Mean Channel Shift Research Articles

Antitrophoblast antibodies are associated with recurrent miscarriages

To investigate whether antitrophoblast antibodies are associated with unexplained recurrent miscarriages, we used choriocarcinoma cells JEG-3, since these cells are negative for class I and II antigens, but they do express HLA-G, resembling an antigen expression of endovascular and interstitial trophoblasts. Case-control study. Academic research center. One hundred ninety-four patients with two or more consecutive, idiopathic recurrent miscarriages (RM; <20 weeks of gestation) were compared with 110 controls with normal pregnancies and without pregnancy complications. Anti-JEG-3 reactivities were measured by using flow cytometry and comparisons with two in-house standards antibody samples of low and high reactivity. Anti-JEG-3 reactivities above the 95% confidence interval of controls were defined as positive. Sera of RM patients reacted significantly stronger with JEG-3 cells than that of controls. In addition, RM patients significantly more often had positive anti-JEG-3 reactivities (17.5%) than controls 5%. This difference was markedly increased with a subgroup of 80 RM patients who had three or more miscarriages, as 27 of these women (34%) were anti-JEG-3 positive. Antitrophoblast antibodies show significantly more mean channel shift reactivities, and positive reactivities are significantly more prevalent in RM patients as compared with controls. Such antibodies may be involved in mechanisms affecting pregnancies.

Antibody Testing Strategies for Deceased Donor Kidney Transplantation After Immunomodulatory Therapy

Immunomodulatory protocols including intravenous immunoglobulin/rituximab (IVIG/R) are employed to decrease anti-human leukocyte antigen (HLA) antibody levels for patients broadly sensitized to HLA and increase chances for transplantation with a compatible deceased donor (DD). The aim of our study was to identify the optimal antibody levels allowing for selection of compatible DDs for these sensitized patients. From January 2006 to December 2009, 108 patients broadly sensitized to HLA who had reached the top of the DD waitlist were treated with IVIG/R. Antibody levels were monitored monthly by Luminex-based single-antigen bead assay. The antigens identified to produce positive complement-dependent cytotoxicity crossmatches (XMs; >200,000 standard fluorescence intensity [SFI]/10,000 median fluorescence intensity [MFI]) were determined to be unacceptable and entered into the United Network for Organ Sharing database generating the calculated panel reactive antibody (CPRA). The mean CPRA (mCPRA) for this group was more than 80. DDs were selected based on T-cell flow XMs (FXMs) less than 250 MCS and B-cell FXMs less than 300 mean channel shifts (MCS). Monthly Luminex-based single-antigen assays showed that the IVIG/R therapy decreased antibody levels for a period of 30 to 120 days. Of the 108 patients treated, 80 (74%) were transplanted and 28 (26%) were not (mCPRA 96 ± 11). Forty-two (53%) patients were transplanted with a positive FXM; 28 (35%) patients (mCPRA 84 ± 25) were transplanted with a negative FXM; and 10 patients (12%; mCPRA 90 ± 19) received zero HLA ABDR mismatched grafts. After therapy, careful selection of acceptable DD involves the antibody profiling strength and XM results. These approaches provide patients broadly sensitized to HLA with an opportunity for compatible DD transplantation.

Use of Intravenous Immune Globulin and Rituximab for Desensitization of Highly Human Leukocyte Antigen-Sensitized Patients Awaiting Kidney Transplantation

