1. The biphasic nature of the hemolysin associated with
PCH requires that serum be incubated with cells at
cold temperature and then at 37°C.
2. Active complement is essential for demonstration of
the antibody. Because patient with PCH may have
low levels of serum complement, fresh normal serum
should be included in the reaction medium as a source
3. To avoid loss of antibody by cold autoadsorption before
testing, the patient’s blood should be allowed to clot
at 37°C, and the serum separated from the clot at this
Chequerboard Titration for Quality Control of
Anti-IgG Potency in Polyspecific AHG Reagent and
Evaluation of Complement Potency with
Reagents and Materials Required for Chequerboard
1. Anti-D IgG reagent with albumin titer 256–512.
3. Freshly collected O RhoD positive cells
5. 12 ×100 mm and 12 × 75 mm test tubes
352 Concise Book of Medical Laboratory Technology: Methods and Interpretations 7. Table centrifuge
9. Water bath or laboratory incubator.
Preparation of 3% cell suspension
1. Collect 2 mL of freshly drawn venous blood in a clean
12 × 100 mm test tube with suitable anticoagulant.
2. Centrifuge at 3000 rpm for 2–3 minutes to form a cell
4. Resuspend the cell button in 5 mL of normal saline.
5. Centrifuge at 3000 rpm for 2–3 minutes.
6. Repeat the washing of cells (steps 4 and 5) twice more
so that the cells are washed three times.
7. After the final centrifugation, remove the supernatant
without disturbing the cell button.
8. Take 0.75 mL of packed cells and resuspend them in
24.25 mL of normal saline to get a 3% cell suspension.
Dilutions of Anti-D (IgG) Reagent
1. Take a set of ten, 12 × 100 mm test tubes and number
2. Add 2 mL of normal saline to each of the tubes from
3. Add 2 mL each of anti-D (IgG) reagent to tube number
4. Mix the content of tube number 2 and transfer 2 mL
of diluted reagent to tube number 3 with the help of
5. Continue this serial dilution till tube number 10.
6. Discard 2 mL of diluted reagent from tube number 10.
1. To each of the above dilutions of anti-D (IgG), add
2 mL of well mixed freshly prepared 3% cell suspension.
2. Mix well all the tubes and cover them with aluminum
3. Incubate the tubes at 37°C for 30 minutes, with
4. Centrifuge the tubes at 3000 rpm for 2–3 minutes.
5. Remove the supernatant and resuspend the cell button
6. Centrifuge at 3000 rpm for 2–3 minutes.
7. Repeat the washing (Step 4 and 5) at least four times.
8. Resuspend the cell button from each tube in 2 mL of
normal saline to get a 3% suspension of sensitized cells.
Thorough washing of sensitized cells (after incubation) is
very important as even slight traces of free anti-D IgG can
lead to false negative results.
Dilutions of Anti-human Globulin Reagent
1. Take a set of six, 12 × 100 mm test tubes and number
2. Add 2.5 mL of normal saline to each of the tubes from
3. Add 2.5 mL of polyspecific AHG reagent to tube
4. Mix the content of tube number 2 and transfer 2.5 mL
of the diluted reagent to tube number 3.
5. Continue this serial dilution till tube number 6.
6. Discard 2.5 mL of diluted reagent from tube number 6.
Preparation of Complement-Coated Cells
Reagents and material required:
3. O group red blood cells 50% suspension
7. Water bath or laboratory incubator.
Preparation of 50% Cell Suspension of
1. Collect 1 mL of freshly drawn venous blood in a
clean 12 × 100 mm test tube containing suitable
2. Centrifuge the tube at 3000 rpm for 2–3 minutes to
4. Resuspend the cell button in 5 mL of buffered saline.
5. Centrifuge at 3000 rpm for 2–3 minutes.
6. Repeat the washing of cells (steps 4 and 5) twice more
so that the cells are washed three times.
7. After the final centrifugation, remove the supernatant
without disturbing the cell button.
8. Add 1 mL of buffered saline to the packed red blood
cells to get a 50% O group red cell suspension.
Collection of Inert O Group Serum
1. Collect 2 mL of freshly drawn venous blood in a clean
2. Immediately centrifuge at 3000 rpm for 2–3 minutes.
3. Collect 0.5 mL of serum in a clean test tube.
Sensitization of O Group Red Blood Cells
1. Place 8.5 mL of LISS into a 20–25 mL container.
2. Add 0.5 mL of fresh inert O group serum to it.
3. Mix well and add 1 mL of 50% O group red cell
4. Mix thoroughly and incubate at 37°C for 30 minutes
with occasional further mixing.
Blood Banking (Immunohematology) 353
5. Centrifuge at 3,000 rpm for 2–3 minutes to form a cell
6. Discard the supernatant and resuspend the cell button
7. Centrifuge at 3,000 rpm for 2–3 minutes.
8. Repeat the washing of cells (steps 6 and 7) three more
times so that cells are washed four times.
9. After the final centrifugation, remove the supernatant
without disturbing the cell button.
10. Add 14.5 mL of buffered saline to packed red blood
cells to obtain 2–3% suspension of complementcoated cells.
Chequerboard Titration (Table 11.8)
1. Take a set of sixty, 12 × 75 mm test tubes, number and
arrange them as shown below in the Table 11.8:
2. Add 0.2 mL each of N (neat) AHG in the respective
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