IMMUNOHEMATOLOGY MT

PREFINALS
LABSHEET BASED 306

ACTIVITY NO. 8 COOMB’S TEST

MATERIALS: 2. Place 1 drop of the 3%–5% red cell suspension to

be tested in a tube (e.g., donor cells,reagent
• Phlebotomy Kit screening cells).
3. Add recommended number of drops of
• Test Tube
enhancement media (e.g., LISS, PEG) if indicated.
• Personal Protective Equipment
4. Incubate at 37° C for the time period indicated for
• Test Tube Rack
the assay being performed (from 15–60 minutes).
• Centrifuge Machine 5. If indicated in the procedure, centrifuge for 15-20
• Test Tube Brush seconds (Immediate Spin) at 3400 rpm and
• Labeling Permanent Marker examine the serum for hemolysis and the cells for
• Pasteur Pipette agglutination or hemolysis after gently dislodging
• Parafilm the cell button.
6. Whether step 5 is performed or not, continue by
REAGENTS: washing the tube(s) three to four times with normal
saline.
• Antihuman Globulin Reagent 7. Blot tubes dry after the last wash.
• Coomb’s Check Cells 8. Add 2 drops of antiglobulin sera (polyspecific or
• EDTA Blood Sample monospecific anti-IgG) to all tubes.
• Physiologic Saline (0.85-0.90% NaCl) in Wash Bottle 9. Centrifuge for 15-20 seconds (Immediate Spin) at
3400 rpm and examine for agglutination or
hemolysis after a gentle dislodging of the cell button.
PROCEDURES:
Protocol may include optional microscopic
examination to confirm macroscopically negative
A. Direct Coomb’s Test
tests.
10. Add check cells to all negative tubes.
1. Prepare a 3%–5% suspension in normal saline of
the red cells to be tested. NOTE: In every tube method, always add first the clear
2. Add 1 drop of red cell suspension to a tube and
solution. This is to ensure that all needed solutions are added.
wash three to four times with saline.
3. Blot the tube after the last wash.
4. Add 2 drops of polyspecific AHG (anti-IgG + anti-
complement).
5. Centrifuge for 15-20 seconds (Immediate Spin) at INTERPRETATION:
3400 rpm and examine for agglutination or
hemolysis after gently dislodging of the cell button. • IAT POSITIVE- Presence of agglutination.
Protocol may include optional microscopic o A positive IAT indicates the presence of
examination to confirm macroscopically negative atypical antibodies in the patient’s serum
tests. coating red cells in vitro.
6. Add check cells to all negative tubes (see Quality • IAT NEGATIVE- Absence of agglutination.
Control of the Antihuman Globulin Test section). o A negative IAT indicates the absence of
atypical antibodies in the patient’s serum
NOTE: For DAT, blood samples should be collected in EDTA to coating red cells in vitro.
prevent fixation of complement in vitro by clinically insignificant
cold autoagglutinins

INTERPRETATION:

• DAT POSITIVE- Presence of agglutination.

o Accurately grade the reaction by holding the
tube with a good light source. Never place the
tube in the test tube rack if the grading is not
yet done.
o It is important to always remember that
hemolysis is a positive result and a strongest
reaction in hemagglutination reaction.
• DAT NEGATIVE- Absence of agglutination.
o Before concluding a negative test, do
notforget to add the QC cell or the Coomb’s
Check cells.

B. Indirect Coomb’s Test

1. Add 2–3 drops of patient serum or the
recommended number of drops of commercially
prepared anti-sera to each tube.

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 ACTIVITY NO. 9 WEAK – D TESTING

