Evaluation of three different laboratory methods to detect preformed human leukocyte antigen antibodies in a South African kidney transplant population.

BACKGROUND: Anti-human leukocyte antigen antibodies (anti-HLA) play a crucial role in graft. Detection of anti-HLA, both pre- and post-transplant is a crucial investigation in clinical organ transplantation. OBJECTIVES: Three methodologies for the detection of lymphocytotoxic antibodies were compared to establish which of these is best suited to optimise pre-transplant donor-recipient matching. METHODS: Serum samples from 15 renal transplant patients were tested for the presence of anti-HLA by i) cytotoxic-dependent cross-match (CDCXM), ii) flow cytometric cross-match (FCXM) and iii) Luminex-based donor specific antibody cross-match (DSAXM) method, Confirmatory tests for the presence of preformed HLA antibodies were tested using Luminex methodology. RESULTS: Two (13%) of the 15 patients had positive HLA Class I antibodies (Ab) using all 3 methods. An additional 2 HLA Class I Ab were identified with FCXM/CDCXM. DSAXM identified 1 HLA Class I positive, not indicated by CDCXM/FCXM.High HLA Class II positivity (40%), identified by CDCXM, while DSAXM and FCXM identified two and one patients, respectively. CDCXM produced 4 false-positive results confirmed by lymphocyte single antigen (LSA) assay. CONCLUSIONS: The DSAXM method appears to add value in pre-transplantation screening to identify pre-sensitised patients that may not reject the donor graft due to the absence of donor-specific antibodies.

Relationship between plasma rabbit anti-thymocyte globulin concentration and immunosuppressive therapy response in patients with severe aplastic anemia.

OBJECTIVES: Patients with acquired aplastic anemia (AA) without HLA-matched sibling donors or aged >40 years receive immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG). We investigated the relationship between plasma rabbit ATG (r-ATG) concentration and IST response. METHODS: From May 2012 to October 2017, 81 patients with severe AA who required initial IST were included. A 1:1 block randomization was employed for 2.5 and 3.5 mg/kg doses of r-ATG. RESULTS: No significant difference in response rates was observed between the 2.5 and 3.5 mg/kg groups (63% vs. 58%, P = .894). Median r-ATG concentrations on days 14 and 28 after IST were 15.2 (0.0-97.7) and 1.8 (0.0-74.9 µg/mL), respectively. According to r-ATG concentration, response rates were significantly higher in the group with higher r-ATG concentration than in those with lower r-ATG concentration (day 14, 88% vs. 52%; P = .006 and day 28, 79% vs. 46%; P = .005). In multivariate analysis, higher r-ATG concentrations at day 28 were independent predictors of favorable response to IST at 6 months (odds ratio, 0.29; 95% confidence interval, 0.09-0.93; P = .037). CONCLUSIONS: The present data indicate that higher r-ATG concentration at day 28 resulted in improved IST response.

Removal of Anti-Thymocyte Globulin by Plasma Exchange in ABO-Incompatible and Positive Crossmatch Kidney Transplant Recipients.

BACKGROUND: Recipients of ABO-incompatible (ABOI) and positive crossmatch (PXM) kidney transplants are at high risk for antibody-mediated acute rejection. Despite aggressive immunosuppression in high-risk patients, the incidence of acute rejection remains considerably higher than in other groups. No published studies have examined plasma concentrations of anti-thymocyte globulin (ATG) in patients undergoing plasma exchange. The objectives of this study were to compare plasma ATG concentrations before and after plasma exchange in ABOI and PXM kidney transplant patients to determine the amount removed. MATERIALS AND METHODS: This prospective pharmacokinetic evaluation enrolled 10 patients undergoing ABOI or PXM kidney transplant at an academic medical center. Blood and waste plasma samples from 5 patients were assayed for total and active ATG concentrations. Patient records were monitored for renal function and rejection rates in the first 6 months post-transplant. RESULTS: Total ATG concentrations decreased a mean of 59.78 ± 13.91% after each plasma exchange session, and active ATG levels decreased a mean of 56.8 ± 17.08%. Mean daily concentrations reflect a lack of expected ATG accumulation. Only 1 of 4 patients had detectable ATG concentrations after 30 days. After 6 months, the incidence of acute rejection in this sample was 44% and graft survival was 89%. CONCLUSIONS: This is the first study to show that plasma exchange removes a substantial amount of ATG in high-risk kidney transplant patients. Based on these results, we believe these high-risk patients have been traditionally underdosed.

