Lec. Medical lab.

Techniques Department
Class 13 Blood transfusion

Anticoagulants in blood transfusion

A brief history ‫لالطالع فقط‬

*1916 first anticoagulant preservative –Rons and turner.it consisted of a
citrate-glucose solution.
*Rouns turner’s solution was used for storage of human blood during the
First World War.
*1943-during the second world war, acidified citrate dextrose (ACD)
solution was introduced-loutit and mollison.
*1957-Gibson and others developed citrate – phosphate – dextrose
(CPD).
CPD eventually replaced ACD and became commonly used preservative
for storage of blood /red cell in liquid form.
Shelf-life of blood stored in CPD at 2-4C was21day
*1978-citrate-phoshate-dextrose with adenine (CPDA-1).
The addition of adenine improved the synthesis of ATP in the stored
blood, which prolonged the storage of blood/ red cell at 2-4 C to 35days.

Anticoagulant preservative solutions

Blood collection packs are designed to hold 450 ±45ml of blood mixed
with 63 ml of:
1. Acid citrate Dextrose (ACD)
2. Citrate phosphate dextrose (CPD)
3. Citrate phosphate dextrose Adenine (CPd-1) and (CPd-2).
Purpose
1. To prevent coagulation,
2. To preserve the life and survival of RBCs so as to have the maximum
post transfusion survival.

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Action of ingredients of anticoagulant solution

1. Citrate: Acts by chelating calcium.
2. DEXTROSE; necessary for the metabolism of stored RBCs. It passes
from plasma into the red cell and utilized for energy production.
3. Citric acid; prevents caramelization of glucose in citrate dextrose
solution during autoclaving.
4. Adenine; improves the viability of red cells.
ADDITIVE SOLUTIONs
1. Additive solutions are preserving solutions that are added to the
RBCs after removal of the plasma with/without platelets.
2. Reason for their development –removal of the plasma component
during the preparation of RBCs concentrates removed much of the
nutrients needed to maintain RBCs during storage.
3. Also overcome the problem of high viscosity of RBC concentrates.
4. With CPD anticoagulant in the primary bag , the additive solution
used is SAGM(saline,adenine,glucose,mannitol)
ADVANTAGES
1. Extends the storage of RBCs.
2. Lowers the viscosity of packed red cells for ease of transfusion.
3. Maximum amount of fresh plasma is harvested.

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Class 4 Blood transfusion
Autologous blood transfusion
An autologous blood donation is one in which a person donates blood
for their own use prior to or during a scheduled surgery. When the blood
is given back, it is called an autologous blood transfusion. This is in
contrast to an allogeneic blood transfusion, in which blood from another
from a person is used.

Types

There are four types of autologous blood transfusion procedures:

• Preoperative autologous donation (PAD) is a procedure in which

blood is collected weeks in advance of the surgery, stored in a
blood bank, and transfused back into the donor when needed.
• Acute normovolemic hemodilution (ANH) involves the removal
of blood right after anesthesia is given for surgery. To maintain
normal blood volumes and blood pressure, the same amount of
replacement fluid is transfused into the body.
• Intraoperative cell salvage is a technique in which blood lost
during the surgery is immediately returned into circulation using a
machine called a cell saver. The machine not only filters the blood
of debris but adds an anticoagulant to keep it from clotting.
• Postoperative cell salvage involves the collection of lost blood
during surgery via wound drains. The blood is treated in the same
way as an intraoperative cell salvage but is reinfused after the
surgery rather than during

Indications
Autologous blood transfusions may either be recommended based on
the type of surgery you are undergoing or an elective procedure used in
people concerned about blood borne infections, allergy, or an
autoimmune response.

Recommended Use
If there is a possibility that you will need a blood transfusion during or
after surgery, your surgeon may recommend an autologous blood
donation prior to surgery. Cell salvage transfusions can also be done in an
emergency situation.

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This is especially true for surgeries in which blood loss can be massive,
such as the resection of a lung or liver in people with cancer. In cases like
these, person need as much as 10 units of packed red blood cells. An
autologous donation can be used in addition to allogeneic donations to
relieve pressure on the community blood supply.

