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458
Medical Laboratory Technology: Volume 1
appendix
Appendix 17.1
REQUEST FOR BLOOD TRANSFUSION
Hospital Ward
Family name of patient First name
Sex Date of birth Age Address
Test Requested: (circle) Grouping Cross-matching Titre Other
Grouping and Compatibility Tests
Material Requested:
Units of whole blood Units of packed red cells
Date and time when blood (or its component) required
Date Time AM/PM
Diagnosis and reason for transfusion
Previous transfusion history
Untoward reactions to previous transfusions
Note If the patient is a woman, please record history of previous pregnancies, if any, and state
whether she ever had a miscarriage, a stillbirth, or an infant that suered from haemolytic
disease of foetus or newborn.
Details of last Hb cone. g/dL, Hct value %
Aending physician/Medical ocer
Nurse Date
Blood Transfusion Services and Clinical Approach to Haemolytic Disease of the Newborn
Pampee Paul Young and Jay S Raval
Chapter Outline
• Introduction to Blood Transfusion Services
• Pretransfusion Testing
• Release of Blood for Transfusion
• Blood Transfusion Therapy
▪ Blood transfusion process
• Transfusion Reactions
▪ Investigation of transfusion reaction
• Haemolytic Disease of the Foetus and/or Newborn
▪ HDFN caused by Rh incompatibility (HDFN-Rh)
▪ HDFN caused by ABO incompatibility (HDFN-ABO)
▪ Treatment of HDFN
• Review Questions
18
IntroductIon to Blood transfusIon servIces
The application of immunohaematology principles in a clinical laboratory is usually carried
out in the blood bank or the transfusion services department. The commonly used designation
‘blood bank’ comes from the fact that blood units have traditionally been stored or banked
in one location.
On a daily basis, the technologists working in a blood bank are responsible for typing
patient’s blood, testing blood for unexpected antibodies, matching compatible blood units
to patients for transfusion and providing other blood components (such as platelets) for
transfusion. In addition, blood banks are also responsible for the collection, storage and
supply of blood and its components to the patients for transfusion therapy. These functions
leave lile room for error and any mistake can be fatal.
PretransfusIon testIng
Details of pretransfusion tests have been presented in the previous chapter. Here we will
provide an overview of the basic facts in order to relate pretransfusion testing with blood
transfusion.
The principle behind the immunohaematological testing process is that the antibodies,
which are proteins and present in the serum, react with antigens on the surface of red
460
blood cells to abnormally remove them from circulation or cause a serious clinical condition.
These antibodies can either be acquired naturally or through immunization with allogeneic
red cells. They are of usually IgM and IgG types, respectively. The immune antibodies (IgG)
are smaller in size than the naturally-occurring antibodies (IgM). All antibodies to red cell
antigens, other than naturally occurring anti-Α and anti-B, are considered unexpected. They
may be alloantibodies directed against foreign non-ABO system antigens or autoantibodies
directed against self-antigens (such as in autoimmune diseases). Although less common,
unexpected antibodies in donor plasma may destroy recipient red cells, whereas antibodies
in the recipient may cause accelerated destruction of transfused donor red cells. In pregnant
women, such antibodies may cross the placenta and cause haemolytic disease of the fetus
and/or newborn (HDFN).
Antigen–antibody reactions with red cells and serum include direct haemagglutination,
sensitization or haemolysis of red cells when incubated in the presence of complement. The
sensitized cells are detected by the antihuman globulin (AHG) test. If the sensitization has
occurred within the body, the direct AHG test will be able to detect this. For antibody screening,
the sensitization is done in the laboratory by incubation followed by indirect AHG testing.
After the discovery of ABO and Rh blood group systems, numerous other antigens have
been assigned by the International Society for Blood Transfusion (ISBT). These inherited
antigens are distributed across >30 blood systems.
A healthy donor donates blood which is ultimately transfused to a patient who is the
recipient of the donated blood. Blood collection agencies conduct the initial ABO and D typing
as well as perform tests for infectious diseases. The blood is collected in plastic bags with
satellite connections to other bags for blood component preparation under aseptic conditions.
The donor bag also carries small test tubes with or without anticoagulant to provide whole
blood or cloed blood. A portion of the extended plastic tube is segmented and contains
aliquots of blood for laboratory testing.
