoxication from alcohol or drugs should also be noted
Physical examination, weighing and/or measurement of vital signs
(pulse, blood pressure) are part of the basic health check and are
carried out at this stage.
The venipuncture site should be examined to check that the donor’s
veins are accessible and suitable to enable easy venipuncture.
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MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
The basic health check also enables an assessment to be made of
any physical disabilities that may impede the donation process, such
as:
• Mobility: the donor should be able to easily access the donor
bed or couch
• Sight or hearing impairment: assistance should be provided
by a staff member.
Issues that require special attention during donor health and risk
assessment include:
• The prevalent culture and context of the environment for
donation; in some situations, a donor may simply be
overawed by the medical setting and procedures
• The provision of sufficient privacy and assurance of
confidentiality to make the donor comfortable when
answering probing and sensitive questions
• Identifying and overcoming language barriers or lack of
understanding of questions in the donor questionnaire
• Ensuring good communication by using simple jargon-free
language and explaining any medical terms.
Informed consent
Informed consent is a voluntary agreement given by the prospective
donor to the donation of blood, to the testing of a blood sample for
TTI, for the transfusion of the donated blood to patients and if
required, for the use of the blood for additional tests, quality
assurance or research purposes.
To obtain informed consent, the BTS should provide the following
minimum information to the potential donor:
• The donation process and potential adverse donor reactions
• The tests that will be performed (TTI and others) on the
samples taken from the donated blood and the reasons for
these tests
• Confidentiality of all personal information, including test
results.
The donor should sign and provide informed consent to the
donation of blood or blood components on a voluntary basis.
Informed consent signifies that the donor has understood the
questionnaire, has provided accurate answers and is willing to
donate blood.
Page 11 of 16

It also indicates that the donor understands the blood donation
process, the possibility of adverse reactions to blood donation, the
risks of the transmission of infections through donated blood and
the implications of any abnormalities that may be detected during
the donation process and blood screening, and is providing consent
for post-donation notification and counselling, if detected to have a
positive viral infection marker or any other abnormality.
Donor Deferral:
Donors who do not to meet the selection criteria should be
deferred on a temporary or permanent basis.
All deferred donors should be treated with respect and care in a
confidential manner and should be given a clear explanation of
the reason for deferral and an opportunity to ask questions.
They should be informed whether the deferral is to safeguard
their own health and/or that of the recipient.
It is the responsibility of the BTS to ensure that donors who are
deferred due to medical conditions are referred for further
investigations and management, as appropriate.
Temporarily deferred donors should be advised on when they
could donate and encouraged to return
A donor deferral registry (DDR) is a confidential list of donors
who are positive for a transfusion-transmissible infection and
who have been permanently deferred.
A DDR is used to monitor the incidence and prevalence of such
infections in the donor population and may also assist in
identifying areas that require strengthening in the donor
selection process.
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MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
Criteria for Blood Donor Selection
Age
The lower age limit for blood donation in most countries is 18 years,
although in some countries national legislation permits 16–17 year-
olds to donate provided that they fulfil the physical and
haematological criteria required and that appropriate consent is
obtained.
• Adolescents of either gender are at risk of iron deficiency
during the pubertal growth spurt when the average daily total
requirement of absorbed elemental iron is 1.50 mg/day for
males aged 15–17 years and 1.62 mg/day for females
Upper age limits for blood donation of between 60 and 70 years
have been implemented in the past because of concerns regarding
the increasing incidence of cardiovascular disease with age and the
potential risk of adverse reactions, which are more likely in first-
time donors.
• The usual upper age limit for blood donation is 65 years
• First-time donors older than 60 years and regular donors over
the age of 65 may be accepted at the discretion of the
responsible physician
• First-time donors over 60 years should make their first
donation at a donation site where a physician is available
DONOR APPEARANCE AND INSPECTION
Prospective donors should be accepted only if they appear to be in
good health and comply with donor selection criteria
The prospective donor should appear generally well and should not
be febrile, breathless or suffering from a persistent cough.
The colour of exposed skin and mucous membranes should be
normal, with no jaundice, cyanosis, flushing or pallor, and no signs
of skin infection, rash or obviously enlarged lymph nodes.
• If body piercings or tattoos are present, the risk of
transfusion-transmissible infections (TTI) should be assessed
MINOR ILLNESSES
Individuals with a history of recent infection: defer for 14 days
following full recovery and cessation of any therapy, including
antibiotics
Page 12 of 16

• Minor non-specific symptoms (e.g. general malaise, pain,
fever, headache, cough, diarrhoea) may indicate the presence
of an acute infection that may be transmissible by transfusion.
• Donors should be asked to confirm that they are free from
such symptoms on the day of donation and that they have
fully recovered from any recent infection(s).
• Individuals suffering from minor illnesses and not feeling well
should not donate blood.
WEIGHT
Prospective donors of whole blood donations should weigh at least
45 kg to donate 350 ml ± 10% and 50 kg to donate 450 ml ± 10%
Prospective donors of apheresis platelet or plasma donations should
weigh at least 50 kg
Prospective donors of double red cell apheresis donations should
have an estimated blood volume of more than 5 litres; this
requirement is generally met by non-obese individuals weighing
more than 70 kg.
VITAL SIGNS
PULSE
• A normal pulse rate of 50–100 per minute and a regular
rhythm are indicators of good health; many BTS recommend
that these are examined prior to donation.
• The ability to detect significant abnormalities of pulse rate or
rhythm depends on the skill and experience of staff.
BODY TEMPERATURE
• A prospective donor who is febrile – defined as a core oral
temperature more than 37.5°C– is by definition unwell and
should be deferred.
• Fever can indicate any number of medical conditions and
infections, but is usually associated with other symptoms
BLOOD PRESSURE (BP)
• A normal blood pressure (systolic 180 mmHg, diastolic 100
mmHg) is generally regarded as an indicator of good health.
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MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
DONOR IRON STATUS
Haemoglobin screening safeguards anaemic individuals from
donating blood and also protects returning donors from donation-
induced iron deficiency (DIID), the depletion of iron stores by
repeated donations.
Collecting a unit of blood from a donor with a normal haemoglobin
level also provides good quality blood components, with adequate
and consistent haemoglobin content in the collected blood.
In determining the lower limits of haemoglobin for whole blood
donation and implementing haemoglobin screening, the BTS should
consider:
• A haemoglobin level of not less than 12.0 g/dl for females and
not less than 13.0 g/dl for males as the threshold
Only sterile disposable lancets should be used for blood sampling
FLUID INTAKE AND FOOD
Most BTS guidelines recommend that donors should maintain their
usual food and fluid intake before donation but should avoid heavy
or fatty meals which may result in a lipaemic donation that may
need to be discarded
The BTS should consider providing 500 ml drinking water to donors
before donation to minimize the risk of vasovagal reactions
PREGNANCY, LACTATION AND MENSTRUATION
The average woman needs about 350–500 mg additional iron to
maintain iron balance during pregnancy.
Female donors should be deferred during pregnancy and for a
sufficient time after delivery (or following abortion or miscarriage)
and during lactation to allow for the recovery of iron stores.
Menstruation is not a reason for deferral. However, women who
report regular excessive menstrual bleeding and are found to have
low haemoglobin levels should not donate blood and should be
referred for medical assessment.
Contracting and relaxing the muscles in the legs, arms and abdomen
during donation may reduce the risk of vasovagal reactions,
particularly among female donors
The BTS should encourage donors to practise applied muscle
tension during blood donation
Page 13 of 16

Accept female donors during menstruation, provided that they feel
well and meet the minimum haemoglobin level for blood donation
Defer female donors during pregnancy and up to 6 months after
delivery or termination of pregnancy
Defer female donors during lactation
Reducing the risk of transfusion-associated acute lung injury (TRALI)
The gender of the donor may influence the type of blood
component prepared from the donation.
Plasma-rich blood components from multiparous women are
more likely to cause TRALI and related disorders than those from
males, because plasma from such women is likely to contain
alloimmune-reactive antibodies; these include antibodies to
human leucocyte antigens (HLA) or to human neutrophil antigens
(HNA), which are transferred passively during transfusion, to the
possible detriment of a recipient who possesses the
corresponding antigen.
Frequency of Donation
The minimum interval between donations of whole blood should be
12 weeks for males and 16 weeks for females
The minimum interval between donations of platelets should be 4
weeks
The minimum interval between donations of plasma should be 2
weeks
The minimum interval before an apheresis platelet or plasma
donation should be 4 weeks following a whole blood donation, an
apheresis red cell donation or a failed return of red cells during
apheresis
In determining the frequency of donation and whether iron
supplementation is given, the BTS should consider:
• The need for longer donation intervals for young donors and
female donors of childbearing age
• Assessing the feasibility and affordability of providing iron
supplementation to donors susceptible to donation-induced
iron deficiency, especially women, adolescents, and repeat
and regular donors
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MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
NOTE:
One unit of whole blood can be broken down into one unit of
packed red cells, one unit of platelets, and one unit of fresh
frozen plasma/cryoprecipitate.
Methods for collection of blood for preparation of blood components:
Single whole blood donation
After collection of a unit of whole blood in the primary bag, blood
components can be separated from one another by differential
centrifugation due to differences in their specific gravities.
After their separation, various components can be transferred from
one bag to another in a closed circuit thus avoiding exposure to the
external environment and maintaining the sterility.
Blood should be processed for component separation within 6 hours of
collection
Apheresis
Apheresis donation uses a specialized programmable machine that
processes over 3.5 L of the donors' blood and separates whole blood
into the blood component that is required (either up to 750 mL plasma
or 100–400 mL platelets), returning the remainder to the donor
(sometimes with saline fluid replacement).
Depending on the component that is separated and removed, the
procedure is called plateletpheresis, leukapheresis, or plasmapheresis.
Blood is withdrawn and returned via the 16-gauge apheresis needle
(single arm/needle procedure).
Whole blood is mixed with anticoagulant and the required blood
component (plasma or platelets) is collected into the attached plastic
blood bag.
Red cell products can also be collected via apheresis, although this is
less common than plasma and platelet apheresis.
Apheresis donation takes an average of 1 hour.
Upon completion of the apheresis collection, the blood component
requires no additional processing and has been depleted of the
majority of white blood cells during the collection via filtration and/or
centrifugal conditions built in to the apheresis procedure.
Page 14 of 16