The article by Vo et al. (1) about transplanting highly sensitized patients is one of their most recent contributions in trying to tackle this recalcitrant problem. The authors used a rituximab and intravenous immune globulin desensitization protocol, which is simple to administer. This, however, together with the fact that the Cedars-Sinai group is well respected and that the protocol is proposed for highly human leukocyte antigen-sensitized patients (as shown in the title) is promoting its use worldwide with no results to justify the expense. Their definition of “highly sensitized” patients by flow panel reactive antibody is questionable. This test is sensitive but can give high percentage values with low or no sensitization (in contrast to the traditional cytotoxicity panel reactive antibody where high values usually go together with high titers). In addition, they do acknowledge that a number of their transplanted patients had flow crossmatches of less than 250 mean channel shifts, which they defined as “acceptable positive.” Many centers transplant these patients with increased immunosuppression but no desensitization. The authors mention that in some cases, patients received the treatment but the donor-specific crossmatch with the pretransplant specimen turned out to be low by chance. True, but this is certainly not a case of success because of the protocol. The authors also state that they would not transplant such patients without using their protocol, but they have not shown that patients with a weakly positive flow crossmatch do better with the protocol than without. Thus, it is not clear from the data how many patients would have had acceptable positive crossmatches before desensitization, or how many patients were treated and not transplanted. In addition, the effect of rituximab itself on alloantibody, and its indications in transplant patients, has not been established, as one of the authors acknowledged recently (2). Although a strict quantitative evaluation on the effect of rituximab plus intravenous immune globulin on human leukocyte antigen antibody levels remains to be done, the patients transplanted were obviously not highly sensitized against their donors. Indeed, at the end of the discussion, the authors themselves state that patients with lower donor-specific antibody levels benefit more and that no protocol deals well with highly sensitized patients. Some readers may miss this statement, but no one will miss the title. In summary, it has to be made clear that this protocol does not represent a solution for highly sensitized patients. Shehzad Rehman1 Herwig-Ulf Meier-Kriesche1 Juan Scornik2 1 Department of Medicine University of Florida Gainesville, FL 2 Department of Pathology University of Florida Gainesville, FL

The strength of donor-specific antibody is a more reliable predictor of antibody-mediated rejection than flow cytometry crossmatch analysis in desensitized kidney recipients

The aim of this study was to evaluate the utility of donor-specific antibodies (DSA) and flow cytometry crossmatch (FCCM) as tools for predicting antibody-mediated rejection (AMR) in desensitized kidney recipients. Sera from 44 patients with DSA at the time of transplant were reviewed. Strength of DSA was determined by single antigen Luminex bead assay and expressed as mean fluorescence intensity (MFI). T- and B-cell FCCM results were expressed as mean channel shift (MCS). AMR was diagnosed by C4d deposition on biopsy. Incidence of early AMR was 31%. Significant differences in the number of DSAs (p = 0.0002), cumulative median MFI in DSA class I (p = 0.0004), and total (class I + class II) DSA (p < 0.0001) were found in patients with and without AMR. No significant difference was seen in MCS of T and B FCCM (p = 0.095 and p = 0.307, respectively). The three-yr graft survival in desensitized patients with DSA having total MFI < 9500 was 100% compared to 76% with those having total MFI > 9500 (p = 0.022). Desensitized kidney transplant recipients having higher levels of class I and total DSA MFI are at high risk for AMR and poor graft survival. Recipient DSA MFI appears to be a more reliable predictor of AMR than MCS of FCCM.

Use of Intravenous Immune Globulin and Rituximab for Desensitization of Highly HLA-Sensitized Patients Awaiting Kidney Transplantation