The bag containing the donor’s blood bears a label with identity and date of collection. It
is unacceptable to correct an incorrectly labeled sample. Cloed blood is the specimen of
choice in performing various laboratory tests. Red cell suspensions are made from the blood
clot and serum provides information about the antibodies present. Blood samples used for
compatibility testing, including donor red cells, must be kept at 4°C for at least one week
after each transfusion. This ensures that appropriate samples are available for investigational
purposes, should an adverse response to transfusion occur.
Two methods are commonly used for ABO and D typing: slide method and tube method.
The tube method is more reliable but requires special equipment. Between these two methods
of testing, antibody screening can only be performed by the tube method. The tube method
allows the addition of various additives and one can detect agglutination more accurately.
Since 1990, a new method (gel phase testing) has emerged and is becoming popular. This
methodology has not yet reached the peripheral laboratories of developing countries.
Medical Laboratory Technology: Volume 1
release of Blood for transfusIon
Request form
A request form indicating the intended recipient’s name, unique identication number and
ABO and Rh type must be completed for each unit of blood to be released. This form must
also include the donor unit identication number, its ABO and Rh type, the interpretation
of the cross-match, and the identity of the person performing the test. If, as in an emergency,
pretransfusion testing is incomplete, the form must indicate the current status of serologic
testing.
Blood Transfusion Services and Clinical Approach to Haemolytic Disease of the Newborn
461
Label
Before blood is released, blood bank personnel must aach a label or tag to the unit containing
essentially the same information that appears on the transfusion form, namely, the recipient’s
rst and last names, unique identication number, the donor unit number, cross-match
interpretation and the name of the person performing the test.
Inspection
The unit must be inspected visually before it is released. In case there is any abnormality in
colour or appearance, it must be noted and the unit should not be issued. Make a record of the
result of inspection. The expiration date must also be checked to avoid issuing an outdated
unit.
Blood unit release record
The laboratory should maintain a log book in which the name of the individual who is
releasing the unit of blood, the patient for whom the blood was released, the date and time of
issue and the person to whom blood was issued or destination of the unit are recorded.
Bedside check
A mistake in blood transfusion could prove fatal for the patient. Hence, all care must be
taken to avoid clerical error. Yet, it does happen in spite of all precautionary measures. Clerical
errors are the most common cause of ABO incompatible transfusions. Before administering
blood, the physician’s wrien order should be reviewed to verify the request for transfusion.
The nurse who administers the blood is responsible for this and for performing a nal-error
check. Verication of the following must be recorded on the transfusion form:
• Recipient identication: The name and identication number on the patient’s wristband
must be identical to the name and number on the form aached to the unit.
• Unit identication: The unit number on the blood container must match the unit
number on the transfusion form.
• ABO/D: The ABO and D type on the donor unit primary label must agree with that
recorded on the transfusion form.
• Expiration date: The expiration date of the unit should be checked and the unit veried
as acceptable for transfusion.
Quality assurance program
Pretransfusion testing and blood transfusion constitute a quality assurance program designed
to detect serologic incompatibility between donor unit and the intended recipient and to
prevent both clerical and technical errors that may have serious consequences. Assurance
of quality requires proper performance of each task. There can be no substitute for proper
patient identication, proper sample labelling and proper performance of serologic tests.
Donor recipient issues
• Red blood cells of donor’s whole blood, selected for transfusion, should be compatible
with the serum of the intended recipient.
• Group-specic blood is more desirable than using group О blood.
• Group О individuals must receive group О red cells but AB individuals can receive red
cells of any ABO type.
• D-negative individuals, particularly women with childbearing potential, should receive
D-negative blood.
• D-positive individuals may receive either D-positive or D-negative blood components.
462
• There should be no agglutination of donor’s red cells in the major cross-match when
reacted with the recipient’s serum in any phase.
• If the cross-match shows positive agglutination in the above test, unexpected antibodies
are suspected to be present in the recipient’s serum.
• All eorts have to be made in identifying the unexpected antibody.
• When the antibody is identied, select the red cells for transfusion that lack the
corresponding antigen or antigens. Examples of potentially signicant antibodies
include those directed towards Rh, Jk, Kell and Fy system antigens. It also includes Ss
antigens of the MNS system, as well as most other antibodies active at 37°C.