Double red blood cell donation
A person donates twice as many red blood cells as with a single
donation of whole blood.
This double donation is possible because the person gives only red
blood cells rather than whole blood.
Whole blood is drawn from the donor, and a machine that separates the
blood into its components selectively removes the red blood cells and
returns the rest of the blood components (platelets and plasma) to the
donor.
Some fluid is also given to the donor intravenously because otherwise,
the donor's blood pressure could become low enough to cause
symptoms, such as light-headedness or loss of consciousness.
After double red blood cell donation, people may be less able to
exercise vigorously for a few days.
Double red blood cell donation can be done as often as once every 112
days (every 16 weeks).
Some experts recommend that people take iron supplements after
double red cell donation so that their body can replace the donated red
blood cells more rapidly.
Autologous transfusion
Donors are recipients of their own blood.
The person takes iron pills after donating the blood to help the body
replenish the lost blood cells before surgery.
Also, during some types of surgery and in certain kinds of injuries, blood
that is lost can be collected, washed, and immediately given back to the
person (intraoperative blood salvage).
An autologous transfusion eliminates the risks of incompatibility and
blood-borne disease (unless the wrong blood is given by mistake).
However, doctors do not use this technique as often as standard
transfusion because the general blood supply is very safe due to
rigorous donor screening and testing.
In addition, older people may not tolerate donating blood befor surgery
because they are more likely to have side effects during donation such
as low blood pressure and fainting.
Older people are also more likely to have fewer blood cells than normal
(a low blood count) to begin with.
Also, autologous transfusion is more expensive than standard
transfusion.
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MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
Directed or designated donation
Family members or friends can donate blood specifically for one
another if the recipient's and donor's blood types and Rh factors are
compatible.
For some recipients, knowing who donated the blood is comforting,
although a donation from a family member or friend is not necessarily
safer than one from an unrelated person.
Blood from a family member is tested, as are all blood samples, and
then treated with radiation to prevent graft-versus-host disease, which,
although rare, occurs more often when the recipient and donor are
related.
Whole Blood
One unit of donor blood collected in a suitable anticoagulant-
preservative solution (citrate phosphate dextrose adenine or CPDA-1).
Whole blood donation involves collection of 450 mL (±10%) blood from
the antecubital vein via a 16- gauge needle into the attached specialized
PVC plastic blood collection bag typically containing 63 mL
anticoagulant.
Whole blood donation usually takes 5–10 min.
The donated whole blood is then processed into components including
red cell concentrate, platelet concentrate and plasma, via centrifugation
Processing of whole blood must commence within 24 hours of blood
donation in order to maintain maximum quality of the components.
Whole blood is stored in an approved blood bank refrigerator at 1°-6°C.
If collected in ACD or CPD, shelf-life is 21 days and CPDA-1 is 35 days
It does not contain functionally effective platelets and labile coagulation
factors (Factor V and Factor VIII).
Transfusion of whole blood should commence within 30 minutes of
removal from the refrigerator and should be complete within 4 hours of
starting.
Transfusion of one unit raises hemoglobin by 1 gm/dl or hematocrit by
3%.
Indications
• Acute blood loss with hypovolemia
• Exchange transfusion in neonates
• Non-availability of red cell concentrate or suspension
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
APPENDICES Sample Problem # 2:
 Mathew, weighing 95 lbs, wants to donate blood for a friend. How
Sample Problem # 1: much must be removed from the blood bag?
 Given: 40kg individual that to donate a blood
a. Volume to Collect:  50 kg = 110 lbs
40 𝑘𝑔 a. Volume to Collect:
= 𝑥 450 𝑚𝐿
50 𝑘𝑔 95 𝑙𝑏𝑠
= 𝑥 450 𝑚𝐿
110 𝑙𝑏𝑠
 360 mL volume of blood to be collected
 388.64 mL volume of blood to be collected
b. Reduced Volume of Anticoagulant:
360 𝑚𝐿 d. Reduced Volume of Anticoagulant:
= 𝑥 63 𝑚𝐿
450 𝑘𝑔 388.64 𝑚𝐿
= 𝑥 63 𝑚𝐿
 50.4 mL 450 𝑚𝐿
 54.4 mL
c. Volume of Solution to be removed: e. Volume of Solution to be removed:
= 63 𝑚𝐿 − 𝑟𝑒𝑑𝑢𝑐𝑒𝑑 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐴𝑛𝑡𝑖𝑐𝑜𝑎𝑔𝑢𝑙𝑎𝑛𝑡 = 63 𝑚𝐿 − 54.4 𝑚𝐿

 Answer: 12.6 mL  Answer: 8.6 mL

-END OF TRANSCRIPTION-

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
MLS 18, April 13, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
RATIO 8:1, 8 parts of the circulating blood & 1 part of the
Legend: anticoagulant
Transcription Bullet TRANSPORT 1°-10° C
Notes/Module Packet Bullet TEMPERATURE
 PPT SHELF-LIFE ACD, CPD, CPD2D: 21 DAYS,
CPD-A1: 35 DAYS,
MODULE OUTLINE CPDA-2/S: 42 days, (S indicated for additive sol’n.)
I. Organizations that Regulate or Accredit the Immunohematology Heparin 2 days/48 hours
Laboratory ADENINE- is related with the energy production, it
II. Blood Donor Selection increases the phosphorylation and ATP production in
III. Types of Donation order to provide the needed energy of our cells
IV. Blood Component
V. Safe Storage of Blood
Cellular Components
VI. Packing Blood Components for Transportation

LEARNING OUTCOMES Legend

At the end of this module, the student shall be able to:  Red cells
1. Identify the organizations that regulate or accredit the o Packed red cells
immunohematology laboratory. o Red cells in additive solution (Red cell suspension)
2. State the minimum acceptable levels for the following tests in o Leukocyte-poor red cells
allogenic & autologous donation o Washed red cells
3. Identify the medical history information that would cause o Frozen red cells
permanent deferral or temporary deferral and state the length of o Irradiated red cells
deferral period.  Platelets
4. Explain the reasons for permanent or temporary deferral given the o Platelet concentrate (Random donor platelets prepared
various medical conditions. from whole blood unit)
o Apheresis platelets (Single donor platelets) (SDP)
 Granulocytes
BLOOD COMPONENTS o granulocyte concentrate
Whole Blood
Cellular Components: Red Cells
INDICATION To increased volume and RBC mass, meaning that both PACKED RED CELLS
RBC mass/count and plasma volume is reduced or
decreased INDICATIONS:
STORAGE 1°-6° C is applicable to the whole blood and RBC • Anemia: Chronic severe anemia, severe anemia with
components congestive cardiac failure, anemia in elderly
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
Do the heavy spinning at 5000 xg for 5 minutes in order to extract
INDICATION To increase RBC mass
platelet concentrate as well as the plasma
STORAGE 1°-6°C
TRANSPORT 1°-10°C LIGHT SPINNING
SHELF-LIFE Open system: 24 hours, without the use of centrifuge If you want a platelet concentrate and a packed RBC at the same
- Manually transfer the pocked RBC to the other time, then do the LIGHT SPINNING. 3200xg for 2-3 minutes and
side of the satellite bag thru the use of plasma the temperature for platelet is set at 20°-24° Celsius.
separator After light spin it will have a platelet rich plasma, from the
- Only viable for 24 hours platelet rich plasma, platelet concentrate can be separated from
Closed system: same as whole blood, commonly used the plasma
- Follows the shelf-life of the anticoagulant If you want to extract cryoprecipitate from the plasma, plasma
preservative that is used unit can be subjected to further centrifugation
DOSAGE Increase hemoglobin by 1g/dL
Increase Hematocrit by 3%
QC ≤ 80% of the Hct RED CELLS IN ADDITIVE SOLUTION (Red cell suspension)
• Acute blood loss (transfused along with a crystalloid or a INDICATIONS:
colloid solution) • Anemia: Chronic severe anemia, severe anemia with
congestive cardiac failure, anemia in elderly
Packed red cells are prepared by removing most of the plasma from • Acute blood loss (transfused along with a crystalloid or a
one unit of whole blood colloid solution)
Whole blood is either allowed to sediment overnight in a
refrigerator at 1-6°C or is spun in a refrigerated centrifuge. These are red cells with minimal residual plasma and an additive
Supernatant plasma is then separated from red cells in a closed solution (SAG-M which contains saline, adenine, glucose, and
system by transferring it to the attached empty satellite bag. mannitol).
Red cells and a small amount of plasma are left behind in the This increases shelf life from 35 days to 42 days.
primary blood bag. After collection of whole blood in the primary collection bag
Packed red cells have a high viscosity and therefore the rate of (containing CPDA-1), maximum amount of plasma is removed (after
infusion is slow. centrifugation) and transferred to one satellite bag.
Transfusion of one unit of red cells increases hemoglobin by 1 gm% The additive solution from the second satellite bag is transferred
(or increases hematocrit by 3%). into the primary collection bag (containing packed red cells) in a
closed system.

LEUKOCYTE-POOR RED CELLS

2 OPTIONS IN PREPARING PACKED RBCs
INDICATION To increased RBC mass as
HEAVY SPINNING
STORAGE 1°-6°C
If you want to prepare a packed RBC without platelet
TRANSPORT 1°-10°C
concentration, then do the HEAVY SPINNING with the whole
SHELF-LIFE Open system: 24 hours
blood sample.
Closed system: same as whole blood
Heavy spinning at 5000 xg for 5 minutes and set the temperature
DOSAGE Increase hemoglobin by 1g/dL
of the centrifuge at 4° Celsius.
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
Increase Hematocrit by 3% Washed red cells are blood cells remaining after washing with saline
QC ≤ 85% RBC recovery, WBC should fall ≤5x10^6 solution to remove plasma
- Most commonly used blood component to o Open System
reduced HLA alloimmunization and CMV • needs to be transfused within 24hrs
transmission • can also be used w automated cell washers (Close
- the RBCs must be in patient suffering from System)
severe and recurrent febrile non-hemolytic
transfusion reaction. This is due to the FROZEN RED CELLS
presence of leukocyte antibody INDICATION Storage of rare blood and
autologous units
INDICATIONS: STORAGE -65 or -120°C
• Prevention of HLA immunization in patients who are likely to SHELF-LIFE 10 years
receive allogeneic bone marrow transplantation
• Prevention of febrile nonhemolytic transfusion reactions in If a cryoprotective agent such as glycerol is added, red cells can be
persons receiving multiple transfusions stored frozen for up to 10 years.
• Prevention of transmission of cytomegalovirus. This method can be used for storage for donor red cells with rare
blood groups, for future autologous transfusion, and for individuals
Leukocyte-poor red cells contain < 5 × 106 white cells per bag. who have repeated febrile nonhemolytic transfusion reactions.
Methods for leukocyte depletion are: used in the long-term storage of rare blood units for
• Leukocyte-reduction filters autologous units and units for special purpose in cases of the
• Removal of buffy coat intrauterine transfusion
In Frozen RBC, one cannot directly transfuse frozen RBC in the
WASHED RED CELLS patient.
INDICATION To increased RBC mass in patient Cryoprotective agent classifications
with allergic, anaphylactic, febrile o Penetrating Agent
and urticarial reactions  small molecules
STORAGE 1-6°C  can enter the cell and prevent cell dehydration as the ice
SHELF-LIFE Open system: 24 hours forms; ex. Glycerol & DMSO
DOSAGE Increase Hb by 1g/ dL o Non-penetrating Agent
Increase Hct by 3%  includes Hydroxyethyl starch, glucose, polyvinyl-povidone
QC ≤ 70% Hct  large molecules; does not enter the cell
 forms shell around the cell thereby preventing the loss of
Red cells can be washed with normal saline to remove plasma
water
proteins, white cells, and platelets.
Prepared by washing packed RBC by multiple batch processing THREE METHODS IN FREEZING
through the process of centrifugation and decanting 1. High Glycerol Method
supernatant saline solution uses 40% of weight per volume of the final concentration
Such red cells are used for IgA-deficient individuals who have To slow uncontrolled freezing and most common method used
developed anti-IgA antibodies, as exposure will lead to anaphylaxis. RBC units can be stored at -80C using mechanical reader
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
2. Low Glycerol Method
uses 20% of weight per volume
more rapid and controlled compared to High Glycerol
freezing temp is much lower at -120C and uses liquid nitrogen
freezer
3. Agglomeration
employs deglycerolization technique in which the presence of
low ionic strength solution, the cells will agglomerate, forming
large clumps that will sink at the bottom of the bag
o the supernatant can be removed and wash procedure can
be reinstituted
Whole blood can be pulled into batches of 6-8 units prior to
transfusion
PRP is then transferred to the attached satellite bag and spun (high
spin) to get platelets at the bottom and supernatant plasma.
Most of the supernatant is returned back to the primary collection
bag or to another satellite bag, leaving behind 50-60 ml of plasma
with the platelets.
Platelets are stored at 20°-24°C with continuous agitation (in a
storage device called platelet agitator).
22-24 deg C; Units = 5.5x1010 platelets, must contain
sufficient plasma to maintain pH =  6.2

Maximum period of storage is 5 days.

