9. Depending on the speed necessary for completing

the test, one of the two alternatives may be chosen:

(i) the test tubes may be left at room temperature for

at least 2 hours, or (ii) after 2 to 3 minutes, the tubes

may be centrifuged (1 minute at 1500 rpm in a clinical

centrifuge).

10. After incubation or centrifugation, the red cell

suspensions are redispersed by tapping the tubes.

Presence of agglutination is checked with the naked

eye, with the scanning lens of a microscope and if the

results are doubtful or negative, microscopically (10X

objective) after placing a drop on a slide.

Interpretation

The results of an ABO grouping test can be accepted as

valid only if the findings obtained in the first three test

tubes with known antisera agree with those obtained in the

second three test tubes with known red cell suspensions;

this second, part of the test is called confirmation, check

or reverse grouping. Given on previous page are the result

in the four main ABO groups in such tests when both

the unknown red cells and unknown serum are tested.

Agglutination is indicated by the +sign, –sign indicates no

agglutination.

Absent or weak agglutination with the unknown serum

may be simulated by hemolysis of the known red cells,

this suggests the presence of hemolytic anti-A or anti-B

antibody. The assumption that hemolysis is due to anti-A

or anti-B must be confirmed by repeating the test with

the serum after its inactivation in a water bath at 56oC for

30 minutes—use of the inactivated serum is expected to

result in agglutination instead of hemolysis.

If discrepancies in the two parts of ABO grouping are

observed, the test should be redone. If a second test reveals

the same discrepancy, the following possibilities are to be

considered:

1. Cold agglutinins may cause agglutination of the known

cells by the unknown serum regardless of its ABO

group. Confirm this by testing the unknown serum

with its own red cells at 4°C for 1 to 2 hours. Under

those conditions cold agglutinins produce agglutination, which disappears after transfer of the specimen

to a water bath at 37°C for 5 to 10 minutes.

2. Agglutination of the unknown red cells by known

antisera that conflict with the results obtained in the

confirmation grouping may be due to coating of the red

cells by autoantibodies. Confirmation of this phenomenon is obtained by performing on the unknown red

cells the direct antiglobulin test and obtaining positive

results. In order to obtain in such cases, a reliable

ABO grouping, the red cells should be washed several

times with large amounts of isotonic saline solution,

following which they are more likely to give adequate

result.

3. Unexpected agglutination obtained with the

unknown serum may reflect presence of irregular

agglutinins, such as Rh antibodies, which react with

the corresponding blood factor in the suspensions of

the known red cells.

FIG. 11.3: ABO blood grouping—tube method

Blood Banking (Immunohematology) 331

4. Discrepancies in the two parts may sometimes be

present due to A subgroup. In which anti-A1 serum,

anti-A2 serum, and A1 and A2 reagent red cells are used

for confirmation.

5. Serum of newborn and young infants may not contain

the isoagglutinins expected from the reactivity of

their red cells; hence, in infants, the use of unknown

serum is not a reliable method. Much less frequently

isoagglutinins may be absent in older children and

adults due to hypo or agammaglobulinemia or for

unknown reasons.

Briefly the Reasons can be Described as:

a. Improper identification of specimen.

b. Improper techniques like:

• Failure to add proper reagent

• Incorrect cell to serum ratio

• Failure to identify hemolysis

• Incorrect reading, recording or interpretation of

test results.

c. Failure of equipment.

d. Poor standardized or stored reagent.

e. Patients problems.

• Patient may fail to express ABO antigens on red

cells, e.g.

1. Age (newborn or old age).

2. Disease states, i.e. leukemia or lymphomas.

This Leads to False Negative Results

¾ Acquired B-antigen can occur

1. Gram-negative septicemia

2. Carcinoma colon.

This may Cause False Positive Reaction

¾ Rouleaux formation: This is an aggregation of red cells

in the form of piles of coins and can be misinterpreted

as agglutination

¾ Acquired antibodies, e.g. anti-A1, in A2 persons anti-H

in Bombay phenotype, cold autoantibodies, all

unexpected antibodies

¾ Absence or weakening of antibodies, e.g. immune

deficiency states, agammaglobulinemias, etc.

Solving Problems of Discrepancies

Once a discrepancy is detected in ABO cells and serum

grouping, repeat the test before additional investigations

are carried out. Quality assurance of reagent, correct

technique, careful observation and interpretation of

results resolve many problems.

Repeat Preliminary Procedures

1. Obtain a fresh blood sample from donor unit or

patient to rule out discrepancy due to contamination

or unidentified samples.

2. Wash the cells 3–4 times in normal saline to rule out

rouleaux formation and prepare 2–5% cell suspension.

3. Perform direct antiglobulin test on the cells, to detect

if cells are coated with antibody as in HDN and AIHA.

4. Retest the cells with fresh and potent anti A, anti-B,

anti-AB, anti-A or anti-H as appropriate for individual

problem.

5. Test the serum against appropriate A1, A2 and B cells.

Group O cells and autologous cells should be used as

controls to detect alloagglutinins and autoagglutinins.

6. Use group O cord cells if anti-I is suspected.

RH BLOOD GROUP SYSTEM

It had been suspected for a long time that cause of many

transfusion reactions was due to specific differences in

blood other than the four main blood groups originally

described by Landsteiner. In 1939, Levine and Stetson

described an antibody in the serum of a group O mother

who delivered stillborn fetus and subsequently developed