BACKGROUND.: We have shown that high-dose intravenous immune globulin (IVIG; 2 g/kg x2 doses)+rituximab (1 g x2 doses) was effective in lowering anti-human leukocyte antigen (HLA) antibodies and improving rates of transplantation. The aim of this report was to evaluate the efficacy of IVIG+rituximab on reduction of anti-HLA antibodies to a level that was permissive for living donor (LD) or deceased donor (DD) transplantation without incurring the risk of antibody-mediated rejection and immediate graft loss. METHODS.: From July 2006 to February 2009, 76 HLA-sensitized (HS) patients who met strict sensitization criteria received kidney transplants after desensitization using IVIG 2 g/kg (days 1 and 30)+rituximab (1 g, day 15). Parameters evaluated included rates of transplantation, previous transplants, panel reactive antibodies, donor specific antibody, crossmatches (CMXs), patient and graft survival, acute rejection, serum creatinines, and infections. RESULTS.: Seventy-six HS CMX treated patients (31 LD/45 DD) were transplanted. For LD and DD recipients, significant reductions were seen in T-cell flow cytometry CMXs from pretreatment (T cell 183.5+/-98.4 mean channel shifts (MCS) for LD and 162.8+/-41 MCS for DD) to time of transplant (T cell 68.2+/-58 MCS for LD [P<0.00006] and 125+/-49 for DD [P=0.05]), respectively. Time on wait list for DD recipients was reduced from 95+/-46 months to 4.2+/-4.5 months after treatment. Twenty-eight patients (37%) experienced acute rejection (29% C4d/8% C4d). Patient and graft survival up to 24 months was 95% and 84%, respectively. The mean serum creatinines, at 12 and 24 months were 1.5+/-1.1 and 1.3+/-0.3 mg/dL, respectively. Viral infections were seen in six patients. CONCLUSIONS.: IVIG and rituximab seems to offer significant benefits in reduction of anti-HLA antibodies allowing improved rates of transplantation for HS patients, especially those awaiting DD, with acceptable antibody-mediated rejection and survival rates at 24 months.

Acceptable Donor-Specific Antibody Levels Allowing for Successful Deceased and Living Donor Kidney Transplantation After Desensitization Therapy

The aims of this study were to determine the level of donor-specific antibody (DSA) that allows for successful transplantation after desensitization with IVIG and rituximab and to identify patients at risk for antibody-mediated rejection (AMR). Pre- and posttransplant sera from 16 patients with DSA before desensitization were tested. Strength of DSA was determined by single antigen Luminex bead assay and results expressed as standard fluorescence intensity (SFI). T-cell flow crossmatch results were expressed as mean channel shifts (MCS). AMR was determined by biopsy and C4d deposition. Six had negative pretransplant flow crossmatches with a mean DSA of 8,805 SFI. Five had positive flow crossmatches (78-192 MCS) with mean DSA of 55,869 SFI. No patients in either group had AMR. Five had positive flow crossmatches (222-266 MCS) with mean DSA of 118,063 SFI. Three experienced AMR. The MCS and DSA levels for patients with AMR were significantly higher than patients without (P < or = 0.001). For patients without complications (n=7), DSA remained less than 10(5) SFI and usually decreased to approximately 10(4) SFI posttransplant for both class I and II. For patients with AMR (n=3), predominant increases in class II DSA more than 10(5) SFI were observed. All three patients continue to have DSA approximately 10(5) SFI with stable creatinine after treatment for AMR. Approximately 63% of patients were transplanted with a positive flow crossmatch. The results show that patients with DSA more than 10(5) and FCM more than 200 MCS are at higher risk for AMR. Treatment of AMR improves renal function without significant changes in DSA.

New Crossmatch Technique Eliminates Interference by Humanized and Chimeric Monoclonal Antibodies

Humanized and chimeric antilymphocyte antibodies (Ab) are used to prevent and treat rejection and for treatment of human disease. Rituximab (RIT, anti-CD20), daclizumab (DAC; anti-CD25), alemtuzumab (ALE; anti-CD52), or infliximab (IFX) may interfere with Ab detection methods such as complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM). These agents are recognized as anti-human Ab or fix complement and are not differentiated from anti-allo-Ab. A new enzyme-linked immunosorbent assay crossmatch (XM) utilizing class I and II HLA antigens from donor cells called Transplant Monitoring System (TMS; GTI, Waukesha, Wisc) potentially precludes interference by eliminating non-major histocompatability complex antigens. To test this, normal sera (nonsensitized volunteers) were supplemented with 0.1 or 10 μg/mL of RIT, DAC, IFX or ALE, and were tested using three methods: the TMS T-cell CDCXM with antihuman globulin (AHG); and B-cell CDCXM without AHG; and FCXM with mean channel shifts of 45 and 150 indicating positive T-cell and B-cell crossmatch, respectively. No reactivity occurred with normal sera using any crossmatch technique. At 0.1 and 10 μg/mL, RIT interfered with CDC B-cell, but not T-cell crossmatch. RIT at 10, but not 0.1 μg/mL interfered with B-cell FCXM. No interference occurred with RIT in T-cell FCXM or TMS. ALE interfered with B-cell and T-cell CDC and FCXM but neither class I nor II TMS. DAC did not interfere with CDC or FCXM at 0.1 μg/mL, but gave false positive B-cell FCXM and CDCXM with some samples. No interference by DAC occurred using TMS. TMS may be useful to differentiate de novo donor-specific Ab after treatment with humanized or chimeric Ab.

