PRETRANSFUSION COMPATIBILITY
as follows:
TESTING
SELECTING WHOLE BLOOD FOR TRANSFUSION:
Before proceeding to cross-match the donor and recipient blood
samples, the following should be carried out:
A. DONOR BLOOD:
1. Using the blood in the unit segment attached to
the donor bag, determine the following using
standardized techniques (e.g., tube method, gel
or column agglutination method):
a. ABO blood group – shall be determined by
forward-typing the donor cells using Anti-A
and Anti-B, and reverse-typing the donor
plasma from the segment with known A1, B,
and O cells. Do not depend on the blood bag
label for the blood group of the donor. The
responsibility for the crossmatch is the
medical technologist’s. BE CERTAIN OF THE
DONOR’S BLOOD GROUP.
b. Rh blood group – shall be determined using
the Anti-D slide test or high-protein serum,
and carry out the antiglobulin test on all Rh-
negative tubes to ensure that the blood is Du
negative.
2. Carry out an indirect antiglobulin test (IAT) on
all donor blood units, using either a pool of
commercially obtained group O red cells or
making a pool composed of at least ten (10)
different cells.
3. Carry out a test to determine the five (5) DOH-
mandated transfusion-transmissible infections.
B. RECIPIENT BLOOD:
1. ABO blood group – shall be determined by
forward-typing the donor cells using Anti-A and
Anti-B.
2. Recheck the forward ABO blood group by reverse
or back typing, using the recipient’s serum and
freshly prepared 3%-5% suspensions of known
A1, B, and O red cells.
3. Rh blood group – shall be determined using the
Anti-D tube method.
NOTES:
▪ All blood grouping/typing procedures should be carried out
in TUBES in routine testing. Slide technique should not be
used because weak reactions cannot be determined
accurately.
▪ If you have demonstrated compatibility within the ABO
groups and the blood samples are identical with regard to
the presence or absence of the Rh factor (D antigen),
proceed by screening the patient’s serum for the presence
of atypical or unexpected antibodies by the indirect
antiglobulin/Coombs’ test (IAT).
▪ A serologic crossmatch is not required if the patient’s
serum does not contain unexpected antibodies.
▪ If a crossmatch procedure, however, is carried out, proceed
MAJOR CROSSMATCHING (COMPATIBILITY TESTING)
Principle: Human red blood cells contain antigenic blood
group substances such as the ABO, Rh, Kell, Duffy, and
others. The serum of most individuals contains antibodies,
which specifically react with the red cell antigens of others,
but not react with their own (self-antigens). In a major
serologic crossmatch (antibody test), the PATIENT’S
SERUM (the source of unexpected antibodies) is mixed
with the 3%-5% suspension of the DONOR’S RBCs
(contains blood group antigens; used as the test cells).
The procedure for the INDIRECT antiglobulin test (IAT,
Indirect Coombs’ Test) will be followed.
When such a person who has antibodies in his/her serum
is given a blood transfusion of blood, which contains the
blood group antigen against which those unexpected
antibodies are active, a hemolytic transfusion reaction
usually develops.
Procedure (Conventional Tube Method):
A. Preparing the 3%-5% RBC Suspension
1. Label a 12-mm test tube with the name of the
patient (will be used for the autocontrol), and
another test tube with the blood bag segment
(non-clotted pilot tubing) number of each donor
blood unit.
2. Into each labeled test tube, place 1-2 mL of
normal saline solution and add 100 µL of patient
and donor RBCs. Cover with paraffin sealing film
and gently mix by inversion. Centrifuge and
carefully remove the supernatant liquid. Repeat
this step until a total of three (3) washings has
been made.
3. Prepare a 3% to 5% RBC suspension of the
patient and donor’s packed red blood cells in
normal saline solution.
B. Compatibility Testing (Major Crossmatch)
