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Abstract
Background
The lymphocytes separated from whole blood are used in HLA flow cytometry cross-match (FCXM) for renal transplantation. In this study, the methodology of whole blood flow cytometry was applied to FCXM, omitting lymphocyte separation step.
Methods
In the 20 cases (including positive 5 cases) of T cell FCXM for renal transplantation, the standard assay using the separated mononuclear cells (MNC) was compared with the two variant assays using whole blood. In the latter assay, the donor whole blood was incubated with the excessive recipient serum. The red cells were lysed (lysed whole blood, LWB). Otherwise, instead of red cell lysis, the signals of T cells among whole blood (WB) were acquired using fluorescence triggering. The sample/negative control mean fluorescence intensity (MFI) ratio was calculated for the interpretation.
Results
The MFI ratio of the 20 cases by MNC, LWB and WB assay were 4.9±8.1, 5.4±9.7 and 4.8±7.8, respectively. Both LWB and WB assay were not significantly different from MNC assay (P=0.313, 0.831, respectively, paired t-test). The qualitative determinations were concordant in all cases, except for one case which was weakly positive with MFI ratio 2.2 by LWB assay.
References
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Fig. 1.
Analysis of T cell flow cytometry crossmatch. (A) Lymphocyte scatter gate (R1) in FSC/SSC plot. (B) T cell gate (R2) in CD 3PE/ SSC plot of gated lymphocytes. (C) Anti-IgG FITC histogram of gated T cells. This overlay histogram shows a representative example of the calculation of MFI ratio.
Abbreviations: FSC, forward scatter characteristics; SSC, side scatter characteristics; MFI, mean fluorescence intensity.
Fig. 2.
Event flow through gates during acquisition. By a technique of fluorescence signal triggering, only events with parameter values above a trigger signal are acquired and unwanted events eliminated. This increases the efficiency of the flow cytometric analysis when the number of unwanted cells overwhelms that of cells of interest likewise whole blood flow cytometry.
Fig. 3.
The correlations of sample/control MFI ratio among the three assays: MNC, LWB and WB. Panel A shows 15 negative cases and panel B 5 positive cases. The MNC assay was a standard assay using separated mononuclear cells. The LWB and WB assay were variant assays using whole blood. Red cells were lysed in the LWB assay. In the WB assay, the signals of T cells among whole blood cells were acquired using fluorescence triggering instead of red cell lysis. The resutss were concordant in all cases, except for one case which was weakly positive with MFI ratio 2.2 by LWB assay.
Abbreviations: MNC, mononuclear cells; LWB, lysed whole blood; WB, whole blood.
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