Quantification of T Cell Binding Polyclonal Rabbit Anti-thymocyte Globulin in Human Plasma with Liquid Chromatography Tandem-Mass Spectrometry.

The addition of rabbit anti-human thymocyte globulin (ATG) to the conditioning regimen prior to allogeneic hematopoietic cell transplantation has significantly reduced the risk of graft-versus-host disease (GvHD) and graft failure. However, ATG has a small therapeutic window. Overexposure of ATG post-HCT hampers T cell immune reconstitution and has been associated with increased relapse rates and viral reactivations, whereas underexposure has been associated with an increased incidence of GvHD, both of which lead to increased mortality. Therapeutic drug monitoring of T cell binding ATG plasma levels provides a means to optimize dosing for patients at high risk for graft failure to ensure timely T cell immune reconstitution and subsequently increase survival chances. This manuscript describes the first liquid chromatography tandem-mass spectrometry (LC-MS/MS) method to quantify the pharmacologically active fraction of polyclonal ATG in plasma. This was achieved through immunoaffinity purification of active ATG from plasma with Jurkat T cells. After the binding and washing, samples were eluted, denatured, and trypsin-digested. Signature peptides originating from the IgG constant chain were measured with LC-MS/MS. Critical method parameters were optimized, and the method was successfully validated following European Medicines Agency (EMA) guidelines. The method covered the therapeutic range of ATG and was validated at a lower limit of quantification (LLOQ) of 1 AU/mL with an overall CV and bias of 11.8% and - 2.5%, respectively. In conclusion, we developed a LC-MS/MS-based method to quantify active polyclonal rabbit ATG in human plasma. We suggest that this novel assay can be used to monitor and optimize dosing of ATG in clinical practice.

DSA-FXM: Accelerated Donor-specific Flow Crossmatch Discriminating Class I and II Antibody Specifically and Only to Donor HLA for Determining True Incompatibility.

BACKGROUND: Worldwide, a final crossmatch is the gold standard for determining compatibility between patient and donor before solid organ transplantation and preventing hyperacute rejection. In the absence of autoantibodies, an incompatible crossmatch in a sensitized patient is attributed to mismatched donor HLA. However, current physical crossmatch methods cannot distinguish reactivity to HLA from other clinically irrelevant cell surface targets nor the class of HLA if it is the target. Result interpretation is difficult or impossible when autoantibodies, alloantibodies, or therapeutic antibodies coexist. METHODS: Herein, we describe a unique donor-specific flow crossmatch (DSA-FXM) that distinguishes HLA class I or II donor-specific antibody bound to HLA antigens on the donor cell surface in their native conformation that is not impacted by rituximab, anti-thymocyte globulin (after absorption), or autoantibodies. It is HLA specific. RESULTS: We compared the results of single-antigen antibody testing, autoreactive and alloreactive flow cytometry crossmatches (FXM) using traditional FXM and our DSA-FXM method from 94 patients (enriched for auto+/allo+ pairs; n = 64) against 110 donors (338 tests) and show that, in our cohort, positive traditional FXM results are not directed to donor HLA 60.25% of the time and negative traditional FXM results are missing HLA donor-specific antibody 36.2% of the time based on the DSA-FXM. CONCLUSIONS: We demonstrate that the DSA-FXM is able to define categorically distinct and clinically important HLA antibody profiles in half the time required for the standard FXM, potentially shortening cold ischemia time and providing clinicians with unambiguous essential information regarding HLA compatibility when time is critical.

The Relationship between High Iodine Consumption and Levels of Autoimmune Thyroiditis-Related Biomarkers in a Chinese Population: a Meta-Analysis.

