Flow cytometric evaluation of pregnancy-induced anti-HLA immunization and blood transfusion-induced reactivation.

BACKGROUND: Pregnancy-induced alloimmunization (PIA) may decrease to a level that becomes undetectable by complement-dependent cytotoxicity (CDC). Nevertheless, such alloimmunization may provoke acute rejections after kidney transplantation and lead to broad-spectrum immunizations after transfusion. Flow-cytometry (FC) was used to estimate the frequency of low-level PIA and to evaluate its influence on posttransfusion alloimmunization profiles. METHODS: To evaluate the frequency of low-level PIA, the sera of 36 women, free of CDC-detectable anti-HLA IgG (CDC-IgG- negative), were cross-matched by FC against their husband's or offspring's lymphocytes and further analyzed with human leukocyte antigen (HLA) Ag-coated microbeads (Flow-PRA One-Lambda, Canoga Park, CA). To evaluate the influence of low-level alloimmunization on posttransfusion appearance of CDC-IgG, pretransfusion sera of a second cohort of 43 women, also CDC-IgG-negative and included in a transfusion protocol, were analyzed by Flow-PRA. Posttransfusion sera were analyzed for the development of cytotoxic IgG. RESULTS: Ten of the first cohort of 36 (27.8%) CDC-IgG-negative women showed a positive FC cross-match against the husband or offspring lymphocytes. Flow-PRA analysis confirmed that 9 of 10 positive cross-matched sera contained anti-HLA IgG. Among the 43 transfused patients, 11 of 16 (68.7%) of the women who were CDC-IgG-positive after blood transfusion showed FC-detectable IgG before transfusion; although 2 of 27 (7.4%) of the patients who remained CDC-IgG-negative after transfusion showed FC-detectable IgG before transfusion (P <0.001). CONCLUSION: Most of the de novo anti-HLA immunizations detected by CDC after transfusion in previously pregnant women can be detected by Flow-PRA before transfusion.

Incidence and risk factors of anti-HLA immunization after pregnancy.

Pregnancy is the only natural source of anti-HLA immunization. The exact frequency of this immunization remains undetermined as prior studies either used methods with a low sensitivity or were performed late after delivery. We present here the first study on women at delivery evaluating anti-HLA immunization by Luminex. We also attempted to isolate factors influencing immunization, such as soluble HLA-G (sHLA-G) levels and genetic polymorphisms. With Luminex, anti-HLA immunization was observed in 54.4% of the women. As expected, immunization frequency increased with the number of children, reaching 74% in women with >2 deliveries. Among immunized women, strong cytotoxic Ab (as detected by Complement Dependent Cytotoxicity) were associated with a lower level of sHLA-G. None of the studied polymorphisms influenced immunization rate in the whole cohort. Among 94 first pregnant women with no history of miscarriage, the -174 IL-6 gene promoter mutation (G/C) appeared more frequently in non immunized women (69% vs. 45% in immunized ones, p=0.02). Lastly, the occurrence of a miscarriage before the first live delivery significantly decreased immunization. These results may help to understand mechanisms of pregnancy induced immunization. They also have an impact in the management of previous pregnant women requiring organ or hematopoietic stem cell transplantation.

Misleading de novo detection of serum anti-HLA-A3 antibodies in kidney recipients having received ATG before transplantation.

Anti-HLA antibody (Ab) monitoring is essential for the follow-up of transplant patients. However, it can be affected by drugs, especially Ab infused as a conditioning regimen for transplantation. ATG Fresenius, commonly used in this setting, is a polyclonal rabbit Ab raised against the Jurkat human T cell line (HLA-A3, 32; B7, 35). We report here the de novo detection by CDC and flow cytometry (Luminex) of anti-HLA-A3 Ab in the serum of kidney recipients treated with ATG Fresenius. The Ab, of rabbit origin, was detected in every assessable patient (n = 16), with the exception of the HLA-A3 recipients and/or recipients receiving an HLA-A3 graft, before becoming undetectable, at latest, at day 102 after transplantation. It is of major importance that transplantation monitoring laboratories bear in mind the possibility of therapeutic Ab detection when interpreting anti-HLA Ab results.

Hyperacute rejection after lung transplantation caused by undetected low-titer anti-HLA antibodies.

Hyperacute rejection is an early complication of transplantation, caused by the presence in the recipient of pre-formed donor-directed antibodies (Ab). We report a case of fatal early graft dysfunction after lung transplantation in a female recipient with no anti-HLA Ab detected before transplantation. Further immunologic studies revealed the presence, in the pre-transplant serum, of low-titer anti-HLA Ab directed against the donor's HLA, detectable only by flow-cytometry screening for panel-reactive antibodies. This observation emphasizes the importance of sensitive Ab detection in patients awaiting lung transplantation. Women should be specifically targeted for such sensitive Ab detection.