Standardization of flow cytometric crossmatch (FCXM) for investigation of unexplained habitual abortion.

PROBLEM

To define a positive flow cytometric crossmatch (FCXM), in terms of channel shift, for maternal IgG and IgM (n = 28) against paternal T and B lymphocytes.

METHOD

A reference range study. Mononuclear cells were obtained from 28 healthy volunteers using density gradient separation of heparinized blood, followed by pre-incubation with goat immunoglobulin. A total of twelve tubes were prepared for each volunteer. Primary incubation was with negative control serum, positive control sera (either IgG or IgM) and individual AB sera. Secondary incubation was with four combinations of fluorochromes: CD3 PE/IgG-Fc F(ab')2FITC, CD3 PE/IgM F(ab')2FITC, CD20 PE/IgG-Fc F(ab')2FITC and CD20 PE/IgM F(ab')2FITC. The cells were then analyzed with an EPICS Profile flow cytometer, using 256-channels and a four decade log scale.

RESULTS

The linear mean channel fluorescence of the negative control serum was subtracted from the individual AB sera (channel shift) for each of the four combinations of fluorochromes. By determining the 95% one-sided upper reference limits of the negative control serum for each of the four trimmed data sets, we clinically defined a positive FCXM for bound IgG or IgM to T lymphocytes as a shift of 10 or more channels, and for bound IgG or IgM to B lymphocytes as a shift of 25 or more channels, above the linear mean channel shift of the negative control serum.

CONCLUSION

Positive FCXMs were defined for maternal IgG and IgM against T and B lymphocytes, in terms of channel shift above the linear mean channel fluorescence of the negative control serum. By standardizing the dual-color FCXM methodology, the clinical significance of alloantibodies in the maintenance of pregnancy could be addressed in a collaborative manner.