BB Book 2017

The Basic Blood Banking
ACKNOWLEDGEMENT
I feel greater pleasure in expressing my sincere and heart-felt gratitude to Dr. Sadia
Sultan, who gives lectures on this work, for helping me in statistical calculation and
many worth-while suggestions in both the experiment and in the preparation of this
assignment.
I am also thankful to Prof. Dr. S.M Irfan, Head of the Department of Blood bank,
Liaquat National Hospital and Medical College, for his cooperation, valuable
suggestion and encouragement.
I am also acknowledges the cooperation of other staff members specially our

Deputy Manager Mr. Amjad Baig and all other technical staff of the Blood bank for
their friendly attitude throughout the work.
Yours Obediently
Mr. Partab_Rai
Assistant Medical Technologist at Blood Bank
Liaquat National Hospital and Medical College, Karachi
Department of Blood Bank, Liaquat National Hospital and Medical College, Karachi
Table of Contents
Acknowledgement
S.No. Topic Page No.
1 - Introduction of Blood Bank--------------------------------------------------------1

2 - History of Blood Bank--------------------------------------------------------------1
3 - Blood Bank Regulatory Agencies-------------------------------------------------5
3.1 - American Association of Blood Banks (AABB)
3.2 - Food and Drug Administration (FDA)
3.3 - College of American Pathologists (CAP)
3.4 - Joint Commission on Accreditation of Healthcare Organizations (JCAHO)
3.5 - National Committee for Clinical Laboratory Standards (NCCLS)
4 - General Directions for Safety in Blood Bank-----------------------------------7
5 - Blood Introduction-------------------------------------------------------------------8
5.1 - Cellular part (Formed Elements)
5.2 - Non cellular part (Plasma)
5.2.1 - Organic Substance
5.2.2 - Inorganic Substances
6 - Red Blood Cells (Erythrocytes) Morphology----------------------------------10
7 - Functions of Blood-----------------------------------------------------------------11
8 - Source and Need of blood---------------------------------------------------------11
9 - Anticoagulant-----------------------------------------------------------------------12
9.1 - Heparin
9.2 - Ethylene Di-amine Tetra-acetic Acid (EDTA)
9.3 - Citrate glucose
9.4 - Acid-citrate-dextrose
9.5 - Citrate-phosphate-dextrose (CPD)
9.6 - Citrate-phosphate-dextrose-adenine (CPDA-1)
9.7 - Saline-adenine-glucose-mannitol (SAGM)
10 - Calculation of Amount of anticoagulant needed------------------------------14

11- Low Volume Blood Bags Bleed------------------------------------------------14
12 - Blood Sample Collection and Identification----------------------------------15
13- Blood Donations------------------------------------------------------------------16
13.1 - Types of Blood Donation
13.1.1 - Voluntary blood donation
13.1.2 - Exchange Donation
13.1.3 - Directed Donation
13.1.4 - Paid Donation
13.1.5 - Autologous Donation
13.2 - Methods of Blood Donation
13.2.1 - Whole blood Donation
13.2.2 - Aphaeresis Donation
13.2.2.1 - Plateletpheresis: (SDP / Mega unit Donation)
13.2.2.2 - Plasmapheresis: (Donation of Plasma)
13.2.2.3 - Erythrocytaphaeresis :( Red Cell Donation)
13.2.2.4 - Leukapheresis: (Donation of leukocytes)
14 - Post Donation Advice------------------------------------------------------------25

15 - Health Benefits to Donor of Blood Donation---------------------------------25
16 - Adverse Donor Reactions--------------------------------------------------------26
16.1 - Fainting or vasovagal syndrome
16.2 - Nausea and vomiting
16.3 – Hematoma (Bruise)
16.4 - Convulsions
16.5 - Serious cardiac difficulties
17 - Therapeutic Plasmapheresis-----------------------------------------------------28
18 - Quality Assurance----------------------------------------------------------------29
18.1 - Pre-analytical stage
18.2 - The analytical stage
18.3 - Post analytical stage
19 - Quality Control--------------------------------------------------------------------30
19.1 - Quality Control of Reagents
19.2 - Quality Control of Blood Components
19.2.1 - QC of Whole Blood
19.2.2 - QC of Packed RBCs
19.2.3 - QC of Packed RBCs in preservative solution (Adsol/SAGM)
19.2.4 - QC of Platelet Concentrate
19.2.5 - QC of MEGA Unite Platelet
19.2.6 - QC of Cryoprecipitate
19.2.7 - QC of Fresh Frozen Plasma
20 – Mean, Median, Mode, Range and Standard Deviation----------------------36
20.1 - Mean
20.2 - Median
20.3 - Mode
20.4 - Range
20.5 - Standard Deviation
20.6 – West-gard Rules
21- Dilutions of % Solutions----------------------------------------------------------43
22 - Estimation of Hemoglobin by Copper Sulfate--------------------------------44
22.1 - Stock Solution
22.2 - Preparation of Working Solution
22.3 - Error in technique
22.4 - Q/C OF Copper Sulfate
23 - Antigen-----------------------------------------------------------------------------46
23.1 - Red cell Antigen (Blood Group Antigen)
23.2 - Non Red Cell Antigen
24 - Antibodies-------------------------------------------------------------------------47
24.1 - IgG Antibody
24.2 - IgM Antibody
25 - Antigen and Antibody Reactions-----------------------------------------------50
26 - ABO Blood Grouping System--------------------------------------------------51

27 - ABO Inheritance and genetics--------------------------------------------------52
28 - Rhesus Blood Group System----------------------------------------------------53
29 - Preparation of Red Cell Suspension--------------------------------------------53
30 - Preparation of Control IgG Sensitized Cells (Check Cells)-----------------55
31 - Blood Grouping-------------------------------------------------------------------56
Table -1: Interpretation of Results of ABO & Rh Testing
Table -2: Grading of Results in Red Cells Agglutination Test
32 - Rh-Control for AB Positive-----------------------------------------------------58
33 - Weak-D or D u Testing For Anti-D Negative---------------------------------58
34 - Bombay Blood Group------------------------------------------------------------60
35 - Sub Groups of A------------------------------------------------------------------61
36 - ABO Discrepancy-----------------------------------------------------------------61
37 - Resolving ABO Discrepancies--------------------------------------------------63
38 - Direct Anti-Globulin Test for Poly-specific by Tube Method--------------65
39 - Positive Direct Anti-Globulin Test for Monoclonal: (By Tube Method)--67
39.1 - Test with Anti- IgG
39.2 - Test with Anti-C3d
40 - Direct Anti-Globulin Testing By Column Agglutination Technique------68
(By Gel Technique)
41 - Inspecting Gel-cards Before Use-----------------------------------------------69
42 - Compatibility Test (Cross Matching) By Tube Method---------------------69
43 - Compatibility Test (Cross Match) By Column Agglutination Method----72
(By Gel Technique)
44 - Compatibility Test (Cross Match) for Neonates------------------------------74
45 - Incompatible cross match--------------------------------------------------------75
45.1 - Rouleaux formation
45.2 - Saline replacement technique
46 - Blood Transfusion Reaction-----------------------------------------------------77

46.1 - Acute (within hours) Transfusion Reactions
46.2 - Delayed (>24 Hours) Transfusion Reaction
47 - Instruction for Blood Transfusion Reaction-----------------------------------79
48 - Re-cross-matching after Transfusion Reaction-------------------------------80
49 - Hemolytic disease of the Newborn (HDN)------------------------------------80
50 - Antibody Screening---------------------------------------------------------------81
51 - Antibodies Identification---------------------------------------------------------84
52 - Blood Components---------------------------------------------------------------88
52.1 - Whole Blood
52.2 - Packed Red Cells
52.3 - Washed red blood cells
52.4 - Fresh frozen plasma (FFP)
52.5 - Platelet Concentrates
52.6 - Cryoprecipitate anti hemophilic factor (AHF)
52.7 - Irradiated Blood Products
53 - Storage of Blood------------------------------------------------------------------96
Table - 3: Blood and Blood Component Storage Conditions
Table - 4: Time Need for Blood Component Issuing
Table - 5: Emergency release of blood
54 - Rate of Infusion-------------------------------------------------------------------99
55 - NAT Testing-----------------------------------------------------------------------99
56 - Laboratory Waste Management and Disposal-------------------------------100
57 - Reference-------------------------------------------------------------------------101
58 - Abbreviation---------------------------------------------------------------------102
The Basic Blood Banking 1
1 - Introduction of Blood Bank:

A Blood bank is a bank of blood and their components, gathered as a result of blood
donation, where it is stored with preservative for later use in transfusion.
Blood banks not only collect and save the blood for transfusion but it also performs the
testing to determine the blood group of patients as well as donors, their cross match test and
donor screening tests.
2 - History of Blood Bank:

These days blood transfusion is essential part of modern health care, use correctly it can
save life and improve health. But it doesn’t always perfect, it can possible by many
experiments.
In 1628 English physician William Harvey described that blood is continuously circulating
in the body through the veins and arteries by the pumping action of heart, he also defined
that liver convert food into blood.
Harvey did not perform any blood transfusion experiment but he did advance to inject some
medicine and liquids directly into the circulatory system. It was the major step for the help
of blood transfusion.
In 1665 the first recorded successful blood transfusion performed by Physician Richard
lower in England, he keeps dog alive by transfusion of blood from a young dog to another
old dog via a tied artery.
In 1666 Richard Lower assisted by Dr Edmund King worked to transfuse sheep's blood into
a 32 years old man named Arthur Coga, who was mentally ill. He infuses fresh blood from
sheep to this man by removing their old blood.
After first successful transfusion Lower wanted to treat Coga several times but he refused.
In 1667 the first fully documented human blood transfusion was administered by Jean-
Baptiste Denys in France, he successfully transfuse blood from lamb to 15 years old boy via
carotid artery, who had suffered from fever for many months and he had bleed many times.
Denys performed another transfusion into a labourer, who also survived. Both instances
were likely due to the small amount of blood that was actually transfused into these people,
which allowed them to withstand the allergic reaction.
In 1667 Denys administered transfusions of calf's blood to Antoine Mauroy, a madman.

Mauroy died during the third transfusion. Much controversy surrounded his death. Mauroy's
wife asserted Denys was responsible for her husband's death, and Denys was charged with
murder. Denys quit the practice of medicine.
After Denis and some others transfusion experiments on animal to human shows life
threatening reactions, which means transfusion from animals to humans was found to be
unsuccessful and in 1670 the procedure was banned.
In 1818 James Blundell, a British obstetrician, performs the first successful transfusion of
human blood to a patient for the treatment of postpartum hemorrhage during child birth.
Using the patient's husband as a donor, he extracts approximately four ounces of blood from
the husband's arm and, using a syringe, successfully transfuses the wife. The patient died
after initially showing improvement.
Between 1825 and 1830, he performs 10 transfusions, five of which prove beneficial to his
patients, and publishes these results.
James Blundell also made various instrumental devises for performing transfusions.
In 1901, Karl Landsteiner, an Austrian physician discovered the first human blood groups,
which helped transfusion to become a safe practice. By performing experiments in which he
mixed blood samples taken from his staff, Landsteiner discovered blood groups A, B, and C.
Blood type C was later changed to O. For this great discovery in 1930 He was awarded with
the Nobel Prize in Physiology & Medicine and established the basic principles of ABO
compatibility.
His colleagues Alfred Decastello and Adriano Sturli add AB, the fourth type, in 1902.
In 1907 Hektoen suggests that the safety of transfusion might be improved by

crossmatching blood between donors and patients to exclude incompatible mixtures. Reuben
Ottenberg performs the first blood transfusion using 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.
In 1916, Francis Rous and J.R.Turner introduce an anticoagulants such as citrate glucose
solution were found to prolong shelf life of blood for several days and refrigeration also
proved to be an effective means of preserving blood.
In 1917 Allowing for blood to be stored in containers for later transfusion allows for the
establishment of the first blood depot. Oswald Robertson, an American Army officer, is
credited first blood depot with type “O” blood group for the causalities in World War I.
In the 1920’s and 30’s, the voluntary donation of blood for storage and transfusion service
was started. The term “Blood Bank” is coined.
In 1940 Edwin Cohn, a professor of biological chemistry at Harvard Medical School,

develops cold ethanol fractionation, the process of breaking down plasma into components
and products. Albumin, a protein with powerful osmotic properties, plus gamma globulin
and fibrinogen are isolated and become available for clinical use. John Elliott develops the
first blood container, a vacuum bottle extensively used by the Red Cross.
In 1940 The Rhesus (Rh) blood group system is discovered by Karl Land Steiner, Alex
Wiener, Philip Levine, and R.E. Stetson and is soon recognized as the cause of the majority
of transfusion reactions.
In 1940 The United States government first time uses plasma separation method after a
shortage of plasma during World War II.
In 1943 The introduction by J.F. Loutit and Patrick L. Mollison of acid citrate dextrose
(ACD) solution, which reduces the volume of anticoagulant, permits transfusions of greater
volumes of blood and permits longer term storage.
In 1947 The American Association of Blood Banks (AABB) is formed to promote common
goals among blood banking practitioners and the blood donating public.
In 1948 – First plastic bag developed for blood collection.
In 1971 – Blood banking becomes regulated by the FDA.
In 1972 Apheresis is used to extract one cellular component, returning the rest of the blood
to the donor.
In 1979 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
among blood banks.
In 1980’s – Many blood recipients develop a disease which is now called AIDS, then to
prevent in future the more sensitive test have been developed to screen donor’s blood from
that called HIV test.
In 1983 introduce Saline-adenine-glucose-mannitol (SAGM) an Additive solution extend

the shelf life of red blood cells to 42 days.
In 2002 Nucleic acid amplification test (NAT) for HIV and HCV was licensed by the Food
and Drug Administration.
3 - Blood Bank Regulatory Agencies:

Following some common agencies that are regulates the blood banks worldwide.
3.1 - American Association of Blood Banks (AABB):
The AABB is a United States-based professional body and standards

organization that was founded in 1947 as the American Association
of Blood Banks. The organization is now international with
members in 80 countries and has taken on a broader scope to include
all of transfusion medicine as well as cellular therapies, specifically
ones based on hematopoietic stem cells.
3.2 - Food and Drug Administration (FDA):
From 1971 – Blood banking becomes regulated by the FDA. The FDA is responsible
for protecting and promoting public health through the regulation and supervision of
blood banking industry.
A person's suitability to donate blood depends on two general considerations: that the
donation will not be injurious to the donor, and that the donated blood will not be
unnecessarily hazardous to the recipient.
3.3 - College of American Pathologists (CAP):
The College of American Pathologists (CAP) inspects and accredits labs by using a
“checklist”; accreditation occurs every year.
The CAP Laboratory Accreditation Program’s goal is to improve patient safety by

advancing the quality of pathology and laboratory services through education, standard
setting, and ensuring laboratories meet or exceed regulatory requirements.
 Donor and recipient / patient records (10 years)
 Records of employee signatures, initials, and identification codes ( 10 years )
 Quality control records ( 5 years )
 Specimens from blood donors units and recipients ( 7 days post-transfusion )
3.4 - Joint Commission on Accreditation of Healthcare Organizations (JCAHO):
Inspects entire hospitals every other year – focuses on

healthcare delivery.
3.5 - National Committee for Clinical Laboratory Standards (NCCLS):
A not-for-profit membership organization, the

Clinical and Laboratory Standards Institute
(CLSI) brings together the global laboratory
community for a common cause: fostering
excellence in laboratory medicine. We do so by
facilitating a unique process of developing
clinical laboratory testing standards based on
input from and consensus among industry,
government, and health care professionals.
4 - General Directions for Safety in Blood Bank:

The salient general blood bank directions which must be obeyed by all are:
 Blood bank is full of potential hazards that can cause serious injury and or may damage
to the equipment.
 It is your responsibility to know the location of the fire extinguisher, eye wash, and
safety shower in your blood bank and also know how to use them during an emergency.
 All the equipment to be used safely don’t misuse or play with them and clean the
equipment before leaving the bench.
 If a piece of equipment fails while being used, report it immediately to your supervisor.
Never try to fix the problem yourself because you could harm yourself or damage the
equipment.
 When working on bench work wear gloves, laboratory coats, and safety shoes. Never
allow a reagent and patients sample to come in contact with your skin.
 Keep the working area clean from all material expect those needed for your work.
 Long hair should be bound back neatly away from shoulders. Do not wear any jewellery
to blood bank sessions.
 Never eat, drink, or smoke in blood bank working area. Keep fingers, pen etc. out of
your mouth.
 Wash your hands properly before leaving the blood bank and before eating anything.
 Do not discard glassware’s, pipettes, tubes or slides in wastepaper basket or garbage can.
 Immunize the laboratory workers against vaccine-preventable diseases such as hepatitis

B etc.
 Avoid re-capping of syringes or use one-handed technique for recapping and dispose off
all sharp objects or needles in rigid containers appropriately.
5 - Blood Introduction:
An adult human has about 5–6 litters of blood circulating in the body (about 7% of the
human body weight). Blood is a specialized type of body fluid. It is made up of two parts,
cellular part (formed element) and non cellular part (plasma). Blood consist of 45 % formed
element and 55 % plasma.
5.1 - Cellular part (Formed Elements):

 Red blood cells (erythrocytes)
 White blood cells (leukocytes)
- Granulocytes
 Neutrophils
 Eosinophils
 Basophils
- Agranulocytes
 Lymphocytes
 Monocytes
 Platelets (thrombocytes) Fig: A scanning of electron microscopic image
of a normal red blood cell, a platelet and
a white blood cell.
5.2 - Non cellular part (Plasma):
Plasma is pale yellow color, consists of a fluid part (water 91%) and solid part (9%).
Solid part of plasma contains:
5.2.1 - Organic Substance:

