Presence Of HLA Antibodies Research Articles

The complement interference phenomenon as a cause of underestimation of high titer anti-HLA antibodies in a renal transplant recipient

Single antigen bead assay is the most sensitive laboratory tool for the anti-HLA antibody (Ab) identification in renal transplant recipients. However, this assay was shown to be prone to the prozone effect (PrE), giving false negative results in sera with high titer anti-HLA Ab. The ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement was described to eliminate the PrE allowing the anti-HLA Ab identification. A mean fluorescence intensity (MFI) ⩾ 2 fold increase in neat versus EDTA treated sera was considered relevant to PrE. We report a case of chronic Ab mediated rejection in a renal transplant recipient with anti-HLA-DQA1∗05:05,DQB1∗03:01 donor specific Ab (DSA), revealed after treatment of the sera with EDTA. Female patient, 36 years old, diagnosed with juvenile nephronophthisis, received a first graft from a living related donor in 1991. During the first year post transplantation the patient experienced two biopsy proven reversible rejection episodes and until 2010 she had no other evidence of graft malfunction. De novo DSA were detected in 2010, as shown on the table. In 2015, due to inconsistency between the sudden elevation of serum creatinine levels and absence of DSA, the patient’s sera were further analyzed after EDTA treatment. DSA with high MFI were detected post-EDTA treatment. The presence of DSA was confirmed with positive B-Flow crossmatch with the donor. Also, a kidney biopsy on 03/26/2015 showed chronic antibody mediated rejection with C4d deposition. Taking into account that the follow up post-transplantation is based on HLA Ab detection and identification, these results highlight the importance of using patients’ sera with EDTA treatment to abolish the PrE before the performance of single antigen bead assay, in order to detect the presence of HLA antibodies. Download : Download full-size image

HLA Antibody testing- it’s not a one stop assay

HLA laboratories rely on single antigen (SA) assays to determine unacceptable antigens and perform virtual crossmatches (vXM) that can immunologically guide clinicians in the identification of an appropriate donor. Although solid phase antibody detection assays are sensitive, specific and robust-instances arise when further testing is needed to verify results or resolve inconsistencies between these tests. Patient S.T. is a 50 year old male diagnosed with heart failure. Routine work-up in our laboratory is to evaluate % FlowPRA and compare those results to solid phase assays on a Luminex platform which measures the breadth and strength of antibodies in a patient’s sera. Patient S.T. had two samples tested three months apart with a class I FlowPRA of 58% and 62%, respectively. However, The Luminex SA results consistently demonstrated only 5 class I (all B15) antibodies in the positive MFI range leading to a cPRA of about 15%. Moreover, the internal negative control values were > 2500 MFI leading us to believe that there may be additional specificities masked by the high background. How should we deal with these contradictory test results? Can we rely on a vXM to accept a heart for this patient? To reconcile these seemingly contradictory results and to determine if the antibodies detected by Luminex assays were relevant, surrogate FCXM were performed. Surrogate Donor 1 (B15 positive) was FCXM positive. Surrogate donor 2 (B15 negative) was FCXM negative indicating that non-specific binding/false-positivity due to high background should not affect FCXM interpretation, Importantly, testing using phenotype beads were not affected by high background and further confirmed the presence of B15 antibodies. Due to the presence of HLA antibodies and the high background exhibited by this patient, a prospective FCXM must be performed prior to accepting an organ. In conclusion, this is an example in which different tests provide contradictory (or non-consistent) results - and our approach to resolve these ambiguities to support successful organ transplantation.

Predictive factors of allosensitization in renal transplant patients switched from calcineurin to mTOR inhibitors

Conversion of kidney-transplant recipients from calcineurin inhibitors to mTOR inhibitors has been suggested to be a risk factor for increased alloimmune response. We have analyzed the development of new HLA-antibodies (HLA-Abs) early after conversion in 184 patients converted in stable phase at our hospital and compared with a control group of nonconverted comparable 63 transplants. Using single-antigen solid-phase immunoassay analysis, a preconversion and a 3-6months postconversion sera were prospectively analyzed in every patient for the appearance of new HLA-Abs. Renal function at 2years postconversion and cumulative graft survival were compared between groups. In 16 patients, new HLA-Abs (3-DSA and 13-NonDSA), not present at the moment of conversion, were detected (8.7% vs. 3.1% in the control group). The type of mTORi used, type of CNI preconversion, the presence of steroids, time of conversion, or indication for conversion did not have influence on this effect but the presence of HLA-Abs before conversion highly correlated with the appearance of new specificities. Patients with de novo HLA-Abs showed a trend to worst graft function and survival. In conclusion, conversion to mTORi can be followed by early appearance of de novo HLA-Abs, especially in patients with HLA-Abs preconversion, and this complication should be screened early after conversion.