To comprehensively evaluate the relationship between high iodine concentration and biomarker abnormalities related to autoimmune thyroiditis in a Chinese population. Medline, PubMed, and Embase electronic databases were searched for articles published domestically and internationally on the relationship between high iodine concentrations and thyroid hormone antibodies and thyroid-stimulating hormone in China before March 2019. Articles published in Chinese were searched in the China Biology Medicine (CBM) disc, Wanfang Database, and China National Knowledge Infrastructure (CNKI). A total of 16 cross-sectional articles were included in this study, including 9061 participants. A meta-analysis was conducted in Stata 14.0. The binary categorical and continuous variables used odds ratios (ORs) and standardized mean differences (SMDs) with the corresponding 95% confidence intervals (CIs) as the effect statistics, respectively. The results showed that high iodine concentrations had a minimal association with the abnormal rates of thyroid peroxidase antibody (TPOAb) (OR = 1.274, 95% CI (0.957, 1.695), P > 0.05) and thyroglobulin antibody (TGAb) (OR = 1.217, 95% CI (0.911, 1.626), P > 0.05) in the entire population. The thyroid-stimulating hormone (TSH) level in the high iodine group was greater than that in the adaptive iodine group (SMD = 0.202, 95% CI (0.096, 0.309), P < 0.05). The results of the subgroup analysis showed that the abnormal TPOAb rate in pregnant women (OR = 1.519, 95% CI (1.007, 2.291), P < 0.05) and children (OR = 3.365, 95% CI (1.966, 5.672), P < 0.05) in the high iodine group was greater than that in the adaptive iodine group, and the abnormal TGAb rate of children in the high iodine group was greater than that in the adaptive iodine group. The TSH levels of lactating women (SMD = 0.24, 95% CI (0.053, 0.427), P < 0.05), pregnant women (SMD = 0.301, 95% CI (0.176, 0.426), P < 0.05), and children (SMD = 0.25, 95% CI(0.096, 0.309), P < 0.05) in the high iodine group were higher than those in the adaptive iodine group. Egger's and Begg's tests showed no significant (P > 0.1) publication bias. High iodine can increase the risk of abnormal levels of TPOAb, TGAb, and TSH related to autoimmune thyroiditis in pregnant women, lactating women, and children in China.

Role of complement in patients with autoimmune hemolytic anemia and platelet transfusion refractoriness.

The complement is a key player of the innate immune response. It provides defense mechanisms that are not specific, but very efficient at neutralizing any invader, accounting for 4% of the proteins in the peripheral blood. Nevertheless, there is a dark side to the complement system, as it may activate its machinery against healthy cells such as peripheral blood red blood cells and platelets resulting in undesired hemolysis and thrombocytopenia, respectively. Understanding and identifying the role of complement in these settings allow physicians to adjust their diagnostic and therapeutic modalities accordingly. The role of complement in the pathophysiology and management of autoimmune hemolytic anemia and of alloimmune-mediated thrombocytopenia is under investigation and discussed.

Anti-HLA Antibodies After Precocious Transplantectomy by Vascular Thrombosis.

BACKGROUND: Our objective in this study was to determine the effects of early renal transplantectomy on patients and the production of anti-human leukocyte antigen (anti-HLA) antibodies. METHODS: Between January 2003 and May 2017, we analyzed a group of patients for the presence of specific HLA class I and/or II donor-specific antibodies (DSA), their panel-reactive antibodies (PRA), and the time period in which the antibodies were still detectable after transplantectomy. RESULTS: Anti-HLA antibodies were detected in 60.8% of patients, 60.8% and 52.2% of those patients had anti-class I and anti-class II antibodies, respectively. DSA were detected in 91.7% of the anti-HLA class I patients. Class II DSA were detected all of the patients with anti-HLA class II antibodies. The average (mean ± SD) PRA levels in our patients after transplantectomy was 60 ± 34% in class I and 63 ± 36% in class II. CONCLUSION: Anti-HLA antibodies can be detected well after transplantectomy. Even if the kidney allograft had been transplanted for only a short time, when the intensity of immunosuppression was the highest, many patients developed anti-HLA antibodies. The patients who continued with immunosuppression after transplantectomy did not develop anti-HLA antibodies.