IRRADIATED RED CELLS Transfusion of one unit will raise the platelet count in the recipient
INDICATION Prevent TA-GVHD by about 5000/μl.
RADIATION SOURCE Cesium (137 Cs), Cobalt (60 Co) 1 RBC unit can increase blood platelet count by 5,000 to 10,000
SHELF-LIFE 28 days from irradiation or per uL in a typical 70g human
original expiration date
The usual adult dose is 4-6 units of platelet concentrate (or 1
unit/10 kg of body weight).
Gamma-irradiation of red cells inactivates lymphocytes and These units (which are from different donors) are pooled into one
prevents graft vs. host disease. bag before transfusion.
Irradiated red cells are indicated for intrauterine or premature This dose will raise the platelet count by 20,000 to 40,000/μl.
neonate transfusions, and in individuals with immunodeficiency,
and in those receiving blood from first-degree relative donors.
APHERESIS PLATELETS (Single donor platelets)
In platelet pheresis, a donor is connected to a blood cell separator
Cellular Components: Platelets machine in which whole blood is collected in an anticoagulant
INDICATIONS: solution, platelets are separated and retained, and remaining
• Bleeding due to decreased platelet production components are returned back to the donor.
• Bleeding in hereditary disorders of platelet function With this method, a large number of platelets can be obtained from
• Massive blood transfusion a single donor (equivalent to 6 units of platelet concentrate).
PLATELET CONCENTRATE (Random donor platelets prepared from This method is especially suitable if HLA-matched platelets are
whole blood unit) required (i.e. if patient has developed refractoriness to platelet
Platelet concentrates can be obtained from single donor units or by transfusion due to the formation of alloantibodies against HLA
plateletpheresis. antigens).
One unit of whole blood is centrifuged (light spin) to obtain platelet-
rich plasma (PRP). Through the use of apheresis
Whole blood must be processed within 6-8hrs after collection 22-24 °C; Shelf life = 5 days

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
11
Units = 3.0 x 10 platelets per unit; equivalent to 6-10 units of FFP- Plasma units that were prepared within 6hrs after
random donor platelet collection with ACD as anticoagulant, within 8hrs if CPD or
Used to increase the platelet count from 30,000 to 60,000 uL CPDA1 was used
Why do we need constant Agitation? Plasma is separated from whole blood by centrifugation, expressed
 That is to maintain maximum biological function. into the attached satellite bag, and rapidly frozen at –20°C or at
 To prevent clotting. lower temperature.
 To prevent clumping of platelets. FFP contains all the coagulation factors.
FFP can be stored for 1 year if temperature is maintained below –
 For the oxygen to be sufficient in the storage.
25°C.
 To facilitate oxygen transfer into the platelet bag and oxygen
Stored at -80°C for 1 year
consumption.
If wanted to prolong shelf life, it can be stored in a much
lower temperature
o Usually: -65 °C
Cellular Components: Granulocytes
Granulocyte Concentrate When required for transfusion, FFP is thawed between 30- 37°C
and then stored in the refrigerator at 2-6°C.
Plasma Components must be placed in a plastic bag to prevent any
Legend contamination
 Fresh frozen plasma (FFP) Since labile coagulation factors rapidly deteriorate, FFP should be
 Cryoprecipitate transfused within 2 hours of thawing.

Fresh frozen plasma (FFP) Plasma Frozen 24

INDICATIONS: Plasma Frozen within 24 hours or PF24
• Multiple coagulation factor deficiencies: liver disease,
warfarin overdose, massive blood transfusion Cryoprecipitate
• Disseminated intravascular coagulation
• Inherited deficiency of a coagulation factor for which no INDICATION Fibrinogen deficiency, hemophilia
specific replacement therapy is available a, vWF disease, Factor XIII
• Thrombotic thrombocytopenic purpura deficiency
STORAGE ≤ -18
Plasma collected from platelet concentrates or from the platelet CONTENT Fibrinogen: 150 mg
rich plasma preparation can either be prepared as: AHF: 80 IU
1. FFP or Fresh Frozen Plasma vWF, FXII
2. Plasma Frozen 24 SHELF-LIFE 1 year
NOTES White mass of precipitate in 15
FFP or Fresh Frozen Plasma mL plasma
FFP is prepared from whole blood within 6 hours of collection
because after this time labile coagulation factors are lost. INDICATIONS:
• F VIII deficiency (if F VIII concentrate is not available)
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
• Von Willebrand disease Thawed units cannot be refrozen
• Deficiency of fibrinogen.

Cryoprecipitate is prepared from plasma that has been freshly Plasma Derivatives
separated (within 6 hours of collection) by rapidly freezing it at - Legend
20°C or lower and thawing it slowly at 4-6°C.  Human albumin solutions
A white flocculent precipitate and plasma are obtained.  F VIII concentrate
The mixture is centrifuged and supernatant plasma is removed  F IX concentrate
leaving behind sediment of cryoprecipitate suspended in 10-20 ml  Prothrombin complex concentrate (PCC)
of plasma.  Immunoglobulins
The unit is then refrozen (-20°C or colder) and can be stored at this o Non-specific immunoglobulins
temperature for 1 year. o Specific immunoglobulins
When needed, cryoprecipitate is thawed at 30-37°C, required Plasma derivatives are manufactured by fractionation of large
donations are pooled and transfused to the patient. volumes of pooled human plasma.
Cryoprecipitate contains
• F VIII
• von Willebrand factor Human albumin solutions
• fibrinogen Albumin is prepared by cold ethanol fractionation of pooled
• F XIII plasma and is sterilized during manufacture to destroy viruses and
• fibronectin. bacteria.
Albumin is used as a replacement fluid in therapeutic plasma
Prepared from FFP, allow FFP to thaw then do thawing in a slower exchange, and for treatment of diuretic-resistant edema of
method hypoproteinemia.
Place FFP in a refrigerator at 1-6 °C in order to allow the ice formed
crystal in FFP to cool down Both the normal serum albumin and plasma protein fractions are
o Usually takes hours 14-16 hours in slow thawing used in treatment of hypo bulimic and hypo proteinimic cases and
In using cryoprecipitate thaw bath set at 4 °C it will only take 4 patients who are in shock or have severe burns
hours to cool down Have higher amount of Albumin than TPF, 96% albumin and 4%
o Endpoint of thawing is when presence of slushy precipitate globulin.
is present in plasma. Stored at 2-10 °C for 5 years
o Centrifuge using hard spin to separate the supernatant Granulocyte products used to treat severe neutropenia and
plasma from the precipitate you leave approximately 10- granulocyte dysfunction. Stored 20-24 °C for 24 hours
15ml of the plasma on the precipitate is now the
Cryoprecipitate
Freeze within 1 hour from the time the plasma reaches the slushy F VIII concentrate
state.
Supernatant fluid can be refrozen and label as cryo-poor plasma. INDICATION Prevents or control bleeding in
Thawed unit must be kept at room temperature until transfusion. If Hemophilia A
several bags of precipitate are pulled blood must be infused or STORAGE 1-6°C
transfused within 4 hours
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
Non-specific immunoglobulins:
Freeze-dried F VIII concentrate is prepared by fractionation from
large pools of fresh frozen plasma.
To reduce the risk of transmission of viral infections, it is treated
with heat or chemicals during manufacturing process.
F VIII concentrate is the treatment of choice for treatment of
hemophilia A and severe von Willebrand disease.
Specific single factor concentrates were also used in treatment of
diseases
factor VIII used in treatment for hemophilia A and factor IX for
hemophilia
Usual factor concentrates were VIII, IX, XIII are actually lyophilize
Specific immunoglobulins:
products prepared from pulled plasma using con ethanol method.
o Lyophilization must be in order to prevent transmission of
viruses
Pasteurization method, solvent and detergent method, monoclonal
purification method, and ultraviolet irradiation method
F IX concentrate
Prothrombin complex concentrate (PCC)
MAIN USES/ INDICATIONS:
• Deficiency of F IX
• Deficiency of F VIII with development of inhibitors against F
VIII
• Inherited deficiency of factors II, VII, and X.
PCC contains factors II, VII, IX, and X, and also protein C and S.
A serious risk of PCC is thrombotic complications due to the
presence of small amounts of activated coagulation factors.
Immunoglobulins
Immunoglobulins are obtained by cold ethanol fractionation of large
pools of human plasma.
They are of two types: specific and nonspecific.
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INDICATIONS:
• passive prophylaxis of viral infections like hepatitis, rubella,
and measles
• treatment of hypogammaglobulinaemia,
• autoimmune thrombocytopaenic purpura to induce a rise
in platelet count
• neonatal sepsis
These are derived from the pooled plasma of non-selected
donors.
They are obtained from donors who have selected high titer IgG
antibodies.
Anti-RhD immunoglobulin is prepared from plasma of Rh-
negative donors who have produced anti-D following
immunization; it is used for prevention of sensitization to RhD
antigen in Rhnegative women giving birth to a Rh-positive baby.
Other specific immunoglobulins include hepatitis B immune
globulin, varicella-zoster immune globulin, and tetanus immune
globulin that are used for passive prophylaxis of infections.
SAFE STORAGE OF BLOO D
Whole blood
Whole blood and red cells must always be stored at a temperature
between +1°C and +6 °C.
The main reasons for giving a blood transfusion are to restore or
help to maintain the body’s oxygen-carrying capacity and the
volume of blood circulating around the body.
• If blood is not stored at between +1 °C and +6 °C, its oxygen-
carrying ability is greatly reduced.
• The anticoagulant/preservative solution in the blood bag
contains nutrients for the blood during storage and stops the
blood from clotting.
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
• The red cells can only carry and deliver oxygen if they remain
viable: that is, if they retain the same properties as they have
during their normal circulation in the body.
Another important reason for storing blood between +1 °C and
+6 °C is to keep the growth of any bacterial contamination in the
unit of blood to a minimum.
• If blood is stored above +6 °C, bacteria that may have
inadvertently entered the unit during collection may grow to
such an extent that transfusion of the contaminated blood
could be fatal.
The lower limit of +1 °C is also very important.
• This is because red cells are very sensitive to freezing.
• If they are allowed to freeze, the red cell membranes rupture
and the haemoglobin is released; that is, the cells are
haemolysed.
• The transfusion of haemolysed blood can also be fatal.
Fresh frozen plasma
Fresh frozen plasma (FFP) is plasma that has been separated from a
unit of whole blood within 6 to 8 hours of collection, and has been
rapidly frozen and maintained at all times at a temperature of –
20 °C or lower.
There is no lower temperature limit for the storage of FFP,
although the optimal temperature is –30 °C or lower
Plasma contains water, electrolytes, clotting factors and other
proteins (mostly albumin), most of which are stable at refrigerator
temperature, i.e. +1 °C to +6°C.
Factor V and Factor VIII, however, which are essential in the clotting
mechanism, will deteriorate and diminish in quantity if they are not
stored at –20 °C or lower and greatly reduce the clotting activity of
the plasma.
FFP may be given to a patient to restore or help to maintain
coagulation factors such as Factor V or Factor VIII.
Plasma should not be used as a volume expander unless crystalloids
and colloids are unavailable
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Cryoprecipitate
Cryoprecipitate is the cold insoluble portion of plasma remaining
after FFP has been thawed between +1 °C and +6 °C and is useful for
correcting certain coagulation defects.
It contains approximately 50% of Factor VIII and von Willebrand
Factor, 20–40% of fibrinogen and some of the Factor XIII originally
present in the fresh plasma.
Plasma is separated from red cells within 6 to 8 hours of collecting
blood.
The plasma is frozen solid rapidly, certainly within 30 minutes of
separation from the cells.
The plasma is then thawed slowly at below +4 °C.
In order to get the maximum yield of Factor VIII in the
cryoprecipitate from a blood unit it is important to adhere strictly to
the standard procedures for the collection, storage and processing
of the component.
The stability on storage is dependent on the storage temperature
available.
• The optimal storage temperature is below –30 °C.
Platelet concentrates
Platelet transfusions are used to prevent spontaneous bleeding or
to stop bleeding in patients with established thrombocytopenia or
platelet dysfunction
• Ex., hypoplastic anemia or bone marrow failure – due to
replacement with malignant cells or to the effects of
chemotherapy.
Both manual and automated methods can be used in the
preparation of platelet concentrates.
Lower temperatures adversely affect platelet function and viability.
• For this reason, whole blood should be kept at between
+20 °C and +24 °C until it is processed into platelet
concentrates and other blood components.
Platelet-rich plasma must be separated from whole blood by
centrifugation within 8 hours of phlebotomy.
Additional centrifugation and removal of most of the supernatant
plasma may then concentrate the platelets.
Platelet concentrates should be stored at a temperature of between
+20 °C and +24 °C with continuous agitation.
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
• This is essential to prevent platelet aggregation which results
in loss of viability.
The shelf life and transport conditions differ according to the type of
plastic bag used to store the component.
Platelet concentrates stored at between +20 °C and +24 °C maintain
their function and viability better than refrigerated platelet
concentrates.
Current plasticizers used in the manufacture of plastic bags allow for
storage of up to five days, because gaseous exchange takes place
between the container and the environment and this results in the
maintenance of pH in the component, which is critical for platelet
storage.
Note:
If no platelet agitator or rotator is available, it is not possible to store
platelets.
Once prepared, they must be transfused immediately unless the
blood bank is equipped with:
an air-conditioned facility with a temperature monitoring system
that will maintain an ambient temperature of between +20 °C
and +24 °C or
- a platelet incubator that will keep the platelet concentrates at a
temperature of between +20°C and +24 °C.
Since platelet concentrates are stored at room temperature, they
pose a greater risk for bacterial proliferation.
SOPs on the cleaning of the venipuncture site prior to donation
must be strictly followed, and the disinfectant in use must undergo
regular quality control checks.
Storage conditions and expiry dates should also be strictly adhered
to in order to prevent septic shock for the recipient.
After the hermetic seal is broken, platelet concentrates should be
transfused as soon as possible, but definitely within a maximum of 4
hours of storage at between +20 °C and +24 °C.
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Plasma derivatives
Unlike blood components, plasma derivatives such as albumin or
immunoglobulin are concentrated, sterile specific proteins,
obtained from large pools of donor plasma through a complex
pharmaceutical process called plasma fractionation.
They are used to treat patients with specific protein deficiencies or
requirements for passive immunity.
PACKING BLOOD COMPONENTS FOR TRANSPORTAT ION
The following general observations must be kept in mind:
Label the container THIS WAY UP with an arrow.
Ice should be placed above the blood because cool air moves
downwards.
o Cubed wet ice may be better than chipped or broken ice for
long distance shipments of blood because it melts more
slowly.
o Ice packs can be used at –5 °C or below.
The recommended storage conditions must be maintained when
blood is moved from one location to another, including:
o from a mobile or satellite collection site to the laboratory
o from the blood bank to a different facility (to a hospital or
clinic or another blood bank)
o from the blood bank to hospital wards or operating rooms
Red cell components:
at no point should ice be allowed to come into direct contact with
the blood as the red cells nearest to the ice may freeze and
hemolyze.
o In boxes shipped long distances or at high environmental
temperatures, the volume of ice should at least equal that
of the blood.
o In an insulated container, the temperature can be
considered to be in the +2 °C to +10 °C range as long as
unmelted ice is still present on arrival at destination.
Plasma
There should be at least as much wet ice in the cold box as there is
plasma.
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
o It is important to protect the frozen plasma units during
transportation.
o If possible, they should have been placed in cardboard
boxes before freezing to protect the bags from developing
small cracks.
o A simple method to determine if plasma units have thawed
and refrozen is to place a rubber band around the unit at
the time of preparation.
o Once the unit freezes it leaves an indentation at the sides.
o If the unit has thawed, or thawed and refrozen, the
indentation will not be there.

