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Table 14.2 Expected coagulation test results for some disorders of haemostasis
Common haemostatic disorders Laboratory test results
Acquired
Vitamin К deciency (II,VII, IX, X) and
liver disease
Anticoagulant therapy Normal Prolonged Prolonged
DIC Prolonged Prolonged Prolonged Low
Idiopathic thrombocytopenia Prolonged Normal Normal Low
Inherited
Haemophilia A
Haemophilia В Prolonged Normal Abnormal
von Willebrand’s disease Prolonged Abnormal Abnormal
Inherited platelet disorder Prolonged Normal Normal Low
Vascular disorder Prolonged Normal Normal
Medical Laboratory Technology: Volume 1
Bleeding time PT APTT Platelet count
Normal Prolonged Prolonged
Prolonged Normal Abnormal
Two basic tests—APTT and PT—are done according to the procedure described earlier.
Citrated patient’s plasma is then mixed with aged plasma or adsorbed plasma in equal
proportion and degree of correction noted. The results and their interpretation are presented
in Table 14.1.
Preparation of reagents for substitution tests
Reagents for the substitution tests can be prepared in the laboratory and can be purchased
from suppliers of laboratory products. Following methods for in-house preparation of these
reagents may be helpful for laboratories of developing countries.
Preparation of aged normal serum and plasma
The aged normal serum is rich in factors VII, IX, X, XI and XII but decient in the labile factors
V and VIII. The aged normal plasma contains factors II, VII, IX, X, XI, and XII, and is decient
in the labile factors V and VIII. When aged-normal plasma is mixed with aged normal serum
(as done here), the mixture will be rich in factors II, VII, IX, X, XI, and XII. The mixture is
used as a reagent in the substitution test or correction test shown in Table 14.1. If correction
is accomplished, it is concluded that one of the factors present in the aged serum (or plasma)
is decient in the patient.
Principle
The labile factors (V and VIII) are readily removed by the process of aging. Serum does not
contain factor I (brinogen) while plasma has this factor. Thus when they are mixed, factor I
is added to the mixture.
Procedure
1. Clot a tube of normal blood.
2. Incubate the cloed blood for a period of 4 h at 37°C.
3. Add an equal volume of citrated plasma from a normal subject to the preceding tube of
serum.
4. Allow the incubation to continue for an additional two hours at 37°C.
5. Centrifuge for 10 min at 2500 rpm.
6. Remove the supernatant solution and store in the freezer compartment of the refrigerator
(–10°C)
7. Prior to use, thaw and dilute with 0.85% sodium chloride solution (1:5, i.e., one part
serum and four parts sodium chloride solution).
Laboratory Investigation of Bleeding Disorders
Preparation of adsorbed normal plasma
The adsorbed plasma is rich in factors V, VIII, XI and XII but decient in factors II, VII and X
(vitamin К dependent factors). It is used as a reagent in the substitution test. If mixing of the
adsorbed plasma with the patient’s plasma improves (or corrects) the original coagulation
time (АРТТ or PT) determined with the patient’s plasma, it is concluded that one of the
factors present in adsorbed plasma must be decient in the patient.
Principle
Certain insoluble alkaline salts such as barium sulphate or aluminium hydroxide can remove
prothrombin from normal plasma (along with other vitamin К dependent factors). Barium
sulphate is used for oxalated blood and aluminium hydroxide is used for citrated blood.
Reagent
Aluminium Hydroxide Gel Preparation
1. Dilute 50 mL of ammonia (sp.gr. 0.88) with 50 mL of distilled water.
2. Pour the above into 600 mL of water at 63°C containing 22 g of ammonium sulphate and
bring the temperature rapidly down to 58°C.
3. Stir the mixture vigorously and pour at once into a solution of 76.7 g of ammonium
alum dissolved in 1000 mL of water at 58°C.
4. The temperature of this mixture rises to 61°C. Continue to stir the mixture for 10 min
without leing the temperature fall below 58°C.
5. Centrifuge the precipitate and wash ve times with water. For the rst wash, use water
containing ammonia (300 mL of water containing 0.44 mL of ammonia; dilute the
ammonia solution of Step 1 by two-fold). For the second wash, use water containing
0.88 mL of ammonia (see Step 1). Subsequent washings (3, 4 and 5) are done with water.
6. After completing the washing, the nal precipitate is suspended in the least amount of
water that is required to make a gelatinous suspension that can be pipeed.
7. For use as an adsorbent, one volume of the gel is mixed with nine volumes of plasma.
Considering the volume required, one drop of the adsorbent can be mixed with nine
drops of plasma and this will be sucient for one test.