1. Label a 12-mm test tube with the name of the
patient (will be used for the autocontrol test),
and another test tube with the blood bag segment
number of each donor blood unit.
2. Into each labeled tube, place 2 drops of the
patient’s serum.
3. Place one drop of the 3%-5% donor’s RBC
suspension into the tube labeled “DONOR.”
4. Place one drop of the 3%-5% patient’s RBC
suspension into the tube labeled
“AUTOCONTROL.”
5. Add three drops of enhancement medium (30%
bovine serum albumin or low-ionic-strength
saline) into each labeled tube.
37°C INCUBATION PHASE:
6. Mix the contents shaking the tubes gently and
incubate both tubes at 37°C for 30 minutes.
7. After incubation, centrifuge the tubes for 30
seconds at 3,600 rpm. Examine the supernatant
of both tubes for hemolysis, and read visually or
microscopically for hemagglutination.
8. If there is no hemolysis or hemagglutination,
continue with the antiglobulin (AHG) phase.
ANTIGLOBULIN (AHG) PHASE:
9. Shake the tubes to free all red cells that are
adherent to the bottom of the tube.
10. With the saline squeeze bottle under pressure, fill
the tubes with saline. Be sure that no pellet or
button of cells remains unsuspended in the saline.
11. Wash the red cells in both tubes with saline for
three times.
12. After the last wash, carefully remove the
supernatant and add a drop of polyspecific AHG
reagent (aka Coombs serum; contains antibodies
to IgG and complement) into each tube. Shake
the tube gently to mix the contents.
13. Centrifuge the tubes for 30 seconds at 3,600 rpm.
14. Examine the supernatant of both tubes for
hemolysis, and read visually or microscopically for
hemagglutination.
15. Quality Control: if there is no agglutination, add
a drop of Coombs control cells (aka Check Cells)
into each tube (with negative AHG). Shake to mix
the contents.
16. Centrifuge the tubes for 30 seconds at 3,600 rpm.
17. Examine both tubes for agglutination of the
Coombs’ control cells. If there is no agglutination,
then the test is INVALID or UNSATISFACTORY. The
major crossmatch and autocontrol tests should be
REPEATED.
Causes of unsatisfactory test (no agglutination in
the Coombs control cells:
- Neutralization of the AHG reagent caused by
inadequate washing.
- Inactive or expired AHG reagent.
- Omission or failure to add the patient’s
serum or AHG reagent.
- The test was read with inadequate
gentleness to detect an actually positive
crossmatch.
Preparation of Coombs Control (Check) Cells:
1. Incubate group O, Rh-positive red blood cells in
1:100 saline dilution of Anti-D antiserum reagent
for 1 hour (1-part Anti-D + 99-parts saline).
2. Wash three (3) times with saline.
3. Prepare a 3% to 5% suspension.
Interpretation of Results:
▪ “COMPATIBLE WITH PATIENT’S SERUM”
- If there is no agglutination or hemolysis in
any of the tubes (Major Crossmatch and
Autocontrol), the donor’s RBCs are
compatible with the patient’s serum, and may
be released for transfusion.
▪ “INCOMPATIBLE WITH PATIENT’S SERUM”
- If there is agglutination or hemolysis in any
of the tubes (Major Crossmatch and
Autocontrol), it indicates the presence of
single or multiple unexpected antibodies.
Hence, the donor’s RBCs are incompatible
with the patient’s serum, and must NOT be
released for transfusion.
▪ “DOUBTFUL”
- When agglutination occurs in the Autocontrol
tube, as well as one or more of the other
tubes, 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.
- Do not issue the blood for transfusion until
the cause of the positive Autocontrol is
established.
- Determine the antibody specificity using 3 or
more screening cell panels.
INVESTIGATION OF “DOUBTFUL” CROSSMATCH:
1. When agglutination appears in the Autocontrol tubes, the
agglutination may be due to an AUTOANTIBODY (i.e., an
antibody directed against self-antigens), such as those
that occurs at times in acquired hemolytic anemias and