 Proteins:
o Albumin
o Globulin
o Fibrinogen
o Pro thrombin ( clotting factors )
o Antigen and antibodies
o Plasma complements system. Consists of approximately 20proteins.
 Nitrogenous substances:
o Urea
o Uric acid
o Creatinine
o Amino acid
o Ammonia
 Non nitrogenous substances:

o Glucose
o Cholesterol
o Galactose
o Phospholipids
o Triglyceride
 Pigment
o Bilirubin
 Enzymes
o Amylase
o Lipase
o Phosphatase
o Carbonic anhydrase
5.2.2 - Inorganic Substances:

o Sodium
o Potassium
o Chloride
o Calcium
o Bicarbonate
o Iodine
o Magnesium
o Phosphorus
o Chlorine
Human blood is bright red in color, when its hemoglobin is oxygenated. Blood is circulated
around the body through blood vessels by the pumping action of heart. Medical terms
related to blood often begin with hemo 0r hemato.
6 - Red Blood Cells (Erythrocytes) Morphology:

RBCs are red hemoglobin containing, none nucleated cells present in blood. They are elastic
and biconcave disc in shape, Average diameter of 7.5 microns with a range of 6 – 9 microns,
2.0 micrometer in thick at the ends and 1 micro meter in thick at the center.
The life span of RBCs is about 110 to 120 days in adults and 70 – 90 days in neonatal. There
is a fine balance between synthesis and destruction of red cells in the body. The body
recycles the breakdown components of red cells.
In the early developing fetus, the yolk sac, the liver and the spleen are forming blood cells.
By the seventh month of gestation and into adulthood, only the red bone marrow makes
blood cells unless something happens.
Red Blood Cells Membrane:
Fig: Structure of normal red cell membrane
About 50% of membrane is protein, 40% is fat & up to 10% is carbohydrate. RBCs
membrane comprises a lipid bi-layer (which determines the membrane fluidity); the bilipid
layer is composed of a class of molecules called phospholipids. Phospholipids consist of
hydrophilic poly head and hydrophobic tail. It is natural that hydrophobic tail hidden within
the bilipid layer and hydrophilic heads are on the surface of bilipid layer.
The protein molecules (which are responsible for flexibility) attached in bilipid layer are
most of the sugar molecules (oligosaccharides). Some sugar molecule attached directly to
the external surface (peripheral) of bilipid layer, the structure is called glycolipid and when
sugar molecule attached to the interior bilipid layer protein molecule (integral) the complex
is called glycoprotein.
When RBCs reach the end of their lifespan, the globin is degraded to amino acids (which are
reutilized in the body), the iron is released from heme and also reutilized, and the
tetrapyrrole component of heme is converted to bilirubin, which is mainly excreted into the
bowel via the bile.
7 - Functions of Blood:
 Transport of Gases, nutrients, waste products
 Regulation of pH by osmosis from 7.35 – 7.45
 Maintenance of body temperature at 98.6 °F
 Protection against foreign substances
 Clot formation to prevent from bleeding
8 - Source and Need of blood:

Even with all of today’s technology, there is no substitute for blood; the only source of
blood is human. Humans donate blood to help the fellow humans as an act of charity.
Blood is always needed for:-
 Accident victims due to excess blood loss
 Cancer patients
 Blood disorder patients i.e. anemia
 Surgery patients
 Pre-mature, pre term babies
 And in many other cases
9 - Anticoagulant:
Definition: Anticoagulant is a chemical agent that prevents blood from clotting by
suppressing the synthesis or function of various clotting factors and it is also used as a
preservative in blood banks for blood products.
Serum: When blood is collected in a plain vacuum tube or serum separator tube (red top
contain zinc clot activator). Allow the blood sample to clot for approximately half an hour
then centrifuge and serum was separated.
Plasma: When blood is collected in an anticoagulant containing tube which prevents from
clotting, therefore the coagulation factors are not removed. After centrifugation the
suspended red cells settle down at the bottom of the tube because they are heavy and plasma
was separated.
Some of the commonly used anticoagulants in blood bank are:
9.1 - Heparin:
It may be considered to be a natural anticoagulant because it is already present in the blood,
but in concentrations less than that required to prevent clotting in freshly drawn blood.
Heparin prevents coagulation by increasing the activity of anti-thrombin III (natural blood
thinner), an inhibitor of thrombin (convert fibrinogen into fibrin).
9.2 - Ethylene Di-amine Tetra-acetic Acid (EDTA):

It is an anticoagulant which acts by virtue of removing calcium ions by chelating. A
concentration of the EDTA of 2 mg /ml of blood is sufficient. Concentration even greater
than this produce no detectable change in erythrocyte volume.
K2EDTA tube is used for Complete Blood Counts (CBC), preparation of blood films for cell
differentiation and Malarial Parasite test.
9.3 - Citrate glucose:

In 1916, Francis Rous and J.R.Turner introduce an anticoagulants such as citrate glucose
solution were found to prolong shelf life of blood for several days and refrigeration also
proved to be an effective means of preserving blood.
9.4 - Acid-citrate-dextrose:
In 1943 J.F. Loutit and Patrick L. Mollison introduce anticoagulant acid citrate dextrose
(ACD) solution. It contains citric acid, sodium citrate, and dextrose. It reduces the volume of
anticoagulant as compare to citrate glucose, which permits transfusions of greater volumes
of blood.
Shelf life is 21 days but due to acidic pH it does not help in maintaining 2, 3-DPG levels
therefore it is no longer use for red cells storage but it still use in aphaeresis procedure.
2, 3-diphosphoglycerate: Hemoglobin binds not only to oxygen but to other substances

such as hydrogen ions (which determine the acidity, or pH, of the blood), carbon dioxide
and 2, 3-DPG (a salt in the red blood cells that plays a role in liberating oxygen from
hemoglobin in the peripheral circulation).
9.5 - Citrate-phosphate-dextrose (CPD):

Citrate phosphate dextrose (CPD) a solution containing sodium citrate, sodium phosphate,
and dextrose that is the primary anticoagulant used for preservation of whole blood or red
blood cells for up to 21 days. The official USP name is anticoagulant citrate phosphate
dextrose solution.
 Citrate – Calcium-chelating agent, preventing coagulation

 Phosphate – During storage the red cells continue their glycolytic activity, which is
usually slow and produces Lactic acid. This Lactic acid decreases pH of the stored
blood. Sodium bi phosphate is a buffer which prevents from dropping pH
 Dextrose – provides nutrients to RBCs during storage
9.6 - Citrate-phosphate-dextrose-adenine (CPDA-1):

1979 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
among blood banks.
Addition of adenine helps in maintaining 2, 3-DPG by alkaline pH which improved the

synthesis of ATP, allowing longer shelf life to 35 days (at 2 – 8 °C).
Whole blood is collected in clear plastic bags that contain CPDA-1

63 mL in 450 mL bag
70 mL in 500 mL bag
9.7 - Saline-adenine-glucose-mannitol (SAGM):

1983 introduce Saline-adenine-glucose-mannitol (SAGM) an Additive solution extend the
shelf life of red blood cells up to 42 days. The system consists of a primary bag containing
anticoagulant CPD solution and one satellite bag containing SAGM, available in Triple and
quotable bags.
Whole blood (450 mL ± 10%) was collected from healthy volunteer donors into CPD
anticoagulant (63 mL). After collecting blood in CPD containing bag, separate plasma
containing platelets from red blood cells by centrifugation and added approximately 100 ml
of SAGM to the packed red cells bag. Other advantage is by removing maximum amount of
plasma from blood is used for the manufacture of factor VIII (cryoprecipitate) or FFP and
platelets.
10 - Calculation of Amount of anticoagulant needed:

If he / she posses a rare blood group such as Negative Groups but their weight is less than 50
kg otherwise healthy and fulfils all other criteria of blood donation can donate blood after
approved by medical director of blood bank.
Amount of blood to be drawn calculated by:
Volume to draw (A) = weight in kg X 500

50
Amount of anticoagulant needed (B) = A X 14
100
Amount of anticoagulant removed = 70 – B
Note: The volume drawn should not be exceeding 10.5 ml / kg body weight.
11 - Low Volume Blood Bags Bleed:

The routine blood bags have volume enough for collection of 450 ml + 45 ml of whole
blood (i.e. 405 - 495 ml) with volume of Anti-coagulant: 70 ml
If 300 to 404 ml blood is collected in the blood bag which is designed for 500 ml collection;
other blood components should not be prepared from this low volume unit. Red cells can be
used.
If whole blood collection is less than 300 ml into a blood bag design for 500 of donor blood.
This unit is not fit for human consumption. Therefore it should be discarded.
12 - Blood Sample Collection and Identification:

 The phlebotomist must positively identify the patient/donor.
 Do not apply the tourniquet too tightly or for too long time period.
 Do not collect the blood from an arm into which an intravenous (IV) infection is being
giving. Select a vein for phlebotomy which can be seen or felt easier.
 Check the syringe and needle are dry and that the barrel and plunger of the syringe fit
well.
 Do not moving the needle once it is in the vein and also not drawing the blood too
rapidly. Allow the blood to run gently down to the inside wall of the container.
 Adding the correct amount of blood to anticoagulant and Always use new tubes or
bottles for the collection of blood and allowing sufficient time for the blood clot.
 Forthing or shaking of the blood must be avoided because it causes hemolysis but gently
mix it with the anticoagulant. Because hemolysis of red blood cells is more affected on
our result.
 Information on the request form must be matched with the sample labeling. Such as
patient name, unique identification number and date of collection.
 Clot sample should be collected in red top tube (4 – 6cc) and anticoagulant sample in
EDTA purple top tube (2 – 4cc). Clot sample in syringe or in yellow top are not
acceptable.
 Before performing the test make sure sample is clotted and centrifuge the clot blood
sample for minimum period of time at high speed in centrifuge machine.
 Sample can be stored for three days after the date of collection. Don’t store whole blood
sample in the freezing refrigerator.
 An acceptation sample can be stored up to 7 days at 2 – 8 °C if the patients having no
history of pregnancy or history of blood products transfusion in past three months. The
cross match with that sample is valid for until the date of surgery (Expiry of blood
product should be noted).
13 - Blood Donations:
The donor is a healthy adult person defined by a set criterion who donates blood voluntarily
without any discrimination of color, sexual or religious, and without being under any kind of
pressure and not motivated by any financial profit such as time off, T-shirts, coffee cups and
pens are not considered direct payment for donation.
13.1 – Types of Blood Donation:
There are several types of blood donation such as:
13.1.1 - Voluntary blood donation:

Most of blood units come as a result of whole blood donation from the volunteers, who have
no connection to the person who will get the blood. Donors are not paid for donation as a
result this maximizes blood safety. Only donor meets the general donor selection criteria for
voluntarily blood donation.
13.1.2 - Exchange Donation:

Donating blood for a family member, friend or other specified patient is called exchange
donation. The donor must meet the general donor selection criteria for exchange blood
donation. Blood banks received blood from donor on the basis of exchange and provide
patients specific blood group. If the person doesn’t need it, the blood bank will use it for
someone else.
13.1.3 - Directed Donation:

Donating blood for a family member, friend or other specified patient is called directed
donation. The donor doesn’t only meet the general donor selection criteria but also must full
fill the patient’s requirement. I.e. Mega Unit Platelet
If the person doesn’t need it, the blood bank will use it for someone else.
13.1.4 - Paid Donation:
Paid donors actually professional donors who’s selling their blood, giving wrong medical
history and most of them are drug addictive. Blood from paid donors cannot be used in the
world wide for transfusion purpose. Some donation also should be consider as indirect paid
donation such as giving time off, coffee cups, pens and T-Shirts etc so avoid it.
13.1.5 - Autologous Donation:
Donating your own blood for later use is called autologous donation. For autologous
donation generaly no need to follow donor selection criteria except if Hct < 33 cannot
donate for themselves. Also their screening tests and cross match will be performed as per
normal donation.
The last blood donation at least 3 days before surgery which helps blood cells count goes
back to normal after blood donation, or can be donating 2 bottles in a week with 3 days
interval by taking iron supplements. Hct is every times checked before donation and label
clearly state “For Autologous Use Only”.
This is generally thought to be safest form of blood transfusion because you are getting your
own blood back. No any transfusion transmitted disease risk or any transfusion reaction
chance but there is always a very small chance of bacterial contamination or clerical error
can be happen. Mostly whole blood is used for autologus. If blood not required during or
after the patient surgery normally is discarded.
13.2 - Methods of Blood Donation:
13.2.1 - Whole blood Donation:

Un-separated blood collected into an approved container containing an anticoagulant
preservative solution
Donors Selection Criteria:
Required information:
 Date and time of donation
 Name
 Gender
 Address:
 Telephone: Residence, business or mobile.
 NIC number
 Age or date of birth. Blood donors must be at least 18 years of age and not more than
55 years old.
Note: Donation must be accomplished in such a way that the safety of both the donor and
the potential recipient is assured.
 Donors Weight: ≥ 50 Kg
 Hemoglobin level ≥ 12.5 g/dL
 Donors body Temperature: 98.6 0F ± 0.5
 Pulse: 60 – 90 beats per minute.
 B.P:
Systolic: 110 mmHg - 140 mmHg
Diastolic: 70 mmHg - 100 mmHg
 Donors are allowed to donate whole blood if the period of previous donation is ≥ 3
months.
Donors Deferral Criteria:
Donor deferral criteria are as follow;

 Donors who have taken drugs containing Asprin (loprin, disprin) within the last 72
hours should be deferred.
 Donors who are using Antidiabetics or currently using Antibiotics / medication
should be deferred.
 Donors who give history of to take drugs or sharing needles, multiple needle
puncture marks if seen at the site of vein puncture should be deferred.
 Donors who had a previously reported positive test for Anti HIV I / II, Anti HCV or
HBsAg should be deferred.
 Donors with history of malaria within the last 12 months.
 Donors with any systemic illness like hypertension, diabetes, asthma or other disease
involving respiratory tract, central nervous system, cardiovascular disease and
bleeding disorder should be deferred.
 Donors who have been transfused with blood or any of its products within the last 12
months should be deferred.
 Donors who give history of any major surgery during the last 12 months should be
deferred.
 Donors who give history of jaundice during the last 12 months should be deferred.
 Persistent fever of ≥ 100 0F from ≥ 10 days and / or un-explained decrease in weight
of ≥ 5 kg noticed by the donor before donation; should be deferred.
 A donor took vaccines for cholera, polio; mumps, rabies and hepatitis-B etc are
should be deferral for 21 days. Note: If the person have been bitten by Dog and took
rabies vaccine should be deferral for 12 months.
 A donor must wait for next 8 weeks (56 days) after donating platelets or plasma
before donating whole blood.
 Pregnant women should be deferred.
 Females who have delivered or lactating in last one year should be deferred.
 Females who have aborted during the last 6 weeks should be deferred.
 Women during menstrual cycles should be deferred.
 Asking questions helps determine the overall suitability of the donor; from this, the
donor will be:
Accepted
Temporarily deferred
Permanently deferred
Whole Blood Donation Procedure:
The donor room is an area where the donor is appropriately interrogated in a relaxed
atmosphere and a short clinical examination is performed. An adequate explanation of the
procedure is given to the donor to maintain his trust and respect.
After the donor is selected and properly remitted, he lies down on a comfortable couch
specially designed for the purpose of blood donation. A trained phlebotomist proceeds with
the whole procedure. Trainees are not allowed to do the phlebotomy of the donor.
An identification number is allotted to the donor. This number relates to all the donor record
and it is labeled on the donor’s history Performa, blood bag, screening test tubes and in the
screening/component register. For all further references related to a particular donation this
identification number is very important and extreme caution is necessary to avoid any mix-
up or duplication of the numbers.
Take triple or double or single bag and write donor name, donor number, date of collection
and date of expiry on blood bag.
Place bag carefully on bio mixer being sure that it is below the level of the donor’s arm.
Clamp the donor bag tubing.
Inspect both arms; use a tourniquet or a blood pressure cuff inflated to 40-60 mm Hg and
ask the donor to close the fist to make the vein prominent.
Select the vein and scrub the area at least 1.5 inches in all directions from the intended site
of vein puncture with 0.7% povidone iodine.
After the skin has been prepared, it must not be touched again. The arm need not be dry
before the next step (maximum 30 seconds).
Remove the needle cover. Place your free hand well below the prepared area in order to pull
the skin taut over the puncture site.
Holding the needle at a 450 angle, puncture the skin with a quick thrust. Lower the angle of
the needle to 100-150 angles and advance the needle to pierce the vessel wall. Remove the
clamp. Blood should now flow freely into the pack. A clean skillful venipuncture is essential
for collection of a full, clot free unit. Tape the tubing to hold needle in place.
Ask the donor if the arm feels comfortable. Also ask the donor not to twist or turn the arm
and to make a fist and release every ten seconds in order to encourage a better flow.
Keep the donor under observation throughout the collection of blood. Check biomixer move
adequately during collection.
Collect unit within10 to 15 minutes to prevent activation of coagulation system. At the end
of the donation procedure the blood bag volume is 500 ml with whole blood and
anticoagulant then beep the biomixer by clamp the tubing.
Apply pressure over gauze remove needle and tell the donor to raise his / her arm.
Recap the needle by using one hand technique.
Remove the clamp and write duration of bleeding on unit as indicated on biomixer.
Next, collect blood into sample tubes for screening.