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Aim “Interfering factors” mask the presence of HLA antibodies detected in solid phase assays, especially in highly sensitized patients. In this study, we evaluated the effect of EDTA on HLA antibody detection using sera from 20 wait list patients (each with a cPRA of > 80%). Methods Briefly, 5ul of a 6% solution of EDTA was added to 95ul of patient sera. Paired samples (untreated vs EDTA treated) were then tested on FlowPRA® and LABScreen® Single Antigen Beads (SAB) for class I and II HLA reactivity. Results Among 20 patient sera tested by SABs (untreated or EDTA treated), HLA antibody profiles were identical in 7/20 paired samples and significantly different in 13. In 4/13 samples, antibody specificities detected at moderate levels (2000-7000 MFI) without EDTA increased to >19,000 MFI with EDTA treatment. In 9/13 sera, antibody specificities not detectable in untreated sera (0 MFI) presented with MFI values ranging from 18,000-25,000 following treatment with EDTA. When one such serum was crossmatched with informative HLA-A2 positive target cells (i.e., no other unacceptable antigens expressed), T and B cell crossmatches were strongly positive (>300,000 MESFs) with EDTA treated or untreated sera. Surprisingly, all sera, with or without EDTA treatment, had identical staining patterns by FlowPRA®. Conclusions These findings demonstrate that EDTA sensitive interfering factors can mask the detection of HLA antibodies that result in strongly positive crossmatches. While the untreated serum reported here was unequivocally vXM negative with the selected HLA-A2 positive target cells, the actual crossmatch against these cells was strongly positive. Interestingly, the FlowPRA® assay does not appear to be affected by these factors. As the transplant community embraces virtual crossmatching in lieu of physical crossmatching, sera from highly sensitized patients should be treated with EDTA (or other such approaches) to unmask HLA antibodies not detectable by SABs due to “interfering factors.”

Impact of donor-specific anti-HLA antibodies on graft failure and survival after reduced intensity conditioning-unrelated cord blood transplantation: a Eurocord, Societe Francophone d'Histocompatibilite et d'Immunogenetique (SFHI) and Societe Francaise de Greffe de Moelle et de Therapie Cellulaire (SFGM-TC) analysis

Graft failure is a major complication after unrelated cord blood transplantation. Presence of HLA-antibodies before cord blood transplantation may impact graft failure. To analyze the effect of anti-HLA antibodies on unrelated cord blood transplantation outcomes, we analyzed 294 unrelated cord blood transplant recipients after reduced intensity conditioning regimen. The majority of the patients (82%) were transplanted for malignancies, 60% with double-unrelated cord blood transplant, 63% were HLA mismatched. Retrospectively, pre-unrelated cord blood transplant serum was tested for HLA-Ab using Luminex™ platform. Results were interpreted as mean fluorescence intensity (MFI) against donor-specific mismatch. Among 62 recipients (23%) who had anti-HLA antibodies before unrelated cord blood transplant, 14 patients had donor specific anti-HLA antibodies (DSA) (7 were donor-specific anti-HLA antibodies for single unrelated cord blood transplant and 7 for double unrelated cord blood transplant). Donor specific anti-HLA antibodies threshold ranged from 1620-17629 of mean fluorescence intensity (MFI). Cumulative incidence of Day-60 neutrophil engraftment was 76%: 44% for recipients with donor specific anti-HLA antibodies and 81% in those without donor specific anti-HLA antibodies (P=0.006). The cumulative incidence of 1-year transplant related mortality was 46% in patients with donor specific anti-HLA antibodies and 32% in those without antibodies (P=0.06). The presence of donor specific anti-HLA antibodies was associated with a trend for decreased survival rate (42% vs. 29%; P=0.07). Donor specific anti-HLA antibody in recipients of unrelated cord blood transplant is associated with graft failure and decreased survival. Patient's screening for donor specific anti-HLA antibodies before unrelated cord blood transplantation is recommended before choosing an HLA mismatched cord blood unit. Whenever possible it is important to avoid selecting a unit for which the patient has donor specific anti-HLA antibodies.

