Definition
A crossmatch or compatibility test is a test between a patient or
recipient of a blood transfusion and his prospective or donors.
Purpose
1. To prevent any possible transfusion reaction
due to antibody.
2. To ensure maximum benefit to the recipient.
Two kinds of crossmatch
1. Major crossmatch – uses patient’s serum and
donor’s red cells (PS–DR). The purpose
of the major crossmatch is to prevent a transfusion reaction by detecting
antibodies in the patient’s serum which would reduce the survival of the
donor’s cells and to ensure maximum benefit to the recipient.
2. Minor crossmatch – uses patient’s red cells
and donor’s serum (DS–PR). This detects antibodies in the donor which may be
capable of affecting the recipient's’ red cells.
The crossmatch will detect the following:
1. Most recipient antibodies directed against
antigens on the donor red blood cells.
2. Major errors in ABO grouping, labeling and
identification of donors and recipients.
The crossmatch will not:
1. Guarantee normal donor cell survival
2. Prevent recipient immunization
3. Detect errors of Rh and ABO typing
4. Detect all antibodies unless antibody is
specific to the donor antigen being crossmatched
5. Detect antibodies to white blood cells or
platelets
6. Detect errors of labeling and collection of
sample
Specimen
1. Fresh, not inactivated serum, less than 48
hours old must be used for the crossmatch. If plasma is used, fibrin clots may
form and interfere with the test and complement activation.
2. Hemolyzed patient samples should be avoided,
because they may ask hemolysis of donor red blood cells.
3. A washed red blood cell suspension may be
prepared.
Pre–transfusion procedures in a Blood Bank:
1. Reviewing blood bank records of testing of
previous samples of the recipients.
2. ABO grouping, Rh typing and unexpected
antibody screening on each recipient sample sent for compatibility testing.
3. ABO grouping, Rh typing and unexpected
antibody screening on the unit of blood.
4. Crossmatching
Techniques used in crossmatching
1. Saline Technique
Certain antibodies react at body
temperature in vivo; however, they may give optimum reactions in vitro
temperatures below that of the body. Saline techniques are designed to detect
IgM antibodies which react optimally at room temperature (22oC) or
lower.
Agglutination or hemolysis in the
saline procedure at 22oC will detect
a. ABO incompatibility, often due to incorrect
typing.
b. Cold agglutinin or autoagglutinins.
c. Saline acting antibodies such as anti–M, anti–N,
anti–S, anti–Lu, anti–F and anti– Le.
The saline crossmatch at 37oC
detects:
a. Saline acting anti–Rh antibodies.
b. Some anti–M, anti–N and anti–S antibodies.
c. Some anti–K and anti–Le antibodies
Agglutinins produced by cold
agglutinins will disappear or diminish.
2. Albumin Technique
The addition of bovine albumin to
the crossmatch presents ideal conditions for the detection of Rh antibodies.
Bovine albumin act to increase the dielectric constant of the medium, thus
allowing IgG antibodies to be demonstrated. Most IgG antibodies are detected.
The albumin crossmatch at 37oC
detects:
a. Most anti–Rh antibodies
b. Some anti–M, anti–N and anti–S antibodies
c. Some anti–Le antibodies
3. Antiglobulin Technique
This is probably the most
important and most widely used serologic procedure in modern blood banking.
Compatibility testing should be considered incomplete unless the antiglobulin
technique is included as part of the test. Most antibodies that result in
transfusion problems are detectable only by the antiglobulin test. It detects:
a. IgG antibodies whose donors do not react in
either saline of high protein media or which react weakly or variably in these
media.
b. Antibodies which bind complement but which
may not be capable of binding themselves to the cells in sufficient amount to
give a positive test.
c. Anti–Fy, anti–JK and most anti–K antibodies.
4. Enzyme Technique
Enzyme techniques have proved
valuable in crossmatching. They serve as an important “back up” test for the
indirect antiglobulin test and detect many clinically significant IgG and
saline–inactive IgM antibodies.
Enzyme used are:
a. Trypsin
b. Papain
c. Ficin
d. Bromelin
5. Broad Spectrum Compatibility
Testing – developed
to be an all-inclusive test, one which would detect clinically significant
antibodies without sacrificing speed – so essential in an emergency, or
simplicity so important in prevailing errors.
