ACTIVITY NO.

5: ‘A’ CELL SUBGROUPING

• MATERIALS:
o Phlebotomy Kit
o Personal Protective Equipment (PPE)
o Centrifuge Machine
o Permanent Marker
o Parafilm
o Test Tube
o Test Tube Rack
o Test Tube Brush
o Pasteur Pipette
o Dropper
• REAGENTS:
o 3-5% Type A Red Cell Suspension
o Physiologic Saline (0.85-0.90% NaCl) in Wash Bottle
o Anti-A reagent serum
o Anti-A1 Lectin
• Group A subgroups are generally more common than B
subgroups. It consists of 2 subgroups, A1 and A2
Phenotypes. 80% of all group A individuals are A1 and
the remaining 20% are A2 or weaker subgroups. The
differences between A1 and A2 are both quantitative and
qualitative. Lectins are seed extracts that agglutinate
human cells with some degree of specificity. This reagent
agglutinates A1 cells but does not agglutinate A2 cells. It
is used to differentiate the 2 principal subgroups by
agglutinating specific A cell subgroup. Lectins used in
Blood Banking are Dolichos biflorus which agglutinates
A1 or A1B cells, Grifonia simplicifolia formerly called
Bandeiraea simplicifolia agglutinates B cells, and Ulex
europaeus which agglutinates O cells with H antigen
specificity and other blood groups depending on the
amount of H antigen available.
PROCEDURE:
1. Make a 3-5% Type A RBC Suspension
2. Prepare 2 clean tubes and label with A and A1
respectively.
3. Place 2 drops of Anti-A Reagent Serum in tube A and 2
drops of Anti-A1 lectin on tube A1. [always place FIRST
the clear one to avoid confusion]
4. Add 2 drops of freshly prepared 3-5% Type A RCS to both
tubes.
Follow the tabulated procedure below.
Note: In every tube method, always add first the clear
solution. This is to ensure that all needed solutions are added.
Tube Content A Tube A1 Tube
Anti-A rgt. Serum 2 drops -
Anti-A1 Lectin - 2 drops
3-5% RCS 2 drops 2 drops
5. Cover the tubes with Parafilm.
6. Mix the solution and spin for 15 seconds at 3,400 rpm.
Gentle mixing is recommended.
7. Carefully dislodge the cell button by gently mixing it and
examine for agglutination.
o Grade the reaction, record and interpret the results.
• Interpretation:
o SUBGROUP A1. Agglutination in tubes A and A1
o SUBGROUP A2. Agglutination in tube A only
SUBGROUP TUBE A TUBE B
A1 + +
A2 + 0