High Immunological Risk Recipients Displaying Persistent and Contiguous DSA

The presence of HLA antibody (Ab) or donor specific Ab (DSA) pre/post transplantation (Tx) has been associated with poor renal graft outcomes. It is unclear, however, which sensitized patient will or will not experience rejection or graft loss. The purpose of our study was to try to identify recipients (recips) at greatest immunological risk for post Tx rejection and/or graft loss. Methods: We evaluated two hundred and seventy seven (277) renal allograft recips because we had sufficient pre/post Ab data on these patients. These included 23% living donor and 77% deceased-donor recips who received either Prograf, Myfortic and Pred or Cyclosporine, Rapa, and Pred treatment. Antibody mediated rejections (AMR) were biopsy confirmed and treated with Thymoglobin, Pheresis and Rituxin while cellular rejections were treated with steroids. All donor-recips were ABO compatible and transplanted following a negative donor-specific cell based flow cytometry crossmatch (FCXM) using historical and pre-Tx recip sera. Recips were risk-ranked according to whether they had or did not have HLA and/or DSA post-Tx. A subgroup of patients with persistently, contiguous (presenting at least two or more times) HLA or DSA Ab was identified. HLA Ab and/or DSA was identified using SAB (One Lambda, Inc.) and a Luminex based solid phase assay platform. Mean Fluorescence Intensity (MFI) of 1,000 was considered positive. Results: Of 277 recips undergoing renal Tx, 92 presented with no HLA or DSA post-Tx (no Ab post-Tx) while 185 recips had positive Ab post-Tx. Only 1 recip with no Ab had DSA present pre-Tx but not post-Tx. Of the 185 recips with Ab post-Tx, 31% (58/185) presented with DSA. The frequency of AMR for no Ab vs positive Ab post-Tx was significantly different 8% (7/92) vs 21% (39/185), p < 0.01 as was the three year graft survival of 92% (82/92) vs 87% (164/185), p < 0.02 for the same patients. There were 58 patients with positive post-Tx Ab that were DSA positive, however, only 39 of these recips displayed persistent and contiguous Ab and only 54% (21/39) of these lost their grafts. Moreover, 50 recips that presented with positive DSA pre-Tx would not have been considered for a donor crossmatch or transplanted using the virtual crossmatch. Conclusion: These data suggest that not all post-Tx Abs that are DSA positive result in AMR or graft loss. Recips presenting with persistent, contiguous DSA appear to be at a higher immunological risk for AMR and/ or graft loss. Finally, patients with pre-Tx DSA but who display negative donor specific cell based FCXM's can be safely transplanted.

High Immunological Risk Recipients Displaying Persistent and Contiguous DSA

The presence of HLA antibody (Ab) or donor specific Ab (DSA) pre/post transplantation (Tx) has been associated with poor renal graft outcomes. It is unclear, however, which sensitized patient will or will not experience rejection or graft loss. The purpose of our study was to try to identify recipients (recips) at greatest immunological risk for post Tx rejection and/or graft loss. Methods: We evaluated two hundred and seventy seven (277) renal allograft recips because we had sufficient pre/post Ab data on these patients. These included 23% living donor and 77% deceased-donor recips who received either Prograf, Myfortic and Pred or Cyclosporine, Rapa, and Pred treatment. Antibody mediated rejections (AMR) were biopsy confirmed and treated with Thymoglobin, Pheresis and Rituxin while cellular rejections were treated with steroids. All donor-recips were ABO compatible and transplanted following a negative donor-specific cell based flow cytometry crossmatch (FCXM) using historical and pre-Tx recip sera. Recips were risk-ranked according to whether they had or did not have HLA and/or DSA post-Tx. A subgroup of patients with persistently, contiguous (presenting at least two or more times) HLA or DSA Ab was identified. HLA Ab and/or DSA was identified using SAB (One Lambda, Inc.) and a Luminex based solid phase assay platform. Mean Fluorescence Intensity (MFI) of 1,000 was considered positive. Results: Of 277 recips undergoing renal Tx, 92 presented with no HLA or DSA post-Tx (no Ab post-Tx) while 185 recips had positive Ab post-Tx. Only 1 recip with no Ab had DSA present pre-Tx but not post-Tx. Of the 185 recips with Ab post-Tx, 31% (58/185) presented with DSA. The frequency of AMR for no Ab vs positive Ab post-Tx was significantly different 8% (7/92) vs 21% (39/185), p < 0.01 as was the three year graft survival of 92% (82/92) vs 87% (164/185), p < 0.02 for the same patients. There were 58 patients with positive post-Tx Ab that were DSA positive, however, only 39 of these recips displayed persistent and contiguous Ab and only 54% (21/39) of these lost their grafts. Moreover, 50 recips that presented with positive DSA pre-Tx would not have been considered for a donor crossmatch or transplanted using the virtual crossmatch. Conclusion: These data suggest that not all post-Tx Abs that are DSA positive result in AMR or graft loss. Recips presenting with persistent, contiguous DSA appear to be at a higher immunological risk for AMR and/or graft loss. Finally, patients with pre-Tx DSA but who display negative donor specific cell based FCXM's can be safely transplanted.

