Elutions Prior to Antigen Typing

The second main purpose of elutions is to obtain intact red cells that have a negative DAT. The cells can then be used for antigen typing by an IAT method [typing for K, Fy^a, Jk^a, etc. where neither saline nor chemically-modified antisera exist]. The cells can also be used to autoadsorb warm autoantibodies so that the patient's autoadsorbed serum can be used to detect possible alloantibodies that were hidden by the pan-reacting autoantibodies. Three methods exist that can save intact red cells. None of the three are useful to obtain antibody for identification because the antibody yield in the eluate is poor.

Modified Heat Elution (40°- 45°C)

Unlike the regular heat elution at 56°C, the modified heat method uses lower temperatures that will not denature antigens. A disadvantage is that the DAT may not become negative until the temperature is so high that antigens may be denatured.

Chloroquine Diphosphate (CDP)

CDP is the method of choice for obtaining cells with a negative DAT for antigen typing. The method can be described in general as follows: (1) wash the red cells; (2) add CDP solution, mix and incubate at RT for 30 min; (3) test a small aliquot with anti-IgG antiglobulin serum; (4) if DAT negative, wash cells well and use as required; if still DAT positive, repeat the DAT at 30 min intervals until negative or almost negative. Most, but not all, antibodies will dissociate within 2 hrs.

ZZAP

ZZAP is a reagent consisting of cysteine-activated papain and dithiothreitol (a sulfhydryl agent). Its method is similar to that of CDP, except that the treated cells are incubated at 37°C instead of RT. Because of its papain component, ZZAP will denature MNS Fy^a Fy^b antigens; papain and DTT together also denature antigens in the Kell BGS.

Quality Control (CDP, ZZAP and Modified Heat Elutions)

When these methods are used to obtain DAT negative cells for antigen typing, routine QC includes setting up positive and negative antisera controls. The red cells chosen for these controls must be treated in exactly the same way as the patient's cells, e.g., heated or treated with the chemical for the same time as the test cells. This is required because the treatment/manipulation may denature some antigens (causing false negatives), or uncover latent/hidden antigens (causing false positives).

Elution (antigen typing)