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448
Medical Laboratory Technology: Volume 1
Figure 17.13
donor’s 4% red cell suspension in a test tube. Centrifuge and observe any haemagglutination
suspected, (d) If no agglutination is seen, proceed to incubate for 90 min at 37°C, (e) Add
22% albumin and reincubate for 20 min, (f) Look for haemagglutination reaction. Donor’s
blood is considered to be compatible if there is no agglutination at any of the reaction phases.
Note: AHG phase testing is not performed routinely in laboratories of developing
countries.
Routine Laboratory Procedures in Blood Bank
3. Antihuman globulin (AHG) phase (or Coombs’ phase): In this phase the incubated
cells are thoroughly washed (to remove free globulin) and reacted with AHG reagent
(Coombs’ reagent) at 37°C.
449
Emergency Cross-Match
Occasionally the laboratory is asked to do an emergency cross-match. One or more steps in
the cross-match procedure can be eliminated to gain speed; however, each omission produces
a small but denite risk of missing an unexpected antibody that is present.
In their 1984 Standards, AABB has omied Coombs’ stage of the cross-match as well as the
high-protein (or LISS) stage, relying instead on ABO and D typing and the antibody screen
of the recipient serum plus an ‘immediate spin’ saline cross-match procedure for possible
ABO incompatibility. The immediate spin method entails centrifugation and examination
without incubation, which takes only about 5 min. A traditional crossmatch procedure would
be performed only when the antibody screen detects an abnormality.
This protocol saves a signicant amount of time and money, since ABO typing and recipient
serum antibody screen are usually performed well in advance of the actual order to transfuse.
Studies indicate that only a very small percentage of recipients have an antibody that will
be missed and, even if present, are unlikely to be life-threatening. However, there is still
considerable debate about how far to go in altering the traditional cross-match procedure and
whether or not to perform it in all cases. Though there is no doubt that abbreviating the crossmatch saves time, there is some controversy whether the risk is suciently compensated for
by the speed of blood availability.
Principle
Serum of the recipient is tested against red cells of the donor under dierent conditions
in order to establish their compatibility or non-agglutination. Agglutination in any of the
conditions indicates the presence of incompatible antibody in the patient, natural or immune.
The three phases of compatibility testing are listed as follows and illustrated in Figure 17.12—
saline (immediate spin) phase, protein/thermophase and AHG phase. ABO incompatibility is
recognized in the saline phase, while agglutination in other phases indicates the presence of
immune, incomplete or irregular antibodies. If agglutination is not seen in any of the above
phases, donor red cells and recipient serum are considered to be compatible.
Specimen
Donor’s cloed blood specimen is available from the pilot tube. Donor’s red cells are taken
out of the clot, repeatedly washed with saline and a 5% v/v suspension is made in saline
(0.1 mL packed red cells mixed with 1.9 mL of saline). Patient’s blood is drawn fresh and
collected in a sterile pre-labelled dry container without any anticoagulant. Separate the serum
promptly. Patient’s serum is used for major cross-matching and cell suspension (3 to 5% in
saline) for the auto-control.
Procedure (see Figure 17.13)
1. Take two small tubes (10 mm × 75 mm) and mark them as 1 and 2. Tube 1 will be used
for crossmatching while tube 2 will be the auto-control. Tube 2 receives all treatments of
tube 1, except that it does not contain donor’s cells.
Note Figure 17.13 illustrates the handling of tube 1 only.
2. Add 2 drops of patient’s serum in both the tubes.
3. Add 1 drop of 5% saline suspension of donor’s cells in tube 1 and 1 drop of patient’s cell
suspension (5% in saline) in tube 2.
Note The auto-control is the suspension of patient’s red cells in its own serum.
4. Mix and centrifuge at 1500 rpm (150G) for 1 min.
450
Medical Laboratory Technology: Volume 1
5. Gently dislodge the cell buon and examine for agglutination and haemolysis. Record
the results. If agglutination is noted in tube 1, ABO incompatibility is suspected.