A NEW CROSSMATCH TECHNIQUE ELIMINATES INTERFERENCE BY HUMANIZED AND CHIMERIC DRUGS

P3 Aims: Humanized and chimeric anti-lymphocyte antibodies (Ab) are being used as immunosuppressive agents to prevent or treat rejections. The presence of drugs such as rituximab (RIT) (anti-CD20), daclizumab (DAC) (anti-CD25), and alemtuzumab (ALE) (anti-CD52) in serum interfere with standard detection methods such as complement dependent cytoxicity (CDC) and flow cytometric crossmatch (FCXM). These agents are recognized as anti-human antibody and/or they fix complement and cannot be differentiated from alloantibodies. Magnetic beads may be used to remove interfereing xeno anti-lymphocyte preparations. Removal of humanized or chimeric antibodies using magnetic beads without removing patient immunoglobulin would require beads bound to anti-idiotype Ab. A new ELISA crossmatch technique utilizing class I and class II HLA antigens from lysed donor cells, Transplant Monitoring System (TMS) (GTI, Inc.,Waukesha, WI), potentially precludes interference by eliminating the interfering antigens. Methods: To test this hypothesis, sera from nonsensitized volunteers alone or sera supplemented with 0.1 mcg/mL or 10 mcg/mL of RIT, DAC, or ALE (to mimic trough and peak clinical levels) were tested using three crossmatch techniques. T-cell CDC crossmatches used two washes with anti-human globulin (AHG); B-cell CDC crossmatches used one wash and no AHG; FCXM used a standard 3-color protocol with a mean channel shift of 45 indicating a positive T-cell result and mean channel shift of 150 indicating a positive B-cell result. TMS was performed as per manufacturer’s instructions. Equivalent donor cells were used as targets. Results: No reactivity was seen when testing sera without added drug with any of the three crossmatch techniques. At both 0.1 mcg/mL and 10 mcg/mL RIT interfered with CDC B-cell, but not T-cell crossmatches. RIT at 10 mcg/mL, but not 0.1 mcg/mL interfered with the B-cell FCXM. No interference was seen with RIT in T-cell FCXM or TMS. ALE interfered with B-cell and T-cell CDC and FCXM, but neither class I nor class II TMS. DAC did not interfere with CDC or FCXM at 0.1 mcg/mL, but caused a false positive B-cell result in both FCXM and CDC with some samples. No interference by DAC was seen with TMS. Conclusions: While further testing is needed, TMS may be a useful alternative method to differentiate de novo donor specific antibodies after treatment with humanized or chimeric immunosuppressive agents.