MONTEMOR, DJ. 8
 ACTIVITY NO. 6 SALIVA TESTING FOR SECRETOR STATUS
• MATERIALS:
o Phlebotomy Kit
o Personal Protective Equipment
o Centrifuge Machine
o Parafilm
o Test Tube
o Test Tube Rack
o Test Tube Brush
o Pasteur Pipette
o Dropper
• REAGENTS:
o Saliva
o Human polyclonal anti-A and anti-B serum
o Anti-H lectin from Ulex europaeus
o Physiologic Saline 3% to 5 % washed group A, B,
and O cells
• Secretor Substances
o ABO gene and Sese (secretory) gene
o synthesized on type 1 precursor chains
o Secretion of A, B, and H Ag in body fluids
o 78%) in saliva and gastric juice
o Readily detected
o Capable of hemagglutination
• A, B, and H Soluble substances
o Secreted substances are glycoprotein
▪ Beta 1-3 linkage
o Synthesized on type 1 precursor chain
▪ A-2-L fucosyltransferase enzyme
• Fluids in which A<B< and H substances can be
detected in Secretors
o Saliva
o Tears
o Urine
o Digestive Juices
o Bile
o Milk
o Amniotic fluid
o Pathologic Fluids: Pleural, Peritoneal, Pericardial
and Ovarian Cyst
Formation of soluble A, B, and H substances or secretor
substances is the same as that described for the formation of
A, B, and H antigens on the RBCs, except for a few minor
distinctions such as Secreted substances are primarily
synthesized on type 1 precursor chains compared to ABH on
RBCs which are syntheziesd on type 2 precursor chain. The
demonstration of A, B, and H substances in saliva is evidence
for the inheritance of ABO gene and Sese (secretory) gene.
The term “secretor” refers only to secretion of A, B, and H
soluble antigens in body fluids. Certain blood group
substances occur in soluble form in a large proportion (78%)
of individuals in secretions such as saliva and gastric juice.
These individuals are termed “secretors” (they possess the Se
gene) and secrete ABH-soluble antigens. These water-
soluble blood group substances are readily detected in very
minute quantities because they have the property of reacting
with their corresponding antibodies and thereby neutralizing
or inhibiting the capacity of the antibody to agglutinate
erythrocytes possessing the corresponding antigen. The
reaction is termed hemagglutination inhibition and provides a
means of assaying the relative activity or potency of these
water-soluble blood group substances.
PROCEDURE:
A. SALIVA COLLECTION AND PREPARATION:
1. Collect about 2 to 3 mL of saliva in a test tube.
2. Centrifuge at 900 to 1000 g for 8 to 10 minutes.
3. Transfer supernatant to a clean test tube, and place
stoppered tube in a boiling water bath for 10 minutes.
This inactivates enzymes that might otherwise
destroy blood group substances.
4. Recentrifuge and collect clear supernatant into a
clean tube.
B. TEST FOR SECRETOR STATUS
1. Dilute saliva with an equal volume of saline
(undiluted saliva contains nonspecific glycoproteins
that can inhibit antisera and lead to incorrect results).
2. Add one drop of diluted antiserum to an appropriately
labeled tube (anti-A, anti-B, anti H).
For dilution, titrate anti-H, anti-A, and anti-B, testing
against appropriate cells at immediate spin. Select the
dilution giving 2+ agglutination, and prepare a sufficient
quantity to complete the test.
3. Add one drop of supernatant saliva to each tube. Mix
and incubate at room temperature for 8 to 10
minutes.
4. Add one drop of the appropriate indicator cells (A, B,
or O cells) to the properly labeled tube.
5. Mix and incubate at room temperature for 30 to 60
minutes.
6. Centrifuge. Observe for macroscopic agglutination.
• CONTROL:
1. One drop of saline is used in place of dilute saliva.
Test in parallel with the saliva.
2. Test saliva from a known secretor and a nonsecretor
in parallel with test saliva.
• Interpretation:
o NONSECRETOR: Agglutination of RBCs by
antiserum-saliva mixture; control tube positive.
o SECRETOR: No agglutination of RBCs by antiserum
and saliva mixture; control tube positive. The
antiserum has been neutralized by the soluble blood
group substances or antigens in the saliva, which
react with their corresponding antibody. Therefore,
no free antibody is available to react with the
antigens on the reagent RBCs used in the testing.
This negative reaction is a positive test for the
presence of ABH-soluble antigens and indicates that
the individual is a secretor.
• Procedure Notes:
1. Dilution prevents zonal reactions due to either
excessive amount of antigen (post zone) or
excessive amount of antibodies (prozone).
2. The procedure follows the hemagglutination
inhibition or neutralization reaction, which provides a
means of evaluating the relative strength or potency
of the water soluble blood group substances.
MONTEMOR, DJ. 9
 ACTIVITY NO. 7 RH TYPING
• MATERIALS a. Label a tube with patient initials and “D ctrl”
o Phlebotomy Kit below initials.
o Personal Protective Equipment b. Place 1 drop of patient RBC suspension in the
o Centrifuge Machine tube
o Permanent Marker c. Add 1 drop of Rh control reagent to the labeled
o Parafilm tube and mix
o Test Tube Rack d. Serofuge for 15-20 seconds
o Test Tube Brush e. Shake gently to resuspend
o Pasteur Pipette f. AB Positive - If tube is negative, it confirms the
o Dropper patient is AB Pos; however, if agglutination is
• REAGENTS observed, further testing is required before a
o Anti-D Typing Sera blood type can be determined. STOP HERE for
o 3-5% Known Red Cell Suspension AB Pos.
o Physiologic Saline (0.85-0.90% NaCl) in Wash Bottle g. D Negative – continue with Weak D test.
• Rh Blood Group • Interpretation:
o Required when testing donor and patient’s blood o Blood Type of the patient is the tube or slide showing
o IgG AGGLUTINATION. There is no reverse typing in Rh
o Reacts optimally at 37C since there is no innate or preformed antibody
o Can cross the placenta against Rh antigens. Antibody production will only
o Rh (+): D or weak-D test is positive happen when there is exposure to Rh antigen.
o Rh (-): Neg for both slide/Tube and IAT testing
Determining the D status of an RBC sample is required when
testing donor bloods. Blood for transfusion is considered Rh
positive if either the D or the weakD test is positive. Any donor
blood sample that is typed Rh0 (D)-negative by the slide or
rapid tube method must be tested further by an indirect
antihuman globulin technique. If both tests are negative, the
donor sample is considered Rhnegative. If the donor sample
tests positive in any phase of Rh0 (D) testing, the sample is
considered Rh-positive. Blood for transfusion is considered
Rh-positive if either the D or weak-D test is positive; if both the
D and weak D tests are negative, blood for transfusion is
considered Rh-negative. Most Rh antibodies are IgG
immunoglobulins and react optimally at 37C or following
antiglobulin testing; exposure to less than 0.1 mL of Rh-
positive RBCs can stimulate antibody production in an Rh-
negative person.
PROCEDURE:
A. Slide Method
1. Prepare 1 clean slide and label with “Anti-D”
2. Perform capillary puncture. Remember to wipe out
first before placing a drop of blood to the slide.
[contains capillary juices might interfere the
agglutination process]
3. Place a drop of anti-D typing sera just beside but not
touching the blood.
4. Carefully mix the two solutions by using a clean
applicator stick
5. Record the result.
B. Tube Method
1. Make a 3-5% RCS of both Patient (Px) and Donor
(Do
2. Prepare 2 clean tubes and label with PX, DO
respectively
3. Place 1 drop of anti-D typing sera on to each tube.
4. Add 1 drop of Patient RCS to tube PX and Donor
RCS to tube DO.
5. Cover the tubes with Parafilm.
6. Mix the solution and spin for 15 seconds at 3,400
rpm. Gentle mixing is recommended.
7. Carefully dislodge the cell button by gently mixing it
and examine for agglutination or hemolysis.
8. Grade the reaction and record the results. Note: In
every tube method, always add first the clear
solution. This is to ensure that all needed solutions
are added.
• MAKING D-CONTROL TUBE: D CONTROL
o If anti-D tube is negative OR all tubes in the forward
type are positive (patient appears to be AB positive)
a negative D (Rh) control tube must be run.

MONTEMOR, DJ. 10