Platelets
Containers for transporting platelets should be equilibrated at a
temperature of +20 °C to +24 °C before use.
o If outdoor temperatures are extremely high, special
chemical, coolant pouches are available that may be
shipped with platelets and will maintain temperatures of
approximately +20 °C to +24 °C for up to 12 hours.
o Also available are containers with a power source that
maintains temperatures between +20 °C and +24 °C.
o Platelets should reach their destination within 24 hours,
which is the maximum time allowed without agitation.

-END OF TRANSCRIPTION-

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 3)
MLS 18, April 6, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`

Legend:
Transcription Bullet
Notes/Module Packet Bullet
 PPT
MODULE OUTLINE
I. Test required for donations
II. Therapeutic Apheresis
III. Transfusion Therapy
MODULE OUTLINE
At the end of this modules, the student shall be able to:
1. Differentiate among the four different methods of autologous blood
donation.
2. Describe the procedure for a whole blood donation and post
phlebotomy care instructions for the donor.
3. Differentiate mild, moderate, and severe donor reactions and list
recommended treatments for each.
4. List the tests required for allogenic, autologous, and apheresis
donortions.
5. State the acceptable interval of donation for allogenic and apheresis
donors.
6. List the labeling criteria for a unit allogenic and autologous blood.
7. Define apheresis; leukapheresis, platelepheresis, plamapheresis, &
erythcypheresis.
8. Compare and contrast the procedures of continuous flow
centrifugation and intermittent flow centrifugation.
9. List the components that can be collected using apheresis technology.
10. Explain the rationale for the basic types of therapeutic apharesis.
11. Explain the adverse effects of apharesis.
12. Describe the blood products that are currently available for
therapeutic us - the composition and approximate volume of each.
13. Select the appropriate blood product for patient with specific
disorders.
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14. State the expected incremental increase of a
a) patient’s hematocrit level following transfusion of each unit of RBCs
and
b) platelet count following transfusion of each unit of platelet.
15. Identify the group of recipients who are at high risk of infection from
transfusion of cytomegalovirus-positive RBcs or platelets.
16. Explain the role of irradiation in the prevention of tansfusion-
associated graft- versus-host disease
17. State the respective blood products of choice for treating von
Willebrand’s disease, hemophilia A, and hemophilia B
18. List the main advantages and disadvantages of autologous
transfusion.
19. Identify the most important factors to consider when emergency
transfusion is indicated.
20. Define massive transfusion.
TESTS REQUIRED FOR DONATIONS
ABO/Rh
Testing for the donor’s ABO group must include both forward and
reverse grouping.
The ABO group must be determined by testing the donor RBCs with
anti-A and anti-B reagents and by testing the donor serum or
plasma with reagent A1 cells and B cells
The donor’s Rh type should be determined by testing with anti-D
reagent at the immediate spin phase.
Antibody Screen
Donors with a history of pregnancy or transfusion must be tested
for unexpected antibodies to RBC antigens
Uses a pooled cell reagent:
o This reagent contains two or three individual cells pooled
into a single reagent.
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
o The pooled screening cell reagent contains all of the Deferral
required antigens and is capable of detecting the presence  A positive result for anti-HBc along with a positive HBsAg would place
of the clinically significant alloantibodies. the donor in a permanently deferred category.
The antibody screen performed on a recipient prior to transfusion  A positive test for anti-HBc without an accompanying positive test for
uses two or three individual reagent cells HBsAg or HBC is not cause for donor deferral unless it occurs on more
than one occasion or on two consecutive tests.
HBsAg o The products will not be used for transfusion even if the donor is not
Methods currently available include: deferred for a positive result
o Enzyme immunoassay (EIA)
o Chemiluminescence (ChLIA)
o Nucleic acid amplification (NAT) Anti-HCV
Is able to detect small amounts of viral nucleic acid in blood before
Deferral antibodies or viral proteins such as HCV core antigen are detectable
 If the EIA or ChLIA tests are initially positive, they must be repeated by current methods.
in duplicate and a confirmatory (neutralization) test must be Screening tests for anti-HCV involve:
performed. o EIA
o If the confirmatory test is positive, the donor is considered to be o ChLIA
infected (acute or chronic) with the hepatitis B virus (HBV) and must o NAT
be permanently deferred Confirmatory methods include:
 NAT is not mandated for donor screening o RIBA (recombinant immunoblot assay)
o If the initial HBsAg testing is reactive but the confirmatory testing is o HCV RNA
not (unconfirmed positive), all the current products are discarded
but the donor need not be permanently deferred, provided the Deferral
antibody to hepatitis B core (anti-HBc) testing is also nonreactive.  An individual who is positive by RIBA is considered to have the
o The donor is deferred for 8 weeks and may be reinstated if the next HCV antibody and would be indefinitely deferred as a blood
HBsAg testing is nonreactive. donor
 Donors who test repeat reactive for HCV screening tests must be
deferred, and the components or products prepared are
Anti-HBc discarded.
The methods employed are similar to those for HBsAg.  If the confirmatory RIBA or NAT testing are nonreactive, the
Presence of HBc antibody in the donor serum suggests the donor may be considered for reentry.
possibility of HBV infection, either acute or chronic.

Anti-HIV-1/2
All donor units must be screened for the presence of the human
immunodeficiency virus (HIV-1/2) antibody using an FDA-approved
method.

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
o If the initial screening test is negative, the unit is suitable for Any individual units that test reactive should be discarded, and the
transfusion; donor should be deferred from further donations for 120 days
o If it is positive, the test must be repeated in duplicate.
Screening tests include: Syphilis
o EIA A serologic test for syphilis is done because the disease is
o ChLIA characterized as being sexually transmitted and places the donor at
o NAT (run using 16 to 24 donation samples per pool) higher risk for possible exposure to hepatitis and HIV
Confirmation tests for HIV include: Screening tests include:
o Western blot (Wb) o Rapid plasma regain (RPR)
o Immunofluorescence assay (IFA) o Venereal Disease Research Laboratory (VDRL).
o Results are expressed as positive, negative, or indeterminate o Both tests are based on Reagin, or antibody directed toward
cardiolipin particles.
Anti-HTLV-I/II o Cardiolipin-like antibodies have been documented in
HTLV-I virus or human T-cell lymphotrophic virus type I is the persons with untreated syphilis infections.
causative agent of adult T-cell leukemia o Antibody will agglutinate cardiolipin carbon particles in the
Associated with a neurological disorder called HTLV-associated form of visible flocculation.
myelopathy. The confirmatory test for syphilis is:
HTLV-II has been shown to have about 60% homology with type I o FTA-ABS or fluorescent treponemal antibody absorption
and is prevalent among intravenous drug users in the United States. test
Persons can contract both viruses from transfusion via infected o Indirect immunofluorescence is used to detect antibodies to
lymphocytes the spirochete T. pallidum, the agent that causes syphilis
Screening test methodologies include: o If the STS or VDRL screening test is reactive or
o EIA indeterminate, the components from that donation must be
o ChLIA discarded and the donor deferred unless a confirmatory
Confirmatory tests include: FTA-ABS test is nonreactive.
o Western blot o If the FTA-ABS test is nonreactive, the donor may be
o RIPA reentered and the components labeled as reactive with the
o NAT testing screening test.
o The donor of a confirmed reactive test may be reentered
WNV RNA into the donor pool after 12 months and documentation of
Test include: completion of treatment
Mini-pool (MP-NAT)
o Uses pools of 6 to 16 donor samples T. cruzi (Chaga’s Disease)
o Is routinely used when risk of WNV infection in the Chagas’ disease is a parasitic infection that is endemic to Mexico
geographical area is low and Central and South America
The infection is generally mild but persists, without symptoms,
Individual donor (ID-NAT) method throughout the infected individual’s life and can be transmitted
o Recommended when the risk is high through blood transfusions
All donors (allogeneic and autologous) are to be tested once.