Autologous blood transfusions are generally considered when it is

anticipated that 20% or more of a person's blood may be lost during
surgery. In addition to major cancer surgeries, autologous blood
transfusions are often needed for major joint replacement
surgeries, vascular surgeries, or cardiothoracic surgeries.

Preoperative autologous donations are generally avoided if less than

10% of blood is expected to be lost during surgery

Elective Use
Autologous transfusions may also be used as an elective procedure for
people who are concerned about getting a blood borne infection
like hepatitis C or HIV. Although transmission by this means is extremely

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rare due to blood screening protocols, donating and receiving your own
blood eliminates the risk entirely.

Autologous blood transfusions can also mitigate the risk of

an allergic or autoimmune response that can occur on rare occasions with
allogeneic transfusions. This includes hemolytic transfusion reactions in
which the immune system attacks and destroys donated blood cells.

Advantages and Disadvantages

Advantages
• Reduced risk of blood borne infections
• Reduced risk of allergic or hemolytic transfusion reaction
• Safer in people with rare blood types
• Safer in people with multiple autoimmune disorders
• Reduces the demands on the community blood supply
Disadvantages
• More costly than an allogeneic blood transfusion
• Risk of an unnecessary blood transfusion
• Unused blood is wasted
• Risk of anemia and cardiac complications following the donation
• Risk of bacterial infection due to improper storage of blood

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Disorders of Hemostasis Dr. Jalal Alwan

A. Thromboembolytic Disorders (undesirable clotting)

1. Thrombus - blood clot in normal blood vessel

2. Embolus -blood clot/gas bubble floating in blood
a.TPA tissue plasminogen activator streptokinase -
can dissolve a clot
b.aspirin - inhibits Thromboxane formation
c.heparin - inhibits thrombin & platelet deposit
d.dicumarol - anticoagulant, blocks Vitamin K

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B. Bleeding Disorders
1.thrombocytopenia - reduced platelet count; generally below
50,000 per cubic millimeter; can cause excessive bleeding from
vascular injury
2.impaired liver function - lack of procoagulants (Clotting Factors)
that are made in liver a. vitamin K - essential for liver to
make Clotting Factors for coagulation
3.hemophilias - hereditary bleeding disorders that occur almost
exclusively in males
a. hemophilia A - defective Factor VIII (83%)
b. hemophilia B - defective Factor IX (10%)
c. now genetically engineered TPA and Factor
VIII are produced so patients do not need transfusions as
often.

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Hemolytic anemia

Hemolytic anemia is a form of anemia due to hemolysis, the

abnormal breakdown of red blood cells (RBCs), either in the blood
vessels (intravascular hemolysis) or elsewhere in the human body
(extravascular). This most commonly occurs within the spleen, but also
can occur in the reticuloendothelial system or mechanically (prosthetic
valve damage). Hemolytic anemia accounts for 5% of all existing
anemias.

Signs and symptoms

Symptoms of hemolytic anemia are similar to the general signs of
anemia. General signs and symptoms include fatigue, pallor, shortness of
breath, and tachycardia. In addition, symptoms related to hemolysis may
be present such as chills, jaundice, dark urine, and an enlarged
spleen. Certain aspects of the medical history can suggest a cause for
hemolysis, such as drugs, medication side effects, autoimmune disorders,
blood transfusion reactions, the presence of prosthetic heart valve, or
other medical illness.
Chronic hemolysis leads to an increased excretion of bilirubin into
the biliary tract, which in turn may lead to gallstones. The continuous
release of free hemoglobin has been linked with the development of
pulmonary hypertension (increased pressure over the pulmonary artery);
this, in turn, leads to episodes of syncope (fainting), chest pain, and

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progressive breathlessness. Pulmonary hypertension eventually causes
right ventricular heart failure, the symptoms of which are peripheral
edema (fluid accumulation in the skin of the legs) and ascites (fluid
accumulation in the abdominal cavity).

Cause
Intrinsic cause
Hereditary (inherited) hemolytic anemia can be due to :

• Defects of red blood cell membrane production (as in hereditary

spherocytosis .
• Defects in hemoglobin production as in thalassemia .
• Defective red cell metabolism (as in glucose-6-phosphate
dehydrogenase deficiency and pyruvate kinase deficiency).