369
Note Aluminium hydroxide gel can be purchased commercially. Follow the manufacturer’s
instructions. Usually 1 g of gel is diluted with 4 mL of distilled water.
Procedure
1. To nine volumes of citrated plasma add one volume of aluminium hydroxide gel
suspension.
2. Mix well and incubate for 5 min at 37°C.
3. Centrifuge at 2000 G to obtain a clear supernatant and perform the PT test. If adsorption
is satisfactory, the time should be more than 60 s.
Additional information
Factors present in adsorbed normal plasma: I, V, VIII, XI, XII
Factors present in aged normal serum or plasma: VII, IX, X, XI and XII
Note Calcium (Factor IV) is present in serum but not in plasma.
TheraPy of Bleeding disorders
Transfusion of various blood components—normal plasma, platelet rich plasma, coagulation
factor concentrates, brinogen and cryoprecipitate-rich plasma—are some of the common
practices for the treatment of bleeding problems. Table 14.2 gives a list of haemostatic
disorders and their expected laboratory test results.
370
Cryoprecipitate is a frozen blood product prepared from normal plasma. It is also called
cryoprecipitated anti-haemophilic factor or ‘cryo’ in short. It is rich in factor VIII and brinogen.
It is used in the treatment of haemophilia, von Willebrand’s disease and hypobrinogenaemia
(low brinogen levels). The product is manufactured by slowly thawing a unit of freshfrozen plasma (FFP) at temperatures just above freezing (1–6°C), typically in a water bath or
a refrigerator. The product is then centrifuged to remove the majority of the plasma and the
precipitate is resuspended in the remaining plasma or in sterile saline. The product may be
pooled and frozen or frozen as individual units.
Medical Laboratory Technology: Volume 1
review QuesTions
1. After a long ight from Chicago to Kolkata, Robin Menta complained of chest pain with
cough and cold that he contracted on the way. His pain in the right calf also troubled him
at the time of landing. He had been sleeping most of the time during the ight without
much physical activity. Before reaching home he called his physician. The physician
asked him to get admied to the hospital and the emergency department was ordered
to perform the D-dimer test. What was the diagnosis of the physician?
(Answer DVT that may lead to pulmonary embolism.)
2. What causes pulmonary embolism?
3. List two tests used to monitor heparin therapy.
4. What is the origin of D-dimer in the body? What is the clinical signicance of D-dimer testing?
5. Describe the principle of latex agglutination test. How is this applied in the determination
of D-dimer?
6. Is brinolysis a natural process? What happens during brinolysis?
7. Compare the physical dierences between brin monomer and brin polymer.
8. What is the clinical signicance of bleeding time determination? How is this determined?
9. What would you infer from a prolonged whole blood cloing time (Lee–White)? What
is the clinical signicance of clot retraction test and the test for lysis time?
10. What is the most commonly used specimen for coagulation studies? What are the
precautions taken for preparing specimens (blood) for coagulation studies?
11. How would you perform one-stage prothrombin time (PT) test? What is the PT time for
a normal citrated plasma specimen? What would you infer from the prolonged PT time
of a patient’s plasma specimen?
12. How is APTT performed? What is its clinical signicance?
13. How would you prepare the following reagents?
(a) Adsorbed normal plasma, (b) Aged normal plasma, (c) Aged normal serum, (d)
500 mL of 0.025 M calcium chloride
14. How is thrombin time determined? What is its normal value? What would you infer
from a prolonged TT?
15. Which tests will be aected by platelet disorder?
16. Which of the coagulation tests will be aected by liver disorder and vitamin К deciency?
17. How does the physician identify von Willebrand’s diseases? What treatment course is
adopted?
18. How is the deciency of factor VIII diagnosed in the laboratory?
19. State some of the causes of vascular defect. How is the vascular defect diagnosed in the
laboratory?
20. What is cryoprecipitate? How is this prepared? Under what conditions does the
physician order the administration of cryoprecipitate?
21. A retired army surgeon was reported to have developed cirrhosis (liver disease) due to
excessive alcohol consumption during his active life. Which haemostatic tests will be
abnormal?