certain other disorders. Such agglutination may usually be
clarified as follows:
▪ Incubate an equal volume of well-washed (37°C
saline) patent’s RBCs and serum for 30 minutes to 1
hour at 4°C. Separate the serum from the red cells by
centrifugation and add an equal volume of washed
patients RBCs (37°C saline). Re-incubate at 4°C for
an additional 30 minutes to 1 hour.
▪ Re-crossmatch this absorbed serum with the original
donor cells.
▪ If the crossmatch is still incompatible, elute the
antibodies from the patient’s RBCs by multiple
washings at 37°C. the blood should not be transfused.
The antibody specificity should be investigated and
determined using 3 or more screening RBC panel
reagent cells, and testing at the temperature and with
the same methodology that originally detected it in
the crossmatch. It is unnecessary to include a room-
temperature (20°C to 25°C) incubation or immediate
spin phase. A high-protein Coombs and/or enzyme-
Coombs test (both at 37°C) suffice for routine
compatibility testing if used.
2. ROULEAUX FORMATION occasionally is mistaken for true
agglutination. It is exhibited by a serum having an increase
in plasma proteins, especially in an inverted albumin-
globulin ratio (e.g., multiple myeloma, Waldenström’s
macroglobulinemia, nephritis, etc.).
▪ Add 4 drops of saline to each tube and gently rotate
to mix.
▪ Examine for agglutination. If the agglutination clears
in both tubes (provided that the Coombs’ test is
negative), the blood is reported as “Compatible with
Patient’s Serum.” If the agglutination clears in the
Autocontrol tube, but not in the Major Crossmatch
tubes, with identical handling, the blood is reported as
“Incompatible with Patient’s Serum.”
Crossmatching a Newborn Patient:
▪ Because the baby’s immune system is still developing, it
usually lacks the ABO isoagglutinins until 3 to 6 months of
age. Expected incompatibilities for A and B on the major
side of crossmatching may not appear. However, infants
may have passively transferred maternal antibodies, which
 should be reckoned with in doing direct crossmatch on the
newborn.
▪ It is important to obtain the blood of the mother of a
newborn requiring blood transfusion.
▪ Crossmatch the donor’s RBCs with the MOTHER’S serum.
▪ The infant will only have antibodies derived from the
mother. The concentration of such antibodies will be
greater in the mother’s serum than in the infant’s.
OTHER BLOOD BANKING TECHNIQUES:
A. ABSORPTION OF ANTIBODIES:
▪ Nonspecific antibodies can be removed by absorbing
them from the serum by allowing the blood to clot at
the optimum temperature of the antibody.
▪ Absorption techniques are helpful in the following
situations:
(a) To remove non-specific antibodies;
(b) To separate mixtures of antibodies to aid in their
identification; and
(c) To determine the presence of a specific
alloantibody as well as a nonspecific autoantibody
in the patient’s serum with acquired hemolytic
anemia.
▪ Procedures:
1. The test cells are washed 3-4 times in saline. The
residual saline is decanted and an equal volume
of the test serum is added to the test cell button
or pellet. NOTE: If the absorption procedure is to
be carried out at refrigerator temperature (0-
4°C), the test cells and serum should be prechilled
before mixing them together.
2. The cell-serum mixture is then incubated at the
appropriate temperature for 30 minutes and the
cells are continually shaken gently to ensure
maximum absorption. NOTE: Nonspecific cold-
reacting antibodies are incubated at 4°C; warm-
reacting antibodies at 37°C; Rh, Fy, Jk, K, etc.).
3. After the incubation, the cells are separated by
centrifugation and the patient’s serum is tested
by adding the appropriate cells.
4. It may be necessary to repeat the absorption
several times before all antibody is successfully
removed from the serum.
5. Variation in the length of time that each