6. To both tubes add a drop of 22% bovine albumin, mix and incubate at 37°C for 30 min.
Alternatively Incubate both tubes at 37°C for 15 min and add a drop of 22% albumin
along the side of each tube. Mix and re-incubate for another 20 to 30 min.
7. Centrifuge at 1500 rpm (150G) for 1 min. Examine for agglutination and haemolysis.
Record the results of agglutination with grading.
Note Figure 17.13 does not show AHG reaction because it is not done routinely in
developing countries. The Coombs’ reagent (AHG) is expensive and unstable in hot
climates.
8. Wash the cells 3 to 4 times with saline, decant completely after each wash and add
2 drops of AHG serum to the sedimented cells. Shake the tubes to mix the contents
and then centrifuge at 1500 rpm (150G) for 1 min. Examine for agglutination and grade
the agglutination reaction. Also look for haemolysis. Some of the antibodies bind
with complement and cause haemolysis, which should be considered as evidence of
immunologic reaction (positive).
9. If there is no agglutination reaction (negative), add a drop of pre-sensitized check cells.
The sensitized or check cells must agglutinate if the cells are adequately washed and
AHG is reactive.
Interpretation (See Table 17.5)
• Agglutination should not be visible in any phase, including auto-control.
• Incompatibility in the saline phase should be investigated in the line of ABO grouping.
Haemolysis suggests the presence of cold-reacting antibodies.
• If a patient’s screen is negative, then only saline (‘immediate spin’) testing is done. If the
screen is positive, the AHG phase is performed to provide cross-matched compatible
units.
• Many laboratories have eliminated the thermophase in order to save time. All the
thermophase reacting antibodies are detected in the AHG phase.
• Haemolysis at the thermophase with protein might indicate the presence of immune
antibodies directed against A and B, anti-Le and Rh antibodies.
• Haemagglutination at the AHG phase detects such antibodies directed against Fy, Jk,
K, and antigens of other blood systems. Some of the hard-to-detect Rh antibodies and
antibodies of warm autoimmune haemolytic anaemias are also found in the AHG phase.
Additional information
If the donor’s red cells pass through all the phases of compatibility testing, the donor unit
can be considered to be safe for transfusion to the recipient. If the donor’s cells indicate
incompatibility, run a screening test with the reagent О cells in search of possible irregular
antibody in patient’s serum. The reagent О cells go through all of the above phases in place of
donor’s cells. Following screening and identication of any antibody, a donor is sought who
lacks the corresponding antigen. If the phenotypes of dierent donor units are not known,
choose dierent donor units at random. Reference laboratories use reagent О cells of known
antigenic composition to identify unexpected antibodies. These laboratories are also capable
of identifying donor red cell antigens by using known antibodies. Because of the high cost of
reagent cells and antisera, routine laboratories cannot handle the identication and hence test
against dierent donor units for compatibility.
Routine Laboratory Procedures in Blood Bank
Table 17.5 Probable incompatible antibody present in patient’s serum in case of agglutination and/
or haemolysis
Phase in cross-matching Probable antibody in serum
Saline phase at room temperature
(“immediate spin”)
Thermophase with protein anti-D, anti-C, anti-E, anti-c, anti-e
anti-Α, anti-B, anti-I, anti-Η, anti-P, anti-M, anti-N, anti-Le, anti-Lu
Note Many laboratories have eliminated the thermophase for saving
time. These antibodies are then detected in the following phase.
451
AHG phase
or Coombs’phase
anti-D, anti-C, anti-c, anti-Ε, anti-e, anti-S, anti-s, anti-K, anti-Fy, anti-Jk,
anti-Le, anti-k
Summary
1. Request for compatibility tests labelled as ‘Stat’ or ‘emergency’ must take precedence
over all other work in the laboratory.
2. The aending physician should indicate the urgent nature of the situation and, if
unmatched, crossmatched or incompletely cross-matched blood is required, should be
made aware of the inherent risks involved.