 6cc clotted sample for Syphilis, Hepatitis B surface antigen (HbsAg) / Anti HCV and
Anti HIV I/II.
 1cc sample is collected in EDTA anticoagulant for MP by peripheral blood film
examination. (Note: If the EDTA sample is already collected before the donation for
hemoglobin check, uses that one for MP).
 Note: Now a day’s donors all screening sample will be collected directly from donor
before donating blood to safe the bag contamination (Closed System).
Seal the tubing attached to the bag.
The donor is in structured to lie down on the couch for 15 minutes and juice is served at the
end of the session.
Thank you for Your Blood donation

We hope to see you again soon!!
Note: Good communication skills and public relations helps in recruiting new donor and
for first time donor to become regular blood donors.
13.2.2 – Aphaeresis Donation:
Aphaeresis is a Greek word which means “to separate” or “to carry away”, in blood bank it
is termed as hemaphaeresis (to separate the blood). In hemaphaeresis whole blood is
withdrawn from a donor or patient in anticoagulant solution and separated into their
components.
There are two types of hemaphaeresis / aphaeresis:
A) Manual aphaeresis:
In this aphaeresis whole blood is collected from donor in a bag which is attached with
multiple bags and centrifuged off-line to separate blood components, involves great care
to ensure from bacterial contamination and also that the bags are labeled correctly.
B) Automated aphaeresis:
In this aphaeresis we used an automated aphaeresis machine which separating specific blood
component and returning the rest of blood to the donor or patient.
It is used for both component preparation and therapeutic applications of aphaeresis.
Component preparation: (Apheresis when done on healthy donors)

• Plateletpharesis (SDP)
• Plasmapharesis (Collection of Plasma)
• Erythrocytapheresis (Red cell Donation)
• Leukapheresis (Collection of leukocytes)
• Peripheral stem cell apheresis (Stem cell collection for Bone marrow transplant)
The advantage of this type of donation is most of the blood is returned, a large amount of
needed component can be collected.
Therapeutic applications: (Apheresis when done on patients)

• Plasmapharesis (TPE)
• Leukapheresis ( e.g. Leukemia’s; WBCs level increase > 200 x 103/μl)
• Erythrocytapheresis ( e.g. sickle cell disease or Malaria)
• Plateletpharesis ( e.g. thrombocytosis: Plt count is raised > 1,000 x 103 /cumm)
13.2.2.1 - Plateletpheresis: (SDP / Mega unit Donation)
Objective is to get platelets concentrate for patients who have deficient. This unit is
especially selected due to lower allo-immunization and transfusion of viruses to the patients.
Selection of Donor for SDP:

A general selection criterion for plateletpheresis is same as random donor accept:
• Inspect the both arms for the vein suitability.
• The donor deferral if the donor’s platelet count is less than 150 x 103 /cmm
• The interval between last single unit platelet donations should be ≥ 72 hours and 7
days for double unit platelet donation.
• Donors should not undergo plateletpheresis more than twice in a week or more than
24 times in a year.
• IF the duration of last donation (unit of Whole Blood) is less than 3 months should
reject for platelets donation.
• IF donor donate Mega unit he/she cannot donate whole blood for next 8 weeks (56
days).
• Also if it becomes impossible to return the SDP donor’s red cells (more than 200 cc)
during plateletpheresis, donor should not be accepted for subsequent plateletpheresis
for next 3 months.
• The donor deferral if the person has taken aspirin containing medication within 72
hours.
• Discuss the nature of the procedure with the donor its expected benefits and possible
risk.
Investigation:
• CBC
• Test for ABO grouping and Rh Type
• Markers for transfusion transmitted diseases (MP, Syphilis, HbsAg, Anti HCV and
HIV)
• Unexpected alloantibody (Red cells antibody screening)
Procedure:
• Install the SDP kit on machine and start the procedure.
• Generally no replacement fluid is required but ACD is used as an anticoagulant. May
need a rubber ball in hand to blood pumping for better flow.
• During the procedure don’t leave the donor alone, monitor closely for any type of
reaction.
• If any reaction occurs, inform the blood bank Doctor and if no one is available and
reaction is sever shift the patient to the emergency room for further management.
• After completation of procedure label the product bag with the patients name, unique
identity number, ABO and Rh blood grouping of patient as well as donor, date of
collection / expiry and name of product.
• If RBCs shows in MEGA Unit platelet then perform cross-match before issuing.
• Take sample from pouch (mini bag) not from main unit for the QC of platelet count
after 30 – 45 minutes of agitation. Yield is 3 to 5 x 1011 /µL.
Yield = Product Volume (ml) X Product count (103) X conversion factor (1000 μl/ml)
• A pH paper is used to check the pH of product which should be > 6 (it used to detect
bacterial contamination)
• The volume of SDP is 150 – 300 ml
• Stored MEGA Unit at room temperature at 20 to 24 °C on agitator
• Expiry only last for 5 days (Day of collection should be consider as zero)
• 1 Mega Unit (platelet pharesis) an average of the equivalent of 6 – 8 units of
random platelets
• 1 dose of Mega Unit should raise patient’s counts by 50 – 80 x 103 /μl after 1 hour
of transfusion.
Note: Platelets Count returns the pre donation levels within 48 hours of donation.
13.2.2.2 - Plasmapheresis: (Donation of Plasma)
• The collection of plasma (the liquid suspension medium of blood) from whole blood
and return back all cellular components to the donor are called plasmapheresis.
• The donor selection criteria is same as routine donors, in additional to check total
serum protein level which should not be below 6.0 g/dl.
• The minimum interval between plasmapheresis is 48 hours with normally return of
remaining cellular components and not more than two procedures in a week.
• If the donor of such procedure donates a unit of blood or if it is not possible to return
red blood cells, the donor should not undergo platelet or plasmapheresis for 3
months.
• The volume of plasma obtained from a donor having weight ≥ 50 kg should not be
exceeding 500 ml with serum protein normal levels, and not more than 1000 ml per
month with a maximum of 12 liters per year.
13.2.2.3 - Erythrocytaphaeresis :( Red Cell Donation)
• The collection of red blood cells from whole blood and return back the remaining
blood components to the donor. A single or double unite of RBCs can collected at
the same time with the automated aphaeresis machine.
• Aphaeresis RBCs product have standard volume and hematocrit.
• For single RBCs donor selection criteria is same as for routine donors, but to be
eligible for donate double red blood cells donor must have:
 Weight: > 65 kg
 Hight: > 5 feet’s
 Hb%: >13.5 g/dl
 And hematocrit > 40%
 Good Venous access.
 The next interval of blood donation is must be 4 months for double RBCs
unit donor.
13.2.2.4 - Leukapheresis: (Donation of leukocytes)
• The donor selection for leukapheresis is same as routine donors except before leuka
pheresis donor total white blood cells count should not be less than 4,000 /μl with
normal differential count.
• Leukocytes concentrates should contain at least 10 x 103 /μl leukocytes.
• They should be transfused as soon as possible, preferably within 6 hours not more
than 24 hours at 2 to 6 °C.
• Transfusion should not be given through micro aggregate filter.
14 - Post Donation Advice:

After the donation you are requested to fallow these instructions:
• Rest for at least 10 – 15 minutes and in the end juice is served.
• Drink lots of fluids for next 24 hours.
• Avoid smoking for 30 minutes and alcoholic cold drink till after meal.
• Avoid operating heavy machine, exercise and games for a day.
• Remove band-aid after 20 minutes, if bleed reapply it and hold your arm above the
heart level.
• If you felt dizzy, lie down and put your feet above the level of head. You will be
alright within 15 – 20 minutes.
• If any type of reaction is persist, return to blood bank or contact your nearest doctor.
Note: Before donating blood or their component get a good night sleep, eat a well balanced
meal and drink extra fluids that are non alcoholic and non caffeine.
15 - Health Benefits to Donor of Blood Donation:
Regular donation (2-3 times a year)

 Lowers cholesterol
 Lowers lipid levels
 Decreases incidence of heart attacks, strokes
Body recovers the Blood very quickly after Donation:

 Blood plasma volume– within 24 - 48 hours
 Platelets & White Blood Cells – within 48 – 72 hours
 Red Blood Cells – in about 3 weeks (21 days)
16 - Adverse Donor Reactions:

The blood donation process is highly safe but adverse reactions can occur. Most donors
tolerate giving blood very well, but adverse reactions occur occasionally. Donor’s attendant
must be trained to recognize adverse reactions and to provide initial treatment. Special
equipment to handle emergency situations must be available.
General: If signs of a reaction occur during the phlebotomy, remove the tourniquet and
place two or three sterile gauze squares over the vein puncture site, withdraw the needle
from the arm and apply firm digital pressure for 10 minutes, with the donor’s arm held
above the heart level.
If signs of a reaction occur after the phlebotomy, then hold the donor on the chair or bed, if
not possible to move then place the donor on the floor.
Note: Some donors who experience prolonged hypotension so they need to an infusion of
normal saline but this decision to initiate such therapy should be made by the donor center
physician or in a policy stated in the facility’s SOP manual.
16.1 - Fainting or vasovagal syndrome:

A vasovagal reaction is a general feeling of discomfort and sweating, weakness, dizziness
and nausea, which may progress to loss of consciousness (Faint) and involuntary passage of
feces or urine. It may be caused by the watching blood, by watching other donors, or due to
first time donation or donation excitement; it may also happen for unexplained reasons.
Whether caused by psychological factors or by neurophysiologic response to blood
donation.
On occasion, the skin feels cold and blood pressure falls. Sometimes, the systolic blood
pressure levels fall as low as 50 mm Hg or cannot be heard with the stethoscope. The pulse
rate often slows significantly. This can be useful in distinguishing between vasovagal attacks
and cardiogenic or hypovolemic shock, in which cases the pulse rate rises.
 Place the donor on his or her back, with their legs raised above the level of head.
 Loosen tight clothing; be sure the donor has an adequate airway.
 Apply cold compresses to the donor’s forehead or the back of the neck.
 Extremely nervous donors may hyperventilate. Divert the donor’s attention by
engaging in conversation, to interrupt the hyperventilation pattern.
 Monitor blood pressure, pulse, and respiration of the donor, if donor does not lead to
rapid recovery, call the blood bank physician appointed for such purposes.
16.2 - Nausea and vomiting:

 Instruct the donor who is nauseated to breathe slowly and deeply.
 If the donor vomits, turned him to the side because due to difficulty in aspiration,
provide a suitable receptacle and have cleansing tissues or a towel ready.
 After vomiting has ended, give the donor some water to rinse out his or her mouth.
 Apply cold compresses to the donor’s forehead and/or back of neck.
16.3 – Hematoma (Bruise):

 A haematoma is an accumulation of blood in the tissue outside the vessels.
 Remove the tourniquet and place two or three sterile gauze squares over the
venipuncture site, withdraw the needle from the arm and apply firm digital pressure
for 10 minutes, with the donor’s arm held above the heart level.
 During the phlebotomy hematomas occur then identify that should an arterial
puncture (blood color is bright red and pulse is palpable) or due to needle pass out
from the vein (interior bleeding).
 Should an arterial puncture be suspected, check the presence of a radial pulse. If
pulse is not palpable or is weak, call a donor center physician.
 Apply ice to the area for 3 to 4 times after every 5 minutes, if desired.
16.4 - Convulsions:
 Call for help immediately. Notify the donor center physician. Prevent the donor from
injuring himself or herself and others. During severe seizures, some people exhibit
great muscular power and are difficult to restrain. If possible, hold the donor on the
chair or bed; if not possible, place the donor on the floor.
 Be sure the donor has an adequate airway. A padded device should separate the jaws
after convulsion has ceased.
16.5 - Serious cardiac difficulties:

 Call for medical aid and/or an emergency care unit immediately.
 If the donor is in cardiac arrest, begin CPR immediately and continue it until help
arrives.
Note: The nature and treatment of all reactions should be recorded on the donor’s record or
a special incident report form. This should include a notation of whether the donor should be
accepted for future donations.
17 - Therapeutic Plasmapheresis:
Therapeutic plasmapheresis should be done only at the written request of patient’s

physician.
Indication: Plasmaphaeresis is done as a part of initial treatment to reduce the patient’s load
of a pathologic substance to the normal levels that drugs could become really effective.
• Para proteins (multiple myeloma, Waldenstrom’s, macroglobulinemia)
• Autoantibody (myasthenia Gravis, GB syndrome, good pasture’s syndrome)
• Thrombotic thrombocytopenic purpura (TTP)
• Lipid (Familial hyper cholestrolemia)
• Toxin or drugs
• Lupus nephritis
There is no need to follow uniform selection or deferral criteria and also no screening
required but can require some investigation to perform the procedure, such as:
Weight: _____ (Kg) Height: ________ (cm) Blood Group: _________ Diagnosis: ________
Hb: ________ gm/dl HCT: __________% TLC____________ Platelets: _______________
PT: ____ APTT: ____ Calcium: ____ Albumin: _____ mg/dl Corrected Calcium: ________
Formula for corrected Calcium: [(4.5 – Serum Albumin) x 0.8] + Serum Calcium
Patients physician should inform to the consent family member in particular explained the
benefits and outcomes of procedure. Also informed about the need of repeat procedure in
some circumstances
It is important to maintain the blood volume during the procedure and some kind of
replacement (mostly protein containing) fluid is always used. Depending on patient it can be
FFP, CS for TTP only, Normal saline, Ringers, 5 % albumin or combinations.
In the end of therapeutic plasmapharesis procedure heparinise the DL line with the heparin
of ratio 1/10 in 0.9% of normal saline.
Caution: Do not use heparin in acute CVA or thrombocytopenia patients (PT > 15, APTT >
48, or Platelets <100 X103).
Complications of Aphaeresis:
• Citrate toxicity or Hypocalcaemia due to citrate
• Haemolysis
• Vasovagal reaction
• hypovolemic reaction
• Air embolism
• Allergic reaction
• Bleeding is rare
18 - Quality Assurance:
Quality assurance (Q.A) mean to provide blood and their products that are affordable and
test result are reliable and timely to the patient. Quality assurance includes all those activity
both inside and outside the blood bank that are required to achieve and sustain the objective
of quality assurance.
There are three stages of quality assurance.
18.1 - Pre-analytical stage:

The pre-analytical stage covers the reason for performing a particular test, collection of
specimen, the request form, and how and when the specimen reaches the Blood bank.
Factor regarding the collection of blood specimen that can be affected on our result:
 Incorrect vein puncture technique.
 Do not apply the tourniquet too tightly or for too long time period.
 Do not collect the blood from an arm into which an intravenous (IV) infection is being
giving.
 Select a vein for phlebotomy which can be seen or felt easier.
 Check the syringe and needle are dry and that the barrel and plunger of the syringe fit
well.
 Do not moving the needle once it is in the vein and also not drawing the blood too
rapidly.
 Removing the needle from the syringe before dispensing the blood into the specimen
container. Allow the blood to run gently down to the inside wall of the container.
 Adding the correct amount of blood to anticoagulant and Always use new clean dry
glass tubes or bottles for the collection of blood and allowing sufficient time for the
blood clot.
 Forthing or shaking of the blood must be avoided because it causes hemolysis but gently
mix it with the anticoagulant. Hemolysis of red blood cells is more affected on our
result.
 Centrifuge blood sample for minimum period of time. Don’t store whole blood sample
in the freezing refrigerator.
18.2 - The analytical stage:
The analytical stage covers the principle of the test method, the reagents, and standard,
details of the test method and quality control method / procedure.
18.3 - Post analytical stage:
The post analytical stage include the reporting, interpretation and checking (verifying) of
test result and issuance of blood and their components.
19 - Quality Control:
A major role of the blood bank is performing the screening of blood, blood grouping and
their cross match tests for the purpose of diagnosis, prevention from disease, and for greater
understanding of the blood transfusion reactions.
To fulfill these aims the data generated has to be reliable for which strict quality control has
to be maintained.
Quality control involves consideration of a reliable analytical method.
Reliability of the selected method is determined by its accuracy, precision, specificity and
sensitivity; with major emphasis of QC being laid on monitoring the precision and accuracy
of the performance of analytical methods.
Accuracy has to do with how close the mean of a sufficiently large number of
determinations on a sample is to the actual amount of substance present and is dependent on
the methodology used.
Precision refers to the extent to which repeated determination on an individual specimen

vary using a particular technique and is dependent on how rigorously the methodology is
followed.
Specificity is the ability of an analytical method to determine solely the analyze it is required
to measure.
Sensitivity is the ability of an analytical method to detect small quantities of the measured
analyze.
Analytical methods require calibration; the process to standardize the instrument with the
sample of known concentration is called calibration.
Two major types of errors may occur in analytical methods:
Random errors that arise due to inadequate control on pre-analytical variables, patient
identity, sample labeling, sample collection, handling and transport, measuring devices etc.
Systemic errors that occur due to inadequate control on analytical variables; e.g. due to
error in calibration, impure calibration material, unstable/ deteriorated calibrators, unstable
reagent blanks etc.
19.1 - Quality Control of Reagents:

Reagents quality control must be performed and documented every day and whenever a
new vial is opened.
QC is performed to check certain critical control points in testing are within specific
parameters and reagents are used according to manufacturer's instructions and facility's
SOPs.
Only monoclonal and high titer anti-sera should be used in blood banks. All the reagents
when not in use should be stored at 2 – 6 °C.
Reagent may be made on site or purchased from manufacturer; both of them should be
tested each day. If the results are incorrect repeat it if still incorrect, vial should be
discarded and a new vial must be tested.
The control ABO cells used for reverse grouping should be prepared by pooling three
ABO group red blood cell samples of the same blood groups.
FDA and AABB recommend QC on day of use according to manufacturer instructions:
Reagent Reaction
• Anti A + A1 cell 4+
• Anti A + B cell 0
• Anti B + B cell 4+
• Anti B + A1 cell 0
• Anti A,B + A cell 4+
• Anti A,B + B cell 4+
• Anti A,B + O cell 0
• Anti D + SC 1 2+
• Anti D + SC II 2+
• Anti D + SC III 0
• AHG + Check Cells 2+
• AHG + O Cells 0
• Albumin 22% + B Cells 0
• Albumin 6% + B Cells 0
• Check Cells + Normal Saline 0
• Check Cells + AHG 2+
Note: Anti-D anti-sera positive QC can be checked by O positive cells and negative QC
can be checked by O negative cells
As we know albumin is a protein media (No any antigen or antibodies inside) so there is no
any positive control and negative control QC can be checked with any type of ABO cells.
The titer of anti-A & anti-B should be 1:256, anti-D should be 1:128 and anti-AB titer is
1:28 each with A & B cells.
19.2 - Quality Control of Blood Components:
Definition: Ensuring safe and efficacious supply of blood and there components by testing
of random components with specific standards.
Sampling:
Non-destructive sampling methods usually involve use of pack tubing, mixing of product
and stripping of lines are vital methods needed to be standardised.
For platelet count samples should be taken into an EDTA tube to induce disaggregating.
Note: Sample not to be taken from the last part of the tube because this section is difficult to
strip properly therefore the last 2 cm should be cut off after stripping the rest of the line.
The QC of Blood component should be performed at least 4 units / month, if quantity of

blood component is more than 100 unites.
Transfusion transmitted diseases (HIV-I & II, HBS-Ag, Anti-HCV, MP, Syphilis) should be
Negative of all the blood components.
Volume (ml) = Weight of bag + Blood components (g) – Weight of empty bag
Specific gravity of component
Specific gravity:
• Whole blood = 1.050
• Packed RBC = 1.093
• Platelets = 1.035
• Plasma = 1.030
Note: The weight of empty plastic bag is 25 grams.
19.2.1 - QC of Whole Blood:

 Volume = 450 ml ± 45 ml
 Storage = 2 – 6 ºC
 PCV (Hct) = 30 – 40% (100% of all units)
 Sterility By culture = Negative
 Minimum 1% or 4 bags per month should be checked for QC
 QC should be performed on the unit weekly of whole blood at near to expiry.
(CPDA-1 having 35 days expiry)
19.2.2 - QC of Packed RBCs:

 Volume = 230 – 250 ml
 PCV (Hct) = >55 – 79 % (100% of all units)
 Minimum 1% or 4 bags per month should be checked for QC
 QC should be performed weekly on the unit of Packed RBCs at near to expiry
(CPDA-1 having 35 days expiry).
19.2.3 - QC of Packed RBCs in preservative solution (Adsol/SAGM):

 Volume = 330 – 350 ml
 PCV (Hct) = 60% + 5 (100% of all units)
 Minimum 1% or 4 bags per month should be checked for QC.
 QC should be performed on the unit weekly of Packed RBCs containing solution
Adsol / SAGM at near to expiry (42 days).
19.2.4 - QC of Platelet Concentrate:

 Volume = 50-70 ml
 Storage = 20-24 ºC (gentle agitation)
 Swirling = Present
 Yield = ≥ 0.55 x 1011 / unit
 pH = ≥ 6.2 in at 90% of the tested donor units.
 Sterility By culture = Negative (It should be performed on last day of expiry)
 QC should be performed on the unit of platelet concentrate after their expiry date (on
6th day).
Calculate the per cent yield by formula:
Yield = Product Volume (ml) X Product count (103) /mm3 X conversion factor (1000 μl/ml)
Note: Check color and consistency for heamolysis and bacterial contamination, infusion of
red cells to be cause immunization.
Positive swirling test is because of discoid shape of the platelets, they move in circular
direction when the bag is tilted upward and downward.
Negative swirling test is when platelets are not moving in circular direction rather moving
straight upward and downward indicating they are function less and should not be
transfused.
19.2.5 - QC of MEGA Unite Platelet: (Platelet Concentrate by Aphaeresis)

 Volume = 150 – 300 ml
 Swirling = Present
 Platelet count = ≥ 3.0-5.0 x 10¹¹ / unit
 pH = ≥ 6.2 in at 90% of the tested donor units.
 QC should be performed on the each unit of MEGA Unite Platelet after collection
(After 30 – 45 minutes of agitation).
 Sterility By culture = Negative (It should be performed after their expiry date on 6th
day).
19.2.6 - QC of Cryoprecipitate:
 Volume = 10-15 ml
 Factor VIII = ≥ 80 IU / ml
 Fibrinogen = ≥ 150 mg / unit
 QC of Cryoprecipitate should be performed after 12 months of storage at -18o C (or
lower) by randomly selected unit.
19.2.7 - QC of Fresh Frozen Plasma:

 Volume = 200 ml ± 50 ml
 PT/APTT = Normal Range
 Factor VIII = ≥ 0.7 IU/ml
 Fibrinogen = ≥ 140 mg / unit
 QC of fresh-frozen plasma should be performed after 12 months of storage at -18oC
(or lower) by randomly selected unit.
20 – Mean, Median, Mode, Range and Standard Deviation
20.1 – Mean:
The mean is the numerical average of the data set
The mean is found by adding all the values in the set, then dividing the sum by the total
number of values.
Mean = (X1 + X2 + X3 … X20)  (n)
Where;
“X” is the one value of set and “n” is the total number of sets.
i.e. 12, 15, 11, 11, 7, 16
First, find the sum of the data.

12 + 15 +11 + 11 + 7 + 16 = 72
Then divide by the total number of data.
72 / 6 = 12
The mean is 12
20.2 – Median:
The MEDIAN is the number that is in the middle of a set of data

Put all numbers in order from lowest to highest value and find the number that is exactly in
the middle.
I.e. #1 15, 11, 36, 19, 27, 17, 33

First arranging values from least to greatest, then find the Median
11, 15, 17, 19, 27, 33, 36
The median is 19
Median always fined with a set of odd number values, which is in the middle.
I.e. #2 15, 11, 19, 27, 17, 33

First arranging values from least to greatest, then find the Median
11, 15, 17, 19, 27, 33
Since there is an even number of values, there are two numbers in the middle
Add these 2 numbers and then divide by 2

17 + 19 = 36
36 / 2 = 18
The Median is 18
20.3 – Mode:
The MODE is the piece of data that occurs most frequently in the data set.
Put all numbers in order from lowest to highest value, which makes it easier to find the
mode and find the most occurring number.
i.e. 15, 17, 11, 27, 33, 27, and 19
First arranging values from least to greatest, then find out the Mode
11, 15, 17, 19, 27, 27, 33
The value 27 appears twice. All other numbers appear just once. Its mean our mode is 27.
A set of data can have:

One mode 15, 16, 18, 18, 20
More than one mode 7, 10, 10, 10, 15, 15, 16
No mode 2, 9, 12, 17, 24
20.4 – Range:
The RANGE is the difference between the lowest and highest values.
Largest number – lowest number = Range
I.e. 16, 12, 26, 17, 23

First arranging values from lowest to highest, and then finds the Range
12, 16, 17, 23, 26
26 – 12 = 14
The Range is 14
20.5 - Standard Deviation:
The standard deviation (S.D) can be determined to a set of repetitive measurements which
indicate how much they are differing from the mean.
The smaller standard deviation is the higher quality of the measuring instrument.
Standard deviation is commonly used for preparing Levey-Jennings chart.
Quality control charts are used to record the results of measurements on control samples, to
determine if there are systematic or random errors in the method being used. The most
common type of chart is the Levy-Jennings chart (often referred to as the L-J chart).
In 1950, S. Levy and E.R. Jennings suggested the use of Dr. Shewhart control chart in the
clinical laboratory.
The Levy-Jennings chart usually has the days of the month plotted on the X-axis and the
control observations plotted on the Y-axis.
The first step is to calculate decision limits.
These limits are ±1SD, ±2SD and ±3SD from the mean.
Formula for SD calculates:

SD = √ ∑ (Xn – X) ²
n–1
Where:
s = standard deviation
x = mean (average) of the QC values
∑ (Xn - X) ² = the sum of the squares of n1 differences between individual QC
values and the mean
n = the number of values in the data set
Q: Find out the standard deviation of the following 20 values (12, 16, 18, 13, 17, 14, 13, 15,
18, 16, 13, 15, 12, 16, 18, 15, 13, 17, 15 & 14) mg/dl.
Answer: Formula
SD = √ ∑ (Xn – X) ²
n–1
Calculation of Mean:
The sum of the values (X1 + X2 + X3 … X20) divided by the total number (n) of
observations
The mean of these 20 observations is (300  20) = 15 mg/dl
(To find ∑ (Xn – X) ² we draw a table which is given below)
When a negative value is multiply with another negative value the result should be in
positive, according to mathematically.
SD = √ ∑ (Xn – X) ²
n–1
= √ 74 = √ 74 =1
20–1 19
Formula: +/- 1 SD = Mean +/- 1(SD)

+1 SD = 15 + 1 (1) = 16
- 1 SD = 15 – 1 (1) = 14
+2 SD = 15 + 2 (1) = 17
- 2 SD = 15 – 2 (1) = 13
Reference range = 12 ----- 18 mg/dl
Mean = lower reference value + upper reference value

2
= 12 + 18 / 2 = 15
SD = upper reference value - lower reference value

6
Whereas 6 are the large squires (boxes) in L.J chart
= 18 – 12 / 6 = 1
20.6 – West-gard Rules:
In 1981, Dr. James Westgard set six rules for L.J chart used to evaluate quality of analytical
control values as acceptable or not
Normal graph is zigzag form around the mean and it should be between +/- 2SD.
Rule: 1 – If our control results cross +/- 2SD it’s a warning rule to trigger careful of control
data.
Rule: 2 – If control result will cross from +/- 3SD, this run is rejected
Rule: 3 – Reject the run when two consecutive observations of the same level fall outside
+/- 2SD in the same direction.
Rule: 4 – Reject the run when consecutive one exceeds + 2SD and other exceeds -2SD limit,
total difference between controls is 4S.
Rule: 5 – Reject run when 10 consecutive control measurements fall on one side of the
mean.
Rule: 6 – Reject run if 7 results falling in a pattern up or down
21- Dilutions of % Solutions:

Dilutions can be prepared from more concentrated solutions by using of the following
formula:
V1×C1=V2×C2
Where V 1 and C 1 represent original volume and concentration and V 2 and C 2 represent
final desired volume and concentration.
Example: 22% albumin is available, but we needed 6% albumin in 2 ml. How should the
albumin be diluted?
Formula:
V1 × C1 = V2 × C2
Data:
V 1 =??
C 1 = 22 %
V 2 = 2 ml
C2=6%
Now put it into formula:
V 1 × 22 = 2 × 6
V1 = 2×6
22
= 12 = 0.54
22
Therefore, mix 0.54 ml of 22% albumin with 1.46 ml saline to obtain 2.0 ml of 6% albumin.
(1ml = 1000 μl and 50 μl = 1drop)
22 - Estimation of Hemoglobin by Copper Sulphate:
Principle: Copper sulphate solution is used to test donor haemoglobin. The method is based
on specific gravity, the specific gravity of whole blood established by allowing small drop of
blood, dropped into copper sulfate solution of known specific gravity. If the drop of blood
has higher specific gravity than the solution it will sink within 15 seconds if not, the drop
will hesitate, remain suspended or rise to the top of the solution. It is not quantitative test; it
shows only whether the hemoglobin is below or above acceptable limits.
Material:
Use solution of copper sulfate with specific gravity at 1.053 equivalents to 12.5 g/dl
hemoglobin.
22.1 - Stock Solution:

A stock solution of Cu SO4 is prepared first. It has a specific gravity of 1.100 at 25 °C. To
prepare a stock solution of 170gm of analytical grade Cu SO4 crystals are added to each liter
of Distilled water. After adding the necessary water the solution is shaking thoroughly in
order of dissolve the Cu SO4 crystal.
Storage:
The stock copper sulfate solution should be stored in tightly sealed container otherwise,
evaporation occurs and readings are affected.
The self-life of copper sulfate is at least one year in tightly sealed container.
22.2 - Preparation of Working Solution:

From stock solution working solution is prepared as follow as per requirement of different
Specific gravity (for different hemoglobin concentrate)
Hb % Specific gravity CuSO4 ml (stock sol.) D / W ml

12.5 1.053 512 488
Check specific gravity directly by hydrometer of copper sulfate solution should be 1.053.
Procedure:
 Into a labeled, clean, dry tube or bottle, dispense at least 80mL of working copper sulfate
solution to allow the drop to fall approximately 3 inches.
 The container with a wide mouth 2.5 to 5 inch in width at least 3 inch deep, A screw-
capped jar is a good container because after the end of testing can be sealed and the
solution, will not be subject to evaporation.
 Change the solution daily or after 25 tests.

 Clean the site of the skin puncture thoroughly with antiseptic solution and wipe it dry
with sterile gauze.
 Puncture the finger firmly, near the end but slightly to the side, with a sterile, disposable
lancet or spring loaded disposable needle system.
 Do not squeeze the puncture site repeatedly because this may dilute the drop of blood
 Collect the blood in a capillary tube
 Let one drop of blood fall gently from the tube at a height about 1 - 2 cm above the
surface of the copper sulfate solution. Observe for 15 seconds.
 Dispose of lancets and capillary tubes in appropriate biohazard containers.
Interpretation:
22.3 - Error in technique:

 In copper sulfate method example in corporation of air bubbles or use inadequate
height for dropping the blood that is more than 2cm.
 In rare cases in which the plasma protein concentration is greatly raised, anemic
donor may be accepted.
 Falsely high positive result may also due to high white cells count associated with
leukemia.
22.4 - Q/C OF Copper Sulfate:
Principle:
Copper sulfate can be checked for suitability in donor screening by observing the behavior
of (sinking or floating) drop of blood of known hemoglobin.
Requirement:
o Copper sulfate (Working Solution)
o Hydrometer
o Capillary tubes
o Work sheet for results
o Known hemoglobin 4 samples
(Samples should includes Hb% with slightly above and below 12.5 g/dl)
Procedure:
 Check specific gravity directly by hydrometer of copper sulphate solution should be
1.053.
 Place a drop of each blood sample into copper sulphate solution and observe the drops
for 15 seconds
 Above at 12.5 g/dl Hb must sink down and below must be float
 Record the date of testing, manufacturer, expiration date of copper sulphate, sample
identity, results and person perform the QC.
Note: Copper sulphate powder is highly carcinogenic and should not be touched without
gloves, if touch without gloves can rapidly be absorbed into the skin and can be very
harmful.
23 - Antigen:
A substance that stimulates the production of an antibody when introduced into the body to
whom it is foreign.
Antigens are:
 Microbes: Capsules, cell walls, toxins, viral capsids, flagella, etc.
 Non-microbes: Pollen, egg white, red blood cell surface molecules, serum proteins,
and surface molecules from transplanted tissue.
 Lipids and nucleic acids are only antigenic when combined with proteins or
polysaccharides.
One virus or microbe may have several antigenic determinant sites (Epitopes), to which
different antibodies may bind. Ability of an antigen to react with the products of an immune
response (antibodies) is called antigenicity.
In blood bank we study mostly two types of antigens:
23.1 - Red cell Antigen (Blood Group Antigen):
There are more than 700 blood group antigens are known to exist. The biological role of
blood group antigens is unknown.
Blood banks routinely check blood of donors and patients for ABO and Rh (D), as these are
the most critical antigens for safe transfusion. Generally do not check for minor antigens—
of the Rh, Kell, Duffy, Kidd, and MNS systems etc—but they do screen plasma for
antibodies against these antigens (antibody screen).
Blood group antigens are located on the surface of red cell membrane. The most important
blood group antigens A & B (ABO) are carbohydrates (sugars) attached to the either protein
(glycol-protein) or lipid (glycol lipid). The D (Rh) antigen is a protein attached with lipid
(lipoproteins).
23.2 - Non Red Cell Antigen:
Some human body fluids contain soluble glycoproteins which have antigen determinants
identical with red cell antigens. When antigens are present in solution the term substance is
applied. Thus there are A and B antigen present on red cells and A and B substances are in
saliva and in other body fluids.
The human red cell surface oligosaccharides chain likes substances also found in animals
and plants. Therefore humans are exposed with these substances as an antigen from the
environment and developed against antibodies in the first few months of life, which is called
naturally occurring antibodies.
24 - Antibodies:
Antibodies are the plasma proteins which are produced by the defense mechanism of the
human body as a response of a foreign antigen. Antibodies are gamma globulins which are
the related to the immune response, and now they are commonly known as immunoglobulin
(Igs).
The first time a person is exposed to a non self antigen, antibodies are produced by the
immune system i.e. IgM and this is called primary response. This will kill the foreign
antigen and eliminate it.
When the same person is exposed by the same antigen a second time, the body produces a
large quantity of antibodies rapidly i.e. IgG by the help of memory cells and this is called
secondary response.
Each antibody has at least two identical sites that bind antigen on antigen binding sites.
Valiancy of an antibody: Number of antigen binding sites
I.e. IgG has 2 binding sites and IgM has 10 binding sites
The Structure of Antibodies:-
.
In the blood bank their are two types of antibodies are important i.e. IgG and IgM
24.1 - IgG Antibody:
IgG is a monomer flexible Y-shaped molecule composed of four glycoprotein chains: two
identical heavy chains and two identical light chains. The four glycoprotein chains are
connected to one another by covalent disulfide (S-S) bonds at the hinge region.
 IgG is a monomer shaped molecule.