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
o If the results are nonreactive, the donor need not be
retested with each donation.
o This is because most donors who are infected in the United
States have a chronic infection that was acquired when
residing in a country where the disease is endemic.
o Repeat reactive donors must be deferred indefinitely and
the products quarantined and destroyed
Platelet Bacterial Detection
Platelet components are particularly at risk because they are stored
at room temperature
For apheresis-derived platelets, it is recommended that a culture
method be used that would usually be performed by the collecting
facility.
For whole blood–derived platelets, there are two choices.
For platelets that are pooled within 4 hours of transfusion, a new
PGD test (Verax) can be used.
o It is a relatively easy test to perform, requires about 45
minutes, and can be done by a transfusion service at the
time of issue.
For platelets that are pooled and stored as a pre-pooled product, a
culture-based test is recommended.
o Typically performed by the collection facility
INTERMITTENT FLOW CENTRIFUGATION (IFC)
PROCEDURES USED TO SEPARATE BLOOD COMPONENTS
Apheresis
A procedure in which whole blood is removed from the body and
passed through an apparatus that separates out one (or more)
particular blood constituent.
Returns the remainder of the constituents to the individual’s
circulation.
It allows a larger volume of specific components to be collected
It permits the removal of disease-causing or unwanted cellular or
plasma constituents from a patient.
It is used to harvest stem cells from the peripheral blood of donors
and patients, avoiding the need for extraction from the bone
marrow
Apheresis can be performed on a donor to collect a specific blood
component (donor apheresis), or it can be performed on a patient
#MLSWATER2022
to remove a particular blood component for therapeutic purposes
(therapeutic apheresis).
The process of removing plasma is termed plasmapheresis. In a
similar manner, platelets (plateletpheresis), red blood cells
(erythrocytapheresis), or leukocytes (leukapheresis) can be
removed (or collected) using apheresis technology.
Citrate is used as the primary anticoagulant in apheresis procedures.
o The binding of calcium ions by citrate inhibits the calcium-
dependent coagulation cascade
o Citrate is mixed with the blood immediately as it is removed
from the donor’s (or patient’s) vein, and effectively
anticoagulates the blood before it enters the apheresis
machine
The variables that are considered during an apheresis procedure
include:
o Centrifuge speed and diameter
o Duration of dwell time of the blood in the centrifuge
o Type of solutions added, such as anticoagulants or
sedimenting agents
o Cellular content or plasma volume of the patient or donor
Methods of Centrifugation
Blood is processed in batches or cycles
Whole blood is drawn from an individual with the assistance of a
pump
To keep the blood from clotting, an anticoagulant is mixed with the
blood as it is pumped into a centrifuge bowl through the inlet port
Can be performed as a single-needle procedure with only one
venipuncture
This is advantageous when collecting apheresis products from blood
donors.
If both arms are used (double-needle procedure: one for
phlebotomy and one for reinfusion), the amount of time for the
apheresis can be reduced
Procedure involved complete a cycle before beginning the next one
Usually smaller and more mobile
A single venipuncture may be used
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CONTINUOUS FLOW CENTRIFUGATION (CFC)
The processes of blood withdrawal, processing, and reinfusion are
performed simultaneously in an ongoing manner.
Because blood is drawn and returned continuously during a
procedure, two venipuncture sites are necessary
Separation of the components is performed by centrifugation, and
the specific component is diverted and retained in a collection bag.
The remainder of the blood is reinfused to the individual via the
second venipuncture site.
Two venipunctures are usually required
Membrane filtration technology can also be used to separate
blood components
Membrane separators are typically composed of bundles of hollow
fibers or flat plate membranes with specific pore sizes
This technology lends itself well to the collection of plasma, since
pores can be sized to prevent the passage of even small cellular
elements.
Filtration has several advantages:
o The collection of a cell-free product
o The ability to selectively remove specific plasma proteins
by varying the pore size
COMPONENT COLLECTION
A qualified, licensed physician must be responsible for all aspects of
the apheresis program
Operators of apheresis instruments may include:
o Medical technologists (clinical laboratory scientists)
o Nurses
o Technicians trained on the job
Written, informed consent must be obtained from the donor
The apheresis procedure is more comfortable for the donor:
o Since the needle used for venous access is typically of a
larger gauge (i.e., smaller size) than for whole blood
collection
o The infusion of saline during the procedure helps reduce
donor reactions due to hypovolemia
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MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
RED BLOOD CELLS
RBCs collected by apheresis are typically collected as a double unit
(termed a 2RBC or double RBC procedure)
A clinical advantage to the collection of apheresis RBCs is reduced
donor exposure for the recipient since the patient can potentially
receive two units from the same individual
Since the volume of RBCs being collected during a 2RBC procedure
is greater than it would be for a whole blood donation, the
requirements for donor hematocrit are more stringent.
o The hematocrit must be at least 40% regardless of gender,
and the level (hemoglobin or hematocrit) must be
determined by a quantitative method;
o The use of copper sulfate is not acceptable
If two units of RBCs are collected by apheresis, the donor must wait
16 weeks before providing another donation that includes RBCs.
If one RBC and one plasma and/or platelet unit are collected, the
donor must wait 56 days before donating another red cell product
PLASMA
In a plasmapheresis procedure, whole blood from the donor is
centrifuged, the plasma is diverted into a collection bag, and the
cellular components (RBCs, platelets, WBCs) are returned to the
donor.
o This allows a larger volume of plasma to be collected from a
donor, such that each apheresis unit (“jumbo” plasma) is
the volume equivalent of at least two whole-blood-derived
plasma units.
Purposes:
o It can be used to augment the inventory of fresh frozen
plasma (FFP) of a particular ABO group, especially group AB
o To prepare immune globulin to provide prophylaxis against
infectious organisms in exposed individuals
o Is used commercially to collect plasma for further
manufacturing into such products as intravenous immune
globulin (IVIG), hepatitis immune globulin, and Rh-immune
globulin.
With infrequent plasmapheresis, donation occurs no more than
once every 4 weeks, and the donor requirements are the same as
for whole blood
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With frequent, or serial, plasmapheresis, donation occurs more
frequently than once every 4 weeks
o There must be at least 2 days between procedures and no
more than two procedures in a 7-day period
PLATELETS
Platelets obtained by an apheresis procedure provide the equivalent
of six to eight whole-blood-derived platelets (random-donor
platelets)
o This significantly decreases the donor exposure for a patient
In a plateletpheresis procedure, platelets along with a portion of
plasma are removed, and the remaining RBCs, WBCs, and majority
of the plasma are returned to the donor
o The platelets are suspended in donor plasma in a collection
bag specifically designed for platelet storage
A routine plateletpheresis procedure typically takes 45 to 90
minutes
Donor selection criteria for the plateletpheresis donor are the same
as for whole blood donation, with two additional requirements
o Prior to each plateletpheresis procedure, a sample must be
collected to determine the donor’s platelet count.
o The platelet count must be at least 150,000/µL in order to
provide an adequate platelet collection and for the donor to
safely undergo the collection procedure.
o If the donor’s platelet count is less than 150,000/µL, he or
she is deferred from platelet donation until a subsequent
count is at least 150,000/µL
Plateletpheresis collections should not be performed on potential
donors taking medications that interfere with platelet function, as
this would result in production of a suboptimal and therapeutically
ineffective patient product
Antiplatelet medications have differing deferral time periods:
o 48 hours for aspirin, aspirin-containing medications, and the
anti-inflammatory drug Feldene
o 14 days for clopidogrel (Plavix), ticlopidine (Ticlid), ticagrelor
(Brilinta), and prasugrel
The interval between plateletpheresis procedures must be at least 2
days with no more than two procedures in a 7-day period
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o To protect the donor, if a double or triple apheresis platelet
is collected, 7 (rather than 2) days must elapse before the
donor is again eligible to provide apheresis platelets
o A donor may undergo no more than 24 plateletpheresis
procedures in a rolling 12-month period
The total volume of plasma collected during any one procedure
cannot exceed 500 mL (or 600 mL if the donor weighs 175 lb or
more) or the volume of plasma cleared by the FDA for the
instrument
GRANULOCYTES
Granulocyte transfusions have been shown to be beneficial in some
severely neutropenic patients (neutrophil count less than 500/µL)
who meet the following criteria:
o Documented (clinically or by culture) infection for 24 to 48
hours that is unresponsive to standard antibiotic or
antifungal therapy
o Bone marrow demonstrates myeloid hypoplasia
o There is a reasonable chance of bone marrow recovery (i.e.,
the neutropenia is reversible)
Granulocyte transfusions have also shown favorable results in the
treatment of neutropenic neonates with sepsis
Collection of granulocytes by apheresis provides a higher yield
product
Apheresis collection requires close communication between the
blood center or apheresis center, the blood bank physician, and the
patient’s physician
A minimum therapeutic dose is 1 × 10¹⁰ granulocytes per day
The granulocyte yield is influenced by the donor’s neutrophil count
and the collection process
During centrifugation of whole blood, granulocytes are found in the
buffy coat between the RBC and plasma layers (see Fig. 14–2).
Adding the red cell sedimenting agent, hydroxyethyl starch (HES),
allows better separation of layers, resulting in an improved yield
with reduced RBC contamination.31,32 Since HES is added directly
to the apheresis circuit, some amount will enter the donor’s
circulation during the collection procedure and ultimately be
removed by the reticuloendothelial system. Immediate side effects
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of HES are due to its colloid properties, including circulatory volume
expansion, with headaches and peripheral edema
THERAPEUTIC APHERESIS (TA)
Like donor apheresis, this involves the removal of a specific blood
component, with return of the remaining blood constituents to the
patient
o However, with TA, since the component being removed is
considered pathological (or contributing to the patient’s
underlying disease state), significantly larger volumes of
blood must be processed in order to remove as much of the
offending agent as possible
The TA procedure is classified according to the blood component
removed:
o A cytapheresis procedure may be used to selectively
remove RBCs, WBCs, or platelets
o A plasmapheresis procedure is used to remove plasma
when the pathological substance is found in the circulation
o The indication categories for therapeutic apheresis are as
follows:
Category I  Apheresis is standard and acceptable, either as
primary therapy or as a first-line adjunct to other
initial therapies
 Efficacy is based on well-designed randomized
controlled clinical trials or a broad base of published
experience
Category II  Apheresis is generally accepted in a supportive role or
as second-line therapy, rather than first-line therapy
Category III  Apheresis is not clearly indicated based on insufficient
evidence, conflicting results, or inability to document a
favorable risk-to-benefit ratio
 Decision-making should be individualized
Category IV  Apheresis has been demonstrated to lack efficacy or
be harmful, and should be discouraged in these
disorders
 Clinical applications should be undertaken only under
an approved research protocol
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Adequate vascular access is mandatory during TA, as larger volumes
of blood are processed and the duration of the procedure is longer
than for donor apheresis
Vascular access can be obtained via:
 Peripheral veins
o If only one to two TA procedures are to be
performed, peripheral access can be used
o In such instances, two venous sites are necessary—
one for removal and one for return
 Central veins
 A combination of both
Therapeutic plasma exchange (TPE) is the removal and retention of
the plasma, with return of all cellular components to the patient
o This is the most common TA procedure performed
o The purpose is to remove the agent in the plasma, such as an
antibody, toxin, or abnormal protein, that is causing the
clinical symptoms
o TPE is also used to replace a normal factor or substance that
may be missing or deficient in the patient’s plasma
o During the TPE procedure, replacement fluids are being
administered to maintain the patient’s intravascular volume,
resulting in dilution of plasma proteins
Therapeutic plateletpheresis can be used to treat patients who
have abnormally elevated platelet counts with related symptoms
o During a plateletpheresis procedure, the platelet count will
be decreased by 30% to 60%
Therapeutic leukapheresis has been used to treat patients with
hyperleukocytosis, defined as a WBC or circulating blast count of over
100,000/µL
Erythrocytapheresis, or red cell exchange, removes a large number
of RBCs from the patient and returns the patient’s plasma and
platelets along with compatible allogeneic donor RBCs
o The procedure is most commonly performed in patients
with sickle cell disease in order to decrease the number of
hemoglobin S–containing RBCs, thereby treating or
preventing the complications (acute chest syndrome,
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
impending stroke, unrelenting painful crisis) associated with
the disease
The therapeutic goal is to decrease the level of hemoglobin S to less
than 30%:
 This is usually accomplished with a single red cell exchange
procedure, requiring from six to ten RBC units, depending on the
patient’s age and red cell volume
 The donor RBCs selected for transfusion should be ABO- and Rh-
compatible, relatively fresh (less than 10 days is preferable to
allow maximum in vivo survival), leukocyte-reduced, negative for
hemoglobin S (donor does not have sickle cell trait), and partially
phenotype-matched for the Rh (C, c, E, e) and K1 antigens to
avoid future alloimmunization
 The procedure can be considered for treatment of overwhelming
malaria or Babesia infections
o Both of these protozoa infect red blood cells, and a
pronounced parasitemia can occur
o Red cell exchange has been shown to be beneficial for
treating these patients when the parasite load is greater
than 10%
o A 1.5- to 2-volume red cell exchange should significantly
decrease the parasite load
 Red cell exchange can be used to remove incompatible RBCs from
a patient’s circulation
TRANSFUSION THERAPY
TRANSFUSION MEDICINE
Was created because there are people who are in need of blood and
other blood components in the treatment of disease
A multi-disciplinary branch of medicine that is concern with the
transfusion of blood and the blood components or products. Including
the proper selection and utilization of blood components as well as the
removal of blood or blood components in the treatment of the
diseases
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Transfusion therapy is used primarily to treat two conditions:
o Inadequate oxygen-carrying capacity because of anemia or
blood loss
o Insufficient coagulation proteins or platelets to provide
adequate hemostasis
Blood and blood products are considered to be drugs because of
their use in the treatment of disease
How do we prepare blood components product in transfusion
therapy?
Whole Blood
Whole blood should be used to replace the loss of both RBC mass
and plasma volume
A definite contraindication to the use of whole blood is severe
chronic anemia
For a 70-kg (155-lb) adult, each unit of whole blood should increase
the hematocrit level 3% or hemoglobin 1 g/dL.
o After transfusion, the increase may not be apparent until 48
to 72 hours when the patient’s blood volume adjusts to
normal
If given that the RBC mass is only affected, plasma volume is
increased already. Transfusing and inducing can’t be done to the
whole blood of the patient
Patients suffering from severe chronic anemia has a reduce RBC
count/mass but even though the RBC mass is reduced but the plasma
volume is increased due to the compensation of the body
The patient will suffer from circulatory volume overload once
transfused with plasma, causing for the patients to develop adverse
reaction such as pulmonary edema and heart failure
It happens mostly in patients with pre-existing heart or kidney failure
Red Blood Cells
RBCs are indicated for increasing the RBC mass in patients who
require increased oxygen-carrying capacity
The decreased RBC mass may be caused by:
o Decreased bone marrow production (leukemia or aplastic
anemia)
o Decreased RBC survival (hemolytic anemia)
o Surgical or traumatic bleeding
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Transfusion of RBCs is contraindicated in patients who are well
compensated for the anemia
RBCs should not be used to treat nutritional anemia, such as iron
deficiency or pernicious anemia, unless the patient shows sign of
decompensation (need for increased oxygen-carrying capacity)
RBC transfusion is not to be used to enhance general well-being,
promote wound healing, prevent infection, expand blood volume
when oxygen-carrying capacity is adequate, or prevent future
anemia.
Each unit of transfused RBCs is expected to increase the hemoglobin
level 1 g/dL and the hematocrit level 3% in the typical 70-kg (154-lb)
human, the same as whole blood
In pediatric patients, a dose of 10 to 15 mL/kg will increase the
hemoglobin about 2 to 3 g/dL or the hematocrit 6% to 9%
Leukocyte-Reduced RBCs
Donor leukocytes may cause:
o Febrile nonhemolytic transfusion reactions
o Transfusion-associated graft-versus-host disease (TA-GVHD)
o Transfusion-related immune suppression
In addition, human leukocyte antigens (HLA) are responsible for HLA
alloimmunization
Leukocytes may harbor cytomegalovirus (CMV)
o To reduce HLA alloimmunization and CMV transmission, the
leukocyte content must be reduced to less than 5 × 10⁶,
which can be achieved by using one of several leukocyte
reduction filters
o With these filters, most RBC units are less than 1 × 10⁶;
some are 1 × 10⁴ leukocytes
Washed RBCs and Frozen/Deglycerolized RBCs
Patients who have severe allergic (anaphylactic) transfusion
reactions to ordinary units of RBCs may benefit from receiving
washed RBCs
The washing process removes plasma proteins, the cause of most
allergic reactions
Washed RBCs are used for the rare patient who has had moderate
to severe allergic transfusion reactions and has anti-IgA antibodies
because of IgA deficiency
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Freezing RBCs allows the long-term storage of rare blood donor
units, autologous units, and units for special purposes, such as
intrauterine transfusion
Platelets and Plateletpheresis
Platelets are essential for the formation of the primary hemostatic
plug and maintenance of normal hemostasis
Patients with severe thrombocytopenia (low platelet count) or
abnormal platelet function may have petechiae, ecchymoses, and
mucosal or spontaneous hemorrhage
The thrombocytopenia may be caused by decreased platelet
production (e.g., after chemotherapy for malignancy) or increased
destruction (e.g., disseminated intravascular coagulation [DIC])
o Massive transfusion may also cause thrombocytopenia
because of the rapid consumption of platelets for hemostasis
and the dilution of the platelets by resuscitation fluids and
RBC transfusion
Platelet transfusions are indicated for patients who are bleeding
because of thrombocytopenia or abnormally functioning platelets
One plateletpheresis should increase the adult patient’s platelet
count to 20,000 to 60,000/µL
o Each unit of platelets from whole blood must contain at least
5.5 × 10¹⁰ platelets and should increase the platelet count by
5,000 to 10,000/µL in a 70-kg human
Granulocyte Pheresis
Newborn infants may develop overwhelming infection with
neutropenia because of their limited bone marrow reserve for
neutrophil production
o In addition, neonatal neutrophils have impaired function
o Studies show granulocyte transfusions to be beneficial for
these patients
For an adult or a child, the usual dose is one granulocyte pheresis
product daily for 4 or more days
o For neonates, a portion of a granulocyte pheresis unit is usually
given once or twice
Granulocyte components should be administered as soon as
possible and within 24 hours of collection
The granulocyte pheresis needs to be crossmatched because of the
significant content of RBCs
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
The neutrophil count will increase to 1,000/µL or more in response
to infusion of granulocyte colony-stimulating factor (GCSF)–
mobilized granulocyte pheresis
Plasma
Plasma includes fresh frozen plasma, plasma 24 (frozen within 24
hours), and thawed plasma
o Plasma and plasma 24 contain all coagulation factors
o After fresh frozen plasma and plasma 24 are thawed, they can
become thawed plasma and stored for 5 days at 4°C
o The 5-day storage reduces outdating and allows rapid response
to urgent orders for bleeding patients
o Thawed plasma after 5-day storage has less factor V and VIII
but is still therapeutic
Plasma can be used to treat multiple coagulation deficiencies
occurring in patients with liver failure, DIC, vitamin K deficiency,
warfarin overdose, or massive transfusion
Plasma is the product of choice for patients with multiple-factor
deficiencies and hemorrhage or impending surgery
Usually 4 to 6 units of plasma will effectively control hemostasis
Congenital coagulation factor deficiencies are rarely treated with
plasma, because the dose requirement for surgical procedures and
serious bleeding is so great as to cause pulmonary edema as a result
of volume overload, even in a young individual with a healthy
cardiovascular system
Plasma is sometimes used as a replacement fluid during plasma
exchange
Plasma should not be used for blood volume expansion or protein
replacement because safer products are available for these
purposes—serum albumin, synthetic colloids, and balanced salt
solutions—none of which transmit disease or cause severe allergic
reactions or transfusion-associated acute lung injury
Plasma should be ABO-compatible with the recipient’s RBCs, but the
Rh type can be disregarded
Cryoprecipitate
Cryoprecipitate is used primarily for fibrinogen replacement
The AABB requires that at least 150 mg of fibrinogen be in each unit
of cryoprecipitate
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Fibrinogen replacement may be required in patients with liver
failure, DIC, or massive transfusion and in rare patients with
congenital fibrinogen deficiency
Cryoprecipitate was used as a source for fibrin sealant, which uses
cryoprecipitate as the source of fibrinogen
Cryoprecipitate was originally prepared as a source of factor VIII
Each unit of cryoprecipitate must contain at least 80 units of factor
VIII
Factor VIII
Patients with hemophilia A or factor VIII deficiency have
spontaneous hemorrhages that are treated with recombinant or
human plasma–derived factor VIII replacement
Factor VIII is treated by different methods, such as pasteurization,
nanofiltration, and solvent detergent, to ensure sterility for HIV,
hepatitis B virus, and hepatitis C virus (HCV)
Both the plasma derived and recombinant factor VIII are stored at
refrigerator temperatures and are reconstituted with saline at the
time of infusion
Factor IX
Factor IX complex (prothrombin complex) is prepared from pooled
plasma using various methods of separation and viral inactivation
The prothrombin complex contains factors II, VII, IX, and X;
however, the product is recommended for factor IX–deficient
patients (hemophilia B), patients with factor VII or X deficiency
(rare), and selected patients with factor VIII inhibitors or reversal of
warfarin overdose
Antithrombin and Other Concentrates
Antithrombin is a protease inhibitor with activity toward thrombin
Heparin accelerates the binding and inactivation of thrombin by
antithrombin.
The hereditary deficiency of antithrombin is associated with venous
thromboses, whereas the acquired deficiency is seen most
frequently with DIC
Thawed plasma is an alternative source of antithrombin
Protein C and protein S are vitamin K–dependent proteins
synthesized in the liver
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Protein S functions as a cofactor for activated protein C, which, in
turn, inactivates factors V and VIII, thus preventing thrombus
formation