Three phases of broad spectrum
compatibility test:
a. Protein or room phase
b. Thermo or incubation period
c. Antiglobulin phase
Interpretation of a positive reaction in each
phase:
1. Protein phase
a. Incompatibility in the ABO system will be
detected at this point. Hemolysis especially may indicate the presence of an
immune anti–A and / or anti–B.
b. An incompatibility due to cold agglutinins.
c. The prozoning anti–Rh antibodies are detected
in a serum albumin mixture on immediate centrifugation.
2. Thermo phase
a. Incompatibility in this phase is usually due
to the presence of a low tittered anti –Rh antibody that does not react on
immediate centrifugation.
b. Certain Rh antibodies (anti–C, anti–E and
some anti–D) occasionally react only in an albumin medium and are non–reactive
in the antiglobulin test.
3. Antiglobulin phase
a. Antibodies are detected here which usually
react only in this test (anti–Fja, anti– Jka, anti–X).
b. These antibodies in the Rh system which react
only in the antiglobulin test (so called “third order” or “panagglutinoid”
antibodies) are noted.
c. Antibodies present in acquired hemolytic
anemia will be found.
Procedure
1. The room temperature phase requires two tubes
labeled 1 and 2. Both tubes receive 2 drops of recipient’s serum and two drops
of an appropriate 5% red blood cell suspension(donor’s cells) in their own
serum or saline. In addition, tube 1 receives 22% bovine serum albumin (BSA) to
enhance the reactivity of certain 7S antibodies. The tubes are spin immediately
in a centrifuge and examined microscopically (by naked eye) for agglutination
and / or hemolysis. Saline agglutinins maybe detected in either tube; albumin
antibodies may be detected in tube 1. If no reaction is seen in either tube,
they should be shaken and tested in phase 2.
2. The warm (thermo) phase is designed to detect
antibodies that react optimally at 37oC. Both tubes from phase 1 are
incubated at 37oC for 30 minutes (can be shortened to 15 minutes),
centrifuged and examined macroscopically for agglutination and / or hemolysis.
If there is still no reactivity, tube 1 should be tested in phase 3.
3. The Coombs phase (or anti–human globulin
phase) should detect almost any blood group antibody that fails to react in
phase 1 or 2. Tube 1 (containing bovine serum albumin for the thermo phase)
should be shaken and thoroughly washed by filling with NSS, mixing thoroughly
and centrifuging until all cells are at the bottom of the tube. Decant the
washed saline and repeat the wash cycle at least two more times. Washing the
cells thoroughly is extremely important to remove all unreacted globulins
(antibodies). If any free globulins remain after washing, they may inactivate
the Coomb’s reagent and result in a false negative reading. This inactivation
involves a blocking antibody phenomenon. Soluble globulins can occupy on the
AHG reagent so that it fails to combine with globulin to the red blood cells.
After the last wash solution is decanted, the remaining drop of cells is
shaken. AHG is added and the tube is centrifuged and read macroscopically and
microscopically. A truly negative compatibility test should leave the AHG
reagent free to react with the globulin–coated 5% saline suspension of washed
Coombs control cells. Thus agglutination of the control cells confirms the
facts that AHG was added to the test and that the washing of cells was
adequate.
In the performance of the compatibility test, four tubes will
actually be needed – two for the major test (as has been described above using
patient’s serum + donor’s cells) and two for the minor test (mixture of donor’s
serum and patient’s cells) using the same technique as that of above.
An auto–control should be run simultaneously in the crossmatch
test, using patient’s cell and patient’s serum. If the auto–control is
negative, a corresponding incompatible crossmatch is probably caused by a
specific antibody. On the other hand, if the auto–control is positive and the
corresponding crossmatch is incompatible, the patient has a non–specific
agglutinin that may be masking the presence of one or more specific antibodies.