Correlation of rabbit antithymocyte globulin serum levels and clinical outcomes in children who received hematopoietic stem cell transplantation from an alternative donor.

We analyzed the correlation between rabbit ATG (rATG) serum levels and clinical outcomes in 37 children who received rATG at a total dose of 10 or 15 mg/kg during HSCT conditioning from an alternative donor. Fourteen patients had advanced malignant diseases, 13 had severe AA, and 10 had inherited disorders. Complete engraftment was achieved in all patients, and no rejection occurred. The cumulative incidence of grades II-IV acute GVHD and extensive chronic GVHD was 27% (95% CI, 12.5-39.6%) and 8.1% (95% CI, 0-23.1%), respectively. Multivariate analysis identified lower rATG levels at week 4 as an independent risk factor in the development of grades II-IV acute GVHD (p = 0.037). Serious infections were not observed in any patient following HSCT. No correlation was found between EBV reactivation and rATG levels at week 2 and week 4 after HSCT. Furthermore, no correlation was found between relapse and rATG levels two and four wk post-transplantation. The probability of five-yr OS among patients was 70.3% (95% CI, 59.8-79.2%). Our results suggest that targeted rATG administration may protect patients from severe acute GVHD without increasing the risk of EBV reactivation or relapse.

Tg in Adults as a Sensitive Biomarker of Iodine Status: A 5-Year Follow up Population Study in Different Levels of Iodine Intake Regions.

This study was to evaluate the usefulness of serum thymoglobulin (Tg) in adults to assess iodine status through a 5-year cohort study which was conducted in three regions with different levels of iodine intake: mild deficiency, more than adequate, and excess, from 1999 to 2004 in China. A total of 3099 subjects over 14 years old with normal serum levels of Tg in 1999 were eligible, of whom 2448 were followed in 2004. Serum levels of thyroid hormones and thyroid autoantibodies as well as urine iodine were measured, and B-mode ultrasonography of the thyroid was performed. A general linear model was performed to determine the determinant factors of serum Tg. Among subjects with mildly deficient iodine intake, those with more than adequate intake, and those with excessive intake, the baseline levels of serum Tg were substantially different (7.5µg/L, 5.9µg/L, and 6.8µg/L respectively, P<0.01), which were associated with age, sex, the rate of positive TgAb, abnormal thyroid volume, abnormal TSH, and positive personal history of thyroid diseases. The data from 1856 subjects with normal range of thyroid parameters but no personal history of thyroid diseases were analyzed to clarify the effect of iodine intake on Tg. Among these three regions, the serum Tg levels were substantially different in both 1999 and 2004, with a similar pattern for increased Tg (ΔTg) (3.1µg/L, 2.5µg/L and 3.5µg/L respectively, P<0.01). The general linear model analysis revealed that age, Tg, and baseline TSH levels were the determinants of ΔTg besides iodine intake. In conclusion, serum Tg in adults, resulting from a time-accumulative effect of iodine exposure, is a useful biomarker of regional iodine intake.

Antilymphocyte antibodies in systemic lupus erythematosus: association with disease activity and lymphopenia.

PURPOSE: We analyzed the prevalence, clinical correlation, and the functional significance of ALA in patients with systemic lupus erythematosus (SLE). METHODS: ALA IgG was detected by indirect immunofluorescence in the serum of 130 SLE patients, 75 patients with various rheumatic diseases, and 45 healthy controls (HC). RESULTS: The sensitivity and specificity of ALA IgG in SLE were 42.3% and 96.7%, respectively. ALA was observed in 55.6% (50/90) of patients with lymphopenia, which was significantly higher than in patients with normal lymphocytes (5/40, 12.5%; P<0.001). Patients with active SLE showed higher ALA positivity (60.9%) than those with inactive disease (24.2%; χ2=17.925; P<0.001). ALA correlated significantly with hypocomplementemia, anti-dsDNA antibodies, and higher SLEDAI scores. The incidences of ALA in SLE patients who were seronegative for anti-dsDNA, anti-Sm, or both antibodies were 32.9% (26/79), 41.0% (43/105), and 32.4% (22/68), respectively. The ALA-positive group also had higher incidences of neuropsychiatric SLE (NPSLE) and lupus nephritis (LN). In multivariate analyses, ALA was independently associated with lymphopenia, higher SLEDAI scores, and increased risk for LN. ALA titers significantly decreased as clinical disease was ameliorated following treatment. CONCLUSIONS: ALA occurred more frequently in patients with active SLE and was independently associated with lymphopenia, disease activity, and LN.