A Novel and Effective Technique of Risk Stratification of Renal Allografts Using the Presence of HLA Antibody

A Novel and Effective Technique of Risk Stratification of Renal Allografts Using the Presence of HLA Antibody

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Aim Single antigen bead (SAB) assay allows in-depth characterization of HLA antibody specificities. In addition to detection of antibodies against natural HLA antigens, the SAB may also detect antibodies against denatured HLA antigens. Recently, we observed antibody reactivity to all DR antigens (pan-DR) in serum samples of two transplant candidates using the Luminex SAB assay. This study was to characterize the pan-DR antibodies detected by Luminex SAB on two serum samples. Methods Serum samples received from two transplant candidates were analyzed in the study. Initial antibody screening and specificity tests were performed using Flow PRA and Luminex SAB. Additional tests using Flow SAB, Flow phenotype bead assay and Flow T and B cell crossmatch were performed to confirm the presence of HLA antibodies. Results Flow PRA was clearly negative for both samples, while Luminex SAB surprisingly revealed antibody reactivity to all DR antigens. No antibodies to DQ and DP were detected. However, no HLA antibodies were detected by Flow phenotype bead assay. In the Flow SAB assay, patient #1 showed reactivity with majority of the DR antigen beads, while patient #2 showed no any binding reactivity. The crossmatch with a donor cell carrying DR4, DR15, DR51, and DR53 was completely negative for both serum samples. No specific antibody removals were observed on serum samples adsorbed using the donor cells in a Luminex SAB assay. Conclusions Flow PRA and Flow phenotype beads are coated with natural HLA antigens extracted from cell lines, while SABs are coated with recombinant HLA antigens. The antibody binding detected by SAB, but not by FlowPRA and Flow phenotype, may indicate the presence of antibodies against denatured antigens. Our results suggest that the pan-DR antibodies detected in these patients were possibly antibodies against denatured HLA antigens, more likely to a shared epitope uncovered on denatured DR antigens. Antibodies to denatured antigens would not result in crossmatch reactivity.

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Aim Presence of anti-donor HLA antibodues is a contraindication to transplantation in most solid organs. In contrast, most patients with high titer donor specific antibodies (DSA) and positive flow cytometric crossmatch (FCXM) still undergo liver transplantation. Antibody mediated rejection (AMR) may or may not develop after liver transplantation in such patients. We speculated that despite positive crossmatch results, cytotoxic function of existing and/or de novo DSA is what will ultimately predict development of AMR. Methods In this study we report two conflicting cases of liver transplant patients with high titer pre-transplant DSA and positive FCXM. Pre-transplant sera of patient A and B were retrospectively tested with a C1q assay to determine the cytotoxic function of DSAs. Results One patient (patient A) developed hyperacute AMR shortly after transplantation while the second patient (patient B) did not develop any signs of AMR and her post-transplant period was uneventful. In patient A, shortly after transplantation, serum amino transaminase and bilirubin levels became elevated. Serum HLA antibody testing showed high levels of class I and class II DSAs that resulted in positive T and B cell FCXM. Positive C4d staining of liver allograft biopsy indicated an episode of AMR. Patient was treated with plasmapheresis (days 1-9), Rituximab and IVIG (days 9-13) and Thymoglobulin (days 2-8). Levels of DSA and positive FCXM decreased overtime and 4 years post-transplant DSA antibodies were no longer present. In contrast, patient B did not develop any signs of post-transplant complications related to presence of HLA antibodies. Consistent with our hypothesis at the time of transplant, DSA of patient A were positive for C1q factor. In contrast C1q was negative on DSA of patient B. Conclusions We conclude that pre-transplant antibody testing and C1q assay should be considered as pre-transplant tests to be performed to liver transplant patients. Larger studies should be performed to confirm our findings.

