and mix.

d. Centrifuge for 15 seconds at 900 to 1000 g. Read

and record the results.

e. Add Coomb’s control cells to each negative tube.

Interpretation of Results for Anti-human Globulin Tests

1. Agglutination/hemolysis after incubation at 37°C

constitutes a positive test.

2. The presence of agglutination after addition of AHG

reagent constitutes a positive test.

3. Anti-human globulin tests are considered negative when no agglutination is observed after initial

centrifugation and positive result with Coomb’s

control cells. If after addition of Coomb’s control cells

a negative result is observed, then the test is invalid

and must be repeated.

4. For the LIM (Low Ionic Medium-Polybrene) procedure, agglutination that persists after addition of

resuspending solution indicates a positive result.

Controls

1. The procedure used for the detection of unexpected

antibodies in transfusion testing should be checked

daily with weak antibodies.

2. When LIM technique is used, test an unknown serum

against reagent red blood cells, an inert serum should

be tested against a random cell sample for comparative

purposes.

Notes

1. The incubation time and volume and concentration

of red cells incubated are those given in literature. In

all cases, the manufacturer’s package insert should be

strictly adhered to.

2. For the PEG procedure:

a. Omit centrifugation after 37°C incubation, as red

blood cells will not resuspend readily.

b. Use monospecific anti-human IgG rather than

polyspecific AHG to avoid unwanted positive reactions due to C3-binding antibodies.

3. LISS additive and PEG solutions are available

from various commercial sources. Manufacturer’s

instruction should be followed when using these

reagent.

Papain—One-stage Enzyme technique/Two-stage

Enzyme Technique

Specimen

Serum to be tested. Preferably, freshly collected serum

should be used.

Reagent

1. Reagent red blood cells.

2. Polyspecific AHG or monospecific anti-human IgG

reagent.

3. Coomb’s control cells.

4. Donor cells/reagent red blood cells.

Procedure for One-stage enzyme technique

1. To an appropriately labeled test tube, add two drops

of serum.

2. Add two drops of 2–5% saline suspension of reagent

red blood cells.

3. Add two drops of papain solution and mix well.

4. Incubate at 37°C for 30 minutes.

5. Centrifuge for 15–20 seconds at 900–1000 g and gently

resuspend the cells, observe for agglutination. Grade

and record the results.

6. Wash the cells four times with saline and completely

decant after the final wash.

7. Add AHG to cell button according to the manufacturer’s

instruction. Mix well.

8. Centrifuge for 15–20 seconds at 900–1000 g and

observe for reaction. Grade and record the results.

9. Confirm the validity of negative tests by adding

Coomb’s control cells.

Procedure for two-stage enzyme technique

1. Add one drop of washed packed cells and one drop of

papain reagent to appropriately labeled tube.

2. Incubate at 37°C for 30 minutes.

3. Wash the papain treated three times with isotonic

saline and prepare 2–5% cell suspension.

4. To an appropriately labeled test tube, add one drop

of papain-treated red blood cell suspension and two

drops of serum under test.

5. Mix well and incubate at 37°C for 30 minutes.

6. Centrifuge for 15–20 seconds at 900–1000 g and gently

resuspend the cells, observe for agglutination. Grade

and record the results.

7. Wash the cells four times with saline and completely

decant after the final wash.

8. Add AHG to cell button according to the manufacturer’s

instruction. Mix well.

9. Centrifuge for 15–20 seconds at 900–1000 g and

observe for reaction. Grade and record the results.

Blood Banking (Immunohematology) 347

10. Confirm the validity of negative tests by adding

Coomb’s control cells.

Antibody Titration Studies

Antibody Titration for Characterizing Type of Antibody in Serum

Specimen

Serum (antibody) to be titrated.

Reagents

1. Red blood cells that express the antigen(s) corresponding to the antibody specificity(ies), in a 2–5%

saline suspension. Uniformity of cell suspensions is

very important to ensure comparability of results.

2. Normal saline (Dilutions may be made with 6%

albumin if desired).

Procedure

The master dilution technique for titration studies is as

follows:

1. Label ten test tubes according to the serum dilution

(e.g. 1 in 1,1 in 2, etc.).

2. Deliver one volume of saline to all test tubes except

the first.

3. Add an equal volume of serum to each of the first two

tubes (undiluted and 1 in 2).

4. Using a clean pipette, mix the contents of the 1 in 2

dilution several times, and transfer one volume into

the next tube (1 in 4 dilution).

5. Continue the same process for all dilutions, using

a clean pipette to mix and transfer each dilution.

Remove one volume of diluted serum from the final

tube and save it for use if further dilutions are required.