3. Type-specic blood should be given whenever possible.
4. Begin with routine cross-match and continue even if the blood is released. The use of
LISS as a suspending medium allows the safe reduction of incubation time.
5. If incompatibility is detected in any phase of the cross-match, immediately notify the
patient’s physician and the blood bank physician.
Sources of Error in Cross-Matching
Correct typing and cross-matching are essential for safe transfusion. Some of the common
causes of anomalous results are discussed further:
False positive reactions
• Rouleaux formation: Here the red cells have the characteristic appearance of a roll of
coins when the cell suspension is seen under the microscope. This is caused by high
concentration of globulin under certain diseased states of the body (such as multiple
myeloma) or previous administration of plasma expanders (like Dextran).
Solution Rouleaux formation is dispersed in the majority of cases by dilution of the serum
with normal saline. If the cells do not disperse by the addition of saline, it should be
considered as haemagglutination. One should bear in mind that in compatibility testing
such a manoeuvre could dilute out a very weak antibody and it may not be detected.
• Panagglutination: Polyagglutinability or panagglutination means simply that a sample
of red cells is agglutinated by many samples of human serum. There are several
circumstances in which red cells become agglutinable because of the exposure of
cryptic antigens (normally not exposed) that form part of the structure of normal red
cell membranes. This is a rare phenomenon. In such a case, donor’s blood should be
rejected.
• Autoagglutination: This represents clumping of an individual’s red blood cells (RBCs or
erythrocytes) by his or her own serum due to the RBCs being coated on their surface by
antibodies. Presence of cold agglutinin and autoantibodies in the recipient’s serum might
cause autoagglutination which is recognized from a positive agglutination reaction in
the auto-control tube. The reason for the production of these autoantibodies is not clear.
If the haemagglutination reaction appears at room temperature and disappears at 37°C,
452
presence of cold agglutinin is conrmed. Presence of cold agglutinin in patient’s serum
is usually ignored. Autoantibodies are also produced in case of haemolytic anaemias
and they are directed towards the patient’s own red cells. Cases of acquired haemolytic
anaemias frequently exhibit a positive direct AHG test demonstrating that the cells are
coated with an autoantibody. Autoagglutination should also be distinguished from
“Rouleaux formation” wherein normal blood tends to form stacks on keeping, which
disperses on dilution with normal saline.
Solution Recognition of the problem is of primary importance. If agglutination is seen
in the auto-control, presence of autoantibody is suspected. In order to conrm this, wash
the cells thoroughly with saline, elute in warm saline and then test with the antisera. No
agglutination will be noted.
• Cord red cells: This gives false positive results due to the presence of Wharton’s jelly.
Solution Wash the foetal cells thoroughly with saline before testing. If Wharton’s jelly
contamination is a common problem, eliminate the problem by advising the delivery
room to collect cord blood samples from the umbilical vein using a syringe and needle
rather than allowing the blood to drain from the cord into the collection tube.
Medical Laboratory Technology: Volume 1
False negative reactions
The serum may be inactive or omied from the tube.
Solution Always run quality control tests at least once a day in order to check the reliability
of reagents in use. Duplicate checking of compatibility testing is recommended in order to
avoid technical errors.
antiBody Screening teSt
In case of incompatible cross-match or in the investigation of a transfusion reaction, a search
for the presence of unexpected antibodies is set up by the blood bank. Antibody screening
is especially important for the obstetric patient in whom the detection and identication of
antibody, prior to delivery, allows adequate time for preparations to be made for the possible
transfusion in a newborn infant.
Principle
Antibody screening is based on testing between patient’s serum and reagent red cells. Reagent
О cells are subjected to all phases of cross-matching. Presence of unexpected antibody will
be recognized by the haemagglutination reaction or haemolysis of О cells. The detection
of clinically-signicant antibodies usually occurs in thermophase (with protein) and AHG
phase. If agglutination of red cells does not occur, it is concluded that the patient’s serum
does not have unexpected antibodies.