In this case, the patient’s blood should be absorbed by allowing a sample to
clot at second sample collected with anticoagulant provides cells for the
autocontrol. These cells should be washed thoroughly with warm saline to remove
cold autoantibodies. If a 5–6% saline suspension of the washed cells is tested
with the absorbed serum and gives no reactivity then the serum is considered
fully absorbed and ready for use in crossmatching or in tests with secretor
cells.
The minor crossmatch is no longer used in many hospitals for
erythrocyte transfusion because of the popular use of erythrocyte concentrates
and routine use of antibody screening of donor blood. However, when a large
volume of plasma is used, as may be the case with platelet or granulocyte or
with fresh frozen plasma, especially for children, a minor crossmatch is
desirable. Advocates of the minor crossmatch also point out that it confirms
ABO typing, reflects a positive direct antiglobulin test and may permit
recognition of a new blood group factor.
Segments from the blood bag are recommended rather than tubes
attached to the bag (pilot tubes) as these segments are the true representative
of the bag’s contents.
Interpretations of results:
1. Compatible – if there is no agglutination or
hemolysis in any tubes, the blood is incompatible and maybe release for
transfusion
2. Incompatible – agglutination or hemolysis in
either or both of the major tubes is evidence of isoantibody. The donor is
incompatible and the blood must not be transfused.
3. Doubtful – when agglutination occurs in the
auto tube, as well as one or more of the others, the reaction is “doubtful” and
certain additional procedures should be carried out before a decision can be
made whether or not the blood is compatible.
Unsatisfactory test – if the
control cells are not agglutinated in the antiglobulin test, the test is
unsatisfactory. Either no Coombs serum was added, it was of inadequate activity
or they washed with inadequate gentleness to detect an actually positive
crossmatch. In any case, the Coombs crossmatch and the auto–test must be
repeated, remedying the error.
Problems encountered in grouping, typing and
crossmatching
1. Rouleaux formation
Cells appear to be agglutinated,
yet they have the appearance of a “stocks of coins.” The formation can be
dispersed or diminished by the addition of a drop of saline to the slide
(diluting the serum–cell mixture with saline). Note, however, that weak agglutination
can also be dispersed in this way; therefore, the practice may be dangerous in
certain cases.
Rouleaux formation is often
encountered in certain disease states – notably myelomatosis and
macroglobulinemia. It is also caused by a raised serum globulin concentration and
is often seen in patients with serum protein abnormalities. Certain synthetic
plasma expanders, such as dextran can also cause rouleaux formation. Fibrinogen
has the greatest influence on rouleaux formation and in grouping; serum is used
rather than plasma because of its lack of fibrinogen.
2. Panagglutination
Panagglutination is simply the
spontaneous clumping of all cells against a given serum. There are two main
types of panagglutination reaction:
a. Bacteriogenic panagglutination (Huebner
Thomsen–Friedenrich phenomenon) – is thought to be due to a bacterial contaminant, which exposes
a latent receptor known as T. All human red cells contain this antigenic
receptor but are usually non–reactive with anti–T. Since all human adult sera
contain anti–T. since all human adult sera contain anti–T. Since all human
adult sera contain anti–T in varying amounts, agglutination would occur with
all such cells. The reaction takes place at 20oC but not at 37oC.
b. Nonbacteriogenic panagglutination may be used by acquired
hemolytic anemia or by rare specific antibodies. The reaction takes place at 37oC
and occasionally in the cold. The direct antiglobulin test is often positive.
3. Polyagglutination
Polyagglutination is the reaction
of red cells with varying proportions of fresh normal allogeneic or intert AB
sera. The reaction takes place at 20oC and becomes weak or inactive
at 37oC. Microscopically, the reaction usually has a “mixed field”
appearance. The autocontrol and direct antiglobulin test are usually negative.
Treatment of cells with papain often abolishes the phenomenon.
This is usually the result of
activation of the T antigen in vivo and it is usually a transient phenomenon.
In the laboratory, it is usually discovered because of “difficult” ABO
grouping.
4. Cold grouping
Cold agglutinins are relatively
common problems in the laboratory. They are simple antibodies reacting at
temperatures between 4oC and 20oC and they may be
specific or nonspecific. Cold agglutinin can often be eliminated by reading
tests on a warmed microscope slide, though the absence of agglutination at 37oC
does not mean that the antibody is clinically insignificant, since the addition
of complement may cause the cells to become sensitized to a suitable
antiglobulin serum at that temperature.