22. Why is a person suering from liver disorder given vitamin K?
3
Immunohaematology
or Blood Banking
Chapter 15: Introduction to Blood Transfusion Therapy
Chapter 16: Collection and Processing of Blood for Transfusion
Chapter 17: Routine Laboratory Procedures in Blood Bank
Chapter 18: Blood Transfusion Services and Clinical Approach to Haemolytic Disease of
the Newborn
Introduction to Blood Transfusion Therapy
Pampee Paul Young and Jay S Raval
Chapter Outline
• Basic Concepts of Immunology and Immunohaematology
▪ Denitions of commonly used terms
• Discovery of Basic Human Blood Groups (ABO)
• Principles of Immunohaematology
▪ Immune system of blood
• Red Cell Antigens
▪ Antibodies in serum
• Recognition of Immunologic Reactions of Red Cells
▪ Stages of immunologic reactions
• Laboratory Methods in Detecting Antibodies
• Human Blood Group Systems
▪ Basic blood group system: ABO
▪ Other variants in ABO blood group system
▪ Plant agglutinins for ABO blood group
• Rhesus (Rh) Blood Group System and Immune Antibodies
▪ Rh antigen
▪ Rh antibody
• Other Blood Group Systems
• Pretransfusion Testing
• Antibody Screen
▪ Major cross-match
• Compatible Blood Groups
• Review Questions
15
Blood is essential for human life. The blood volume of a normal adult in a developing country
is approximately 5 L. The cellular phase (approximately 45%) of whole blood consists of
red cells, white cells and platelets, which perform the vital functions of oxygen transport,
defence of the body through immunologic reactions, and stoppage of bleeding, respectively.
The non-cellular liquid phase (approximately 55%) is called plasma, which contains various
chemicals, antibodies and other proteins and coagulation factors. The cellular components
can only function when they are able to oat in the plasma and move freely through the
374
network of body’s circulatory system. In many diseases and health problems, therapeutic
administration of blood is indicated. The technique of replacing blood and its components is
called blood transfusion.
Blood transfusion is a major medical service which is provided through blood banks.
The functions of the blood bank include collection of blood from healthy donors, testing for
infectious diseases, storing it, processing it and supplying it to those who need it. The blood
bank performs a number of tests for the safety of the recipient. The chosen donor’s blood
should be compatible with the recipient’s blood and free from transmissible diseases. It is a
job that must be done accurately. In case of incompatible transfusion, serious complications
might develop in the recipient (the patient).
The compatibility test is based on the immunologic reaction of the red cells of the donor with
the circulating antibody present in the recipient’s plasma under natural conditions. Compatible
healthy red cells of the donor should not carry any antigen whose corresponding antibody is
present in the patient’s serum. The donor’s red cells should coexist with the recipient’s own
red cells. The laboratory testing procedures are based on the visible immunologic reaction of
haemagglutination and haemolysis. It may be worthwhile discussing here, the basic concepts
of immunology, immunohematology and how they dier from serology which is discussed
later (Chapter 24 of Volume II).
Medical Laboratory Technology: Volume 1
Basic concepts of immunology and immunohaematology
Immunology is the study of the body’s immune system. It developed from the study of
immunity. Early immunologists were physicians who worked to develop ways of providing
immunity to infectious disease. They produced vaccines for bacterial and viral diseases such
as smallpox, diphtheria and tetanus. As knowledge advanced, it became evident that the
immune system had considerably broader functions than just providing protection from
invading micro-organisms. It was recognized that a healthy immune system is fundamental
to overall good health. The immune system is involved not only in preventing or ghting
infectious disease but also providing protection from toxins and tumours (cancers).
With the discovery of human blood group systems and practice of blood transfusion as a
therapeutic measure, a new concept emerged—immunohaematology. Here the antigen (red
cells of the donor) is deliberately introduced into the body of the recipient (patient) with
the goal of making the donor’s blood compatible with the recipient’s blood and avoiding an
immunological reaction. Immunohaematology, or blood banking, can thus be dened as the
branch of immunology that uses immunologic principles to identify and study the blood group
antigens and their respective antibodies. While some blood banking procedures are relatively
simple, such as routine ABO grouping and Rh typing, complex procedure such as compatibility
testing for blood transfusion, antibody identication and tissue typing are also performed.
Serology, on the other hand, was the term rst used for laboratory immunology because
early immunological tests used serum for testing and tests were designed around the antigen–
antibody reactions. Today, serological procedures are more properly called ‘immunologic
procedures’ or immunoassays because they use serum, whole blood, urine and other body
uids, as well as cells and tissues in a variety of test methods. For many years serological
tests were mainly used for the laboratory diagnosis of infectious diseases and hence, they
have been discussed in this publication after the Microbiology section (Chapters 23 and 24 of
Volume II). Today, immunoassays are used by all departments in the laboratory in some way.
Definitions of Commonly Used Term
Before we launch into further discussion, it would be useful to overview some of the basic
denitions which apply to immunohematology.