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Extrinsic cause (acquired)

• Immune-mediated causes could include transient factors as

in Mycoplasma pneumoniae infection or permanent factors as
in autoimmune diseases like autoimmune hemolytic anemia (itself
more common in diseases such as systemic lupus
erythematosus, rheumatoid arthritis, lymphoma, and chronic
lymphocytic leukemia).
• Any of the causes of hypersplenism (increased activity of the spleen),
such as portal hypertension.
• Acquired hemolytic anemia is also encountered in burns and because
of certain infections (e.g. malaria).
• Lead poisoning resulting from the environment causes non-immune
hemolytic anemia.
• Low-grade hemolytic anemia occurs in 70% of prosthetic heart valve
recipients, and severe hemolytic anemia occurs in 3%.
•

Mechanism
In hemolytic anemia, there are two principal mechanisms of hemolysis;
Intravascular hemolysis
Intravascular hemolysis describes hemolysis that happens mainly
inside the vasculature. As a result, the contents of the red blood cell are
released into the general circulation, leading to hemoglobinemia and
increasing the risk of ensuing hyperbilirubinemia. Intravascular
hemolysis may occur when red blood cells are targeted by autoantibodies,
leading to complement fixation, or by damage by parasites.
Extravascular hemolysis
Extravascular hemolysis refers to hemolysis taking place in
the liver, spleen, bone marrow, and lymph nodes. In this case little
hemoglobin escapes into blood plasma. The macrophages of
the reticuloendothelial system in these organs engulf and
destroy structurally defective red blood cells, or those with antibodies
attached, and release unconjugated bilirubin into the blood plasma
circulation. Typically, the spleen destroys mildly abnormal red blood
cells or those coated with IgG-type antibodies, while severely abnormal

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red blood cells or those coated with IgM-type antibodies are destroyed in
the circulation or in the liver. If extravascular hemolysis is
extensive, hemosiderin can be deposited in the spleen, bone marrow,
kidney, liver, and other organs, resulting in hemosiderosis.
In a healthy person, a red blood cell survives 90 to 120 days in the
circulation, so about 1% of human red blood cells break down each day.
The spleen (part of the reticulo-endothelial system) is the main organ that
removes old and damaged RBCs from the circulation. In healthy
individuals, the breakdown and removal of RBCs from the circulation is
matched by the production of new RBCs in the bone marrow.
In conditions where the rate of RBC breakdown is increased, the body
initially compensates by producing more RBCs; however, breakdown of
RBCs can exceed the rate that the body can make RBCs, and so anemia
can develop. Bilirubin, a breakdown product of hemoglobin, can
accumulate in the blood, causing jaundice.

Diagnosis
The diagnosis of hemolytic anemia can be suspected on the basis of a
group of symptoms and is largely based on the presence of anemia, an
increased proportion of immature red cells (reticulocytes) and a decrease
in the level of haptoglobin, a protein that binds free hemoglobin.
Examination of a peripheral blood smear and some other laboratory
studies can contribute to the diagnosis. Symptoms specifically related to
hemolysis include jaundice and dark colored urine due to the presence
of hemoglobin (hemoglobinuria). When restricted to the morning
hemoglobinuria may suggest paroxysmal nocturnal haemoglobinuria.
Direct examination of blood under a microscope in a peripheral blood
smear may demonstrate red blood cells that look like spheres
(spherocytes), and/or red blood cells missing small pieces (bite cells). An
increased number of newly made red blood cells (reticulocytes) may also
be a sign of bone marrow compensation for anemia. Laboratory studies
commonly used to investigate hemolytic anemia include blood tests for
breakdown products of red blood cells, bilirubin and lactate
dehydrogenase, a test for the free hemoglobin binding
protein haptoglobin, and the direct Coombs test to
evaluate antibody binding to red blood cells suggesting autoimmune
hemolytic anemia.
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Treatment
Definitive therapy depends on the cause:

• Symptomatic treatment can be given by blood transfusion, if there is

marked anemia.
• In severe immune-related hemolytic anemia, steroid therapy is
sometimes necessary.
• Association of methylprednisolone and intravenous immunoglobulin
can control hemolysis in acute severe cases.
• Sometimes splenectomy can be helpful where extravascular
hemolysis, or hereditary spherocytosis, is predominant (i.e., most of
the red blood cells are being removed by the spleen).
•