Specimen
Test serum of the patient should be fresh (not older than 72 h) and drawn from cloed blood
within 2 h after its collection. Also prepare a 5% red cell suspension in saline of the patient’s
blood, which will be used in seing up the auto-control.
Procedure
1. Label three test tubes (10 mm × 75 mm) as I, II and Auto. These represent O-positive
reagent cells of group I and group II, and the auto-control which will have the patient’s
own cells.
2. Add 2 drops of test serum in all three test tubes.
3. Add 1 drop of 5% red cell suspension of O-positive reagent cell I in the rst tube (as
marked), 1 drop of 5% reagent O-positive cells of group II in the second tube (as marked)
and 5% patient’s red cell suspension in the third tube (auto-control).
Routine Laboratory Procedures in Blood Bank
4. To all three tubes add 2 drops of albumin along the side of the tube, mix gently and
incubate at 37°C for 30 min.
5. Centrifuge at 1500 rpm (150G) for one minute and look for agglutination and haemolysis
and record the results.
6. If the reaction is negative, wash the cells 3 to 4 times with excess volume of saline to
remove the free globulin. Do not overdo this else the cell-bound antibody may be lost.
The common way to wash is to decant the saline as completely as possible between each
washing, and to re-suspend the cells completely with each addition of new saline (3/4
full each time). Shake the cell buon briskly and add the saline in a forceful stream.
7. If there is no agglutination, check the AHG phase with Coombs’ positive control cells.
Interpretation
Agglutination in any phase indicates the presence of unexpected antibody in the serum
provided the auto-control (Auto) does not show any haemagglutination, i.e., the patient’s
cells do not agglutinate by themselves when exposed to the patient’s own serum due to
the presence of autoantibody. If agglutination is seen in any of the other tubes (I or II), the
presence of unexpected alloantibody is suspected. The autologous control or auto-control
should not normally agglutinate.
Additional information
The test is of great value, but it does have limitations. A negative test does not necessarily
mean that the serum lacks unexpected antibodies. It is possible that the corresponding
antigen is not present on the reagent cells selected. However, since the reagent О cells are
pooled from several donors, the chance of this happening is low. If the screening cells do
not react with the serum antibody, it can be safely considered that the serum does not have
any unusual antibody. If agglutination is seen, the next task is to identify the antibody that is
causing agglutination.
453
identification of unexpected antiBodieS
If the presence of an unexpected antibody is detected in the antibody screening procedure
described earlier, the specicity of the antibody should be determined. Because of the lack of
availability of panel reagent cells of known antigenic composition, it is dicult to identify the
antibody in a routine blood bank laboratory of a developing country. Reference laboratories,
however, are beer equipped.
The procedure is same as the antibody screening test, except that the О cells have known
antigenic composition. Based on reaction characteristics of antibodies in the patient serum
with panel cells, the most likely antibody or antibodies are identied. Once the identity of the
antibody is known, donor red cell units lacking the corresponding antigen are selected for
cross-match.
Elution of Antibodies
When a direct Coombs’ test yields positive results, it is desirable to aempt elution (removal
or detachment) of the antibody from red cells to determine the antigen against which it is
reacting. This is usually done by changing the physical conditions surrounding the antibody
to neutralize the aachment forces. The most common current methods are heat, freeze-thaw
and chemical. Once the antibody is removed from red cells, it can be tested with a panel of red
cells containing known antigenic composition to establish its specicity.
In case of haemolytic disease of a newborn or haemolytic anaemia, the oending antibody
is coated on the sensitized red cells. This can be detected by direct AHG test. In such cases,
454
elution techniques are used to remove the antibody from red cells followed by subsequent
identication of the antibody in the eluate. Elution technique is also applied in antibody
investigation.
Heat elution technique is more commonly followed in laboratories of developing countries
and will be described here. Chemical elution with ether and other organic solvents is
performed under special conditions.