A method for the determination of the number of reticulated platelets from whole blood

Platelets still containing RNA and DNA remnants are considered to be the immediate progeny of megakaryocytes which have matured beyond the proplatelet formation stage. Numerous investigators have shown that platelets which stain with thiazole orange, a nucleic acid dye, have increased RNA content, and therefore are the youngest platelets in the circulation. These immature, or reticulated platelets, are analogous to red blood cell reticulocytes in their level of differentiation, but not their function. Platelet analysis using thiazole orange dye, in combination with flow cytometry, has been recently shown by a number of investigators to be an optimal method for detection of this immature platelet population. However, the many variations in protocols cause results to greatly differ between laboratories. In our studies, we used a similar whole blood analysis approach developed by Matic and colleagues to quantitate the amount of dye needed for optimal staining. Therefore, we used a dose-escalation of thiazole orange (0.25 μg–50 μg) in combination with CD61 conjugated with phycoerythrin without any wash steps, which assures minimal platelet loss. CD61 was used in this setting in order to set an initial gate on the platelet population. There was a direct, dose-response relationship between the amount of thiazole orange added and the number of reticulated platelets observed. A plateau was reached with 5 μg thiazole orange per 5 μL of whole blood, while 12.5 μg was found to be optimal. Increasing amounts of thiazole orange produced a population shift, or mean channel shift, that needed to be accounted for when setting gates for thiazole orange positive events. We observed that nearly 7% of the platelets in normal resting BDF1 mice were reticulated. This compares to a reticulated population in humans of less than 10%. This protocol combines the ease of sample preparation with stability and accuracy for determining the number of reticulated platelets, and should be a useful method in efforts to standardize the enumeration of reticulated platelets from whole blood. Furthermore, the use of thiazole orange for determining reticulated platelet numbers provides a reproducible and validated method for the analysis of platelet precursors, and, thus, may be a useful tool for predicting platelet recovery post-chemotherapy.

Choriocarcinoma cell line resistance to NK lysis mainly involves an HLA-G–independent mechanism

To investigate whether antitrophoblast antibodies are associated with unexplained recurrent miscarriages, we used choriocarcinoma cells JEG-3, since these cells are negative for class I and II antigens, but they do express HLA-G, resembling an antigen expression of endovascular and interstitial trophoblasts.Case-control study.Academic research center.One hundred ninety-four patients with two or more consecutive, idiopathic recurrent miscarriages (RM; <20 weeks of gestation) were compared with 110 controls with normal pregnancies and without pregnancy complications.Anti-JEG-3 reactivities were measured by using flow cytometry and comparisons with two in-house standards antibody samples of low and high reactivity.Anti-JEG-3 reactivities above the 95% confidence interval of controls were defined as positive.Sera of RM patients reacted significantly stronger with JEG-3 cells than that of controls. In addition, RM patients significantly more often had positive anti-JEG-3 reactivities (17.5%) than controls 5%. This difference was markedly increased with a subgroup of 80 RM patients who had three or more miscarriages, as 27 of these women (34%) were anti-JEG-3 positive.Antitrophoblast antibodies show significantly more mean channel shift reactivities, and positive reactivities are significantly more prevalent in RM patients as compared with controls. Such antibodies may be involved in mechanisms affecting pregnancies.

HLA antibodies present in the sera of sensitized patients awaiting renal transplant are also reactive to swine leukocyte antigens.

To determine whether preformed HLA alloantibodies present in the sera of patients awaiting kidney transplantation will be detrimental to a potential porcine xenograft, we tested their cross-reactivity to swine leukocyte antigens (SLA). Sera obtained from patients with varying levels of HLA sensitization (high panel-reactive antibodies > 70%, n= 7; moderate panel-reactive antibodies 30-40%, n=2) were analyzed. Pooled normal human AB sera and sera from nonsensitized patients (n=3) served as negative control. IgG was purified by protein-G chromatography, and xenoreactive natural antibodies (XNA) were depleted by passing the IgG through a series of melibiose and thyroglobulin-agarose columns. The elimination of XNA from HLA IgG preparations was confirmed by GS-IB4 lectin blocking assay and by an ELISA. IgG isolated from normal AB serum and three nonsensitized patients, which was depleted of XNA (HLA-IgG), did not react to human or porcine lymphocytes (peripheral blood mononuclear cells; PBMC) either by flow cytometry or by complement-dependent microcytotoxicity assays. However, HLA-IgG isolated from nine sensitized patients were reactive to a panel of porcine peripheral blood lymphocytes (n=6) by flow cytometry (>50 mean channel shift) and in complement-dependent microcytotoxicity assays in addition to their reactivity to human PBMC. The binding of HLA-IgG to porcine PBMC was significantly reduced by preabsorption with pooled human platelet concentrate. Further, the HLA IgG showed recognition of 45-kDa affinity-purified SLA class I on Western blots. This study demonstrates that HLA antibodies present in the sera of sensitized individuals can cross-react with SLA. Thus, xenotransplantation of porcine organs into HLA-sensitized patients has the potential to be rejected by humoral mechanisms. Testing to avoid such cross-reactive antibodies should be considered.