Medical Laboratory Technology: Volume 1
Blood transfusIon theraPy
In case of blood transfusion therapy, donor’s blood is deliberately introduced into a recipient
for treating various clinical conditions. If the donor’s blood is not compatible, a transfusion
reaction can occur.
Blood Transfusion Process
Following pretransfusion testing (Chapter 19), compatible blood from the donor is dispatched
to the ward by the blood bank where the recipient (patient) receives the blood intravenously
(Figure 18.1). Blood is stored at 4°C. If an additive solution is used, the shelf-life of refrigerated
blood may be extended to 42 days. Once the blood is issued from the blood bank it should be
transfused within 4 h. If a delay is expected, store only at 4°C.
transfusIon reactIons
Incompatible blood from the donor when given to the patient can cause a transfusion reaction,
which can be life-threatening. Although blood transfusions always involve some amount of
risk since identical blood of donor and recipient is impractical, the risk is greatly minimized
by modern blood banking procedures. Unfortunately, transfusion reactions do occur for
various reasons and one of them is human error. All aempts should be made to minimize
human error. Irrespective of the reason, the reaction should be investigated to determine the
cause and to remedy it.
Of the various types of transfusion reactions, only the haemolytic transfusion reaction
could be directly related to a technical error on the part of the blood bank. The technical
error of the laboratory could be due to incorrect blood typing and/or pretransfusion testing.
Consider a situation where a recipient is of blood group A, and receives blood of group B.
The anti-B present in the recipient will immediately react with В cells of the donor and will
destroy them. If the transfusion is not stopped immediately, it can be fatal.
Blood Transfusion Services and Clinical Approach to Haemolytic Disease of the Newborn
463
Figure 18.1 Blood transfusion procedure: (a and b) Blood collected from the donor is given to the
recipient, the patient, through a ‘giving tube’ (c); (d) The ‘giving’ needle is inserted into
the recipient’s vein while the other end goes into the bag of donor’s blood
Other transfusion reactions like allergic reactions and pyrogenic reactions have lile to do
with blood bank techniques (aside from manipulations such as washing and leukoreduction,
respectively), and hence these will not be discussed. The blood bank, however, should
screen the donor’s blood very carefully in order to prevent transfer of various diseases, with
particular reference to syphilis, malaria, viral hepatitis and HIV. -
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Medical Laboratory Technology: Volume 1
Investigation of Transfusion Reaction
If the patient manifests the signs of haemolytic reaction (discomfort, fever, chill, change in
blood pressure, breathing rate, pulse, rash, diculty in breathing), stop further transfusion
and proceed with the investigation. This requires a re-check of every aspect of transfusion—
clerical as well as serological.
Note Cross-matching specimens (pilot tubes, etc.) are retained for at least seven days following
transfusion so that testing may be repeated in the event of a transfusion reaction.
The procedure suggested for following the transfusion reactions are listed below:
• Record clinical signs and symptoms, pre- and post-transfusion vital signs (temperature,
heart rate, respiration rate and blood pressure).
• Check all relevant paperwork—the number of units transfused, identication of the
patient, crossmatching results, records of previous transfusions, etc. Ensure that the
transfusion was without faulty clerical work.
• Collect a 10 mL cloed specimen of blood. Take special care to avoid haemolysis during
blood collection.
• Collect the rst post-transfusion specimen of urine and examine for haemoglobinuria.
• Examine patient’s serum for haemolysis.
• Examine the donor’s blood for possible contamination and haemolysis. Look for cloudy
appearance, purple discolouration and clot formation.
• Take a second post-transfusion specimen of blood after six hours of transfusion, to
determine serum bilirubin and haemoglobin concentrations.
• Perform direct antiglobulin test with the washed red cells of the patient. A weak positive
(mixed eld) direct antiglobulin test in the post-transfusion specimen is strongly
indicative of an incompatible transfusion.
• Perform re-grouping of the donor’s blood, using a fresh pilot tube of the donor’s blood
and repeat the cross-match. Take special note of the results obtained in the ABO and Rh
typing (saline, protein and thermophases) with respect to weak or mixed-eld reactions.
• Examine the peripheral blood lm of the recipient following the haemolytic episode.
Presence of new microspherocytes or red cell fragments is suggestive of incompatibility.