Principle
The oending antibody is loosely bound to the surface of red cells and can be eluted by mild
heat (56°C) that will neither destroy red cells nor antibodies, but will release the antibody into
the saline medium.
Procedure for heat elution
1. Wash the sensitized red cells (0.5 to 1.0 mL of packed cells or free cells from cloed
blood), from which the eluate is to be made, six times in large volumes of normal saline
in order to remove the free-oating antibodies. The nal wash should be performed by
adding a volume of saline equal to the volume of washed packed red cells. Centrifuge
(1500 rpm/150G for 1 min) and recover the supernatant to test for the presence of
residual free-oating antibody. If positive, repeat the wash until the antibody screen is
negative. The supernatant of the last wash is nally tested in parallel with the eluate.
The former acts as a negative control that demonstrates that the residual antibody has
been removed before the eluate is prepared from red cells.
2. Add an equal volume of saline to the washed packed red cells in the centrifuge tube.
3. Place the tube in a 56°C water bath for 10 min. Agitate frequently and strongly during
incubation.
4. Centrifuge in pre-warmed cups at high speed (3400 rpm/340G for 1 min).
5. Remove the haemoglobin-tinted supernatant uid. This is the eluate.
6. Test the eluate for the presence of antibody by the method described earlier. Use the last
saline wash of Step 1 as the negative control.
7. The eluate can be stored overnight at 4°C or at –20°C for the extended period.
Medical Laboratory Technology: Volume 1
titration of anti-d
Titration can be dened as a semi-quantitative means of measuring the amount of antibody
in a serum. A titre, therefore, refers to the strength of an antibody, measured by determining
the greatest dilution of antibody-containing serum (in this case anti-D) that will produce a
detectable reaction with a standard volume of red cells possessing the corresponding antigen.
In this case it will be Rh-positive red cells of blood group O.
Quantitative determination of anti-D is routinely requested during the course of pregnancy
if the mother is a potential candidate for immunization. A rising titre could indicate possible
occurrence of HDFN (haemolytic disease of the foetus and/or newborn).
Principle
Antibody titration involves a serial dilution of the serum in saline and testing of each dilution
against the corresponding red cell antigen. In case of anti-D titration, the chosen red cells carry
D antigen (Rh-positive). Titration scores provide a semiquantitative measure of the amount
of antibody in a serum. Titrations are most frequently performed by preparing progressively
double dilutions of the serum. In the course of two-fold dilutions and subsequent testing
of haemagglutination, a point arrives when the antibody is too dilute to bring about the
immunologic reaction between the agglutinogen and the agglutinin. The highest serum
dilution where the agglutination is observed is the titre. For example, if the dilution is 1:32
(one part of serum in a total of 32), the titre is reported as 32.
Routine Laboratory Procedures in Blood Bank
455
Specimen
Blood specimen containing anti-D is allowed to clot. This is centrifuged and the serum is
separated and stored in refrigerator until ready for titration.
Reagents and equipment
• Saline
• 22% albumin (bovine)
• Rh-positive cells
• Test tubes without rim (5-mL)
• Centrifuge
Procedure
1. Label 10 test tubes as follows: (Figure 17.14)
Tube No. 1 2 3 4 5 6 7 8 9 10
Dilution 1 2 4 8 16 32 64 128 256 512
2. Deliver 0.1 mL of saline in each tube (except the rst tube) with the help of a graduated
serological pipee.
Alternate drop method: Use two identical droppers for dispensing saline and serum.
Count the drops for dilution instead of measuring volume.
3. Add 0.1 mL of test serum in tubes 1 and 2.
Figure 17.14
Note: The above dilution can also be done by the ‘drop method’. Use an identical Pasteur pipette to
count the drops of saline or serum. One drop of saline mixed with one drop of serum gives 1:2 dilutions.
Use ceramic tiles with cavities, instead oftest
4. Mix the contents of tube 2 by blowing through the serological pipee and transfer 0.1
mL of diluted serum from tube 2 to tube 3.