Detection of HNA‐3a and ‐3b antibodies using transfected cell lines and recombinant proteins

HNA-3 is a diallellic system located on choline transporter-like protein 2 (CTL2), defined by a polymorphism at Amino Acid 154. HNA-3a antibodies are of clinical importance in transfusion-related acute lung injury but antibody detection requires labor-intensive granulocyte isolation from HNA-typed donors and the use of techniques such as the granulocyte agglutination test or granulocyte immunofluorescence test. Also, there is no commercial test for detection of HNA-3 antibodies. HEK293 cells were transfected to generate stable cell lines expressing CTL2 fragments (Amino Acids 55-230) and full-length membrane bound CTL2 with HNA-3a and -3b epitopes. Soluble fragments were used in enzyme-linked immunosorbent assays to detect HNA-3 antibodies. The cell lines expressing full-length proteins were trypsin treated to remove HLA antigens and frozen at -80°C. Thawed cells were then used to detect HNA-3 antibodies by flow cytometry. Glycosylated and soluble CTL2 fragments were correctly recognized by 15 of 31 anti-HNA-3a sera and by both available anti-HNA-3b sera. Twenty-one anti-HLA sera reacted variably with untreated cell lines expressing full-length CTL2. After trypsin treatment of the cell lines, reactivity with HLA antisera was abrogated and all 31 anti-HNA-3a and two anti-HNA-3b sera bound to the corresponding cell line. Whereas soluble, glycosylated CTL2 fragments cannot be used for the detection of HNA-3 antibodies, the HEK293 cells expressing full-length CTL2 proteins were useful in the detection of HNA-3 antibodies even in the presence of HLA antibodies. Moreover, the cell lines can be stored for at least 6 months before use.

Clinical relevance of pre and post-transplant immune markers in kidney allograft recipients: Anti-HLA and MICA antibodies and serum levels of sCD30 and sMICA

BackgroundThis retrospective study aims to determine the prognostic values of HLA and MICA antibodies, serum levels of sCD30 and soluble form of MHC class I related chain A (sMICA) in kidney allograft recipients. MethodsSera samples of 40 living unrelated donor kidney recipients were tested by ELISA and Flow beads techniques for the presence of anti HLA and MICA antibodies and the contents of sCD30 and sMICA. HLA and MICA antibody specification was performed by LABScreen single antigen beads to determine whether the antibodies were directed against donor mismatches. ResultsWithin first year post operatively 9 of 40 patients (22.5%) showed acute rejection episodes (ARE) that four of them lost their grafts compared to 31 functioning transplants (P=0.001). The presence of HLA antibodies before and after transplantation was significantly associated with ARE (P=0.01 and P=0.02 respectively). Sensitization to HLA class II antigens pre-transplant was strongly associated with higher incidence of ARE (P=0.004). A significant correlation was found between ARE and appearance of non-donor specific antibodies (P=0.02). HLA antibody positive patients either before or after transplantation showed lower graft survival rates than those without antibodies during three years follow-up (P=0.04 and P=0.02). Anti-MICA antibodies were observed in 8/40(20%) and 5/40(12.5%) of patients pre and post-transplant respectively. Coexistence of HLA and MICA antibodies was shown in 2 of 4 cases with graft loss. A significant increased level of sCD30 at day 14 (P=0.001) and insignificant decreased levels of sMICA pre and post operatively were detected in rejecting transplants compared to functioning graft group. ConclusionOur findings support the view that monitoring of HLA and MICA antibodies as well as sCD30 levels early after transplant has predictive value for early and late allograft dysfunctions and the presence of these factors are detrimental to graft function and survival.