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Coombs test Dr. Jalal Alwan

A Coombs test, also known as antiglobulin test (AGT) is either of

two blood tests used in immunohematology. They are the direct and
indirect Coombs tests. The direct Coombs test detects antibodies that are
stuck to the surface of the red blood cells. Since these antibodies
sometimes destroy red blood cells, a person can be anemic and this test
can help clarify the condition. The indirect Coombs detects antibodies
that are floating freely in the blood. These antibodies could act against
certain red blood cells and the test can be done to diagnose reactions to
a blood transfusion[
The direct Coombs test is used to test for autoimmune hemolytic
anemia—that is, a condition where the immune system breaks down red
blood cells, leading to anemia. The direct Coombs test is used to detect
antibodies or complement proteins attached to the surface of red blood
cells. To perform the test, a blood sample is taken and the red blood cells
are washed (removing the patient's own plasma and unbound antibodies
from the red blood cells) and then incubated with anti-human globulin
("Coombs reagent"). If the red cells then agglutinate, the direct Coombs
test is positive, a visual indication that antibodies or complement proteins
are bound to the surface of red blood cells and may be causing
destruction of those cells.

The indirect Coombs test is used in prenatal testing of pregnant women

and in testing prior to a blood transfusion. The test detects antibodies
against foreign red blood cells. In this case, serum is extracted from a
blood sample taken from the patient. The serum is incubated with foreign
red blood cells of known antigenicity. Finally, anti-human globulin is
added. If agglutination occurs, the indirect Coombs test is positive.

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Mechanism
The two Coombs tests are based on anti-human antibodies binding to
human antibodies, commonly IgG or IgM. These anti-human antibodies
are produced by plasma cells of non-human animals after immunizing
them with human plasma. Additionally, these anti-human antibodies will
also bind to human antibodies that may be fixed onto antigens on the
surface of red blood cells (RBCs). In the appropriate test tube conditions,
this can lead to agglutination of RBCs and allowing for visualisation of
the resulting clumps of RBCs. If clumping is seen, the Coombs test is
positive; if not, the Coombs test is negative

Common clinical uses of the Coombs test include the preparation of

blood for transfusion in cross-matching, atypical antibodies in the blood
plasma of pregnant women as part of antenatal care, and detection of
antibodies for the diagnosis of immune-mediated haemolytic anemias

Coombs tests are performed using RBCs or serum (direct or indirect,

respectively) from venous whole blood samples which are taken from
patients by venipuncture. The venous blood is taken to a laboratory (or
blood bank), where trained scientific technical staff do the Coombs tests.
The clinical significance of the result is assessed by the physician who
requested the Coombs test, perhaps with assistance from a laboratory-
based hematologist.

Direct Coombs test

The direct Coombs test, also referred to as the direct antiglobulin test
(DAT), is used to detect if antibodies or complement system factors have
bound to RBCs surface antigens in vitro. The DAT is not currently
required for pre-transfusion testing but may be included by some

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laboratories Pre-transfusion, patients often have an indirect Coombs
test.

Uses
The direct Coombs test is used clinically when immune-
mediated hemolytic anemia (antibody-mediated destruction of RBCs) is
suspected. A positive Coombs test indicates that an immune mechanism
is attacking the patient's RBCs. This mechanism could
be autoimmunity, alloimmunity or a drug-induced immune-mediated
mechanism

Examples of alloimmune hemolysis

Hemolytic disease of the newborn (also known as HDN or
erythroblastosis fetalis)

o Rh D hemolytic disease of the newborn (also known as Rh

disease)
o ABO hemolytic disease of the newborn (the direct Coombs test
may only be weakly positive)

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o Anti-Kell hemolytic disease of the newborn
o Rh c hemolytic disease of the newborn
o Rh E hemolytic disease of the newborn
• Alloimmune hemolytic transfusion reactions

Examples of autoimmune hemolysis/immunohemolytic hemolysis

Warm antibody autoimmune hemolytic anemia

o Idiopathic
o Systemic lupus erythematosus
o Evans' syndrome (antiplatelet antibodies and hemolytic antibodies)
• Cold antibody immunohemolytic anemia
o Idiopathic cold hemagglutinin syndrome
o Infectious mononucleosis
o Paroxysmal cold hemoglobinuria
Drug-induced immune-mediated hemolysis