 Percentage serum antibodies: 80%
 Location: Blood, lymph, intestine
 Half-life in serum: 23 days
 Warm: reacts best at 37°C
 Can cross the placental barrier
 The distance from one antigen-binding site to the other is about 16nm in IgG.
 Known Functions: Enhances phagocytosis, neutralizes toxins and viruses, and
protects fetus and newborn.
 IgG will frequently cause in-vivo hemolysis. Therefore they are significant in the
Blood Bank.
24.2 - IgM Antibody:

 IgM is a pentamer shaped molecule.
 Percentage serum antibodies: 5-10%
 Location: Blood, lymph, B cell surface (monomer)
 Half-life in serum: 5 days

 Cold: reacts best at 4-10°C
 Cannot cross the placental barrier
 The maximum distance between two antigen binding sites of IgM molecule is
about 30 nm
 Known Functions: First antibodies produced during an infection, Effective against
microbes and agglutinating antigens.
IgM usually do not cause in-vivo hemloysis, as it is cold antibody. The most important of
these exceptions are ABO antibodies.
Structure of IgM Antibody (Pentamer)
25 - Antigen and Antibody Reactions:

IN-VITRO PROCESS (Condition out-side the human body): In-vitro antigen-antibody
reactions that we study and perform during the patient ABO grouping, Rh typing, Antibody
screens and identifications, Cross-matching, etc. The study of reactions between antibodies
and antigens are called serology.
When antibodies bind to the antigen on red cells, they bring the red cells together to form a
visible clump or aggregate which is called agglutination. This is an in-vitro reaction which
not occur in-vivo normally.
Stages of Agglutination:
Stage-1: The antibody attaches to the antigen on the red cell membrane. This is called
sensitization.
Stage-2: The antibody coated red cells i.e. sensitized red cells come close together by
forming bridges created by antibody. This result in aggregation of red cells called
agglutination.
Important; agglutination is a reversible process. Reaction should be read immediately

without delay.
IN-VIVO PROCESS (Condition in-side the human body): When donor red cells containing
non-self foreign protein (antigen) on their membrane enter to the recipient’s, they induce the
production of antibodies.
In certain conditions antibodies bind with the red cells and activate the complement cascade.
This result in the rupture of red cells with release of intracellular hemoglobin resulting in
pink or red supernatant and this process is called hemolysis.
Whenever hemolysis is noted, the possibility of an error at the time of sampling must be
ruled out.
Hemolysis is as important as agglutination because both processes are indicative of antigen-

antibody reaction (positive).
26 - ABO Blood Grouping System:

About 15 blood group systems are recognized;
ABO, Rh, Kell, Duffy, MN, P, Lewis, Lutheran, Xg, Li, Yt, Dombrock, Colton, Public
antigens & Private antigens. However– ABO & Rh account 98% common.
Landsteiner’s discovered the ABO Blood Group System in 1901. He classified blood into 4
main classes according to their phenotypes (A, B, AB, and O)
Landsteiner’s Rule: If an antigen (Ag) is present on a patient’s red blood cell the
corresponding antibody (Abs) will NOT be present in the patient’s plasma, under ‘normal
conditions’.
Landsteiner’s says Individual’s will form immune antibodies (naturally occurring) to ABO
blood group because they were thought to arise without antigenic stimulation (antigens they
do not possess).
27 - ABO Inheritance and genetics:

Two genes inherited, one from each parent. Individual who is A or B may be homozygous
or heterozygous for the antigen.
Heterozygous: AO or BO
Homozygous: AA or BB
The ABO Blood group antigens are expressed by individual’s red cells which are
carbohydrate in nature. The alleles for Blood group are in the same place on the
chromosome 9. However the genes have a different code giving the different blood group.
Rh gene located on short arm of chromosome 1.
A & B blood groups genes are dominant over the O blood group whereas A and B group
genes are co-dominant on each other.
Phenotype is the actual expression of the genotype, i.e., group A
Genotype is the actual inherited genes which can only be determined by family studies, i.e.
AO. Two genes inherited, one from each parent.
Example of Determining Genotype:
Mom’s phenotype is group A, genotype AO

Dad’s phenotype is group B, genotype BO
Then their babies’ phenotypes are:
28 - Rhesus Blood Group System:

Scientists sometimes study Rhesus monkeys to learn more
about the human anatomy. While studying Rhesus monkeys, a
certain blood protein was discovered. This protein is also
present in the blood of some people. Other people, however, do
not have the protein.
The presence of this protein, or lack of it, is referred to as the
Rh (for Rhesus) factor.
If your blood does contain this protein, your blood is said to be
Rh positive (Rh+).
If your blood does not contain this protein, your blood is said to be Rh negative (Rh –). The
function of the Rh polypeptides is not fully known, but it seems that it is involved in cation
transport across the red cell membrane.
The Rh antigens are developed before birth (they are detectable in 6-week-old fetus).
29 - Preparation of Red Cell Suspension:
Principle:
Whole blood sample are collected in EDTA tube, which contain too heavy concentration of
red cells and routine methods that are used for red cell antigen typing or antibody detection
on them require an optimal concentration of red blood cells to obtain valid result.
Sample:
For red cell suspension required sample in EDTA purple top tube (1 – 2cc)
Formula:
Dilutions can be prepared from more concentrated blood sample by using of the following
formula:
V1 × C1 = V2 × C2
Whereas:
V 1 = Initial /Original Volume
C 1= Initial /Original Concentration
V 2 = Final / Desired Volume
C 2 = Final / Desired Concentration.
Example: If 5% suspension of red cell is required with the final volume 2ml. How should
the red cell be diluted?
Answer: According to formula:

V1 × C1 = V2 × C2
Data: Where as:
 V 1 =??
 C 1 = 100 %
 V 2 = 2 ml
 C2=5%
Solution: Now put it into formula:
V 1 × 100 = 2 × 5
V1 = 2×5
100
= 10 = 0.1
100
Result: Therefore, mix 0.1 ml (100 μl) of washed cell (packed red cell) is added to 1.9 ml
(1900 μl) of normal saline to obtain 2.0 ml of 5% red cell suspension. (1ml = 1000 μl and 50
μl = 1drop)
Procedure:
To make 5% approximate red cell suspension in 2ml of normal saline:
 Take one clean test tube and label them with the patient ID number.
 Place 5 drops of the red cells from EDTA tube.
 Wash red cells 1 time with the normal saline at 1000 rpm for 60 seconds but
specimen is grossly lipemic or turbid repeat washing 2 times more. After each
washing complete decent the supernatant.
 Take another clean test tube mark them 5% with patient ID. Put 1.9 ml (1900 μl) of
normal saline and added 0.1 ml (100 μl) of washed cells (packed red cells) to obtain
2.0 ml of 5% red cell suspension.
 Compare the suspension with the standard (screening cells).
30 - Preparation of Control IgG Sensitized Cells (Check Cells):
Principle:
IgG Sensitized Cells (Check Cells) are used to validate all negative results which using anti
human globulin (AHG) reagent. Check cells ensure that the washing was complete (all
unbound proteins removed) and AHG reagent was added.
Procedure:
 Label on clean test tube with CC
 Take 5 segments from different “0” positive packed red cells and take 100 µl from
each into test tube.
 Add 5 drops of anti D (IgG) Human in it.
 Mix well & incubate at 37 °C for 15 minutes. During incubation also mix after every
5 minutes.
 Than wash the cells 3 times normal saline & make a 5 % suspension with saline.
 Then check QC by label 2 tubes one control and second for test.
 In control tube add 2 drop saline and 1 drop check cell.
 In test tube add 2 drops AHG and 1 drop check cell
 Immediate spin (Centrifuge at 1000 rpm for 25 sec), record Results
Interpretation:
The reaction should be ≥ 1 + agglutination in test tube and negative in control tube.
Preservation and Storage:

The 5% suspension of IgG coated cells is stored at 2 – 6 °C in an EDTA anti coagulant tube
for 7 ± 2 days.
The 5% suspension in plain test tube with normal saline can use 12 – 24 hours at room
temperature.
31 - Blood Grouping:
Blood grouping can be performed on the cells as well as on the serum.
When it is performed on the red cells, it is called Direct Grouping (Forward Typing) in
which unknown red cells (test cells) are tested against known anti-sera.
When it is performed on the serum, it is called Indirect Grouping (Reverse Typing) in

which the unknown serum (test serum) is tested against known red cells. Reverse grouping
is perform to check validity of results and to find out ABO discrepancy.
Ideally both tests should be performed on each specimen by Tube Method.
Sample:
Clotted blood sample (4 - 6 cc)
EDTA Sample (1 – 2 cc)
Materials:
 Round bottom glass tubes
 Pasture pipettes
 Isotonic saline 0.9%
 Centrifuge machine
 Blood grouping anti sera for forward grouping
Anti-A
Anti-B
Anti-AB
Anti D
 Known “a”cells, “b”cells and “o”cells (negative group are required, in case of non
availability positive group can be used) for reverse grouping
Procedure:
 Centrifuge Clotted blood sample at 4000 rpm for 5 minutes.
 Prepare 5% red cell suspension of patient / donors blood for forward grouping (from
the EDTA sample).
 Label 8 clean test tubes with patient case no (for patient) or donor no (for donor) and
label Anti A, Anti B, Anti AB, Anti D in 4 forward tubes and label Cell a, Cell b,
Cell o and Auto Control respectively in reverse grouping.
 Add 1 drop of Anti-A, 1 drop of Anti-B, 1 drop of Anti-AB and 1 drop of Anti-D in
their respectively labeled tubes.
 Add 2 drops of patient’s serum into each labeled Cell a, Cell b, Cell o and Auto
Control tubes.
 Add one drop of patients or donor’s 5% red cell suspension to label Anti A, Anti B,
Anti AB, Anti D and Auto Control tubes.
 Place one drop of each known 5% red cells suspension into their respectively label
tubes Cell a, Cell b and Cell o for reverse grouping.
 Centrifuge all the test tubes at 1000rpm for 25 sec to get clear agglutination.
Note: Anti-AB anti-sera are used to detect weak antigens, sub-groups and for confirmation
of O group.
Table -1: Interpretation of Results of ABO & Rh Testing:
Agglutination is positive results and Absence of agglutination in the tube or a smooth cell
suspension after re-suspension the cell button is a negative test result.
Anti-A Anti-B Anti-AB Anti-D A1-cells B-cells O-cells Auto Results

- - - - + + - - O’-ve
- - - + + + - - O’+ve
+ - + - - + - - A’-ve
+ - + + - + - - A’+ve
- + + - + - - - B’-ve
- + + + + - - - B’+ve
+ + + - - - - - AB’-ve
+ + + + - - - - AB’+ve
{+} Positive = Cells are agglutinated. {-} Negative = Cells are not agglutinated.
Table -2: Grading of Results in Red Cells Agglutination Test:
Symbol Agglutination score Description

Cell button remains in one solid agglutinate
4+ 12
macroscopically visible
Cell button dislodges into several large
3+ 10
agglutinates
Cell button dislodges into many medium
2+ 8 size agglutinates with clear background
macroscopically visible
Cell button dislodges into small agglutinate,
1+ 5 turbid background clumps, macroscopically
just visible
Cell button dislodges into fine granules,
(+) or w (Weak) 3
only visible microscopically.
Negative results i.e.; all cells free and
0 0
evenly distributed, No agglutination
Mixtures of agglutinated and in-
Mf(mixed field) -
agglutinated RBCs
H - Complete hemolysis
PH - Partial hemolysis, Some RBCs remain
32 - Rh-Control for AB Positive:

In AB positive blood group to rule out false positive reaction or pan agglutination in anti-D
tube we run Rh-control. Since anti-A and anti-B are crosschecked by reverse grouping
where as no crosscheck for anti-D that’s why it is called Rh-control.
I.e. put one drop of 6% Albumin and one drop of 5% patient’s red cells in the tube,
centrifuge at 1000 rpm for 25 seconds and check agglutination. The result of this tube
should be negative for validation of group AB+ve.
33 - Weak-D or D u Testing For Anti-D Negative:

Principle:
Some RBC’s carry a D antigen that is expressed so weakly that they are not directly
agglutinated by most anti-D reagents, Weak expression of D antigen can be recognized by
the Du Testing after incubating the RBC with anti-D.
Reagents:
 5% red cell suspension
 Anti-D anti-sera
 6% albumin
 Antihuman globulin (Comb’s) reagent
 IgG-coated red cells (Check cells)
Procedure:
 Wash the cells 3 times by normal saline and make 5% red cells suspension.
 Take two tubes & mark them as Du C & Du T.
 Place 1 drop of anti-D in a Du T labeled test tube and 1 drop of the 6% albumin in a
second labeled Du C test tube.
 To each tube, add 1 drop of 5% red cell suspension.
 Centrifuge at 1000 rpm for 25 sec gently resuspend the cell buttons and examine the
tubes for agglutination.
 Mix and incubate both tubes at 15 to 30 minutes at 37 °C.
 Centrifuge at 1000 rpm for 25 sec gently resuspend the cell buttons and examine the
tubes for agglutination.
 If the test red cells are strongly agglutinated in the Du T tube but not in the Du C tube,
record the test result as Du + and do not further proceed.
 If the both tube cells are not agglutinated, keep the Du C tube as it is & further
process the Du T tube.
 Wash the Du T tube cells three times with large volumes of saline, decant completely
after last wash.
 Add 2 drops of antiglobulin (Comb’s) reagent. Mix gently and centrifuge at 1000
rpm for 25 sec.
 Examine the tubes for agglutination, if positive result grade them and record the test
result.
 If the test result is negative conform the validity of negative or weakly positive
results with IgG sensitized (coated) red cells (Check Cells).
 After adding check cells negative result should be converted to positive result.
Note: Anti-D contain low protein media so it does not give false positive result while using
high protein media (e.g. 22% Albumin) therefore we use 6% albumin for control.
34 - Bombay Blood Group:

 The H gene appears to be necessary for the formation of A and B Ags. It is very
common 99.9% of all individuals have an HH or Hh genotype.
 The allele h is very rare and does not produce the L-Fucose transferase, which is
necessary for make to complete H-Structure.
 The genotype (hh) or H null is extremely rare and is known as the Bombay
Phenotype or Oh.
 The Bombay Phenotype was first reported by Bhende 1952 in Bombay, India.
 Bombay RBCs are NOT agglutinated with anti-A, anti-B or anti-D (due to lack of H
antigen) but Bombay serum has strong agglutinating with cell-A and cell-B. Not
recognized until serum tested against group O cells and causes strong agglutination.
Anti-A Anti-B Anti-A,B A1 Cells B Cells O Cells Auto Control
0 0 0 4+ 4+ 4+ 0
 Bombay blood group also confirmed by the reagent anti-H, when it was tested with
Bombay RBCs it not agglutinating.
 People who have Bombay phenotype can donate to any ABO blood group provided
that Rh group is compatible.
 However, people with Bombay phenotype cannot receive blood from any member of
the ABO blood group system (which always contains one or more of A and B or H
antigens), but only from people who have Bombay phenotype.
 The Bombay patient serum has anti-H, which is active over a wide thermal range. It
is an IgM antibody that can bind complement and cause red cell lyses. Because the H
Ag is common to all ABO blood groups, Bombay blood is incompatible with all
ABO donors.
35 - Sub Groups of A:
ABO subgroups differ in the amount of antigen carried on red cells.
Subgroup of A are more commonly encountered than subgroups of B. The two principal
subgroups of A are A1 and A2. Red cells from A1 and A2 persons both react strongly with
reagent anti-A. The serologic distinction between A1 and A2 cells can be determined by
reagents (lectin prepared from dolichos biflorus seeds).
Biflorus lectins act as an anti- A1, and agglutinates A1 not A2 red cells. Approximately 80%
of group A or group AB individuals have red cells that are agglutinated by anti-A, antisera
A1 and thus are classified as A1 or A1B. The remaining 20%, whose red cells are
agglutinated by anti-A but not anti- A1, are called A2 or A2B.
Number of A & B Antigens Sites on Erythrocytes (Adult)
Antigen Number of Antigenic Sites

A1 810,000 to 1,170,000
A2 240,000 to 290,000
A3 35,000
Ax 4,800
460,000 to 850,000 (A)
A1B
310,000 to 560,000 (B)
B 610,000 to 830,000
Compared with the red cells of adults, the red cells of newborn infants have about one-third
the number of A and B antigenic sites.
36 - ABO Discrepancy:
An ABO discrepancy exists when the patient cells reactions will not match with serum
reactions. Do not report the ABO group until the discrepancy is resolved.
Positive reactions between the direct and reverse typing should react 2+ or stronger. If
results are weak or 1+ positive in any ABO direct or reverse typing test require further
investigation (microscopic readings should only be done if mixed field agglutination is
suspected).
ABO discrepancies may be arises due to:
A – Technical error:
 Sample collection error
 Contaminated sample or reagent
 Glass tubes not washed properly
 Equipment performance changes: centrifuge, cell washer, agglutination viewers
B – Human error:
 Manufacturer’s directions not followed
 Failure to recognize hemolysis or weak positive reaction (less technical expertise)
 Test reagent or serum not added
 Inappropriate ratio of sample or reagent is added
 Not centrifuge sufficiently or over centrifuge
 Incorrect interpretation or recording of results
C - True error:
1- Weak-reacting or missing antigens:

 Weak expression of A or B antigen may be due to some diseases such as
gastrointestinal carcinoma or obstruction excessive in which ABH blood group
antigens are neutralized from RBCs and found in the serum which known as
substances
 Sub groups: weaker reactions can also found in those persons who have sub blood
groups of A or B or AB (such as A1, A2, A2B and subgroups of B).
 Transplant history: A mixed field reaction may be observed in a patient who has
had bone marrow transplantation or transfused with ABO and Rh compatible red
cells other than the patient’s own blood group type (i.e. An A or B recipient recently
transfused with group O red cells and Rh negative transfused with an Rh positive
group).
2- Weak-reacting or missing antibodies:

 Antibody production is minimal / weak if Age related <4 – 6 months old or elderly ≥
90 years age.
 Reverse grouping not indicated in newborns
 Decrease antibody reaction in immunosuppressive patients or on chemotherapy

 Prozone: A phenomenon associated with antibody excess, binding all the sites of red
cells antigen, leaving no sites on red cells for cross linking. Visible agglutination
cannot take place. Resolution: if suspect Prozone, repeat the test by diluting the
serum with normal saline
3- Unexpected antigen reaction:

 Rouleux formation; Mostly multiple myeloma or dysproteinemia is responsible for
both unexpected antigen and unexpected antibody reactions, resolved by using saline
replacement technique. Strongest reaction at I.S, weaker at 37 °C and not observed at
AHG phase.
 Wharton jelly in newborn: Primitive tissue of the umbilical cord may contaminate
cord blood and may cause unexpected antigen reaction. Resolution: washing red cell
several times 4-6 or Collect blood from vein.
 Polyagglutination; resolving by using monoclonal antisera
4- Unexpected antibody reaction:

 A2 patients in 8% develop anti A 1 antibodies.
 Cold antibody (auto or allo antibodies); resolved by incubation at 37 °C
 Allo-antibodies such as anti-M,-N,-P1, Lea or Leb may react with the A or B cells at
IS (immediate spin) or RT (room temperature)
Note: If an ABO discrepancy is detected, and transfusion is necessary before resolution,

issue only Group O Rh compatible red cell units until the problem is resolved.
If an Rh typing discrepancy is detected, and transfusion is necessary before resolution, only

Rh negative red cells should be issued until the problem is resolved
37 - Resolving ABO Discrepancies:

 If there is any doubt as to the identity or the quality of the specimen or if the group
differs from the historical result, collect a new specimen and repeat the ABO group.
 Check the label(s) on the reagent vial(s) to ensure the correct reagents were used in
the initial testing. Check the reagent(s) appearance for possible contamination by
comparing the reagent used with an unopened vial of the same reagent. Use a new
reagent vial if the current vial appears contaminated.
 Use new glassware, Prepare a new 5% patient washed red cell suspension. Repeat
the ABO testing.
 If unexpected reaction seen in A-cells only use Anti-A1 to check out A sub groups
(A2, A2B).
 In D negative mothers during 1st D positive baby birth the mother was immunized
through placental barrier and produce Rh antibodies. Due to this might be weaker
reaction is seen in reverse grouping so use negative group cells for reverse grouping.
 If false negative or weaker reaction is suspected, then incubate at 4°C for 15-30
minutes to enhance the reaction.
 If false positive or unexpected reaction is seen, then incubate at 37°C for 15-30
minutes to dissolve the reaction.
 If the problem is resolved, interpret the ABO group and record it on the request form.
If the problem is not resolved, test the serum with screening cells. Obtain review the
patient diagnosis, transfusion and obstetrical history.
 If ABO cannot be determined at this time, consult with the consultant or clinical
pathologist of the blood bank.
Note: People with blood group O are called "universal donors" and people with blood group
AB are called "universal receivers."
38 - Direct Anti-Globulin Test for Poly-specific by Tube Method:

Principle:
In normal healthy individuals there are no antibodies present against own circulating red
cells antigens. The direct anti-globulin test is used to demonstrate whether or not red cells
have been coated with anti-D antibody or other antibodies in vivo.
This occurs in the following circumstances:
 In D negative mothers during 1st D positive baby birth the mother was immunized
through placental barrier and pregnancy was not affected but in the 2nd pregnancy
and onward will be affected because mother’s immune system begins to produce Rh
antibodies. The mother’s Rh antibodies cross the placenta and after entering the
baby's blood stream, these antibodies will attach to the baby's Rh-positive red blood
cells.
 After transfusion reactions

 Drug induced red cells sensitization
 Autoimmune hemolytic anemia
In direct combs test we sensitize red cells by the addition of antihuman globulin and
centrifuge to bring sensitize red cell closer together result in macroscopic agglutination.
Requirements:
 Test tubes
 Pasteur pipettes
 Incubator
 Centrifuge
 6% albumin
 Antiglobulin (Comb’s) reagent
 Check cells
Sample:
 EDTA 2cc venous blood sample
 Prepare 5% red cell suspension
Procedure:
1) Label 2 test tubes as one for Test-DAT and second for NC-DAT (negative control).
Place one one drop of 5% red cells suspension in both tubes.
2) Wash the red cells suspension, three times in large volume of saline. Care should be
taken for adequate removal of the supernatant after each wash.
3) Get dry cell button after third wash.
4) Add 1 drops of anti-human globulin to Test and one drops of 6% albumin in NC tube
mix the contents of the tube thoroughly.
5) Centrifuge the tube for 25 seconds at 1000rpm.
6) Gently re-suspend the cells button and examine for agglutination in agglutination
viewer light.
7) Confirm the validity of all negative reactions with IgG sensitized red cells.
Results:
Positive DAT = Agglutination or hemolysis of red cells at the antiglobulin phase
Negative DAT = No agglutination of red cells at the antiglobulin phase
Note: If NC tube is positive than result should be report as invalid and advise for further
investigation.
39 - Positive Direct Anti-Globulin Test for Monoclonal: (By Tube Method)
If test with poly-specific ant-globulin is positive precede with monoclonal anti-sera IgG and
C3d.
39.1 - Test with Anti- IgG:

 Wash red cells 3 times with normal saline solution and decent the supernatant.
 Prepare a 5% suspension of the washed red cells in normal saline.
 Add 2 drop of the mono-specific IgG and add 1 drop of the red cell suspension into a
clean tube.
 Mix well and incubate for 5 minutes at room temperature (18 – 25 0C).
 Centrifuge for 25 seconds at 1000 rpm.

 Carefully read the result by agglutination viewer light source.
39.2 - Test with Anti-C3d:

 Wash red cells 3 times with normal saline solution and decent the supernatant.
 Prepare a 5% suspension of the washed red cells in normal saline.
 Add 1 drop of the mono-specific C3d and add 1 drop of the red cell suspension into a
clean tube.
 Mix well and incubate for 5 minutes at room temperature (18 – 25 0C).
 Centrifuge for 25 seconds at 1000 rpm.
 Carefully read the result by agglutination viewer light source.
 A positive DAT does not necessarily mean that a person’s red cells have shortened
survival. Small amounts of both IgG and complement are present on all red cells.
DAT Normally in healthy individual range Positive case in Detectable range

IgG 5 to 90 molecules 100 to 500 molecules
C3d 5 to 40 molecules 400 to 1100 molecules
40 - Direct Anti-Globulin Testing By Column Agglutination Technique:

(By Gel Technique)
Principle:
This method has been devised to allow simultaneous performance of several tests. Red
blood cells are filtered through a column containing a medium that separates selected red
cell population i.e. agglutinated from un-agglutinated red cells, based on aggregate size.
Requirements:
 DiaMed ID Centrifuge
 DiaMed ID Diluents 2 (Modified Less)
 Micro typing Cards (Gel Columns)
 Test Tubes and Disposable tips with jester /pipetter
Sample:
 EDTA 2cc venous blood sample
Procedure:
 Allow all reagents too reach room temperature before use
 Put the identification number underneath each column
 Prepare a 0.8% suspension of patient red cells as follows:
10 µl RBC + 1.0 ml ID-diluents 2
OR
20 µl Whole Blood + 1.0 ml ID diluents 2
 Mix well
 Remove the aluminum foil as per test requirement
 Add 50 µl 0.8% suspension patient red cell suspensions to the appropriately labeled
micro tube
 Centrifuge this card for 10 minutes in the ID-Centrifuge (at 1000 rpm).
 Interpret the result.
Interpretation:
Positive = If red cells are on the top constitute Positive result
Negative = If sink down than negative result
Note: Grading is done according to the reactions shown in the kits literature.
41 - Inspecting Gel-cards Before Use:

• Do not freeze or expose cards to excessive heat, Store upright at 2 to 25°C. If the
cards have not been stored in an upright position, centrifuge the cards before use.
• Do not use cards that show signs of drying. A liquid layer should appear on top of
the gel in each micro tube.
• Do not use cards in which the micro tubes show discoloration, bubbles or crystals.
• Do not use the micro tube if the seal of the micro tube appears to be damaged or
opened.
42 - Compatibility Test (Cross Matching) By Tube Method:
According to the AABB Standards:
The cross-match is defined “shall use method that demonstrate ABO & Rh incompatibility
and clinically significant antibodies against to donors red cell antigens”
The purpose of cross match is:
 Selection of safest blood components for transfusion

(It prevent from transfusion reactions)
 Increase in vivo survival of donors red cells
 Confirms ABO compatibility (Double checks for ABO errors)
 Another method of detecting clinically significant unexpected antibodies
But compatible cross match will not guarantee:

 Guarantee normal survival of RBCs
 Prevent patient from developing an antibody
 Detect all antibodies
 Prevent delayed transfusion reactions
The Compatibility testing or pre-transfusion testing or cross matching test is the purpose to
ensure that the select blood components will not cause harm to the recipient and will have
acceptable survival when transfused.
The compatibility test consists of a series of procedures performed to detect any irregular
antibodies in the serum of the recipient that would adversely affect the survival of the donor
red cells after transfusion. Negative results indicate compatibility.
Sample:
 EDTA Sample (1 – 2 cc)
 Clotted blood sample (4 - 6 cc)
 Donor’s blood bag
Donor cells are taken from segments that are attached to the unit itself which
eliminate having to open the actual unit.
Procedure:
Identify patient sample and centrifuge clotted sample for serum separation. Determine
patients ABO and Rh group.
Select unit of blood for cross matching and determine ABO and Rh of this unit (perform
only forward grouping).
The test is performed in three phases and takes about 60 minutes.

 Phase 1 The Immediate spin
 Phase 2 The Incubation 370C / Albumin technique
 Phase 3 The Indirect anti-globulin techniques.
1-The Immediate spin:
The test is performed by mixing donor RBCs with patient serum at room temperature for
macroscopic agglutination. The test takes 1-5 minutes and use to detect ABO
incompatibility.
 Place two drops of patient serum in a labeled test tube with donor and patient case
number.
 Add one drop of washed 5% suspension of the donor red cells. Donor red cells
should be saved in the form of segment until blood unit was transfused.
 Centrifuge at 1000 rpm for 25 seconds
 Examine macroscopically for hemolysis and agglutination through agglutination
viewer. Record results and proceed to next step.
2-The Incubation 37 0C / Albumin technique:
This second phase involves incubation of the first phase reaction at 37 °C in albumin and/or
low-ionic strength salt solution. This aids the detection of incomplete antibodies that are
able to attach to a specific antigen but are unable to cause agglutination in a saline solution.
Solution of 22% bovine albumin is used as potentiators of certain antigen, antibody
interaction. Bovine albumin acts to increase the dielectric constant of the medium there by
reducing the zeta potential and allowing IgG antibodies to be demonstrated. The majority of
IgG antibodies are detectable in this way.
This phase takes 30-45 minutes to complete and this is performed to demonstrate
clinically significant IgG antibodies present in recipient serum to corresponding
antigen on donor cells.
 Add three drops of 22% bovine albumin to tube containing 2 drops of patient serum
and one drop of donor cells
 Mix the contents of the tube thoroughly incubate at 37 0C for 30minutes.
 Centrifuge at 3500rpm for 25 seconds.
viewer. Record results and proceed to next step.
3-Indirect Anti-globulin Technique (IAT):

This third phase of the cross match involves the addition of antiglobulin sera to the
incubated test tubes. With this addition antibodies present in the sera become attached to the
antibody globulin on the RBCs causing agglutination. This phase identifies the most
incomplete antibodies from all blood groups systems including Rh, Kell, Kidd, and Duffy
systems.
The IAT is used for the detection of significant antibodies that may cause red cells
sensitization in vitro. If both IgG antibody and the corresponding antigen are present in a
serum red cell mixture, incubation will cause the antibody to attach to the specific antigenic
receptors on the red cell. After washing, to remove the excess antibody in the serum, the
addition of antiglobulin serum will link the IgG molecules on neighboring red cells,
producing agglutination.
This third phase is only performed on blood yielding a positive antibody screen and requires
10 – 15 minutes.
 Wash the albumin tube cells three times with normal saline being careful to decant
completely after each wash.
 Add two drops of anti-human globulin to the tube.
 Mix the contents of the tube thoroughly and centrifuge at 1000rpm for 25 seconds.
viewer.
 Record results.
 Confirm the validity of negative results with IgG sensitized red cells.
Results:
Positive test = Agglutination of the red cells
Negative test = No agglutination of red cells
43 - Compatibility Test (Cross Match) By Column Agglutination Method:

(By Gel Technique)
This method has been devised to allow simultaneous performance of several tests. Red
blood cells are filtered through a column containing a medium that separates selected red
cell population i.e. agglutinated from un-agglutinated red cells, based on aggregate size.
Sample:
Red Blood Cells of Donor
Serum or Plasma of recipient
Requirements:
 DiaMed ID Centrifuge
 DiaMed ID Incubator 37 °C
 DiaMed ID Diluents 2 (Modified Less)
 Micro typing Cards (Gel Columns)
 Test Tubes
 Disposable tips with jester /pipetter.
Procedure:
 Allow all reagents too reach room temperature before use
 Put the identification number underneath each column (the donor unit number and
the patients identification number)
 Prepare a 0.8% suspension of donor unit red cells as follows:
10 µl PRBC + 1.0 ml ID-diluents 2
OR
20 µl Whole Blood + 1.0 ml ID diluents 2
 Mix well
 Remove the aluminum foil as per test requirement
 Add 50 µl 0.8% suspension donor unit red cell suspensions to the appropriately
labeled micro tube
 Add 25 µl of patients / recipient serum or plasma to the appropriately labeled micro
tube
 Incubate the micro typing card for 15 min at 37 °C in the ID-incubator
 Centrifuge the gel card(s) in the ID-Centrifuge centrifuge at the preset specifications
(10 minutes at 1000 rpm).
 Interpret the result.
Interpretation:
Positive (Incompatible cross match) = If red cells are on the top constitute Positive result
Negative (Compatible cross match) = If red cells sink down than negative result
Note: Grading is done according to the reactions shown in the kits literature.
44 - Compatibility Test (Cross Match) for Neonates:

Neonates (patients under 4 months old) have unique pre-transfusion testing requirements.
Because allo-immunization is extremely rare and repeated testing increases iatrogenic blood
loss, so requires limited number of test.
Sample:
 EDTA 1 cc of infant
 Red top 4 cc of infant and 6 cc of mother serum
A cord blood specimen must NOT be used for pre-transfusion testing. The serum or plasma
of either the mother or the neonate may be used to perform the antibody screen and cross-
match.
Initial testing must include ABO and D typing (forward grouping) on baby red cells only, if
Rh is negative no need for weak D testing. The reverse ABO group is omitted when testing
neonates.
Antibody screening on the mother serum (If mother serum is not available perform antibody
screening on infant sample). If antibody screening is negative, select group specific (infant)
unit, perform only forward grouping on unit, followed by immediate spin cross match.
Release the unit, without proceed to albumin and IAT phase until the baby was admitted or
neonate reaches the age of 4 months.
If positive antibody screen perform:
 Perform DAT
 Complete cross-match with donor red cell unit
Select the appropriate unit for cross-match as all red cell components must be:
 As fresh as possible, must be less than 14 days old
 If any ABO Discrepancy select O Negative red cells unless mother has a significant
antibody that is incompatible with O Negative (e.g. anti-c, anti-e)
 In infants due to small amount of blood is needed so arrange pedi-pack (75ml) blood
bag to save excess blood lose and to prevent from contamination.
 If arrange 2 or three 75 ml pedi-pack and all was from same unit then cross match
with only one of them.
45 - Incompatible cross match:
Agglutination or hemolysis indicates a positive result (incompatiblity)
 IS detect RT reactive IgM antibodies

 IAT phase reactive clinically significant IgG antibodies
If cross match is incompatible:
 Check the correct identification of patient specimen and donors unit

 Visual inspection of patient specimen for hemolysis
 Proceed to antibody screening with auto control
Resolving of incompatible cross match through antibody screening results:
1 - If negative antibody screening and negative auto control, the possibilities for cross-match
incompatibility are:
 ABO Incompatible ( Recheck ABO group donor/ patient )

 If blood group A / AB check with Anti A1 for sub group A2 or A2B
 Examine under microscope for rouleux formation
 Perform DAT on donor red cell if positive discard unit and use another unit for cross
match
 Get history of transfusion ( Passive transfused anti A or anti B )
 Cold alloantibody
 Antibody with dosage effects ( Perform antibody identification )
2 - If positive antibody screening and negative auto control, the possibilities for cross-match
 Alloantibody (Perform antibody identification)

 Also perform cross match with 4 – 6 bags, issue compatible bag
3 - If positive antibody screening and positive auto control, the possibilities for cross-match
 Autoantibody
 Examine under microscope for rouleux formation
 Reagent related check it and any doubt than change reagent
 Alloantibody (Perform antibody identification)
 Passive infused antibody (Get history of transfusion)
 Perform DAT with patient sample
 If DAT is negative than Issue antigen negative unit

 If DAT positive than perform cross match with 4 – 6 bags and issue least
incompatible unit by comparing with auto control.
45.1 - Rouleaux formation:

 Rouleaux formation is a property of serum that causes all cells tested to appear
agglutinated at RT but it does not affect at AHG phase because serum is removed by
washing.
 To confirm that the pseudo-agglutination or rouleaux before washing cells, use the
saline replacement technique.
45.2 - Saline replacement technique:

 Spin tubes down again and remove serum
 Add 2 - 3 drops of saline and re-spin
 Rouleaux formation will disperse with the addition of saline, true agglutination will
not.
46 - Blood Transfusion Reaction:

Any unfavorable transfusion-related event occurring in a patient during or after transfusion
of blood components is considered an adverse effect of blood transfusion. It is also referred
to as a Transfusion Reaction.
Most common cause is clerical error (misidentification of the patient). If the wrong blood
type is used the person’s own immune system immediately attacks the donor’s blood and
causes clots and RBCs destruction that can lead to total kidney failure and death.
Types of Transfusion Reactions:
46.1 - Acute (within hours) Transfusion Reactions:
 Acute hemolytic reactions (rapid destruction of RBCs mostly ABO incompatibility)

 Febrile reactions (leukocyte incompatibility = Recipient antibodies is react with
donor leukocyte antigens)
 Allergic reactions (reaction to plasma proteins—IgM, IgA, IgD, IgG, IgE), usually
involves release of histamine and can pre-treat recipient with anti-histamines before
transfusion.
 Anaphylaxis (Recipient is IgA deficient & has anti-IgA in serum which can react to
even small amounts of donor IgA in the plasma in any blood component and treat
with by using wash blood components to remove plasma).
 Non Cardiogenic Pulmonary Edema (NCPE): When transfusion recipient
experiences acute respiratory insufficiency and/or evidence of pulmonary edema
without evidence of cardiac failure. Primary Suspect that Donor antibodies to
recipient WBCs, another cause is WBC emboli aggregate in the lungs causing edema
also called TRALI (Transfusion Related Acute Lung Injury) and treated by giving
leukoreduced blood products to patients.
 Circulatory overload: Rapid increases in blood volume to patient with compromised
cardiac or pulmonary status (Most at risk are elderly and pediatric patients). Infusion
of 25% albumin is also a cause. Prevented by stopping infusion and place patient in
sitting position. Slow down future infusions.
 Sepsis: Improper handling/contamination
 Hypocalcaemia: When plasma (or platelets) are infused at rate >100 mL/minute or
individuals with impaired liver function citrate is broken down by liver. Seen more
in pediatric and elderly patients. Treated by slowing or discontinuing infusion.
Administration of Calcium is not usually necessary.
46.2 - Delayed (>24 Hours) Transfusion Reaction:
A-Transfusion transmitted infections (TTI):

 Viruses: Hepatitis B, C; HIV I & II; HTLV I & II; CMV
 Bacteria: Treponema pallidum (Syphilis); Salmonella
 Parasites: Malaria; Toxoplasma; Microfilaria
B-Immune sensitisation:
 Delayed Hemolytic Transfusion Reaction: Antibodies that usually do NOT activate
complement to completion: Rh, Kell, etc. Associated with extra-vascular hemolysis.
Prevented by giving antigen negative blood.
 Post-transfusion purpura (PTP): Antibodies to platelet antigens causes abrupt onset
of severe thrombocytopenia (platelet count <10,000/l) 5-10 days following
transfusion. Treated with high dose intravenous immunoglobulin (IVIG).
 Transfusion associated graft-versus-host disease (TA-GvHD): Donor T-