Positive virtual crossmatch with negative flow crossmatch results in two cases

Pre-transplant (Tx) presence of HLA antibodies (HLA-Ab) especially donor specific antibodies (DSA) has been correlated with post-Tx rejection. While crossmatch (XM) is the specific method to identify DSA, logistical reasons prevent performing a prospective XM in all transplants. In such cases DSA as identified by solid-phase assay (SPA) are being used to perform a virtual crossmatch (VXM). We present two cases, a heart-lung transplant and a kidney transplant, for which testing detected a presumptive DSA with discordant results: a negative flow cytometric crossmatch (FXM) and a positive VXM using SPA. The subsequent investigation determined the antibody, in both cases, was presumably directed against an epitope of a HLA-B*44 antigen found on the single antigen beads (SAB) used in the SPA but not against the native form on the donor lymphocytes used in the FXM. Manufacturing of SAB beads results in denaturation of epitopes, majority of which are removed from the final product, but residual amount is present on the final product. Denaturation of majority of antigen epitopes on single antigen beads did not remove the activity of the recipient's antibodies but it did diminish the activity of positive control serum. This indicates denaturation of some of the HLA-B*44 antigen during manufacturing of the SAB may have lead to the reactivity. Antibody mediated rejection does not appear to be associated with the titer of this antibody to denatured antigen in the first case and so clinical relevance of such antibodies is unclear. Subsequently a second case of discordant FXM and VXM was identified in a potential kidney transplant patient who went on to an uneventful transplant. In this case, lymphocytes from the donor were positively shown to express HLA-B*44:02 using known anti- HLA-B*44:02 control serum. Platelets identified as HLA-B*44:02 could adsorb the anti-HLA-B*44:02 from the control serum activity but not from that of the recipient's anti- HLA-B 44 antibody adding evidence that this antibody should best be classified as a false positive finding. The presence of such an antibody if misidentified may result in unnecessary therapy being instituted or the inappropriate denial of an organ for transplantation.

Corrigendum to “The presence of HLA-antibodies in recurrent miscarriage patients is associated with a reduced chance of a live birth” [J. Reprod. Immunol. 87 (2010) 67–73

The Fertility Clinic, University Hospital Copenhagen, Rigshospitalet, Fertility Clinic 4071, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands Department of Clinical Immunology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark Department of Obstetrics and Gynaecology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark

Clinical relevance of circulating donor-specific HLA antibodies

For a long period of time complement-dependent cytotoxicity was the standard assay to demonstrate clinically relevant HLA antibodies in the serum of patients before transplantation. The introduction of more sensitive solid-phase assays to detect HLA antibodies had led to a lot of discussion on the clinical relevance of the presence of these antibodies before transplantation and the appearance of these antibodies after transplantation. This review discusses the current controversies. The old dogma that the presence of donor-specific HLA antibodies before transplantation is a contra-indication for transplantation does not exist anymore. The correlation between the presence of HLA antibodies and clinical outcome is less strict although the presence of circulating donor-specific antibodies, both before and after transplantation, should be considered a risk factor. It is clear, however, that the opinion on the clinical relevance of donor-specific HLA antibodies is very different amongst the transplant centers. The clinical relevance of the presence of donor-specific HLA antibodies before transplantation and the appearance of these antibodies after transplantation is a controversial issue. The lack of consensus between different centers is partly due to the heterogeneity of the HLA antibodies involved. Standardization is essential and future studies must focus on the further characterization of the antibody titers, the immunoglobulin (sub)classes of the antibodies and the epitopes recognized. It remains to be established in which cases the HLA antibodies are the direct cause of or just associated with (chronic) graft failure.

The presence of HLA-antibodies in recurrent miscarriage patients is associated with a reduced chance of a live birth

Anti-paternal HLA-antibodies are considered a harmless phenomenon during most pregnancies, whereas their role in recurrent miscarriage (RM) patients is disputed. In contrast to primary RM, patients with secondary RM have carried a fetus to term pregnancy prior to a series of miscarriages, which increases the chance that allogeneic fetal cells appear in the maternal circulation. This study investigates the frequency of HLA-antibodies in secondary RM, primary RM patients and parous controls and analyzes whether the presence of HLA-antibodies in early pregnancy is associated with pregnancy outcome. Sera from women with secondary RM (n=56), primary RM (n=13) and parous controls (n=24) were tested for HLA-antibodies using an ELISA assay and complement dependent cytotoxicity. Samples were taken at gestational week 4-5 in 62 (90%) of the patients. HLA-antibodies were significantly more frequent in secondary RM patients with a boy prior to the miscarriages (62%) compared to secondary RM patients with a firstborn girl (29%, p=0.03), primary RM patients (23%, p=0.02) and parous controls (25%, p=0.005). Forty-one percent of HLA-antibody positive pregnant RM patients had a live birth compared to 76% of HLA-antibody negative RM patients, p=0.006 (adjusted OR: 0.22 (0.07-0.68), p=0.008). In conclusion, HLA-antibodies are significantly more frequent in secondary RM patients with a firstborn boy than in other RM patients and controls. The presence of these antibodies in early pregnancy is associated with a reduced chance of a live birth. Further exploring this association may increase our understanding of maternal acceptance of the fetal allograft.