Albumin
Albumin is prepared by chemical and physical fractionation of pooled
plasma
Albumin is available as a 5% or a 25% solution, of which 96% of the
protein content is albumin
Albumin may be used to treat patients requiring volume replacement
Albumin can also be used in the treatment of burn patients to replace
colloid pressure

Immune Globulin
Immune globulin prepared from pooled plasma is primarily IgG
Products are available for intramuscular or intravenous
administration
o The intramuscular product must not be given intravenously
because severe anaphylactic reactions may occur
o The intravenous product must be given slowly to lessen the risk
of reaction
Immune globulin is used for patients with congenital
hypogammaglobulinemia and for patients exposed to diseases such
as hepatitis A or measles
For hypogammaglobulinemia, monthly injections are usually given
because of the 22-day half-life of IgG
The recommended dose is 0.7 mL/kg intramuscularly or 100 mg/kg
intravenously
The intravenous preparation of immune globulin is used increasingly
in the therapy of autoimmune diseases, such as immune
thrombocytopenia and myasthenia gravis
Rh immune globulin (RhIg) was developed to protect the Rh-
negative female who is pregnant or who delivers a Rh-positive
infant
Rh immune globulin products, which can be administered
intravenously or intramuscularly, are approved for use in idiopathic
thrombocytopenic purpura patients who are Rh (+)

-END OF TRANSCRIPTION-

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 4)
MLS 18, April 6, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`

Legend:
Transcription Bullet During Transfusion
Notes/Module Packet Bullet Positive identification
 PPT  Identification of the patient and also the patient’s blood specimen
prior to transfusion as well as the blood unit that is used in transfusion
MODULE OUTLINE
I. Adverse Effects (page 4)
Clerical errors represent the main cause of transfusion related death
A. Immediate HTR
and acute hemolytic reaction. Final clerical check is performed at
B. Delayed HTR
II. Other Related Disease (page 5) the bedside patient done by nurse and it also double checks the
A. Transfusion-Transmitted Disease blood bag tag attached to the component to be transfused against
B. Hemolytic Disease of the Newborn the patient’s arm band
C. Autoimmune Hemolytic Anemia The medtech’s job is only in the issuance of the blood, to check if
the blood is viable (check for presence of any contaminants,
LEARNING OUTCOMES bacterial contamination and clots that might cause problem during
At the end of this module, the student shall be able to: transfusion).
1. Define transfusion reaction. The specimen must be collected within 3 days of the scheduled
2. Explain the risks of transfusions. transfusion and the blood can’t be used when it exceeds 3 days. The
3. Compare and contrast immediate and delayed HTR; HTR and non- donor’s blood must be kept stoppered or sealed and stored in 1-6°C
HTR
for at least 7 days post transfusion
4. Differentiate the clinical signs & symptoms of the various types of
Even though the transfusion is done, pre-transfusion blood
transfusion reactions.
5. List laboratory findings associated with acute transfusion reactions specimen is prohibited to put into waste
All specimen that is used in pre-transfusion must be kept for 7 days
because if ever there are adverse reactions during transfusion, there
WHAT TO DO DURING TRANSFUSION? would be specimen that can be used as the basis to compare the
after checking if the donated blood can be transfused to the recipient, result in the post transfusion specimens
proper procedure in the transfusing of the blood must also be done
aside from screening or testing the blood being qualified in transfusion, Venous access
proper procedure in transfusing the blood must be observed Should be established before a blood component is issued
take into consideration the precautionary measures transfusion, as well The selection of the location and the type of access are dependent
as cases where transfusion reaction might occur on the volume, timing expected duration of the transfusion therapy
Peripheral access with an 18 gauge needle or catheter is typically
sufficient