Methyldopa (IgG mediated type II hypersensitivity)

• Penicillin (high dose)

• Quinidine (conquinine) (IgM mediated activation of classical
complement pathway and Membrane attack complex)
Laboratory

The patient's RBCs are washed (removing the patient's own serum) and
then centrifuged with antihuman globulin (also known as Coombs
reagent). If immunoglobulin or complement factors have been fixed on to
the RBC surface in-vitro, the antihuman globulin will agglutinate the
RBCs and the direct Coombs test will be positive. (A visual
representation of a positive direct Coombs test is shown in the upper
half).

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Indirect Coombs test

The indirect Coombs test, also referred to as the indirect antiglobulin test
(IAT), is used to detect in-vitro antibody-antigen reactions. It is used to
detect very low concentrations of antibodies present in a patient's
plasma/serum prior to a blood transfusion. In antenatal care, the IAT is
used to screen pregnant women for antibodies that may cause hemolytic
disease of the newborn. The IAT can also be used for compatibility
testing, antibody identification, RBC phenotyping, and titration studies.

Uses
Blood transfusion preparation
blood transfusion and cross-matching

The indirect Coombs test is used to screen for antibodies in the

preparation of blood for blood transfusion. The donor's and recipient's
blood must be ABO and Rh D compatible. Donor blood for transfusion is
also screened for infections in separate processes.

• Antibody screening

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A blood sample from the recipient and a blood sample from every unit
of donor blood are screened for antibodies with the indirect Coombs test.
Each sample is incubated against a wide range of RBCs that together
exhibit a full range of surface antigens (i.e. blood types).

• Cross matching

The indirect Coombs test is used to test a sample of the recipient's serum
for antibodies against a sample of the blood donor's RBCs. This is
sometimes called cross-matching blood.

Antenatal antibody screening

The indirect Coombs test is used to screen pregnant women

for IgG antibodies that are likely to pass through the placenta into the
fetal blood and cause haemolytic disease of the newborn.

Laboratory method

The IAT is a two-stage test. (A cross match is shown visually in the lower
half of the schematic as an example of an indirect Coombs test).

First stage

Nonpatient, washed red blood cells (RBCs) with known antigens are
incubated with patient serum containing unknown antibody content. If the
serum contains antibodies to antigens on the RBC surface, the antibodies
will bind to the surface of the RBCs.

Second stage

The RBCs are washed three or four times with isotonic saline solution
and then incubated with antihuman globulin. If antibodies have bound to
RBC surface antigens in the first stage, RBCs will agglutinate when
incubated with the antihuman globulin (also known Coombs reagent) in
this stage, and the indirect Coombs test will be positive.

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Titrations

By diluting a serum containing antibodies the quantity of the antibody in

the serum can be gauged. This is done by using doubling dilutions of the
serum and finding the maximum dilution of test serum that is able to
produce agglutination of relevant RBCs.

Coombs reagent

Coombs reagent (also known as Coombs antiglobulin or antihuman

globulin) is used in both the direct Coombs test and the indirect Coombs
test. Coombs reagent is antihuman globulin. It is made by injecting
human globulin into animals, which produce polyclonal
antibodies specific for human immunoglobulins and human complement
system factors. More specific Coombs reagents or monoclonal
antibodies can be used.

Enhancement media

Both IgM and IgG antibodies bind strongly with their complementary
antigens. IgG antibodies are most reactive at 37 °C. IgM antibodies are
easily detected in saline at room temperature as IgM antibodies are able
to bridge between RBC's owing to their large size, efficiently creating
what is seen as agglutination. IgG antibodies are smaller and require
assistance to bridge well enough to form a visual agglutination reaction.
Reagents used to enhance IgG detection are referred to as potentiators.
RBCs have a net negative charge called zeta potential which causes them
to have a natural repulsion for one another. Potentiators reduce the zeta
potential of RBC membranes. Common potentiators include low ionic
strength solution (LISS), albumin, polyethylene glycol (PEG), and
proteolytic enzymes

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