Rabbit anti-T-lymphocyte globulin (ATG) persists with differential reactivity in patients' sera after full hematopoetic regeneration from allogeneic stem cell transplantation.

BACKGROUND: Rabbit polyclonal anti-T-lymphocyte Globulin (ATG-F®, Fresenius) is widely used for GvHD prophylaxis in allogeneic stem cell transplantation (SCT). ATG has a wide epitope spectrum and has been shown to react with all compartments of peripheral blood mononuclear cells (PBMNCs). ATG induces apoptosis in all cellular compartments. In this study we investigated the binding of ATG in sera from ten patients treated with rabbit ATG to PBMNCs and subcellular compartments after full hematopoetic regeneration on day 21 post SCT. METHODS: Sera from ten patients treated with unrelated donor allogeneic SCT for hematologic malignancy were collected after full hematopoetic regeneration on day 21 post SCT and incubated with healthy donor PBMNCs. Rabbit ATG on PBMNCs was detected by staining with fluorochrome labeled anti-rabbit IgG antibody. PBMNC compartments were investigated by counterstaining with lineage markers CD4, CD8, CD14 CD20 and CD56. Positive control was the fresh ATG preparation. RESULTS: We found that patient's' sera retained activity towards PBMNCs in all patients, yet at reduced intensity. When cell compartments were analyzed we found a differential pattern of ATG reactivity within sera. The mean percentage of total cells reacting with serum ATG from ten patients compared to fresh ATG (100%) was 44% of CD4 positive and 58% of CD8 positive T-lymphocytes, 41% of CD56 positive NK-cells, 83% of CD20 positive B-lymphocytes and 98% of CD14 positive monocytes. However, inter-individual variations were high with a wide spread around the mean especially for T-lymphocytes. CONCLUSION: We conclude that upon PBMNC regeneration following SCT and immunosuppressive treatment with ATG subpopulations of T-lymphocytes (CD4, CD8) and NK cells (CD56) are selected that lose epitopes recognized by ATG while B-lymphocytes (CD20) and monocytes (CD14) maintain a homogeneity with respect to epitopes recognized by ATG. This may be due to loss of idiotypes reacting with subpopulations of high frequency and turnover. Further studies should investigate the subphenotype of these populations and functional effects of extremely high or low reactivity with one or more compartments in some patients on GvHD and disease outcome.

Detection of donor-specific-antibodies by solid phase assay and its relevance to complement-dependent-lymphocytotoxicity cross-matching in kidney transplantation.

Presensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allogaft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. Our data reinforce the concept that these two types of assays should be used in parallel for pre-and post-transplantation monitoring of anti-HLA antibodies in recipients of solid organ allografts.

Significance of true-positive and false-positive pretransplantation lymphocytotoxic crossmatch in primary liver allograft outcomes.