6. Label ten 10 × 75 mm or 12 × 75 mm tubes for the

appropriate dilutions.

7. Using separate pipettes for each dilution, transfer two

drops of each diluted serum into the appropriately

labeled tubes, and add one drop of red blood cell

suspension.

8. Mix well and test by serologic technique appropriate

to the antibody.

9. Examine test results macroscopically, grade and

record the reactions. The prozone phenomenon may

cause reactions to be weaker in the more concentrated

serum preparations than in higher dilutions; to avoid

misinterpretation of results, it may be preferable to

examine first the tube containing the most dilute

serum and proceed through the more concentrated

samples to the undiluted specimen.

Interpretation

1. Observe the highest dilution that produces 1 +

macroscopic agglutination. The titer is the reciprocal

of the dilution. If there is agglutination in the tube

containing the most dilute serum, the endpoint has

not been reached, and additional dilutions should be

prepared and tested.

2. In comparative studies, a significant difference in titer

is three or more dilutions. Variations in technique

and inherent biologic variability can cause duplicate

tests to give results that differ by one dilution in either

direction.

3. Titer values alone can be misleading, without

additional evaluation of strength of agglutination.

The observed strength of agglutination can be assigned

a number and the sum of these numbers for all tubes

in a titration study represents the score. The arbitrarily

assigned threshold for significance in comparing scores is

a difference of 10 or more between different test samples

(Table 11.6).

TABLE 11.6: Examples of antibodies tilers, endpoint and scores

Reciprocal of serum dilution

1 2 4 8 16 32 64 128 256 512 Titer Score

Strength: 3+ 3+ 3+ 2+ 2+ 2+ 1+ ± ± 0 64 (256)

Sample 1

Score: 10 10 10 8 8 8 5 3 2 0 64

Strength: 4+ 4+ 4+ 3+ 3+ 2+ 2+ 1+ ± 0 128 (256)

Sample 2

Score: 12 12 12 10 10 8 8 5 3 0 80

Strength: 1+ 1+ 1+ 1+ ± ± ± ± ± 0 8 (256)

Sample 3

Score: 5 5 5 5 3 3 3 2 2 0 33

348 Concise Book of Medical Laboratory Technology: Methods and Interpretations Notes

Titration is a semiquantitative technique and technical

variables greatly affect the results. Hence, care should be

taken to achieve the most uniform possible practices.

1. Careful pipetting is essential. Pipettes with disposable

tips that can be changed after each dilution is

recommended.

2. Optimal time and temperature of incubation, time and

force of centrifugation must be used consistently.

3. The age, phenotype and concentration of test red

blood cells influence the results. When the titers of

several antibody containing sera are to be compared,

all should be tested against red blood cells (preferably

freshly collected) from the same donor. If this is not

possible, the tests should use a pool of reagent red

blood cells from donors of the same phenotype. When

a single serum is to be tested against different red

blood cell samples, all samples should be collected

and preserved in the same manner, and diluted to the

same concentration before use.

4. Completely reproducible results are virtually impossible

to achieve. Comparisons are valid only when specimens

are tested concurrently. In prenatal testing of sequential

serum samples to detect changing antibody activity,

samples should be frozen for comparison with subsequent samples. Each new sample should be tested in

parallel with the immediately preceding sample. In

tests with a single serum against different red blood

cell samples, material from the master dilution must

be used for all tests.

5. Measurements are more accurate with large volumes

than with small volumes, a master dilution technique

gives more reliable results than individual dilutions for

a single test. The volume needed for all planned tests

should be calculated and an adequate quantity of each

dilution prepared.

Antibody Titration Studies for Early Detection of

Hemolytic Disease of the Newborn

Specimen

Serum for titration (containing potentially significant

unexpected antibodies to red blood cell antigens, 1 mL). If

possible, test the current sample in parallel with the most

recent previously submitted (preceding) sample from the

current pregnancy.

Materials

1. Anti-human IgG reagent.

2. Dilute bovine albumin (approximately 6% w/v),

optional: 22% (w/v) bovine albumin, 1 mL; isotonic

saline, 3 mL.

3. Micropipettes or equivalent: 0.1–0.5 mL delivery, with

disposable tips.

4. Red blood cells: Group O reagent red blood cells with

double dose expression of antigen to which the serum

contains antibody (use R2R2 RBCs when titrating

anti-D); wash three times and dilute to a 2% red blood

cell suspension with isotonic saline.

Quality Control

1. Test the preceding sample in parallel with the current

sample.

2. Prepare dilutions using separate pipette for each tube.

Failure to do so will result in falsely high titers due to

carry-over.

3. Confirm all negative reactions with Coomb’s control

cells.