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It is desirable for high volume administration or for long term
therapy
Intravenous transfusion device
Should be set before the blood is issued
Isotonic saline or 5% albumin should be used as an intravenous
solution to dilute the blood components. Because it is not
preferable to transfuse blood that is too viscous since it is not
advisable
Do not use 5% dextrose solution in water also known as D5W (a
hypotonic solution)
o that can cause hemolysis of the RBC
Do not use ringer solution, since it contains calcium which initiates
coagulation
No medication must be added or administered in the same line with
the blood component
Clot-screen filters
All blood components must be filtered to remove the clots by using
the clot screen filters.
70 -170 microns of filters are used to remove gross clots and cellular
debris
Vital signs
Pulse rate, respiratory rate, blood pressure, temperature are
checked by the nurse to note if there would be any signs of
transfusion reaction
Blood warmers
Cold blood can cause hypothermia in the patient
Hypothermia can lead to cardiac arrhythmia and haemorrhage
Blood should be set at 37°C
Blood must not be allowed to warm above 38°C because it can
induce physical or chemical damage in our cells, that can make the
blood unit not viable to transfusion anymore
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Automatic temperature control is set when the blood is warmed at
42°C
Never use water bath as blood warmer or microwave
Rate and length of transfusion
Blood components are infused slowly for the first 10-15 minutes
Approximately 2mL for the first 15mins.
Slow transfusion is needed for the nurses and physician to observe
any sign of transfusion reaction
Subsequently the administration rate maybe increased and the
desirable time to complete the red cell transfusion should be
finished within 2 hours
For platelets and plasma, since it is less in volume compared to red
cell transfusion, the transfusion of platelets and plasma should fall
within 30-60 minutes
NOTE: Any transfusion should be completed within 4 hours of
initiation
o Exceeding to 4 hours would put the patients at risk for
bacteremia or septicemia
At least 75% of the transfused red cells remains in the recipient’s
circulation 24 hours after the transfusion
SPECIAL CONSIDERATION IN TRANSFUSION
Emergency transfusion
If patients Abo and Rh group is unknown, during emergency
transfusion patients are given group O Rh negative red cells since
group O/ blood type O individuals are considered to be as universal
donor (they do not contain any antigen)
Emergency situations or transfusion require group O Rh negative
red cell if the patient’s Abo or Rh is unknown
Massive transfusion
defined as the administration of enough blood or components
within less than 24 hours to reconstitute a complete volume
replacement
in adults, this requires 8-10 units of blood
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 4)
Adverse Effects of Massive Transfusion Intrauterine Transfusion
1. Citrate toxicity and Hypocalcemia done when the baby inside the womb is suffering severe anemia
o Citrate toxicity associated with hemolytic disease of the fetus or the newborn
caused by the presence of citrate in the anticoagulant Is done by:
preservative solution o intraperitoneally inject RBCs into the fetal peritoneal cavity
patients experiencing hypothermia, 2,3-DPG depletion, where RBC can be absorbed to the circulation or perform
coagulation factor depletion, as well as depletion of platelets cordocentesis
due to delusional effects because of extremely massive  Process where donor RBC is directly injected into
transfusion the fetal umbilical vein
there could also be accumulation of biochemical and
microaggregates Purpose of the Intrauterine Transfusion
to maintain fetal hemoglobin above 10g/dL
Neonatal RBC transfusion once initiated, the procedure must be repeated every 2-4 weeks
Done in neonates less than seven days old to reduce the risk of until the 34-36 gestation week is reached or until the fetal lung has
hyperkalemia and to maximize 2,3-DPG matured
For O-negative or blood types compatible with the mother, the
infant is given CMV-negative or leukocyte-reduced blood WHAT TO DO WHEN THIN GS GO WRONG?
o should be hemoglobin S negative for hypoxic and acidotic
newborns Bedside Procedures Transfusion Reactions
Prepare aliquots since neonates require small volume of RBCs Investigation Work up
o dosage: 10-15 mL / kg over 2-3 hours 1. Stop transfusion 1. Clerical checks
2. Keep intravenous line open 2. Visual inspection
Exchange Transfusion
with physiologic saline 3. DAT
used in the treatment for both anemia and hyperbilirubinemia that
4. Repeat ABO /Rh
characterizes hemolytic disease of the fetus and the newborn 3. Notify patient’s physician and
5. Repeat compatibility testing
small amounts of blood are removed from the baby and then blood bank 6. Repeat crossmatch
exchanged with the donor’s blood 4. Take care of the patient per 7. Bilirubin test in blood
Main purpose or the indication: physician’s order 8. Urine test
o to replace the antibody-coated red cells with the compatible 5. Perform bedside clerical checks 9. Hemoglobin and hematocrit
donor cells 6. Specimen to be submitted Post
o to remove bilirubin in order to prevent Kernicterus transfusion venous blood. First
o to remove the circulating maternal antibody in the baby’s
voided urine and blood bags
plasma
7. Document reaction
o to suppress the erythropoiesis or the production of
incompatible red blood cells
Greater than 0.5 mg/dL per hour rise in bilirubin or 10 mg/dL in the
first 24hrs amount of bilirubin would be indicative that the baby is
in need of exchange transfusion

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ADVERSE EFFECTS Transfusion-Related Acute Lung Injury (TRALI)
Transfusion Reaction attributed to the administration of donor plasma containing high
Defined as any transfusion-related adverse event that occurs during concentrations of leukoagglutinins
or after the transfusion of whole blood, blood components or Cause: anti-leukocyte antibodies act on endothelial cells of lungs;
human-derived plasma products. emboli form
They are classified according to the time interval between Remedy: Leukopoor RBCs
transfusion and the presentation of adverse effects
Non-Immunologic Reaction
IMMEDIATE (Acute) – DELAYED Bacterial Contamination
signs and symptoms presenting after 24 hours of transfusion Contamination of blood products from donors with transient
within 24 hours of transfusion. bacteremia during phlebotomy, preparation and processing,
Immunologic Non- Immunologic Non-Immunologic thawing.
Immunologic Cause: endotoxins of psychrophilic organisms; Yersinia
Hemolytic- Bacterial Hemolytic TA- Hemosiderosis enterocolitica, E. coli, Pseudomonas spp.
Febrile contamination TA-GVHD Disease Transmission Prevention: Aseptic techniques, visual inspection and infusion
within 4 hours
Allergic Circulatory Post-
Overload transfusion Transfusion Associated Circulatory Overload (TACO)
TRALI Purpura Associated with rapid infusion of large volumes of blood products.
PCITR Children, elderly patients and cardiac disease patients
Treatment can include intravenous diuretics and therapeutic
Immediate (Acute) Transfusion Reaction phlebotomy
Immunologic Reaction
Febrile Non-Hemolytic Physical or Chemical Induced Transfusion Reaction (PCITR)
Increase in temperature of 1 degree Celsius associated with Causes: mechanical damage (infusion through small bore), osmotic
transfusion that cannot be explained by other condition. or chemical damage, thermal trauma, citrate toxicity
Most common adverse reaction seen
Cause: Anti-leukocyte antibodies Includes physical damage to the RBC due to intravascular lysis
Remedy: Leukopoor RBCs Cause: proliferation of T-cells reacts against foreign tissue of the
host recipient
ALLERGIC VS. ANAPHYLACTIC REACTIONS Prevention: Irradiated blood components
ALLERGIC ALLERGIC
Reaction between recipient Afebrile reaction that occurs only Delayed Immunologic Reaction
antibody and transfused donor after infusion of only few mL of Immunologic Reaction
plasma proteins blood Transfusion-Associated Graft vs. Host Disease (TA-GVHD)
Cause: Donor plasma with foreign Cause: IgA deficient patient with Certain susceptible recipients with compromised immune systems
proteins anti-IgA are transfused with blood or blood components containing
Remedy: Washed RBCs Remedy: Washed RBCs
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 4)
immunocompetent lymphocytes which engraft in recipient’s tissue Hepatitis
and multiply. Inflammation of the liver
Cause: Proliferation of T cells that reacts against foreign tissue of Symptoms: jaundice, dark urine, hepatomegaly, anorexia, malaise,
the host recipient fever, etc.
Prevention: Irradiated blood components
Hepatitis A
Picornaviridae
Post- Transfusion Purpura Small, non-enveloped, single stranded enterovirus RNA
Previously immunized patients to platelet antigens through Most common
pregnancy or transfusion produce antibodies once stimulated, Not screened for blood units
destroying patient’s own platelets.
Cause: Platelet antibodies
Hepatitis B
Therapy: Exchange transfusion, corticosteroids, intravenous
A partially double stranded circular DNA virus of the Hepadnaviridae
immunoglobulin

Hepatitis C
Non-Immunologic Reaction
Flaviviridae virus family. RNA virus.
TA- Hemosiderosis Diagnosis depends on biochemical changes suggestive of HCV,
Iron overload accumulating in the mitochondria of cells in organs detection of HCV RNA or anti-HCV in serum.
like the liver, heart and endocrine gland. EIA, ChLIA and RIBA (Recombinant immunoblot assays)
Individuals that are chronically transfused are at risk example
thalassemia major, sickle-cell anemia and hemoglobinopathies. Hepatitis D, E, G
Iron chelating agent like Desferrioxamine can be used for therapy. HDV is a defective, single-stranded RNA virus that is found only in
the patients with HBV infection.
OTHER RELATED DISEASE o It requires HBsAg in order to synthesize an envelope
Transfusion-Transmitted Disease protein. It was previously called the delta antigen. If HBV
Hepatitis B,C,D and HDV are contracted concurrently, this co-infection.
CMV HEV is a member of the Calciviridae family of nonenveloped RNA
EBV viruses.
HTLV 1 and 2 GB virus C (GBV-C) and hepatitis G virus (HGV) are two genotypes of
HIV the same enveloped RNA virus that belongs to the Flaviviridae
T. pallidum family.
Plasmodium spp.
B. microti
T. cruzi
T. gondii

