Leukocyte Antibody Detection

The flow cytometry crossmatch is an in vitro assay used to detect anti-lymphocyte (lymphocytotoxic) antibodies in serum. These antibodies may be present in individuals who have been previously sensitized to donor antigens, such as organ transplant recipients or women with a history of recurrent spontaneous abortions.

In this assay, recipient serum is incubated with donor lymphocytes. If cytotoxic antibodies (IgG or IgM) are present, they will bind to surface antigens on donor T or B cells. Fluorescently labeled secondary reagents detect this binding, and flow cytometry quantifies the percentage of antibody-positive cells. The degree of fluorescence correlates with the amount of antibody bound to donor lymphocytes.

Donor (male) specimen:

• Specimen Type: Whole blood
• Volume: 30–40 mL
• Collection: Green-top tubes (sodium heparin)

Recipient (female) specimen:

• Specimen Type: Serum
• Volume: One 10 mL red-top tube (no additive) or serum separator tube (gel barrier)

General handling:

• Mix tubes immediately and thoroughly after collection to prevent clotting.
• Transport specimens at room temperature.
• Do not refrigerate or place on ice.
• Specimens must be delivered to the laboratory within 72 hours of collection.

Unacceptable specimens include:

• Cold specimens (refrigerated or shipped on ice)
• Clotted samples
• Hemolyzed samples
• Specimens >48 hours old

If a specimen is older than 48 hours, lymphocytes will be isolated and viability assessed:

• If viability ≥80%, the assay will be performed.
• If viability <80%, the specimen will be rejected.

Flow Cytometry

Negative

For specimens positive by flow cytometry, the percentage of positive T or B cells will be reported for IgM and IgG antibodies.

• Cook, D.J., et al. Transplant Proc. 20: Suppl. 1:81, 1988.
• Gilman-Sachs, A., Sung-Ping, L., Beer, A.E., and Beaman, K.D. J. Clin. Lab. Immunol. 30, 53-59, 1989.
• Gilman-Sachs A, Harris D, Beer AE, Beaman KD. J Reprod Immunol. 17:41–51, 1990. “Inhibition of binding of anti-CD3 antibodies to paternal lymphocytes correlates with failure to immunotherapy for treatment of recurrent spontaneous abortions.” 17, 41-51, 1990.

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