5. Wharton’s jelly
Wharton’s jelly is usually
present in cord samples that have been collected by cutting the umbilical cord
and allowing the blood to drain into a collection tube. Samples contaminated
with Wharton’s jelly often agglutinate spontaneously. All cord specimens should
be thoroughly washed with warm saline (3 to 5 times) before testing to guard
against any false reaction due to the substance. If Wharton’s jelly is a common
problem, it is best to advise the delivery room involved to collect cord
samples from the umbilical vein rather than allowing the blood to drain into a
collection tube. This will eliminate the problem.
6. Prozones
The prozone phenomenon
occasionally occurs in the antiglobulin technique and is thought to be caused
by partial neutralization of the antiglobuin reagent (i.e., the antibody is
loosely bound or does not remain on the cells). The antiglobulin reagent,
reacting with this free antibody, is partially neutralized.
7. Antigen or antibody deterioration
Both antigen and antibody undergo
a diminishing of reactivity on storage. The deterioration of antibodies is
dependent on the specificity of the antibody and the length and method of
storage. Saline–reactive antibodies deteriorate most in the first month of
storage, then appear to become relatively stable. IgG antibodies, with a few
exceptions, are stable. Complement binding antibodies deteriorate owing to
complement inactivation and / or the fact that at –25oC, serum may
acquire certain anti–complementary properties.
The patient who will not crossmatch
Occasionally, a patient is found whose serum persistently
agglutinates in one stage of the crossmatch or another, all donor bloods, while
the “auto” tube remains smooth. In such cases, a screening pooled O cell of a
pool of at least ten fresh group O cells should be used to check the serum by
Coombs test for the presence of atypical antibodies. If positive, a panel of
group O cells of known genotype should be tested against the patient’s serum by
the same techniques used in the crossmatch. If these tests do not identify the
antibody, the agglutination may be due to antibodies against the antigen occurring
in a very high % of the population, the so called “public” antigen. Or it may
be due to a mixture of antibodies which between them agglutinate the cells of
nearly everyone. Under circumstances, proceed as follows:
1. Refer the problem to the physician in charge
of the blood bank section and follow his further instruction.
2. Inform the patient’s physician of the
trouble, so that they will avoid unnecessary demand for blood.
3. Send specimen of whole clotted blood to an
acceptable reference laboratory for identification of the antibody. If it is
identifiable, compatible donors may be obtainable form the rare donor files.
4. Study the blood of as many of the patients
close relatives as possible. You may find among them a person with the same
rare blood group as the patient, who could serve as donor.
Crossmatching the newborn
The newborn child usually lacks isohemagglutinin so that the
expected incompatibilities for A & B on the major side of the crossmatch
may not appear. However, infant may have possibly transferred maternal
antibodies which should be reckoned with in doing direct crossmatches on the
baby. It is important to obtain the blood of the mother of a newborn requiring
blood transfusion; crossmatch the donor’s blood with the mother’s serum. The infant
will have only antibodies derived from the mother. The concentration of such
antibodies will be greater in the mother’s serum than in the infant.
Abbreviated crossmatch
If a patient has no known history of and / or no currently
demonstrable unexpected antibodies, ABO and Rh type specific blood is
crossmatched at the time of need by using an immediate spin–saline abbreviated
crossmatch. The abbreviated crossmatch procedure is done by mixing two drops of
patient’s serum with one drop of a 3 – 4% saline suspension of washed donor
erythrocytes. The patient’s serum and donor erythrocytes are mixed and
centrifuged for 20 seconds. The supernatant is examined for hemolysis and the
erythrocyte button is suspended and examined for agglutination. Once blood is
issued, both 37oC incubation and an antiglobulin crossmatch must be
done using the same tube employed for abbreviated crossmatch. This serves as a
check that clinically significant antibodies have not been overlooked. If
either the 37oC incubation of the antiglobulin phase of testing
becomes positive, the patient’s physician must be notified immediately and the
unit of blood must be stopped.