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o Primary infection with HIV may be asymptomatic or result in a
Hepatitis Route of Transmission mild, chronic lymphadenopathy with symptoms similar to those
seen in infectious mononucleosis.
A fecal/oral  Symptoms may occur within 6 to 12 weeks of infection
and persist for a few days to 2 weeks. HIV enters the cell
B parenterally
by the binding of the virus glycoprotein 120 with cell
surface receptors. Cells possessing these receptors
C parenterally
include CD4+ lymphocytes, macrophages, and other
D parenterally antigen-presenting cells.
 Expansion of donor screening protocols in 1996 to include
E fecal/oral p24 core antigen testing reduced the window period from
an estimated 22 days to 16 days
G parenterally
Refer to Appendices for Decision Tree for Anti-HIV-1/HIV-2 testing of donor
blood.
Diagnosis
HBV DNA first marker to appear- PCR
HBsAg- 2 to 12 weeks post-exposure during acute stage; HTLV I and II (Human T-Cell Lymphotropic Viruses Types I/II)
undetectable in 12 -20 weeks after the development of anti-HBsAg HTLV-I and HTLV-II are RNA retroviruses.
HbeAg appears after the HBsAg in recovering patients, disappears o HTLV-I causes a T-cell proliferation with persistent infection.
before HbsAg. Once the RNA has been transcribed into DNA, it is integrated
HbcAg is present in the serum but is undetectable. randomly into the host cell’s genome.
Once integrated into the DNA, the provirus can either complete its
HIV Type 1 and 2 replication cycle or remain latent for many years.
HTLV-I was the first retrovirus to be associated with a human
HIV is a retrovirus that is spherical in shape, with an approximate
disease.
diameter of 100 nm. That association was with adult T-cell lymphoma/ leukemia (ATL), a
It consists of an envelope of glycoproteins, core proteins, and an highly aggressive, mature T-cell non-Hodgkin’s lymphoma with a
inner core of viral RNA and reverse transcriptase. leukemic phase.
o Infection with the virus causes a slowly progressing immune HTLV-II is similar to type-I and is prevalent in intravenous drug
disorder. users.
o The causative viruses, HIV-1 and HIV-2, are similar in structure,
Refer to Appendices for Flowchart of HTLV Testing
varying primarily in the envelope proteins
o The use of very sensitive serologic testing in screening the blood
supply has resulted in an extremely low risk of HIV transmission
o Positive screening tests are repeated in duplicate, and if at least
one of the duplicates also tests positive, WB or
immunofluorescent antibody assay is used for confirmation.
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West Nile Virus EBV
WNV is a member of the Flavivirus family and is a human, avian, and EBV has been called the “kissing disease” because the virus usually
equine neuropathogen. replicates in the cells of the oropharynx, possibly in infected B cells.
It is a single-stranded RNA lipid-enveloped virion that is common in The virus is shed in the saliva and is most frequently associated with
Africa, West Asia, and the Middle East. infectious mononucleosis.
WNV is usually subclinical but may cause a mild flu-like disease.
Mosquitoes have been found to carry WNV, the genus Culex is the Human Herpesvirus 6 (HHV-6) and 8 (HHV-8) Human herpesvirus 6
chief vector. (HHV-6)
The infection in humans has an incubation period of approximately
Is a very common virus that causes a lifelong infection.
3 to 14 days following the mosquito bite, with symptoms lasting 3 to
After infection, the virus replicates in the salivary gland and then
6 days.
remains latent in lymphocytes, monocytes, and perhaps other
o Other animals can become infected, including horses, cats,
tissues, without obvious pathology.
dogs, bats, chipmunks, skunks, squirrels, and domestic birds
HHV-6 causes roseola infantum, also known as exanthem subitum
and rabbits.
or sixth disease.
CMV
Herpesvirus group Syphilis
CMV can remain latent in the tissues and leukocytes for years, with
Treponema pallidum, the causative agent of syphilis, is a spirochete.
reactivation occurring due to severe immune system impairment.
It is usually spread through sexual contact but can be transmitted
Those at the highest risk of a CMV infection are individuals receiving
through blood transfusions.
allogeneic marrow transplants and the fetus.
Spirochete cannot live in blood stored for 72 -96 hours at 1-6⁰C-
o CMV-seronegative recipients transplanted with
which would make platelets the only component capable of
CMVseronegative allogeneic marrow are at risk if they
transmitting the infection.
receive untested and non-WBC-reduced blood components.
Still part of the test since donor positive for syphilis are considered
high risk individuals.
Parvovirus B19
Human B19 parvovirus (B19) is a small, single-stranded DNA non- Tick-Borne Bacterial Agents
enveloped virus and is the only known pathogenic human
Lyme disease, Rocky Mountain spotted fever (RMSF), and
parvovirus.
ehrlichioses are all bacterial diseases spread by a tick bite.
It causes a common childhood illness called “fifth disease” and is
Lyme disease is caused by the spirochete Borrelia burgdorferi and
usually transmitted through respiratory secretions.
RMSF (Rickettsia rickettsii) and ehrlichioses (Ehrlichia species) are
o Fifth disease presents with a mild rash described as “slapped
caused by bacteria that are obligate intracellular pathogens.
cheek” when occurring on the face and a lacy red rash when
occurring on the trunk and limbs.
o The virus must enter the cell through a specific cell receptor. B19 Transfusion-Associated Parasites
parvovirus enters the red blood cell (RBC) via the P antigen and At least three parasites have been associated with transfusionassociated
replicates in the erythroid progenitor cells. infections:
1. Babesia microti
Babesiosis, a zoonotic disease, is usually transmitted by the bite of
an infected deer tick.
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Infection is caused by the protozoan parasite, Babesia, which infects During gestation, and particularly at delivery when the placenta
the RBCs. separates from the uterus, variable numbers of fetal RBCs enter the
Babesia infection may also be acquired by blood transfusion and maternal circulation
solid organ transplant. These fetal cells, carrying D antigen inherited from the father,
immunize the mother and stimulate the production of anti-D.
2. Trypanosoma cruzi Once the mother is immunized to D antigen, all subsequent
Trypanosoma cruzi is a flagellate protozoan that is the etiologic offspring inheriting the D antigen will be affected.
agent of Chagas disease (American trypanosomiasis). The maternal anti-D crosses the placenta and binds to the fetal Rh-
The reduviid bug bite produces a localized nodule, referred to as a positive cells.
chagoma. The sensitized RBCs are destroyed by the fetal reticuloendothelial
system, resulting in anemia
3. Malaria (Plasmodium species)
Malaria, another intraerythrocytic protozoan infection, may be
caused by several species of the genus Plasmodium (P. malaria, P.
falciparum, P. vivax, and P. ovale).
Natural transmission occurs through the bite of a female Anopheles
mosquito, but infection may also occur following transfusion of
infected blood.

Prion Disease
Creutzfeldt-Jakob Disease (CJD)
Creutzfeldt-Jakob Disease (CJD) is one of the transmissible
spongiform encephalopathies (TSE). These are rare diseases
characterized by fatal neuro-degeneration resulting in spongelike
lesions in the brain.

Hemolytic Disease of the Newborn and Fetus (HDFN)

 destruction of the RBCS of the fetus and neonate by the antibodies
produce by the mother
 Only the antibodies if the immunoglobulin G (IgG) class are actively
transported across the placenta

Usually in Rh(D) HDN, the Rh-positive firstborn infant of a Rh-

negative mother is unaffected because the mother has not yet been
immunized.

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Characteristics of HDN INDICATION
Anemia Antenatal RhIg should be given early in the third trimester, or at
• compensated anemia (increased immature RBCs leads to about 28 weeks’ gestation. The dose does not pose a risk
erythroblastosis fetalis) to the fetus, inasmuch as this amount will cause a titer of
Generalized edema and cardiac feature (hydrops fetalis) only 1 or 2 in the mother.
Increased urinary bilirubin Post-partum The Rh-negative nonimmunized mother should receive
Deposition in brain tissue ( kernicterus) RhIg soon after delivery of an Rh-positive infant. The
Hepatosplenomegaly (extramedullary hematopoiesis) recommended interval is within 72 hours after delivery.

Serological Testing Dose and Administration

Mother Newborn Infant 15 mL of packed RBCs or 30 mL of whole blood
 ABO, Rh, Antibody Screen  ABO This is equal to 300 g of the World Health Organization (WHO)
 Antibody Identification  Rh reference material.
 Paternal Phenotype and  DAT
Genotype The total fetal blood volume is estimated to be less than 5 mL at 12
 Elution weeks.
 Fetal DNA Testing
 Antibody Titers o The microdose can be used for abortions and ectopic
pregnancies before the 12th week of gestation.
RhIg An IV preparation of RhIg is approved for use in the United States.
Active immunization induced by RBC antigen can be prevented by This product also contains 300ug in each vial and can be
the concurrent administration of the corresponding RBC antibody. administered either intramuscularly or intravenously.
This principle has been used to prevent immunization to D antigen Massive fetomaternal hemorrhages of more than 30 mL of whole
by the use of high-titered RhIg. blood occur in less than 1 percent of deliveries.
These massive hemorrhages can lead to immunization if adequate
Mechanism RhIg is not administered.
o A maternal sample should be obtained within 1 hour of
o The administered RhIg attaches to the fetal Rh-positive RBCs in delivery to test for massive fetomaternal hemorrhage by a
the maternal circulation. The antibody coated RBCs are removed
screening test, such as the rosette technique. If positive,
by the macrophages in the maternal spleen. The RBC antigens
quantitation of the hemorrhage must be done by Kleihauer-
are thus unavailable for dendritic cells to present antigen to T
helper cells. Betke or similar test or by flow cytometry

Kleihauer-Betke test
Kleihauer-Betke test, a maternal blood smear is treated with acid and then
stained with counterstain.
Fetal cells contain fetal hemoglobin (Hgb F) that is resistant to acid
and will remain pink.

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The maternal cells will appear as ghosts. Bilirubin at
Normal Elevated
birth
Anemia at
No Yes
birth
DAT Weakly positive/ negative Positive
Spherocytosis Yes Rare
Phototherapy Phototherapy,
Therapy Exchange/ intrauterine
transfusion

Immune Hemolytic Anemia

A simpler way to calculate the dose is to multiply the fetal cell
Shortened RBC survival mediated through immune response.
percentage by 50, which gives the volume of fetomaternal
specifically by humoral antibody
hemorrhage in milliliters.
If needed, additional vials of RhIg should be administered within 72 3 TYPES
hours of delivery or as soon as possible. Alloimmune Patient produces alloantibodies to foreign or non-self
The RhIg must be injected according to the product label. The IV RBC antigens introduced into the circulation, most often
product can also be given intramuscularly. through transfusion or pregnancy
The intramuscular form must be given intramuscularly only.
IV injections of intramuscular preparations can cause severe
anaphylactic reactions because of the anticomplementary activity of Autoimmune Patient produces antibodies against his or her own RBC
these products. antigens
RhIg also contains IgA and may be contraindicated in patients with
anti-IgA and IgA deficiency who have had anaphylactic reactions to Drug-induced production of antibodies to a particular drug or drug
blood products. complex with ensuing damage to the individual’s RBCs

Comparison of ABO vs. Rh HDN Refer to Appendices for Diagram on Drug-Induced Hemolytic Anemia

Characteristics ABO Rh Drug-Dependent or Immune Complex (“Innocent Bystander”)

Non immune/immune IgG Immune IgG anti-D Mechanism
Antibody
anti-A, B The antibody (IgG or IgM) produced recognizes determinants on the
Mother= O Mother= Rh negative drug. If the patient ingests the same drug (or a drug bearing the same
Blood group haptenic group) after immunization, the formation of a drug-antidrug
Baby= A/B/AB Baby= Rh positive
First pregnancy and First pregnancy not complex may occur.
Obstetric The complement cascade may be activated because of this antigen-
subsequent pregnancies affected; rare
history antibody interaction. RBCs are thought to be involved in this process
may be affected
Disease only as “innocent bystanders.”
predicted by No Yes
titers

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The soluble drug-antidrug complex nonspecifically adsorbs loosely to
the RBC surface. Complement, when activated, sensitizes the cell and
may cause lysis.

Membrane Modification (Nonimmunologic Protein Adsorption)

It is hypothesized that the cephalosporins, especially cephalothin
(Keflin), both operate through the drug-adsorption mechanism and are
able to modify RBCs so that plasma proteins (e.g., IgG, IgM, IgA, and
complement) can bind to the membrane.

Autoimmune Hemolytic Anemia (AIHA)

Warm Autoimmune Hemolytic Anemia (WAIHA)
DAT: Positive
Donath- Landsteiner test: Negative
IgG antibody
Optimal temperature: 37 degrees Celsius
Rh, Kell
extravascular hemolysis; splenic

Cold Hemaglutinin Disease

DAT: Positive
Donath- Landsteiner test: Negative
IgM antibody
Optimal Temperature: 0-4 degrees Celsius
I blood group
Extravascular hemolysis; hepatic

Paroxysmal Cold Hemoglobinuria

DAT: Positive
Donath- Landsteiner test: Positive
Involves biphasic IgG
May occur on both 0-4 degrees Celsius; 37 degrees Celsius
P blood group
Intravascular

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APPENDICES

Softcopy version:
https://drive.google.com/drive/folders/12nb8DHOKeTirLYYsWSum79-
QBcUZC-oe?usp=sharing

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