BACKGROUND: At the time of transplantation, a recipient's serum is tested against the prospective donor's lymphocytes to identify specific reactivity and to look for a donor-specific crossmatch (CXM). Here, we investigated the relationship between the pretransplantation lymphocytotoxic CXM results and the long-term outcome of liver transplantation at a single center. METHODS: From October 1998 to April 2011, medical records, laboratory data, and pretransplantation lymphocytotoxic CXM results were collected from 1133 consecutive liver transplant recipients. RESULTS: We performed liver transplantations on 80 (7.1%) patients after a true-positive CXM (t+CXM). The t+CXM group exhibited higher initial aminotransferase levels immediately after transplantation compared with a negative CXM group. However, no significant differences in rejection, biliary or vascular complications, viral disease recurrence, or de novo malignancies were found. Although overall graft and patient survival did not differ between the groups, liver-specific graft survival was inferior in the t+CXM group. It was also found that, in 42 (3.7%) recipients, initially positive results converted to final negative results after the elimination of immunoglobulin M autoantibodies. We defined this subpopulation as a false-positive CXM. Significantly decreased posttransplantation aminotransferase levels with a higher incidence of de novo malignancies were observed in this group compared with negative controls. CONCLUSION: Our findings demonstrate that t+CXM transplants show increased aspartate aminotransferase and alanine aminotransferase peak immediately after transplantation, which influences liver-specific graft outcomes. Additionally, the presence of circulating immunoglobulin M autoantibodies against recipients' own antigens may be protective in liver grafts. However, this may be a predisposing factor for de novo malignancies.

Anti-thymocyte globulins capable of binding to T and B cells reduce graft-vs-host disease without increasing relapse.

Anti-thymocyte globulin (ATG) is polyclonal, containing Ab specificities capable of binding to various immune-cell subsets implicated in the pathogenesis of GVHD, including T cells, B cells, natural killer cells, monocytes/macrophages, neutrophils and DC. We wished to determine which ATG specificities are important for GVHD prevention. We measured day 7 serum levels of 23 ATG specificities in 120 hematopoietic cell transplant recipients whose myeloablative conditioning included 4.5 mg/kg ATG (thymoglobulin). High levels of ATG specificities capable of binding to T- and B-cell subsets were associated with a low likelihood of acute GVHD (aGVHD). High levels of these ATG specificities were associated with increased rates of viral but not bacterial or fungal infections. They were not associated with an increased risk of malignancy relapse; on the contrary, high levels of ATG specificities capable of binding to regulatory T cells and invariant NKT cells were associated with a low risk of relapse. In conclusion, high levels of ATG antibodies to Ag(s) expressed on T and B cells are associated with a low risk of aGVHD and a high risk of viral but not bacterial or fungal infections. These antibodies have neutral or beneficial effects on relapse.

Immune reconstitution after anti-thymocyte globulin-conditioned hematopoietic cell transplantation.

BACKGROUND AIMS: Anti-thymocyte globulin (ATG) is being used increasingly to prevent graft-versus-host disease (GvHD); however, its impact on immune reconstitution is relatively unknown. We (i) studied immune reconstitution after ATG-conditioned hematopoietic cell transplantation (HCT), (ii) determined the factors influencing the reconstitution, and (iii) compared it with non-ATG-conditioned HCT. METHODS: Immune cell subset counts were determined at 1-24 months post-transplant in 125 HCT recipients who received ATG during conditioning. Subset counts were also determined in 46 non-ATG-conditioned patients (similarly treated). RESULTS: (i) Reconstitution after ATG-conditioned HCT was fast for innate immune cells, intermediate for B cells and CD8 T cells, and very slow for CD4 T cells and invariant natural killer T (iNKT) (iNKT) cells. (ii) Faster reconstitution after ATG-conditioned HCT was associated with a higher number of cells of the same subset transferred with the graft in the case of memory B cells, naive CD4 T cells, naive CD8 T cells, iNKT cells and myeloid dendritic cells; lower recipient age in the case of naive CD4 T cells and naive CD8 T cells; cytomegalovirus recipient seropositivity in the case of memory/effector T cells; an absence of GvHD in the case of naive B cells; lower ATG serum levels in the case of most T-cell subsets, including iNKT cells; and higher ATG levels in the case of NK cells and B cells. (iii) Compared with non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT was slower for CD4 T cells, and faster for NK cells and B cells. CONCLUSIONS: ATG worsens the reconstitution of CD4 T cells but improves the reconstitution of NK and B cells.

Effects of different serum-levels of ATG after unrelated donor umbilical cord blood transplantation.