• Perform antibody screening of the patient’s serum for the presence of unexpected
antibodies.
haemolytIc dIsease of the foetus and/or newBorn
Haemolytic disease of the foetus and/or newborn (HDFN, also called erythroblastosis foetalis)
is a disease that starts in utero and causes jaundice, anaemia and enlargement of the liver and
spleen in foetus and infant. The degree of severity of the disease ranges from mild anaemia
to mental retardation or stillbirth.
HDFN Caused by Rh Incompatibility (HDFN-Rh)
HDFN caused by Rh incompatibility between the infant and the mother is more common and
is more severe than ABO incompatibility between foetus and mother and is described later.
The potential candidate for HDFN-Rh is an Rh-positive child whose mother is Rh-negative
and father Rh-positive. During gestation and/or childbirth the Rh-positive red cells of the
foetus cross the placenta and enter the maternal circulation (Figure 18.2). If the mother is Rhnegative, she is sensitized and may form anti-D. The immune antibody (IgG anti-D) returns
to foetal circulation in a subsequent pregnancy by crossing the placental barrier. If the red
cells of the infant are Rh-positive, the immune antibody reacts with the red cells and destroys
them. One incompatible pregnancy is required to immunize the mother and consequently the
rst infant is unaected. The rst infants may be aected in the cases of those women who
Blood Transfusion Services and Clinical Approach to Haemolytic Disease of the Newborn
Figure 18.2 Erythroblastosis foetalis or HDFN (Haemolytic disease of the foetus and/or new-born): (a)
It is a disease that starts in utero. The potential candidate for HDFN-Rh is an Rh-positive
child whose mother is Rh-negative. During gestation and/or childbirth, the Rh-positive
red cells of the foetus cross the placenta and enter the maternal circulation, (b) Mother is
sensitized and produces anti-D without hurting her. (c) The immune antibody (IgG anti-D)
returns to foetal circulation in a subsequent pregnancy by crossing the placental barrier.
If the red cells of the infant are Rh-positive, the immune antibody reacts with the red cells
and destroys them. Hence, the rst infant is unaected while the subsequent infants may
suer from HDFN.
465
have received ‘incompatible’ transfusions (D-negative mother given D-positive blood) or
who have had an abortion with an ‘incompatible’ foetus (D-negative mother with D-positive
foetus). Although all pregnant women with an incompatible foetus can become immunized,
50% of them remain unaected even when exposed to antigenic stimuli.
HDFN-Rh can be successfully prevented by administering Rh immune globulin (anti-D) to
the mother within 72 h after the delivery of an Rh-positive baby. The amount of anti-D to be
injected is pre-determined by the number of Rh-positive cells in maternal circulation. For this,
the acid elution test is recommended.
Acid elution test
Principle
Foetal red cells contain haemoglobin (Hb) F which is resistant to acid elution.
Procedure
Prepare a thin blood lm of mother’s peripheral blood and air dry. Dip the lm in acid
buer (citric acid-phosphate buer, pH 3.3) and stain with Leishman stain or any other stain.
Examine the smear under oil-immersion. Normal cells with Hb A will appear as ghost cells
while foetal cells with Hb F will not be aected and retain their haemoglobin. Distribution of
foetal cells in mother’s blood is determined by the following formula.
Note Normal adults may have 1% or fewer cells with Hb F.
% foetal cells × 50 = mL foetomaternal haemorrhage
The physician will decide the amount of anti-D to be administered, based on the report of
the amount of foetomaternal haemorrhage (mL). The supplier of the anti-D (Ortho-Ethnor,
Bombay) provides instructions along with the reagent supplied.
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Medical Laboratory Technology: Volume 1
Laboratory diagnosis of HDFN-Rh
The laboratory diagnostic tests for HDFN can be divided into prenatal and postnatal tests.
The specimens used for these tests are the cloed blood specimens of the mother (prenatal
and postaatal), father (prenatal) and infant (postnatal). A cord blood specimen is obtained by
needle aspiration. The cord blood specimen should be properly labelled with the mother‘s
name, baby‘s identication (by name or family), hospital number and date. Such samples
should be sent to the blood bank and stored in the refrigerator for at least seven days. The
cord blood is then readily available for testing if the newborn develops signs and symptoms
suggestive of HDFN. The cord blood must accompany the maternal blood sample for
laboratory testing.