MATERIALS: Grading Serological Reactions in Tube

Testing
• Phlebotomy Kit Test Tube
• Personal Protective Equipment Test Tube Rack GRADE MACROSCOPIC OBSERVATIONS
• Centrifuge Machine Test Tube Brush 4+ One solid agglutinate, no free cells
• Labeling Permanent Marker Pasteur Pipette 3+ Several large agglutinates, clear
• Parafilm background
2+ Medium sized agglutinates, clear
REAGENTS: background
1+ Small agglutinates, turbid reddish
• Anti-D Sera background
• Antihuman-globulin Sera 1+w Very small agglutination, turbid
• EDTA-Whole Blood background
• 3-5% Red Cell Suspension W+ or +/- Barely visible agglutination. turbid
• Physiologic Saline (0.85-0.90% NaCl) in Wash background
Bottle 0 Negative - no agglutination, smooth
• 22% Bovine Serum Albumin reddish background
MF Mixed Field - mixture of agglutinated and
PROCEDURES: unagglutinated red cells
H Complete hemolysis, grossly red
1. Prepare 3-5% suspension of the red cell to be tested supernate, no RBCs present
or if you encounter a D-typing tube in the course of PH Partial hemolysis, red supernatant\ but
the procedure for pre-transfusion testing, use the some RBCs remain
negative tube instead of making a new suspension.
2. Label 3 tubes as Px, PC, and NC.
3. In a tube labeled with Px, add 1 drop of anti-D then Interpretation:
1 drop of Red Cell Suspension.
• Individuals with negative Weak-D testing shall be
Note: In every tube method, always add first the clear solution. reported as D NEGATIVE.
This is to ensure that all • In the worksheet, grade properly the result based on
the strength of agglutination.
needed solutions are added.
D TYPING WEAK D TEST
4. In a tube labeled with NC, add 1 drop of 22% Bovine INTERPRETATION
Anti- Rh Weak Weak
Serum Albumin then 1 drop of Red Cell Suspension. OF D ANTIGEN
D ctrl D D ctrl
5. In a tube labeled with PC, add 1 drop of anti-D then
3+ POSITIVE
1 drop of known D-Positive Red Cell Suspension.
3+ 3+ INVALID
6. Gently mix the solutions and cover with parafilm.
7. Incubate the 3 tubes at a water bath maintained at 0 0 3+ 0 POSITIVE*
37 O C for 15 minutes. Px Tube with agglutination 0 0 3+ 3+ INVALID
is regarded as Rh positive while the Px tube without 0 0 0 0 NEGATIVE
agglutination goes to the next step. * Only perform D ctrl if using high protein anti-D reagent OR if
8. Wash the cells 3 times with normal saline solution the patient appears AB positive.
and decant the saline completely after the final ** Only perform Weak D testing if the patient appears D
washing. Negative.
9. Add 2 drops of AHG reagent to all tubes and mix
gently. Cover with Parafilm and spin for 15 seconds
at 3,400 rpm.
10. Gently dislodge each cell button and examine for
agglutination. Read macroscopically and
immediately record GRADED reactions, using the
chart provided, as they are read. You must hold the
tube in your hand in front of the column you will
record the results in, observe the reaction and
record it. DO NOT place the tube in the rack until
you have recorded the results!

NOTE: The manner in which RBCs are dislodged from the

bottom of the tube is critical. Shake the tube gently until all cells
are dislodged from the bottom of the tube. This is most easily
accomplished by holding the tube between thumb and
forefinger and gently agitating, then tilting the tube. Avoid over
shaking which breaks up fragile agglutinates (false negative
reaction) while inadequate mixing may result in a false positive
interpretation. If you get reactions of less than 3-4+ in the D or
reverse typing tubes you are shaking too hard which could
result in a false negative result.