Lymphocytes attack recipient (host) tissues. It risk in Immuno-compromised patients
(Cancer, fetus, neonatal, bone marrow transplant and HIV). Prevented by Irradiation
of blood products.
 Transfusion associated lung injury (TRALI)
 Iron overload: Excess iron resulting from chronically transfused patients such as
hemoglobinopathies, chronic renal failure, etc. Treatment is removal of iron without
reducing patients circulating hemoglobin. Infusion of deferoxamine - an iron
chelating agent has been useful.
 Infectious Complications of Blood Transfusion: At what point is the bacteria

introduced into the donor unit? At time of collection: either from the donor or the
venipuncture site, during component preparation, etc. Most often from platelet
components (room temp). Prevention is maintaining standards of donor selection,
blood collection and proper maintenance of collected blood components.
47 - Instruction for Blood Transfusion Reaction:

If the patient has a transfusion reaction:
 FIRST—Stop the transfusion
 Run normal saline 0.9% to keep the vein open
 Call the Doctor and Stay with the patient.
 Prepare to administer emergency meds
 Return blood bag with transfusion set and transfusion reaction form to the blood
bank and blood samples with proper labeling:
Clotted specimen (4 – 6 cc)
EDTA specimen (1 – 2 cc)
 1st voided urine specimen post transfusion send to laboratory
 Collect blood sample 5-7 hours post transfusion to check for bilirubin
48 - Re-cross-matching after Transfusion Reaction:
I. Repeat ABO Grouping and Rh Typing:

 Perform blood grouping on recipient pre-transfusion sample, post-transfusion sample
and donor pre-transfusion segment and post-transfusion blood bag
II. If blood grouping is correct then re-cross-match:

 Recipient pre-transfusion sample with blood bag and pre-transfusion bag segment
 Recipient post-transfusion sample with blood bag and pre-transfusion bag segment
III. If cross match is compatible then perform DAT on recipient post transfusion sample:
Test Result
DAT
Normal Control
IV. If cross match is Incompatible then perform Antibody screening test on recipient post
transfusion sample:
 If negative auto control and negative antibody screening:

 Perform DAT on donor’s cells if positive discard that unit and try second bag.
49 - Hemolytic disease of the Newborn (HDN):

Usually HDN results from D negative female and D positive male producing in
their offspring, when the baby will probably D positive.
In D negative mothers during 1st D positive baby birth the mother was immunized
through placental barrier and pregnancy was not affected but in the 2nd pregnancy
and onward will be affected because mother’s immune system begins to produce
Rh antibodies.
Most anti-A or anti-B antibodies are of the IgM class (large molecules) so these do
not cross the placenta but Rh antibodies are IgG class (small Molecules) that cross
placenta and cause hemolysis of fetal RBCs by agglutination.
Treatment of HDN:
 To prevent this occurrence the female is administered with Rh-IG.
 The anti-Rh gamma globulin or RhoGAM binds with Rh-antigen and
inactivates them.
50 - Antibody Screening:
Detection of unexpected clinically significant antibodies in patients serum other than
naturally occurring anti – A or anti- B, these unexpected antibodies is known as allo-
antibodies which do not react with antigens present on RBC’s.
Clinically significant antibodies are those that are capable of causing hemolysis as a result of
accelerated destruction of transfused red cells.
The production of these antibodies may follow:

 Pregnancy
 Previous transfusion
 By Transplantation
 Injection of Immunogenic materials
 Naturally occurring (unknown)
All antibodies reactive in vitro at 370C therefore antibody screening is also called the
indirect Coombs test or the indirect antiglobulin test (IAT).
For antibody screening test we use commercially prepared 2 – 3 reagent vials of group O
cell suspensions (Screening cells) with known genotyping and obtained from individual
donors.
In antibody screening, patient serum or plasma is tested with known reagent red cells
(Screening cells) that demonstrate clinically significant antibodies.
Implications:
 All pregnant women at 28 weeks irrespective of blood group
 Patients needing a transfusion (before transfusion to make sure patients has no
unexpected antibodies)
 In repeatedly transfused patients
 Case of transfusion reactions
 If hemoglobin is not built up to the desired levels after transfusion
Sample:
Clotted venous blood sample (4 - 6 cc)
EDTA venous blood Sample (2 cc)
History Performa:
With the request of antibody screening need to fill a certain aspects of history Performa,
such as history of resent or past blood or their products transfused, number of pregnancies or
any history of abortion, exposure of anti-D, or severe jaundice after delivery / blood
transfusion.
Requirements:
 Test tubes: (10x75 mm)
 Pasteur pipettes
 Incubator
 Centrifuge
 Reagent Red cells (3 Screening cells)
 22% albumin
 Antiglobulin (Comb’s) reagent
 Check cells
Note: Screening cells licensed by FDA must express the following antigens: D, C, E, c, e,
M, N, S, s, P1, Le a, Leb K, k, Fya , Fyb , Jk a , and Jkb
Test procedure:
Antibody screening should be performed in 3 phases:
1 – Saline phase:
 Take four test tubes; label them screening 1, screening 2, screening 3 and auto
control along with patient case number.
 Place two drops of serum to be tested in each tube labeled
 Add one drop of screening cell 1 in tube number 1
 Add 1 drop of 5% suspension of patient’s RBC’s in 4th auto control tube.
 Mix all tubes well.
 Centrifuge at 1000rpm for 25 seconds.
 Examine for hemolysis and agglutination, Record results.
 Proceed to next step.
2 – Albumin phase:
 Add three drops of 22% Bovine Albumin in all tubes.
 Mix the contents of the tube thoroughly. Incubate in water bath at 370C for 30
minutes.
 Centrifuge at 1000rpm for 25seconds.
 Examine for hemolysis and agglutination.
 Record results and proceed to next step.
3 – Indirect Antiglobulin phase:

 Wash all tubes four times in large amount of normal saline, being careful to decant
completely after each wash.
 Add two drops of anti human globulin to each tube.
 Mix the contents of the tubes thoroughly and centrifuge.
 Gently re-suspend the cell button and examine for agglutination.
 Record result.
 Confirm the validity of negative results with IgG-sensitized red cells.
Interpretation:
Positive results are consistently seen in the presence of antibodies, which will further require
antibody identification. So subsequently antigen negative blood (O’ negative) will be
transfused to prevent hemolysis.
1-In what phase (s) did the reaction (s) occur?
 Low temperature: Abs of the IgM class will react best at low temperatures and are
capable of causing agglutination of saline-suspended red blood cells (immediate spin
reading (IS). Of the commonly encountered Abs are, anti-N, -I. and -P (IgM)
 Abs of the IgG class will react best at the AHG Phase (37°C). Of the commonly
encountered Abs are; anti-Rh, kell, Kidd, and Duffy (IgG).
 Whereas Lewis, M, Abs may be IgG, IgM, or a mixture of both.
2-Did more than one screening cell react and if so, did they react at the same strength
and phases?
 A single Ab specificity should be suspected when all cells react at the same phase
and strength.
 Multiple antibodies are most likely when cells react at different phases with different
strengths and auto-antibodies are suspected when the auto-logous control is positive.
3-Are the cells truly agglutinated or rouleaux present?
 Serum from patients with multiple myeloma or who have received high molecular
weight plasma expanders (dextran) may cause non-specific aggregation of red blood
cells known as rouleaux.
 Rouleaux cells have “a stacked coin” appearance when viewed microscopically.
 It will not interfere with the AHG phase of testing because the patient’s serum is
washed away prior to the addition of the AHG reagent
51 - Antibodies Identification:
An antibody identification procedure is performed to identify unexpected antibodies, which
are detected in the antibody screen. Once antibody detected must determine specificity and
clinical significance.
It is performed by the using of a panel of 11 vials, group “O” red cells with antigen
configuration on anti-gram.
Each of the panel cells has been antigen typed (shown on antigram)
+ refers to the presence of the antigen
0 refers to the absence of the antigen
Sample:
• For optimal results, the determination should be performed using a freshly drawn
sample.
• Preferably, whole blood sample should be drawn into EDTA 1 – 2 cc tube and
prepare a 3–5% red blood cell suspension in isotonic saline solution.
• For serum instead of plasma blood should be collected into plain 4 – 6 cc tube and
the serum must be well cleared, by centrifugation at 3500 for 5 – 10 minutes, before
use avoid fibrin residues, which may interfere with the reaction pattern.
Test Procedure:
Allow reagent red cells to reach room temperature before testing.

Take 12 test tubes and label 1 – 11 test tubes for panel cells and one for auto-control.
An antibody ID is performed into three phases:
1st step - Immediate Spin (IS):

 Add to each test tube 2 drops of the serum to be tested.
 Add 1 drop of each panel cell into corresponding test tube and the 1 drop of red cell
suspension into the auto-control tube.
 Mix gently and centrifuge for 25 seconds at 1000 rpm.
 Examine the supernatant for hemolysis
 Gently re-suspend each red blood cell button and examine for agglutination
 Examine macroscopically with agglutination viewer glass (an indirect light source)
and record the results on the antibody identification antigram.
2nd step - Albumin phase (37 °C):

 Add to each tube 3 drops of bovine albumin 22%.
 Mix gently and incubate for 15–30 minutes at 37 °C.
 Centrifuge for 25 seconds at 1000 rpm and gently re-suspend each red blood cell
button and examine for agglutination.
3rd step - Antiglobulin phase (IAT):

 Wash contents of tubes 3 times with isotonic saline and remove supernatant.
 Add to each tube 2 drops of ˝DiaClon Coombs-serum˝.
 Mix gently and centrifuge for 25 seconds at 1000 rpm.
 Gently re-suspend each red blood cell button and examine for agglutination
 Confirm all negative results with Coombs control cells (Check cells).
Interpreting Results of Antibody ID Panels:
Negative Test: Absence of agglutination throughout the test procedure indicates that the test
serum does not contain detectable antibodies to any of the antigens present in the reagent.
Positive Test: Agglutination of any of the panel red cells at any phase or hemolysis at the
saline shows the presence of antibodies.
Auto-logous control: Review the reactions obtained with the autologous control to
determine if the antibody is allo or auto in nature.
The autocontrol positivity determines the presence of autoantibody or a combination of both
allo-and autoantibody.
There are a few basic steps to follow when interpreting panels
1. Look for a matching pattern:

Uniform reactions suggest a single antibody and it is easy to recognize because it shows
clear cut pattern of reactions.
Variable reactions suggest either multiple antibodies OR single antibody with dosage
2. Ruling Out:
Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous
antigens that show dosage, includes Rh, Duffy, Kidd and MNS antibodies.
Heterozygous (+, +) = It mean one antigen E (+) is received from father and one antigen e
(+) is received from mother.
Homozygous (+, -) = It mean only one antigen E or e (+) is received from either father or
mother.
3. Circle antigens that not crossed out and cut the antigen that’s at least cutting 2 or more
times.
4. The rule of three must be meet to confirm the presence of the antibody
 Positive with 3 cells with the antigen
 Negative with 3 cells without the antigen
 If not fulfilled than two positive and two negative is also acceptable.
 If still less than this add further extended panel cell to identify
Result: More likely antibody is Anti-C, but possibility of anti-E, anti-Cw, K and Kpa which
could not ruled out. Suggest extended panel.
When pattern fails to indicate specificity other procedures may be performed:

 Enzyme Techniques
 Selected Cells panel (extended panel)
 Temperature reduction
 Increase serum to cell ratio
 Increase incubation time
 Decrease pH
 Prewarmed technique
 Adsorption
52 - Blood Components:
A constituent of blood (red cells, white cells, platelets, plasma) that can be prepared
under such conditions that it can be used directly or after further processing for
therapeutic applications.
52.1 - Whole Blood:

 Un-separated blood collected into an approved sterilize container containing
an anticoagulant preservative solution.
 Clinical indications for use of WB are extremely limited.
 Used for massive transfusion to correct acute hypovolemia such as in trauma
and shock, or for exchange transfusion.
 Whole blood must be ABO identical, have not alternative choice.
 The volume of whole blood is 450 ± 50 ml with 1:7 ratio of anticoagulant.
 Whole blood must be ABO identical, have not alternative choice.
 Blood component therapy makes clinical sense as most patients require a
specific element of blood, such as red cells or platelets and the dose can be
optimized.
 Each component is stored under ideal conditions for their longer use to
become effective (e.g. red cells must be refrigerated, platelets must not), so it
is necessary to use blood components as per need rather than using whole
blood.
Whole blood is separated into following components by different

centrifugation method:
 Packed Red Blood Cell (PRBC)
 Platelets Concentrate (RDP)
 Fresh Frozen Plasma (FFP)
 Cryoprecipitate (Cp)
 Cryo-supernatant (Cs)
Other plasma derivatives manufactured from pooled plasma, such as:

 Albumin
 Plasma Protein Fraction
 Fibrinogen
 Immunoglobulin’s
 Factor VIII concentrate
 Other coagulation Factors
Plasma derivatives are not blood components and not prepared in the blood banks,
so it is only manufactured in plasma fractionation centers.
Specialized blood:
• Saline-washed Red Cells
• Irradiated products
• Frozen PRBC
There are more than 20 different products can be prepared from blood.
General Guidelines for Component Preparation:

• Proper labeling: After donor selection take double or triple bag as you have
required blood components and write donor number, donor name, date of
collection and expiry on blood bag.
• Place bag carefully on bio mixer being and clamp the donor bag tubing
before opening the needle cap.
• Select the vein and scrub the area at least 1.5 inches in all directions from the
site of vein puncture with 0.7% povidone iodine. After the skin has been
prepared, it must not be touched again.
• The arm need not be dry before the next step (maximum 30 seconds).
• Collect unit within 15 minutes to prevent activation of coagulation system.
At the end of the donation procedure the blood bag volume is 500 ml with
whole blood and anticoagulant.
• After collection keep the units vertical on the table for 30 to 45 minutes
(Process all units within 6 hours of blood collection and should be stored
within 8 hours).
• Note: Your working bench must be cleaned and only required things on your
bench, no any extra things.
• Buckets & centrifuge bowls clean with Warm water and mild detergent.
(Use 1% Na hypochlorite after each spill/breakage)
52.2 - Packed Red Cells:
Packed RBCs are ordinarily the component of choice with which to increase Hb.
Collected from whole blood or aphaeresis method.
Preparation:
 It is prepared from whole blood, which should be drawn at least in double
bag.
 Packed red cells are prepared by allowing whole blood to sediment or light
spin centrifugation with subsequent removal of supernatant plasma.
 Centrifuge the whole blood using at a speed of 1600 – 2000 rpm for 5 minutes
at 22 °C (light spin).
 After centrifugation place the mother bag on plasma extractor and break the
integral seal of the tube connecting it to the satellite bag manually and
express the supernatant plasma into the satellite bag. Don’t shift all the
plasma into satellite bag leave about 20 ml into main bag along with red
blood cells. When the plasma shifted seal the tubing and release the spring to
stop the procedure.
 Usually about 230 to 250 ml of plasma is removed.

 The volume of pack cell is about 250 t0 300 ml and Hct is about ≥ 55 – 80%.
 The blood should be used within the expiration date of the bags. Packed
RBCs can be store with CPDA1 at 2 to 6 °C for 35 days
 PRBC (not whole blood) cross match must be compatible with antibodies in
the recipient’s plasma.
 After issuing blood it should be start transfuse within 30 minutes or when it
comes on room temperature and will be finished within 4 hours.
 To be returned blood to stock units must be out of refrigeration < 30 minutes
and temperature must not be >10°C
 One unit of PRBC increase the Hct about 3%, or the Hb about 1 g/dL in a 70-
kg non-bleeding adults.
 Anemia treatable by drugs (iron, vitamin B12, folic acid, or erythropoietin)
52.3 - Washed red blood cells:
 Washed RBCs are free of almost all traces of plasma, most WBCs, and
platelets and micro aggregates which may cause febrile or urticarial
reactions.
 They are generally given to patients who have severe reactions to plasma
(e.g., severe allergies, paroxysmal nocturnal hemoglobinuria, or IgA
immunization).
 Patient requiring this product (W-RBCs) for the IgA deficient patient with
anti-IgA antibodies.
 In IgA-immunized patients, blood collected from IgA-deficient donors may
be preferable for transfusion.
 Prepared by using a machine which washes the cells 3 times with saline.
Also prepared by manually washing with normal saline through Cryofuge
machine.
 Before washing the unit, blood bank should perform the compatibility tests.
The washing procedure is undertaken only after the unit is found to be
compatible with recipient.
 The volume of washed red cell is about 180 – 200 ml and about Hct 75%.
 Saline washing may be performed at any time during the shelf-life of a unit
of blood but, because washing is ordinarily performed in an “open” system,
the resultant red cell component can be stored for only 24 hours at 1–6°C. If
not used discard after 24 hours with standard disposal protocol.
52.4 - Fresh frozen plasma (FFP):
Plasma is pale-yellow color liquid part of blood and about 55 % plasma in blood.
Fresh frozen plasma (FFP) is Contents of:
 Clotting multifactor
 Fibrinogen
 Pro-thrombin
 All the plasma proteins( such as Albumin, globulin and antibodies)
 It also contains the preservative added at the time of collection.
Clinically significant of FFP are:

