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Reagents

You are here : Home/ Blood Bank Zone/ Red Cell Serology/ 6. Reagents

6. Reagents

Blood sample

The sample should be as fresh as possible. Venous blood may be drawn in a clean container and stored at 4°C (if not tested within 12 hours).

Use serum (instead of plasma) for reverse grouping as plasma may lead to non-detection of weak or complement binding antibodies.

Blood grouping antisera

All reagents which need to be restandardized for microplate use should be tested for potency and specificity. High concentration of proteins or polymers such as ficoll, polyvinyl pyrrolidine (PVP) and dextran cause red cells to stick to surface of the well.

Antisera for use in microplate technique should be diluted to take care of the dye and to decrease the effect of additives. Colour of the antisera should be light or colourless otherwise it will interfere with the results on plate reader.

In routine laboratory, ABO and Rh (D) grouping is usually performed in parallel. ABO monoclonal and Anti-D IgM reagents will give excellent results when diluted in phosphate buffer saline containing 1- 3% bovine serum albumin.

Polyclonal anti-D sera available for sLide and rapid tube test are usually unsuitable for microplate use. Usually a dilution of 1:20 of ABO antisera and 1:10 of anti-D antisera give good results, however individual laboratories should standardize the technique depending on the antisera in use.

Reagent and test red cells

The cells can be suspended in phosphate buffer saline (PBS), low ionic strength saline solution (LISS) or isotonic saline. A 3% suspension of red cells may be used.

Enzymes techniques are commonly used in mocroplate blood grouping as the forces involved in resuspending negative reactions in the resuspension technique may lead to fragmentation of weak agglutinates. One-stage (bromelain or papain-cystein) enzyme method is likely to give better results for the weaker phenotypes.

Procedure

Prepare a worksheet for proper layout of samples.

I.
- Arrange test samples in serial number i.e 1-10
- Separate cells and serum of the test samples and arrange them as shown below
- Using a fine tipped Pasture pipette, dispense 1 drop each of anti-A, anti-B, anti-AB, anti-Di and anti-D2 (anti-D from two different firms or batches) into each well of row A,B,C,D,E, respectively.
- Add I drop of 2-3% cell suspension of known A cell, B cells and 0 cells to the well of row F-i, G0i and li-i through 12.

Micropiate A4BO & Rh (D) grouping

1 2 3 4 5 6 7 8 9 10 11 12

Anti-A A

Anti-B B

Anti-AB C

Anti-D1 D

Anti-D2 E

A Cells F

B Cells G

O Cells H

II.
- Starting with test samples 1, dispense 1 drop of serum to well F,G and H vertically i.e. row I
- Transfer 1-2 drop of red blood cells to saline tube labelled-I and prepare 2-3% washed suspension of test cells.
- Repeat with all test and control samples till-12.

III.
- Dispense 1 drop of 2-3% cells from test sample 1 to well A,B,C,D, and E inrowl vertically i.e. row 1.
- Repeat with all test samples and control samples till 12.
- Gently tap the microplate and incubate at room temperature (22-24°C) for 1 hour.

If the microplate centrifuge is available, spin after 15 minute incubation at bog for 40 seconds.

IV.
- Resuspend the red cells using a microplate shaker. Overshaking will reduce the strength of agglutination and this can be avoided by switching off the microplate shaker as soon as a known negative reaction is fully suspended and positive reactions dislodged.
- Record results
- Results are preferrably read by two technician independently, one reads while the other records results in the register and then they reverse roles.
- Typical reaction pattern may be interpreted visually or using an automated or semi-automated reader.

Interpretation

A positive reaction will show the button of cells having a strongly crenated edge, while a negative result will show a compact button with smooth edge that streams on tilting or gets suspended on shaking.

A gentle shake of the plate will often assist in clarifying doubtful results.

If a microplate centrifuge and shaker is not available, after the one hour incubation, tilt the microplate at an angle of 600 and leave for 5 minutes to allow negative reactions to stream. (same can be done in V-bottom plates also).

Predispensed plates

A multichannel pipette or automated microplate dispenser may be used, which can save considerable time. Predispensed plates should be kept covered at 4°C until required. In order to avoid evaporation of reagents, predispensed microplates should not be stored for more than 24 hours.

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