Depressed phagocytosis and oxidative burst in polymorphonuclear leukocytes from individuals with pulmonary tuberculosis with or without human immunodeficiency virus type 1 infection.

Phagocytosis and oxidative burst in whole-blood granulocytes were assessed by flow cytometry with Phagotest and Bursttest kits, respectively. Seventy individuals were included in this study: 15 healthy, normal donors, 18 human immunodeficiency virus (HIV) type 1 (HIV-1)-seropositive patients, 19 patients with pulmonary tuberculosis (TB), and 18 patients co-infected with Mycobacterium tuberculosis and HIV-1 (TB-HIV). Granulocyte phagocytosis was assessed by incubating whole blood with fluorescence-labelled Escherichia coli and measuring the proportion of granulocytes with ingested bacteria and the capacity (fluorescence intensity) of each cell to phagocytose E. coli. The percentage of granulocytes converting nonfluorescent dihydrorhodamine to fluorescent rhodamine 123 on production of reactive oxygen intermediates (ROIs) and the mean channel shift were assessed as a measure of oxidative burst. No differences in the proportion of granulocytes that were capable of phagocytosing or producing ROIs in response to E. coli were observed between any of the study groups. Phagocytosis was significantly enhanced in granulocytes from HIV-1-infected individuals. On the other hand, granulocytes from individuals infected with M. tuberculosis alone or in combination with HIV-1 had a significantly reduced capacity to phagocytose E. coli and to produce ROIs in response to E. coli as an agonist. These results provide evidence that granulocytes from individuals with pulmonary TB with or without concomitant infection with HIV-1 have an impaired ability to phagocytose and to undergo oxidative burst, possibly contributing to the enhanced susceptibility to opportunistic infections in these patients.

Cardiac allograft vasculopathy: IgM antibody responses to donor-specific vascular endothelium.

We have previously demonstrated that heightened cell-mediated immunity to donor-specific endothelium was associated with an increased incidence of cardiac allograft vasculopathy (CAV) at 1 year postcardiac transplantation. We further demonstrated that the development of IgG antibody directed to donor-specific HLA antigens was extremely uncommon and, furthermore, had no relationship to the development of CAV. Subsequent studies have demonstrated a correlation between IgM antibody directed against endothelial cell antigens and the development of CAV. Accordingly, recipient serum obtained between 6 and 8 weeks (early) and 1 year (late) after transplantation were reacted with recombinant human interferon (rhIFN)-gamma pretreated donor-specific human aortic endothelial cells (HAECs) in 10 recipients with and 10 recipients without CAV at 1 year after transplantation. HAEC IgM binding was assessed by flow cytometry and complement fixation and HAEC lysis was measured using standard chromium release assays. Seven of 10 and 5 of 10 patients with CAV had IgM detected by flow at early and late time points, respectively (14+/-2 and 16+/-5 mean channel shift), whereas 5 of 10 and 6 of 10 patients without CAV had IgM detected by flow at early and late time points, respectively (15+/-4 and 14+/-3 mean channel shift). This finding was not different between groups. Despite between 50% and 70% of all patients having detectable IgM binding to ECs, no patient's serum was cytotoxic to its donor-specific HAECs. We conclude that IgM antibody to endothelial cells is common (at low titers) after transplantation. This antibody is not cytotoxic and in this study provided no discrimination between those with and without chronic rejection.