 Coagulopathy due to multiple factor deficiencies or when specific factor
replacements are unavailable
 Liver disease
 Disseminated intravascular coagulation [DIC]
 Reversal of nutritional Vit K deficiency or Warfarin overdose
 Massive transfusion
 When PT and APTT exceed > 1.5 x normal
Preparation:
 Whole blood should be drawn at least in double bag.
 FFP’s are prepared by allowing whole blood to sediment or single light spin
centrifugation at a speed of 1600 – 2000 rpm (light spin) for 5 minutes at 22
°C with subsequent removal of supernatant plasma.
 Also prepared by double centrifugation from the triple bag by the separation
of platelet concentrate. At the same time.
 Usually FFP’s volume is about 200 ml ± 50 ml.
 Frozen within 8 hours of collection, if plasma is separated after 8 hours of
collection label as Factor VIII deficient plasma (FVIIID).
 Shelf life is 12 months when store at -18°C or less and up to 5 years when
store under (– 65- 80 °C)
 When required FFP can be thawed with agitation in 37°C in water path and
used within 4 hours at 20 - 24°C.
 ABO group of the FFP should be identical to that of the patient and Rh group
identity is not important. But in children, should also be Rh compatible.
 The next choice is to give “compatible FFP” such as AB Blood group plasma,
which do not sensitize patient’s RBC.
52.5 - Platelet Concentrates:
Low platelet counts (< 50,000/μL) can cause bleeding. Platelet concentrates are used
to prevent bleeding. The normal life span in is 7 – 9 days in the body, size is 2 – 4
micron and normal count of platelets in adults is 150,000 – 450,000/cumm.
Preparation:
 Platelet concentrate should be prepared by centrifugation of a single unit of
whole blood collected in triple bag with a smooth vein puncture and a
continuous flow of blood.
 Centrifuge the whole blood using at a speed of 1600 – 2000 rpm (light spin)
for 5 minutes at 22 °C.
 Place the mother bag on plasma extractor and label the transfer bag one for
platelet concentrate and other for FFP’s with the unit number, blood group
and date of collection prior to separation from the primary bag.
 Clump the tubing of second FFP’s marks bag and transfer the supernatant
platelets rich plasma into first platelets marks attached bag from the whole
blood, leave about 20 ml of plasma into main bag along with red blood cells,
seal the tubing and release the spring to stop the procedure.
 Put PRP bags in the buckets and balance them. Centrifuge 2’nd spin at a
speed of 4000 – 4600 rpm for 5 minutes (Centrifugation temperature at 22 °C)
to separate the platelet from plasma.
 Place the platelets containing bag on extractor and transfer the plasma into
FFP’s marks bag. Seal the tubing twice. Cut between the two seals.
 Leave the platelet concentrates on the laminar flow for 30 minutes.
 The volume of platelet concentrates is about 50 t0 70 ml, stored platelets at
20-24°C (Constantly agitated). Only for 5 days due to storage at RT it is the
most likely component to be contaminated with bacteria. Considering day of
platelets collection as day zero.
 The concentrate prepared should not be contaminated with red cells. The
degree of reddishness of the concentrate indicates red cell contamination.
The units contaminated with red cells should be used as group specific or
can be issued after cross match (Specially for MEGA Unit)
 The pH at storage temperature should not be lower than 6.0 at the end of
storage period.
 For QC of platelet count samples should be taken into a dry EDTA tube, to
induce disaggregation.1 random donor unit contains yield 0.55 X1011
platelets.
 Used immediately after issuing not more than 30 minutes.
 One unit of platelet concentrate increases the platelet count by about 5 –
10,000/μL in a 70 kg adult, 1 unit platelet concentration infused per 10 kg
body weight and 4 to 6 random donor platelet concentrates are commonly
used in adults.
 Rh-negative woman in child-bearing age should receive ABO group specific
platelets.
52.6 - Cryoprecipitate anti hemophilic factor (AHF):
Contents: Cryoprecipitate is content factor I (fibrinogen); factor VIII, Factor XIII and
von Willebrand factor (vWF).
Cryoprecipitate is used in the treatment of:
 Hemophilia A (Factor VIII deficiency)
 Von Willebrand factor (needed to help platelets work)
 Fibrinogen deficiency (a protein produced by liver needed to form a clot)
 Rare factor XIII deficiency.
 Disseminated intravascular coagulation
In general, it should not be used for other indications.
Preparation:
 Whole blood should be drawn in triple bags and cryoprecipitate are

separated by double centrifugation method.
 Centrifuge the whole blood using a heavy spin at speed of 4000 – 4600 rpm
for 5 minutes at 2 – 6 °C.
 Place the mother bag on plasma extractor and label the transfer bag one for
cryoprecipitate and other for Cryo-supernatant Plasma with the unit
number, blood group and date of collection, prior to separation from the
primary bag.
 Clump the tubing of second Cryo-supernatant marks bag and transfer the
supernatant plasma into first cryoprecipitate marks attached bag from the
whole blood, seal the tubing and release the spring to stop the procedure.
 Collect at least 200 ± 50 ml of cell free plasma for processing into
cryoprecipitate.
 Freeze plasma rapidly. The frozen plasma may be stored at –18 °C or lower
for 24 hours or until the cryoprecipitate is prepared.
 When preparing Cryoprecipitate the frozen plasma is thawed slowly at 2 – 6
°C until it has a mushy consistency ( 18 – 20 hours)
 Centrifuge 2’nd spin plasma for the separation of cryoprecipitate and Cryo-
supernatant plasma by using a speed of 4000 – 4600 rpm for 5 minutes
(Centrifugation temperature at 2 – 6 °C)
 Place the cryoprecipitate containing bag on extractor and transfer the plasma
into Cryo-supernatant plasma marks bag. Seal the tubing twice. Cut between
the two seals.
 The volume of cryoprecipitate is about 10 t0 15 ml.
 The shelf life is 12 month, when store at - 65 °C or low and thawed at 37 °C
prior to use.
 Used as soon as possible after thawing or within 6 hours at 20 - 24°C.
 The component should contain a minimum of 0.8 IU. Of factor VIII and 1.5
mg of fibrinogen per ml.
52.7 - Irradiated Blood Products:

• Irradiated blood products are used for the patients at risk of transfusion-
associated graft-versus-host disease (TA-GvHD). The component irradiated
by gamma or X-rays, which kill the lymphocytes. The lack of T-cells prevents
donor vs. host disease.
• They are used for severely immune-compromised patients, lymphoma
patients, stem-cell/marrow transplants and unborn children undergoing
intrauterine transfusion.
• Red cells may be irradiated at any time up to 14 days after collection, and
thereafter stored for a further 14 days from irradiation.
• Platelets can be irradiated at any stage in their 5 day storage and thereafter
can be stored up to their normal shelf life of five days after collection
53 - Storage of Blood:
In vivo at favorable environment, red cells are carried and protected by the plasma;
therefore RBCs life span is 110-120 days, which helps:
 Regulated temperature
 Controlled pH
 Adequate glucose supply
 Removal of metabolic waste
In vitro lowering of temperature and addition of preservatives can do changes in
blood and known as “storage lesion”
Storage Cause some changes in blood such as:

Decrease Na+ and increase K+ level:
Na+/K+ leak through the red cells. Cells lose potassium and gain sodium.
Low pH:
Whole blood or pack cell should be store at “between” 2 – 6 °C, because glycolysis
is slowed at this temperature. Lactate is produce in the result of glycolysis which
decreases pH. Normal blood pH in body is from 7.35 to 7.45
pH Whole blood Pack cell

Day 0 (CPDA1) 7.60 7.55
Day 35 (CPDA1) 6.98 6.71
Increase Plasma Hemoglobin:

ATP is closely associated with red cell viability. Result of storage decrease ATP
level results RBC loose rigidity, decrease deformability and cause minimum
hemolysis which increases plasma hemoglobin. ATP levels on day 35 of storage is
45% in CPDA-1
Table - 3: Blood and Blood Component Storage Conditions:
Blood Product Approximate Storage Temp. Self life

Volume
Whole blood CPD-1 450 to 500 mL 2 to 6 °C 35 days
Packed RBCs 230 to 250 mL 2 to 6 °C 35 days
Washed RBCs 180 to 200 mL 2 to 6 °C 24 hours
Irradiate RBCs 230 to 250 mL 2 to 6 °C 28 days from date
of irradiation
Fresh Frozen 150 - 250 mL Frozen: -18°C Frozen: 1 year
Plasma Frozen: -65 – 80 °C, Frozen: 7 year
as approved by the
FDA
Thawed: 2 to 6 °C Thawed: 24 hours
Fresh Frozen 500 mL Frozen: -18°C Frozen: 1 year
Plasma(Aphaeresis) Thawed: 2 to 6 °C Thawed: 24 hours
Cryo-supernatant 200 – 250 mL Frozen: -18°C Frozen: 1 year
Plasma Thawed: 2 to 6 °C Thawed: 24 hours
Cryoprecipitate 10 to 15 mL Frozen: -18°C Frozen: 1 year
Thawed: 20 to 24 °C Thawed: 6 hours
Platelets 50 to 70 mL Continuous 5 days
(Random Donor) agitation/20 to 24 °C
Platelets (Single 150 – 300 mL Continuous 5 days
Donor Aphaeresis) agitation/20 to 24 °C
Table - 4: Time Need for Blood Component Issuing:
After arrangement of blood and their products store their respective temperature
and need minimum time to issue:
Component Minimum Time

45 minutes
RBCs: saline-washed
30 minutes
Fresh Frozen Plasma: thawed
15 minutes
Platelets concentrate
15 minutes
CRYO: thawed
05 minutes
PRBC / Whole blood
Table - 5: Emergency release of blood:
Level-1 Level-2 Level-3

No time available If little time available If little more time available
(15 minutes) (45 minutes)
“O” negative packed red Group specific packed Group specific with full
blood cells will be issued red blood cells will be cross match packed red
issued blood cells will be issued
When sample was not Patient as well as Perform patient sample as
received, no need to donors blood group well as donor’s bag blood
perform any test. If “O” will be performed. group and then cross match
negative is not available After issuing blood on gel card.
then “O” positive packed cross match of that bag
red blood cells issued by performed as routine.
removing all plasma.
54 - Rate of Infusion:
The first few ml of blood/blood products must be transfused very slowly. Standard
blood transfusion sets deliver 20 drops per ml of whole blood. The usual
recommended and safe transfusion time for one unit of whole blood is four hours
and three hours for packed red cells.
Example:
Whole blood volume = 450 ml
1 ml = 20 drops
Rate of infusion/minute = 450 X 20 = 40 drops / minute
4 X 60
This mean blood is transfused at 40 drops per minute. Platelet concentrate and fresh
frozen plasma should be transfused as rapidly as possible.
55 - NAT Testing:
NAT is a recently developed technology that allows detection of very small

amounts of genetic material (DNA or RNA) by a process of massive copying
(amplification) of a gene fragment.
Pakistan has given the high rate of sero-positivity of HIV, HCV and HBV. Blood
and blood products are required for many patients which replacement by donors.
However, there is still a "window period" during which a donor can be infected, but
have negative screening tests. It is likely that adding NAT to the current screening
tests will have a very significant reduction in Transfusion Transmitted Infections.
Parameter ELISA tests NAT test methods

HIV 22 Days 11 Days
HBV 56 Days 20 – 30 Days
HCV 70 Days 10 – 12 Days
A single unit of whole blood collected from a donor in the window period of
infection may be transfused into up to three recipients.
56 - Laboratory Waste Management and Disposal:
Laboratory personnel are at risk of exposure to blood-borne pathogens related to

injuries from contaminated sharps, splashes to the eyes or mouth, and unprotected
exposure of blood onto non-intact skin. We are required to follow certain Infection
Control Guidelines in order to reduce inherent risks during manipulation of patient
specimens, contaminated sharps, and diagnostic equipment.
Reusable glass ware is put in specially designated plastic containers labeled with
“Reusable Items Only” with BIOHAZARD symbol. These are daily filled with 1%
bleach (Na Hypochlorite solution). However the reusable slides, pipettes and test
tubes are soaked overnight in 1% bleach in large buckets and then after removal
disinfection material wash with tape water and then these are autoclaved at 121°C
for 15 minutes. All the washing is done by a well trained Staff.
There are two types of waste produced in the blood bank.
a) Solid Waste:
Used syringes, needles, plastic reaction vessels & gloves are stored in puncture
resistant container then dispose off it into incinerator.
b) Liquid Waste:
Laboratory personnel also deal with chemical reagents which include chemicals for
diagnostic and experimental work & disinfecting material. The waste generated
from these chemicals is equally important and should be disposed off with care so
as not to be a hazard for human health.
Also other contaminated materials in blood bank are blood samples; reactive blood
bags and expired blood products are stored in separate labeled container then send
to incinerator for safe disposal.
57 - Reference:
It is difficult to mention the reference of each sentence or topic because I have read
many power point presentations from different education sites and some books
which are mention below:
 Bar Basic of Blood Banking written by Dr. Saba Jamal
 Rudman Text Book of Blood Banking and Transfusion Medicine
 14th Edition Technical Manual of American Association of Blood Bank
After these studying as looking to our practical work during my job I have
summaries them into this assignment form, hope this will be a very useful during
the practical and theory for the medical students as well as doctors and also for
technical staffs of the blood banks.
58 - Abbreviation:
Abbreviation Term
2, 3-DPG 2, 3-diphosphoglycerate
AABB American Association of Blood Banks
AHF Anti-Hemaphilic Factor
AHG Anti-Human Globulin
AHTR Acute Hemolytic Transfusion Reaction (also HTR)
AIDS Acquired Immune Deficiency Syndrome
APPT Activated Partial Thromboplastin Time
ARDS Acute Respiratory Distress Syndrome
BSL Laboratory bio safety levels
BTA Blood Transfusion Authority
BTS Blood Transfusion Service
CAP College of American Pathologists
CBC Complete Blood Counts
CC Check cells
CDC Centers for Disease Control
CHD Coronary Heart Disease
CMV Cytomegalovirus
CPD Citrate, Phosphate, Dextrose
CPDA Citrate, Phosphate, Dextrose-Adenine
CRYO Cryoprecipitate
CSP Cryo-supernatant Plasma (CS)
ELIZA Enzyme Linked Immunosorbent Assay
D5W Dextrose 5% water
DAT Direct Antiglobulin Test also known as Coombs Test
DIC Disseminated intravascular coagulation
EDTA Ethylene Diamine Tetracetic Acid
FDA Food and Drug Administration
FFP Fresh Frozen Plasma
GVHD Graft-Versus-Host Disease
Hb Hemoglobin
HbsAg Hepatitis B Surface Antigen
HBV Hepatitis B Virus
HCT Hematocrit
HCV Hepatitis C Virus
HDN Hemolytic Disease of the Newborn
HIV Human Immunodeficiency Virus
HLA Human Leukocyte Antigen
Abbreviation Term
HTR Hemolytic Transfusion Reaction

IAT Indirect Anti-globulin Test
IgA Immunoglobulin A
IU International Units
IV Intravenous
JCAHO Joint Commission on Accreditation of Healthcare Organization
L.J chart Levy and Jennings chart
MCH Mean Corpuscular Hemoglobin
MCHC Mean Corpuscular Hemoglobin Concentrate
MCV Mean Corpuscular Volume
MP Malarial parasite
MTS Micro Typing System
NAT Nucleic acid testing
NC Negative control
NCCLS National Committee for Clinical Laboratory Standards
NCPE Non Cardiogenic Pulmonary Edema
NS Normal Saline
PH Partial hemolysis
PLTs Platelets
PPM Planned Preventive Maintenance
PRBC Packed Red Blood Cells
PRP Platelet-Rich Plasma
PTP Post-transfusion purpura
PTT Partial Thromboplastin Time
QA Quality assurance
QC Quality Control
RBCs Red Blood Cells
Rh Rhesus factor
RPM Rotation per Minute
RT Room Temperature
SAGM Saline-adenine-glucose-mannitol
SD Standard deviation
SI International System of Units
SOP Standard Operating procedure
TRALI Transfusion Related Acute Lung Injury
TTI Transfusion transmitted infections
vWF von Willebrand factor
WB Whole Blood
WBCs White Blood Cells
..The End…

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The Basic Blood Banking

I am also acknowledges the cooperation of other staff members specially our

S.No. Topic Page No.

1 - Introduction of Blood Bank--------------------------------------------------------1

10 - Calculation of Amount of anticoagulant needed------------------------------14

14 - Post Donation Advice------------------------------------------------------------25

26 - ABO Blood Grouping System--------------------------------------------------51

46 - Blood Transfusion Reaction-----------------------------------------------------77

1 - Introduction of Blood Bank:

2 - History of Blood Bank:

In 1667 Denys administered transfusions of calf's blood to Antoine Mauroy, a madman.

In 1907 Hektoen suggests that the safety of transfusion might be improved by

In 1940 Edwin Cohn, a professor of biological chemistry at Harvard Medical School,

In 1948 – First plastic bag developed for blood collection.

In 1971 – Blood banking becomes regulated by the FDA.

In 1983 introduce Saline-adenine-glucose-mannitol (SAGM) an Additive solution extend

3 - Blood Bank Regulatory Agencies:

The AABB is a United States-based professional body and standards

3.2 - Food and Drug Administration (FDA):

3.3 - College of American Pathologists (CAP):

The CAP Laboratory Accreditation Program’s goal is to improve patient safety by

3.4 - Joint Commission on Accreditation of Healthcare Organizations (JCAHO):

Inspects entire hospitals every other year – focuses on

3.5 - National Committee for Clinical Laboratory Standards (NCCLS):

A not-for-profit membership organization, the

4 - General Directions for Safety in Blood Bank:

 Immunize the laboratory workers against vaccine-preventable diseases such as hepatitis

5.1 - Cellular part (Formed Elements):

5.2.1 - Organic Substance:

 Non nitrogenous substances:

5.2.2 - Inorganic Substances:

6 - Red Blood Cells (Erythrocytes) Morphology:

Red Blood Cells Membrane:

Fig: Structure of normal red cell membrane

8 - Source and Need of blood:

9.2 - Ethylene Di-amine Tetra-acetic Acid (EDTA):

9.3 - Citrate glucose:

2, 3-diphosphoglycerate: Hemoglobin binds not only to oxygen but to other substances

9.5 - Citrate-phosphate-dextrose (CPD):

 Citrate – Calcium-chelating agent, preventing coagulation

9.6 - Citrate-phosphate-dextrose-adenine (CPDA-1):

Addition of adenine helps in maintaining 2, 3-DPG by alkaline pH which improved the

Whole blood is collected in clear plastic bags that contain CPDA-1

9.7 - Saline-adenine-glucose-mannitol (SAGM):

10 - Calculation of Amount of anticoagulant needed:

Volume to draw (A) = weight in kg X 500

11 - Low Volume Blood Bags Bleed:

12 - Blood Sample Collection and Identification:

13.1 – Types of Blood Donation:

There are several types of blood donation such as:

13.1.1 - Voluntary blood donation:

13.1.2 - Exchange Donation:

13.1.3 - Directed Donation:

13.1.4 - Paid Donation:

13.1.5 - Autologous Donation:

13.2 - Methods of Blood Donation:

13.2.1 - Whole blood Donation:

Donors Selection Criteria:

Donors Deferral Criteria:

Donor deferral criteria are as follow;

Whole Blood Donation Procedure:

Recap the needle by using one hand technique.

Next, collect blood into sample tubes for screening.

Seal the tubing attached to the bag.