Introduction to Blood Transfusion Therapy
375
Antigen
Antigen is a substance that causes the formation of its cognate. These are mostly proteins,
carbohydrates or lipids aached to the red cell. Each antigen has a certain chemical
conguration that gives it antibody- provoking ability. This specic chemical group may
become detached from its carrier molecule and temporarily lose antigen power; it is then
called, a hapten. Aachment of a hapten to another suitable molecule leads to the restoration
of antigenic properties.
Antibody
Antibodies are proteins of the globulin class, most often gamma globulins produced by
lymphocytes and plasma cells, in response to antigenic stimulation. They may be specic,
binding only to specic antigen molecules, or non-specic, binding to a variety of antigens.
Agglutinin is an antibody that reacts with its cognate antigens and manifests this activity by
clumping the red cells.
Types of antibodies
There are several types of antibodies, depending on their occurrence and laboratory
characteristics:
Complete (bivalent) antibodies: These antibodies usually agglutinate their cognate red
cells directly. In vitro tests for these antibodies tend to demonstrate beer reaction in saline
medium at room temperature (24°C) or lower. They often x complement.
Cold antibodies: These react best at 4°–10°C.
Incomplete (univalent) antibodies: These antibodies usually cannot agglutinate their cognate
red cells directly but only coat their surface. In vitro tests for these antibodies tend to show
beer in high-protein medium.
Isoantibodies (alloantibodies): These are antibodies produced against antigens coming from
genetically dierent individuals of the same species. These ‘foreign’ antigens are usually
introduced into the body by transfusion or by pregnancy (if foetal red cells containing antigens
that the mother lacks reach the maternal circulation). When isoantibodies are produced, they
do not cause diseases unless they interact with the red cells harbouring the cognate antigens
that the antibodies recognize.
Autoantibodies: Autoantibodies are antibodies produced by the body against one or more of
its own tissues. These antibodies are associated with autoimmune disorders.
Agglutinogen
Agglutinogen is an antigen that is found on the surface of red cells that renders the red cells
to clump when they react with its antibody (agglutinin).
Haemolysin
Like agglutinin, it is an antibody that reacts with its cognate red blood cells, except that in
this immunologic reaction, lysis of aected red cells takes place in lieu of, or in addition to,
agglutination.
discovery of Basic human Blood groups (aBo)
Karl Landsteiner rst discovered the basic ABO blood group system in humans during
1900–1901. He received the Nobel Prize in 1930 for this outstanding discovery. His approach
was simple and logical. He drew blood from several of his co-workers and obtained cell
376
Medical Laboratory Technology: Volume 1
suspensions and serum of each sample. Later the serum of each sample was reacted with
cells of dierent donors. This testing yielded four types of blood (Figure 15.1)—cells carrying
A antigen, cells carrying В antigen, cells carrying both A and В antigens and cells that do
not carry neither A nor В antigen. The laer was designated as ‘O’ (implying zero). These
blood group antigens were established by reacting them with their corresponding antibodies
present in the serum of a dierent blood group. This immunologic reaction resulted in
haemagglutination. As a result, the fundamental concept of blood banking emerged. In
summary, the presence of the antigen on the red cells implied the absence of its specic
antibody in the serum but the opposite antibody will normally be found there. Thus, a
person of blood group A has anti-B in serum, and for blood group В the serum has anti-Α.
If both antigens A and В are present on the red cells (blood group AB), the serum of these
individuals has neither anti-Α nor anti-B. In blood group О individuals, however, the serum
has both anti-Α and anti-B. In subsequent years, exceptions to this general rale were also
discovered like Bombay blood group O (Oh). Unlike the regular blood group O, which carries
H antigen, Bombay О does not carry H antigen on the red cells and the serum contains anti-Η
(Figure 15.2).
Figure 15.1 Haemagglutination reaction of red cell antigens (A, В and AB) with their corresponding
antibodies (anti-A, anti-B and anti-Α, B)
Following the discovery of basic ABO blood groups in humans, Mendelian laws of
inheritance of blood groups were traced. As blood transfusion became more and more
common, the occurrence of immune antibodies to other red-cell antigens became evident.
These immune antibodies develop as a result of incompatible transfusions. With improvement
in blood banking techniques, blood transfusion therapy today is reasonably safe. Yet
there are many potential harmful eects of transfusion therapy (e.g., emerging infections,
alloimmunization, acute lung injury, etc.) such that the benets of blood transfusion should
outweigh the risks.
Introduction to Blood Transfusion Therapy
377
Figure 15.2 Red cell antigens and antibodies of various blood groups Α, Β, ΑΒ, Ο and Bombay (Oh).
Note A person normally does not have the corresponding antibody in the serum to an
antigen that is found on that individual’s red cells.