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 CROSSMATCHING

• In transfusion medicine, the ultimate goal is to • Physiologic Saline (0.85-0.90% NaCl) in Wash
administer the safest components with a survival Bottle
rate in the patient’s blood stream of at least 70%. • Low Ionic Strength Solution
• Pretransfusion test consists of Patient and Donor/s
blood grouping, and major and minor crossmatch. PROCEDURES:
o The said tests are part of Broad
Spectrum Compatibility Testing. A. Immediate Spin Serologic Crossmatch
• Crossmatching is divided into two parts: 1. Label 4 test tubes as ACpx, ACdo, MJ and MN.
o Major Crossmatch 2. Follow the table below:
▪ Involves mixing of donor’s
red cell and recipient serum CONTENT ACpx ACdo MJ MN
(RS-DC) Patient’s 2 gtts. - 2 gtts. -
▪ more significant as it detects Plasma
possible antibodies in a Donor’s - 2 gtts. - 2 gtts.
patient’s blood which could Plasma
lyse the donated red blood 3-5% - 2 gtts. 2 gtts. -
cell. Donor
RCS
o Minor Crossmatch 3-5% 2 gtts. - - 1 gtts.
▪ involves mixing of donor’s Patient
serum with recipient cells RCS
(RC-DS). LISS - - - -
▪ in some laboratories is LEGEND: ACpx- Autocontrol (patient), ACdo- Autocontrol (donor), MJ-
omitted in the compatibility Major Crossmatch, MN- Minor Crossmatch
testing
▪ because it detects only 3. Mix and cover with Parafilm.
possible antibodies from the 4. Spin for 15-20 seconds (15 seconds will do) at 3400
donor which could lyse the rpm.
patient’s red cells. 5. After centrifugation, examine the tube first for
• Hemodilution is the reason why minor crossmatch hemolysis before dislodging gently the cell button.
is sometimes omitted. Grade the agglutination if there is any.
o The donor’s serum will be diluted during 6. If no agglutination or hemolysis is observed,
transfusion by the patient’s blood which proceed to the next phase, the incubation phase.
in turn neutralizes the donor’s possible
reacting antibodies. B. Incubation Phase
• Crossmatching consists of 3 phases: 1. All four tubes will be added with 2 drops of
potentiator- LISS.
1. Immediate spin phase
- which detects cold reacting NOTE: LISS is one example of many different
antibodies potentiators. Each potentiator differs in its incubation
2. Incubation phase period for the incubation phase or thermo phase. If 22%
- detects warm reacting Bovine Serum Albumin is used, 30-min. incubation is
antibodies followed while if LISS is used, the incubation time will be
3. AHG phase shortened into 10 min. only.
- which detects unexpected
antibodies. 2. Mix and cover with Parafilm.
3. Incubate for 10-15 minutes at 37OC water bath or
• Autocontrol is also included in the compatibility
in an incubator exclusively made for Tube
testing.
Crossmatching.
o detects self-reacting
4. Spin for 15 seconds at 3,400 rpm
o antibodies.
5. After centrifugation, examine the tube first for
hemolysis before dislodging gently the cell button.
MATERIALS: Grade the agglutination if there is any.
6. If no agglutination or hemolysis is observed, both
• Phlebotomy Kit tubes will proceed to the next phase, the AHG
• Test Tube and Rack Phase.
• Personal Protective Equipment
• Water bath (Set at 37 O C) C. Coomb’s Phase
• Centrifuge Machine 1. Wash the cells from the four tubes with
• Test Tube Brush physiologic saline for three (3) times.
• Labeling Permanent Marker 2. Decant the saline completely after the last wash.
• Pasteur Pipette 3. Add 2 drops of AHG in each tube
• Parafilm Dropper 4. Spin for 15 seconds at 3,400 rpm
5. After centrifugation, examine the tubes first for
REAGENTS: hemolysis before dislodging gently the cell button.
Grade the agglutination if there is any.
6. Add 1 drop of Coomb’s Check cells to tubes
• Antihuman Globulin Reagent
showing no agglutination.
• Coomb’s Check Cells
• EDTA Blood Sample of Recipient and Donor NOTE: In every tube method, always add first the clear
• 3-5% Red Cell Suspension of Donor solution. This is to ensure that all needed solutions are
• 3-5% Red Cell Suspension of Patient (Recipient) added. BEFORE placing back the tubes into the test

DEMECILLO, ALELI ISABEL E. | 3

 tube rack, grade the reaction. Never place it back if you
haven’t graded the reaction.

Interpretation:

• COMPATIBLE. This is when all tubes shows NO

AGGLUTINATION in all phases.
• INCOMPATIBLE. This is when there is presence of
agglutination in any tube even in at least 1 phase.

DEMECILLO, ALELI ISABEL E. | 4