Note Return the residual uid back to the tube in order to maintain constant volume
in all tubes.
456
Medical Laboratory Technology: Volume 1
5. Mix the contents of tube 3 by blowing and retransfer 0.1 mL of diluted serum of tube 3
to tube 4 and continue the process until the last tube is reached. Mix the contents of the
last tube (‘512’ dilution) and set aside 0.1 mL of the diluted serum, which may be needed
for further dilution.
6. Add 1 drop of 22% albumin in all tubes and mix.
7. Add 1 drop of 2.5% saline suspension of Rh-positive red cells in all the test tubes (1 to
10) and mix.
8. Incubate the tubes for 30 min at 37°C.
9. Examine for haemagglutination and record the result (Table 17.6). The reciprocal of the
highest dilution that shows agglutination is the titre.
10. If tube 10 (1:512) shows agglutination, use the diluted serum (Step 5) of the last tube for
further dilution and repeat the procedure.
11. In order to obtain correct information regarding change in titre, freeze the test serum,
store at –20°C and run the titration again in parallel with any fresh specimens that may
be received from the patient. A dierence of two dilutions is taken as signicant change
of the titre value (Table 17.6, Example 2).
Table 17.6 Examples of antibody titre and score
Tube No: 1 2 3 4 5 6 7 8 9 10 Titre
Dilution: 1 2 4 8 16 32 64 128 2S6 512
Example 1 4+ 4+ 4+ 3+ 3+ 2+ 1+ 0 0 0 64
Example 2 4+ 4+ 4+ 4+ 3+ 3+ 2+ 2+ 1+ 0 256
Example 3 0 0 2+ 2+ 1+ 1+ 0 0 0 0 32
Haemagglutination reactions are graded from 0 to 4+.
Note
In case of prozone eect (example 3) the haemagglutination reaction is weaker in the
lower dilution and gets stronger in the higher dilution. This is due to the excessive amount
of antibody against a limited amount of antigen. Beer balance is aained by diluting the
antibody.
Additional information
The prognostic value of titrations has been the subject of some controversy. This is due to
the lack of clear correlation between the value of titre and occurrence of HDFN. In addition,
technical diculties under the conditions of developing countries make it dicult to obtain
reliable titre values.
review QueStionS
1. What is the signicance of quality control in blood bank? How would you check the
eectivity of the following reagents: anti-D, antihuman globulin, anti-Α, anti-B, A1 cells,
В cells and О cells?
2. What special precautions would you take in cleaning the laboratory glassware used in
blood bank?
3. If you are to perform a slide test for ABO typing what things would you require?
4. What is a titre? How would you determine the anti-D titre in a pregnant woman?
5. What is antihuman globulin? How is this prepared?
6. What is forward typing? What is reverse typing? How are these techniques used in
establishing the blood type of an individual?
Routine Laboratory Procedures in Blood Bank
457
7. What are Coombs’ control cells? How are they prepared for laboratory use?
8. Why is it important to wash the cells before adding AHG?
9. Why is it important to grade the haemagglutination reaction?
10. What kind of specimen would you require for routine blood bank testing?
11. What is cross-matching? What is the dierence between major cross-match and minor
cross-match?
12. What is weak D? How is this determined?
13. What are the characteristics of an immune antibody? How can the laboratory recognize
the presence of immune antibody in a patient?
14. When will you suspect that the blood group of a person is A2?
15. What is Rouleaux formation? How does this interfere in blood typing?
16. What are the causes of false positive results in ABO blood typing?
17. What are the causes of false negative results in ABO blood typing?
18. What is antibody screening test? How is this performed?
19. How would you identify an immune antibody found in a patient?
20. What is elution technique? What is its signicance? How is this performed? How would
you determine the nature of eluted antibody?
21. Describe the method of two-fold dilution technique. If you mix 0.1 mL of serum with
19.9 mL of saline, what is the dilution?