Prevalence and Significance of Pre-Formed Anti-HLA Antibodies in Hematopoietic Stem Cell Transplant Recipients.

Abstract 1174Poster Board I-196 Introduction:The presence of sensitization to HLA antigens has been an important consideration in solid organ transplantation. It is considered a standard process to check for donor-specific allogeneic (allo) antibodies (DSA) and monitor formation of such antibodies post-transplant which could predict early and late graft failure. Most of the current data regarding the importance of anti-HLA (human leukocyte antigen) antibodies is available from renal transplant where presence of HLA antibodies is clearly associated with an increased risk of early graft loss up to the magnitude of 21%. It is routine to perform desensitization to alleviate these antibodies in an effort to enhance their chances of engraftment. The role of and approach to prior sensitization in the hematopoietic stem cell transplantation (HSC) setting is far less clear. This is of unique importance as a wider range of donor cell sources and transplant applications are utilized to treat hematologic diseases. Many of our patients have had multiple transfusions in the past, been pregnant or have had prior HLA mismatched allograft, all of which predispose to development of anti-HLA antibodies. Here we analyze the prevalence of Class I and Class II antibodies as a primary goal and also see if they correlate with graft survival. Methods:52 patients were followed between July 2008 and July 2009 with hematologic malignancies including leukemia's, lymphoma's, multiple myeloma and others. 37/52 underwent transplantation of which 14 were unrelated donor (URD), 5 cord blood (CB) and 8 sibling (sib) transplants. Donors with corresponding HLA were excluded. Post-transplantation with day 100 antibody testing was performed in eligible patients. Antibody determination was done by testing the patients' sera with a panel of fluorescent beads coated with single HLA antigens using a solid-phase Luminex™ platform. Cut-point of 1500 [mean fluorescence intensity (MFI) ≥ 1500 defined as positive] was used for performing statistical analyses. The prevalence of positive antibody levels was compared among the transplant groups using a Fisher's exact test. Level of expression of antibodies was evaluated with MFI <500 considered negative, 500-1500 weak, 1500-3000 intermediate and >3000 strong. High resolution HLA typing was performed. Results:Class I antibodies were positive in 24 out of 52 total (46%) with 95% CI: 32% to 61%.14/37 (38%) who underwent transplantation (95% CI: 22% to 55%), 12/27 (44%) undergoing allo transplant, CB (20%), sib (38%), and URD (57%) were positive. The prevalence did not differ significantly among the transplant groups (p=0.3). Class II antibodies were positive in 8 out of 52 total (15%) with 95% CI: 7% to 28%. 5/37 (14%) who underwent transplantation (95% CI: 5% to 29%), 4/27 (15%) undergoing allo transplant, Sib (0%), CB (20%) and URD (21%) were positive. The prevalence did not differ significantly among the transplant groups (p=0.6). In females, 18/28 (64%) were positive for Class I or Class II antibodies of which 5/6 (83%) underwent URD transplants. Persistent antibody levels were detected in 3 of 4 patients tested at day 100 post transplant. Conclusions:Based on this limited pilot study we conclude that there is a high prevalence of anti-HLA antibodies present in recipients at the time of HSC transplantation. However detection of such antibodies did not jeopardize engraftment from various donor sources when HLA donor specific reactions are excluded. Bray et al showed higher incidence of graft failure associated with DSA. Takanashi et al showed that in CB transplants, antibodies were not significant unless the corresponding HLA was present in the CB unit. Based on these studies, we excluded donors with corresponding HLA. All but one patient, in whom donor specific anti-HLA antibodies were identified, achieved sustained marrow engraftment. The long term implications of antibody evolution and specificity to sustained marrow engraftment, graft vs. host and graft vs. tumor effects remain to be clarified. A larger prospective study will need to be conducted to definitely evaluate these relationships including our own which is currently under way.Class I (MFI)Transplant typen<500500-15001500-3000>3000Pre154146URD142435Sib84112CB52201Auto105320Total5217111014Class II (MFI)Transplant typen<500500-15001500-3000>3000Pre158412URD147421Sib84400CB52201Auto109010Total52301444 Disclosures:No relevant conflicts of interest to declare.