Human serum reactivity to porcine endothelial cells after antisense-mediated down-regulation of GpIIIa expression.

The hyperacute rejection of vascularized grafts exchanged between discordant species is a result of the binding of preformed natural antibodies to the endothelium of the donor organ, and the subsequent activation of the complement system. Human natural antibodies to pig endothelial cell antigens appear to be predominantly directed at carbohydrate epitopes expressed by a variety of porcine integrins, including GpIIIa. The identification of porcine xenoantigens whose recognition by human natural antibodies results in hyperacute rejection would allow for the development of strategies to genetically modify the xenograft reaction. We have used antisense technology to down-regulate the expression of one of seven recently identified xenoantigens from the surface of pig aortic endothelial cells. Down-regulation of GpIIIa on endothelial cells resulted in a 20.8% decrease in the mean channel shift (MCS) of IgM natural antibody binding from pooled human sera, and a 28-35% decrease in the MCS of IgM binding from two high-titer individuals. The MCS for human IgG natural antibody binding to the surface of pig cells decreased by 27%. Natural antibody-mediated cytotoxicity to pig endothelial cells was not significantly altered, as indicated by a 2.5-6% decline in complement-mediated cytotoxicity. These results indicate that down-regulation of GpIIIa alone may not be sufficient to significantly alter xenograft rejection. Our results also suggest, however, that antisense-mediated regulation of a functionally important target antigen is technically feasible and may represent a strategy to prevent the xenograft reaction.

Identification of high- and low-risk second kidney grafts.

The purpose of this study was to identify recipients who are at low or high risk of early cadaveric regraft failure by segregating results of the flow cytometric crossmatch (FCXM) test with previous graft survival time (PGST). Early immunologic kidney regraft failure was analyzed in 103 multicenter recipients by cross-stratifying FCXM negative/positive status with < or =3- and >3-month PGST. T cell and B cell cytotoxicity crossmatches were negative. All were tested retrospectively in the T cell FCXM and 60 of the 103 were also tested in the B cell FCXM. A positive T and B cell FCXM was defined as a mean channel shift of > or = 9 (256 channel log scale) or > or = 40 (1024 channel log scale) for pretransplant crossmatch serum above negative control serum. Recipients received triple immunosuppression therapy and limited-use antilymphocyte induction therapy. Early cadaveric regraft losses were biopsied. Comparably good rates of second kidney graft survival at 3 years were found among three ow risk subsets: 78% for 18 FCXM-positive patients with PGST >3 months, 78% for 49 FCXM-negative patients with PGST >3 months, and 84% for 19 FCXM-negative patients with PGST < or =3 months. in contrast, 53% 3-month and 44% 3-year regraft survival rates occurred in 17 high-risk FCXM-positive recipients with a PGST < or =3 months. The odds ratio for increased relative risk of early second graft loss was 4.5 (confidence interval: 1.32-1.67) for the high-risk versus low-risk subsets (P = 0.009). Within the high-risk subset, 56% (5 of 9) of those who were FCXM T negative B positive experienced early regraft loss. A positive B cell FCXM has an adverse clinical impact only for high-risk regraft recipients. Pretransplant panel reactive antibody levels, pregnancy, number of blood transfusions between grafts, repeat donor HLA mismatches, and regraft recipient HLA mismatches did not correlate with early regraft loss. We conclude that kidney regraft survival rates in low-risk recipients (PGST >3 months/FCXM negative or positive [T and/or B cell] and PGST < or = 3 months/FCXM negative) approach primary graft survival rates and justify retransplantation, but the rate in high-risk regraft candidates (PGST < or =3 months/FCXM positive T and/or B cell) suggests that retransplantation should be performed only with a negative FCXM.

Standardization of Flow Cytometric Crossmatch (FCXM) for Investigation of Unexplained Habitual Abortion

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. 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. 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. 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.