Uncrossmatched blood
Uncrossmatched blood should only be issued when its order is
accompanied by a signed declaration form the doctor stating that he accepts
full responsibility for the transfusion based on the medical emergency. Group specific
(ABO and Rh) blood should be issued. The issue of Group O Rh–negative blood to
individuals who have not been grouped is firmly discouraged, except in extreme
emergency situations.
A segment of blood from the unit should be removed before issue
and the blood tested through full crossmatch procedure. Another unit of blood
should be set up, even when it has not been requested. In the case of
incompatibility, the ward or operating room should be informed immediately and
the transfusion promptly discontinued. A compatible unit should be substituted
as soon as possible.
The selection of blood for transfusion
(routine and emergency)
Whenever possible, blood of the same ABO and Rh group as the
patient should be chosen for transfusion. Group O blood may be used for group A & B recipients when these
groups are not available; however, packed cells should be chosen, since the
presence of a high–titered saline agglutinin in the donor Group O blood may
cause the destruction of the recipient’s cells. If “low tittered” blood (so
called “hemolysis free”) is used, the risk are lessened; however, packed cells
should still be the product of choice, group AB recipients may receive A, B or
O blood in an emergency situation; however, packed cells should again be
chosen. If the recipient belongs to a subgroup of A and Group A blood is to be
transfused, the donor blood need not be of the same subgroup unless anti–A1
is found to be present in the patient’s serum.
With regards to Rh typing, O–negative recipients must receive D–negative
blood. This blood should also be negative for the C and E antigens. In cases of
extreme shortage of emergency, E positive (Du negative) blood may be
substituted when transfusing adult D–negative recipients. C–positive blood
should be given to D–negative patients, as it could stimulate anti–G.
For Du–positive recipients, the transfusion of D–positive
blood appears to involve very little risk of immunization.
Transfusion of D–positive blood to unimmunized D–negative adult
males or females past child–bearing age should be avoided except in case of
emergency.
Cases wherein group specific blood is
unavailable:
1. Acute donor shortages, disasters, military
demands.
2. Time does not permit the ABO grouping of the
recipient.
3. The recipient’s ABO group cannot be
accurately determined.
4. Exchange transfusion of a newborn infant
requires compatibility with the mother.
5. The recipient’s serum contains an antibody
against a high incidence antigenic determinant (e.g., anti–Kpb, anti–Yta,
anti–Vel) and the only available blood that lacks the high incidence antigenic
determinant is group O.
Blood component of choice when group specific
blood is unavailable
(in descending order of choice)
1. Deglycerolized frozen red blood cell.
2. Packed red blood cell.
3. Whole blood shown to lack hemolytic anti–A and
/ or anti–B.
4. Whole blood with saline anti–A and / or anti–B
titers of less than 1:50.
Procedure of the determination of high–tittered
blood
1. Prepare a 1:64 dilution in saline of the
serum to be tested. This may be done by adding 1 ml serum to 6.3 ml of saline.
2. Add 2 drops of the diluted serum to each of
two tubes labeled “A” and “B”
3. Add 2 drops of a 2% suspension in saline of
A1 cells to tube A; add 2 drops of a similar suspension of cells to tube B.
4. Incubate for 15 minutes at room temperature.
5. Centrifuge at 1000 – 2000 rpm for 1 minute.
6. Examine for agglutination.
Interpretation:
Those sera which agglutinate
either on both cell suspensions can be considered to be high–tittered. These
bloods should be transfused only to group O recipients. Those bloods which do
not react should be tested by means of the partial neutralization test using
undiluted serum.
Choices of blood when group specific is
unavailable
Patient’s group Alternate
Donor Group
First
Choice Second Choice
A * O
B O
AB A
or B ** O
A2B with anti–A1 A1 or B ** O
A1B with anti–H A1
or B **
* A1 patients with anti–H should be transfused with
blood group A1. When
required by exceptional
circumstances, group A2 is preferable to group O.
** Either group may be chosen but only one of the two should be
used for a given recipient.
Group A is
usually more readily available than group B. If blood of yet another group
is needed, use
Group O.
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Perfect Explanation about crossmatch or compatibility testing
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