Anti-Human Leukocyte Antigen Antibodies, Vascular C4d Deposition and Increased Soluble C4d in Broncho-Alveolar Lavage of Lung Allografts

The hallmark of humoral rejection is the presence of subendothelial C4d in the allograft. A simultaneous determination of vascular C4d with soluble C4d in broncho-alveolar lavage fluid (BAL) and circulating anti-human leukocyte antigen (HLA) antibodies (HLA-Ab) has not been reported in lung transplantation. Forty-two consecutive lung-transplant patients were included in this cross-sectional study. The presence and specificity of HLA-Ab was determined at the same frequency with transbronchial biopsies. Soluble C4d levels were measured by enzyme-linked immunosorbent assay in all 42 patients. In a subgroup of 32 patients with available timely matched paraffin-embedded tissue sections, the vascular C4d deposition was also assessed. The presence of HLA-Ab in 16 patients was associated with biopsy-proven acute rejection (10/16 vs. 3/16, P<0.01) and increased immunosuppression (13/16 vs. 4/16, P<0.005). Pulmonary function was also decreased in patients with HLA-Ab (mean forced expiratory volume in 1 second=49%) when compared with the control group (mean forced expiratory volume in 1 second=66%, P<0.05). Nine patients exhibited specific vascular C4d deposition and in eight of nine (89%) cases HLA-Ab were detected, versus 8 of 23 (35%) in C4d-negative patients (P<0.05). Soluble C4d in BAL was highly (>0.5 microg/mL) elevated in patients with HLA-Ab and vascular C4d and was moderately (0.2 microg/mL) increased in patients with antibodies but C4d-negative. In contrast, only a slight elevation of soluble C4d (<0.1 microg/mL) was detected in patients without HLA-specific antibodies. The association of HLA-specific antibodies with vascular C4d deposition and soluble C4d in BAL, in addition to the reduced pulmonary function, might constitute a diagnostic triad for antibody-mediated rejection in lung transplant patients.

1: Luminex Detected Pre-Formed Donor Specific HLA Antibodies Predict Lung Allograft Failure

Purpose: Pre-formed HLA antibodies have historically been detected by complement dependent cytotoxicity (CDC) assays. The introduction of Luminex assays to detect HLA antibodies has resulted in increased detection of sensitisation, however, the clinical impact of Luminex detected HLA antibodies is unclear. The purpose of this study has been to analyse pre-transplant Luminex detected HLA antibodies, their ability to fix human C4d and to determine the effect on lung allograft survival. Methods and Materials: Pre-transplant serum from 394 lung transplant patients (144 Heart-Lung, 103 Double Lung and 147 Single Lung) was retrospectively tested for the presence of HLA antibodies using Labscreen (One Lambda) and Lifematch (Tepnel) Luminex assays. All sera had previously been screened by CDC at the time of transplant. C4d deposition on HLA coated Luminex beads was also measured. Results were analysed and correlated with graft survival using Kaplan-Meier and Logrank statistics. Results: Prior to transplantation 6 patients had produced HLA antibodies by CDC, all of which were donor specific (DSA). The Luminex assays detected HLA antibodies in a further 50 patients, 16 of which were donor specific. The one year actuarial graft survival for CDC ve DSA patients (n 6) was 0% compared to 56.3% for Luminex ve DSA (n 16), 82.4% for Luminex non-donor specific (n 34) and 71% for Negative patients (n ), p 0.0001. Ten % of the luminex positive sera (44 tested to date) fixed C4d. The one year graft survival for C4d ve/DSA (n 4) was 0%, C4d-ve DSA ve (n 12) 50%, C4d-ve DSA-ve (n 28) 78.6%, compared with 71% for antibody negative patients(p 0.0004). Conclusions: Detection of Luminex ve donor specific HLA antibodies is a prognostic indicator for poor graft survival. The development of a technique to detect C4d fixing donor specific Luminex detected antibodies may be a useful tool to identify patients at high risk of accelerated graft failure.

A perspective on transfusion‐related acute lung injury two years after the Canadian Consensus Conference

A perspective on transfusion‐related acute lung injury two years after the Canadian Consensus Conference