absorption is carried out will depend on the
strength of the antibody present.
B. ELUTION OF ANTIBODIES:
▪ Antibodies coating the red blood cells can be eluted or
removed from the RBC membrane by a number of
methods:
o Ether elution
o Heat elution
o Digitonin elution
▪ Elution techniques may be used in the following
situations:
(a) To verify the diagnosis of hemolytic disease of
the newborn due to ABO incompatibility;
(b) To identify the antibody causing HDFN from the
infant’s cells, if the maternal serum is unavailable;
(c) To aid in the identification of the antibody
coating the RBCs that are responsible for HTR;
(d) To identify specific antibodies and nonspecific
autoantibodies from the RBCs of patients with
autoimmune hemolytic disease;
(e) To identify mixtures of antibodies by absorption
techniques; and
(f) To remove antibodies from the cell surface to
enable the cells to be phenotyped.
C. PREWARMED CROSSMATCH:
▪ This method is of clinical value in crossmatching in the
presence of cold-reacting antibodies.
▪ It is important to avoid the use of albumin in this
method as it enhances the activity of cold-reacting
antibodies.
▪ Procedures:
1. Incubate a 3% to 5% suspension of donor RBCs
at 37°C for 30 minutes.
2. Incubate an aliquot of the patient’s serum at 37°C
for 10 to 15 minutes.
3. Prewarm the crossmatching test tubes, AHG
reagent, and a container of saline solution at 37°C
for 10 to 15 minutes.
4. Add a drop of prewarmed donor’s RBC suspension
to 2 drops of prewarmed patient’s serum, using a
disposable pipette rinsed in 37°C-prewarmed
saline solution.
5. Incubate the serum-cell mixture at 37°C for 30
minutes.
6. Wash the cells in prewarmed saline three times,
and add 2 drops of prewarmed AHG reagent. Mix
the contents well by gentle shaking.
7. Centrifuge the tube (in prewarmed centrifuge
cups if possible) at 3600 rpm for 15 seconds.
Examine for agglutination. Examine weak
reactions using a microscope.
8. Add a volume of Coombs control cells (check
cells) to all AHG-negative tubes. Agglutination of
the check cells should occur for the test to
become valid.
D. IN VIVO CROSSMATCH:
▪ Disadvantage: risk of patient alloimmunization (HTR)
▪ If the compatibility of a unit of blood is in doubt (e.g.,
in hemolytic anemias), the estimation of the red cell
survival of a small aliquot of the cells can be quickly
assessed by determining the patient’s plasma
hemoglobin levels.
▪ 20 to 25 mL of “most compatible” donor whole blood
is infused into the patient in the IV line (standard Y
set). Saline flow is set at 20 drops per minute (for 15
minutes).
▪ The presence of visible or increased free plasma
hemoglobin indicates that the in vivo crossmatch is
incompatible with the patient’s serum and should
immediately be discontinued.
▪ 25 mL of whole blood when infused will normally
liberate 100 mg of hemoglobin per 100 mL of plasma
and will macroscopically be red in color.
THE NUMBER OF BLOOD UNITS FOR TRANSFUSION:
▪ Only ABO and Rh-compatible units should be transfused to
the patient.
▪ This helps us answer the question of how many packed
RBC units would you need to screen in order to find the
requested number of compatible (i.e., negative for the
corresponding antigen) units in case the patient needs
blood transfusion.
▪ To help you determine the number of compatible units to
be screened, refer to the table shown below:

▪ Given that the frequency of Jka antigen is approx.

77% (Caucasian population frequency), there is a
23% chance of finding an RBC unit that is Jka-antigen
negative.

Probability of finding Jka-negative PRBC units

= 100% – 77% = 23% or 0.23

▪ Thus, if the physician requests for two (2)

compatible units of packed RBC:

2 units /0.23 = 8.69 or 9 (rounded off)

…so you need to screen nine (9) units of packed

RBC in order to find two (2) packed RBC units that
are Jka-antigen negative.