Prenatal laboratory tests
• Determine the ABO and Rh group of the mother and the father. If the mother is Rh-
negative (irrespective of ABO blood group) and father is Rh-positive (i.e., D-positive),
the risk for immunization exists.
• Look for the presence of anti-D in the mother, especially in case of a second or subsequent
pregnancy. Also perform a general antibody screening of the mother’s blood specimen.
If the screening result is positive, the unexpected antibody must be identied to
determine if it is a signicant one.
• If the immune antibody is present (anti-D), determine the titre and nd out whether
there is a rising trend of titre. This is done by determining the titre of anti-D at monthly
or two-weekly intervals.
Postnatal investigation
• ABO and Rh grouping of the mother and the infant.
• Direct antiglobulin test of cord blood (Figure 18.3).
• Elution and identication of antibodies are done if the direct antiglobulin test result is
positive. This helps in the search for appropriate blood for possible exchange transfusion.
Figure 18.3 Laboratory diagnosis ofHDN due to Rh incompatibility: Laboratory recognizes the
presence of sensitized red cells in the foetus by antihuman globulin (or Coombs) reaction
of a well-washed blood specimen obtained from the cord.
Blood Transfusion Services and Clinical Approach to Haemolytic Disease of the Newborn
467
HDFN Caused by ABO Incompatibility (HDFN-ABO)
HDFN due to ABO incompatibility presents a somewhat dierent problem. The maternal
serum already has the potentially reactive antibody against the red cell antigen of the
newborn. This may cross the placental wall and interact with the red cells of the newborn.
Thus, unlike HDFN-Rh, the disease more readily occurs in the rst born infant. Often in
HDFN-ABO, the mother is О with high titre IgG anti-A and foetus is of blood group A (more
common). The IgG anti-Α is capable of crossing the placental barrier and will react with the
foetal red cells bearing the A antigen.
Clinically signicant HDFN-ABO is not common. Clinically signicant HDFN can also be
caused by other red cell antibodies such as anti-Keli, E, с, etc.
Treatment of HDFN
In case of Rh incompatibility the condition can be controlled during pregnancy by following
the anti-Rh-titre of the mother’s blood and the bilirubin concentration of the mother’s blood.
These tests indicate whether the pregnancy should be allowed to go to full term or whether
labour should be induced earlier. If the child is seriously aected, exchange transfusion may
have to be performed.
Exchange transfusion
Exchange transfusion is a potentially life-saving procedure that is done to counteract the
eects of serious jaundice or changes in the blood due to diseases such as sickle cell anaemia.
The procedure involves slowly removing the patient’s blood and replacing it with fresh
donor blood or plasma.
Exchange transfusion is performed with the following points in view:
• Lower the bilirubin concentration in the infant’s blood.
• Remove or dilute the infant’s sensitized red cells in circulation.
• Reduce the amount of immune antibodies in the baby.
The compatibility test in case of exchange transfusion is done in a dierent way from the
routine procedure described earlier. The newborn infants do not have natural antibodies in
their serum. Hence their ABO grouping relies entirely on cell typing (forward). Compatibility
testing of the transfused (donor) cells is done with maternal serum.
There are also some practical diculties in handling a cord blood specimen which should be
mentioned here. The cells are trapped in Wharton’s jelly (a gelatinous intercellular substance
consisting of primitive connective tissue of the umbilical cord). The Wharton’s jelly must
be thoroughly washed or it may lead to false positive results of agglutination. In addition,
accurate Rh testing of cord cells is dicult because of the heavy coating of anti-D with the
occurrence of HDFN-Rh. These sensitized cells may also give false results for D-antigen site.
Procedure of Exchange Transfusion
In case of HDFN-Rh, the use of identical blood group with Rh-negative blood type is
recommended, while for HDFN-ABO, blood group О blood type is the optimal choice. In
either case, the donor’s blood must be compatible with the mother’s serum. If the mother’s
blood is not available, test the donor’s cells with eluate made from the infant’s antibody-
coated cells.
During the process of exchange transfusion, blood is intermiently withdrawn from the
infant and replaced by donor blood. Usually a total of two units of donor blood (900 mL)
are ultimately transfused, which is about four times the volume of infant blood (200 mL).
Transfusion is commonly done through the umbilical cord in the newborn. The choice of
blood group for exchange transfusion is given in Table 18.1.