We determined total rabbit-IgG (r-ATG) levels in serum samples before (day 0) and after (day 11 and day 25) unrelated donor umbilical cord blood transplantation (UCBT). Most patients (27/41) suffered from a haematological malignancy. There were 25 children and 16 adults. All patients received rabbit anti-thymocyte globulin (ATG) at a total dose of 6 or 8mg/kg as part of the conditioning. No correlation between the dose of ATG and serum r-ATG levels post UCBT was found. The cumulative incidence of acute GVHD grades III-IV in patients given the 6 and 8mg/kg ATG dose was 15% and 13% (ns), respectively. Patients with r-ATG≤40µg/mL 11days after UCBT (n=19) had a higher incidence of grades III-IV acute GVHD (32% vs. 0%, p<0.01), higher TRM (69% vs. 7%, p=0.005), less relapse (17% vs. 82%, p<0.01) but similar relapse-free survival (RFS) (10% vs. 18%, p=0.4) compared to those with r-ATG>40µg/mL (n=17). Low serum-levels of r-ATG early after transplantation seem to be a strong predictor for acute GVHD grades III-IV, TRM and a low incidence of relapse in patients treated with thymoglobulin before unrelated donor UCBT.

High rabbit-antihuman thymocyte globulin levels are associated with low likelihood of graft-vs-host disease and high likelihood of posttransplant lymphoproliferative disorder.

Rabbit-antithymocyte globulin (ATG) given with conditioning has the potential to decrease the likelihood of graft-versus-host disease (GVHD) or graft failure and to increase the likelihood of relapse or infections. After a given ATG dose, serum ATG levels are variable. Here we determined ATG levels on days 7 and 28 in 153 patients whose conditioning included 4.5 mg/kg ATG (thymoglobulin). Median follow-up was 547 days (range: 14-1519, minimum for patients who have not died, relapsed, developed second malignancy, or had graft failure, 365). Both high day 7 levels and high day 28 levels were associated with low likelihoods of grade II-IV acute GVHD and chronic GVHD needing systemic immunosuppressive therapy, and a high likelihood of posttransplant lymphoproliferative disorder (PTLD). Patients with day 7 ATG levels above 0.803 mg/L had 0.52-fold risk of developing chronic GVHD needing systemic therapy (P = 0.012) and patients with day 7 ATG levels above 1.436 mg/L had 5.84-fold risk of developing PTLD (P = 0.001) compared to patients with lower ATG levels. There was no association of ATG levels with relapse, death, or non-PTLD infections. Association with graft failure could not be evaluated due to only 4 graft failures in the cohort. In conclusion, patients with slow clearance of ATG have a low risk of GVHD, but a high risk of PTLD. The clearance of this relatively low dose of ATG does not impact the likelihood of relapse, death, or non-PTLD infections.

[An enzyme-linked immunosorbent assay for determining serum anti-themocyte globulin concentration].

OBJECTIVE: To establish an enzyme-linked immunosorbent assay (ELISA) for determining anti-themocyte globulin (ATG) levels in serum samples. METHODS: The microplate was coated with mouse anti-rabbit IgG monoclonal antibody, and sheep anti-rabbit polyclonal antibody conjugated with HRP was used as the second antibody for detecting the serum ATG levels in patients undergoing allogeneic hematopoietic stem cell transplantation. RESULTS: The optimal concentration of the coating antibody and dilution ratios of the serum samples and IgG-HRP conjugate were 0.2 microg/ml, 1:40 and 1:2500, respectively. The lower sensitivity limit of the assay was 31.25 ng/ml for ATG detection. A linear relationship was established within the concentration range from 40 to 1000 ng/ml, with the coefficients of variation of 7.91 within assay and 5.22 between assays, respectively. Seven patients undergoing stem cell transplantation with ATG pretreatment showed gradually decreased concentration of ATG, and after 90 days ATG could still be detected. CONCLUSION: The sandwich ELISA we established provides a specific and sensitive method for quantitative measurement of ATG in the clinical setting. In patients undergoing stem cell transplantation with ATG pretreatment, the ATG concentration gradually decreases but remains detectable 90 days after the administration.