Procedure

1. Using 0.5 mL volumes, prepare serial dilutions of

serum in saline or 6% albumin. The initial tube should

contain undiluted serum and the doubling dilution

range should be from 1 in 2 to 1 in 2048 (total of 12

tubes).

2. Place 0.1 mL of each dilution into appropriately

labeled 10 or 12 × 75 mm test tubes.

3. Add 0.1 mL of red blood cell suspension to each

dilution.

4. Gently agitate the contents of each tube; incubate at

37°C for 1 hour.

5. Wash the tubes four times with saline; completely

decant the final wash supernatant.

6. To the cell buttons thus obtained, add Anti-human IgG

according to the manufacturer’s direction.

7. Centrifuge as for hemagglutination tests.

8. Examine the results macroscopically; grade and record

the reactions.

9. Add one drop of Coomb’s control cells to all negative

tests; recentrifuge and examine the tests macroscopically for mixed field agglutination; repeat

antibody detection tests when tests with Coomb’s

control cells are nonreactive.

Results

The titer is reported as the reciprocal of the highest dilution

of serum at which 1 + agglutination is observed. A titer

greater than or equal to 16 is considered significant and

may warrant monitoring for HDN by cordocentesis, high

resolution ultrasound, or examination of the amniotic

fluid for bilirubin pigmentation.

Notes

1. Titration studies should be performed upon initial

detection of the antibody; save an aliquot of the serum

Blood Banking (Immunohematology) 349

(frozen at _

20°C or colder) for comparative studies with

the next submitted sample.

2. When the titer is less than 16 and the antibody

specificity has been associated with HDN, it is

recommended that repeat titration studies be

performed every 2–4 weeks, beginning at 18 weeks

of gestation; save an aliquot of the serum (frozen at

_

20°C or colder) for comparative studies with the next

submitted sample.

3. When the decision has been made to monitor

the pregnancy by an invasive procedure such as

amniocentesis, no further titrations are warranted.

4. Each institution should develop a policy to ensure some

degree of uniformity in reporting and interpreting

antibody titers.

5. For antibodies to low incidence antigens, consider

using paternal red blood cells.

6. Do not use enhancement techniques (albumin, PEG,

LISS) or enzyme treated red blood cells, because

elevated titers may be obtained.

7. LISS should not be used as diluent in titration studies;

non-specific uptake of globulins may occur in serumLISS dilutions.

8. Failure to obtain the correct results may be caused by

incorrect technique, notably; failure to use separate

pipette tips for each dilution or failure to mix thawed

frozen serum.

Use of Sulfhydryl Reagents to Distinguish between

IgM and IgG Antibodies

Specimen

Two mL of serum to be tested.

Reagents

1. Phosphate buffered saline at pH 7.3.

2. About 0.01 M dithiothreitol (DTT) prepared by

dissolving 0.154 g of DTT in 100 mL of pH 7.3 PBS store

at 2–8°C.

Procedure

1. Dispense 1 mL of serum into each of two test tubes.

2. To one tube, labeled as control, add 1 mL of pH 7.3

PBS.

3. To the other tube, labeled as test, add 1 mL of 0.01 M

DTT.

4. Mix and incubate at 37°C for 30–60 minutes.

5. Test the antibody activity in each sample by titration

against red blood cells of appropriate phenotype

(Table 11.7).

Notes

1. Sulfhydryl reagent used at low concentration may

weaken antigens of Kell system. For investigation of

antibodies in Kell system, it may be necessary to use

alkylation with iodoacetic acid, followed by dialysis.

2. Gelling of serum or plasma sample may be observed

during treatment with DTT. This can occur if the DTT

has been prepared incorrectly, and has a concentration

above 0.01 M. Gelling may also occur if serum and

DTT are incubated too long. An aliquot of the sample

undergoing treatment can be tested after 30 minutes

of incubation, if the activity thought to be due to IgM

has disappeared, there is no need to incubate further.

Gelled samples cannot be tested for antibody activity

because overtreatment with DTT causes denaturation

of all serum proteins.

Elution Techniques

Citric Acid Elution Method

Specimen

Packed DAT positive red blood cells washed six times with

saline.

Reagents

1. Elution solution: Citric acid (monohydrate), 1.3 g,

KH2PO4 0.65 g saline to 100 mL, store at 4°C.

TABLE 11.7: Effect of dithiothreitol on blood group antibodies

Dilution

Test sample 1/2 1/4 1/8 1/16 1/32 Interpretation

Serum + DTT 3+ 2+ 2+ 1+ 0 IgG

Serum + PBS 3+ 2+ 2+ 1+ 0

Serum + DTT 0 0 0 0 0 IgM

Serum + PBS 3+ 2+ 2+ 1+ 0

Serum + DTT 2+ 1+ 0 0 0 IgG + 1gM*

Serum + PBS 3+ 2+ 2+ 1+ 0

* May also indicate only partial inactivation of IgM

350 Concise Book of Medical Laboratory Technology: Methods and Interpretations 2. Neutralizing solution: Na3PO4, 13.0 g; distilled water

to 100 mL; store at 4°C.

3. Supernatant saline from final wash of the red blood

cells to be tested.

Procedure

1. Chill all reagents to 4°C in ice bath before use.

2. Place 1 mL of packed red blood cells in a 13 × 100 mm

test tube.