ELISA—CDC实验方法的研究

目的：为了提高对抗供者HLA-IgG类抗体（DSA）的识别能力,建立酶联免疫吸附-淋巴细胞毒交叉配型（ELISA-CDC）.方法：对已知HLA抗体特异性的PRA阳性血清进行ELISA-CDC实验,以比较ELISA-CDC与ELISA-PRA在同一特异性HLA抗体识别上的吻合率.选自ELISA-PRA阳性血清,24例已知HLA抗体的血清和针对性HLA-A、B、DR基因的淋巴细胞ELISA-CDC实验为第一组;24份已知HLA抗体的PRA阳性血清和无针对性HLA-A、B、DR基因的淋巴细胞ELISA-CDC实验为第二组;PRA阴性血清为阴性对照组.结果：PRA阳性血清与包含针对性HLA基因的淋巴细胞ELISA-CDC均为阳性;PRA阳性血清与不包含针对性HLA基因的淋巴细胞ELISA-CDC均为阴性;PRA阴性血清与上述2组淋巴细胞ELISA-CDC亦为阴性,吻合率100%.结论：ELISA-CDC能够表达出与ELISA-PRA一致的HLA抗体特异性.ELISA-CDC实验不需要活淋巴细胞,并且排除了非供者特异性抗体反应和非HLA抗体反应;并且,可以提纯供者HLA抗原低温长期保存,不仅便于移植前多个受者与单一供者交叉配型,而且是便于移植术后使用同一供者HLA基因进行监测DSA的一种简单、客观而且有效的方法.     

HLA配型在致敏受者肾移植中的应用研究

目的 探讨人类白细胞抗原（ＨＬＡ）配型在致敏受者肾移植中的临床意义。方法 应用莱姆德细胞板通过补体领事微量细胞毒性试验检测受者的群体反应性抗体（ＰＲＡ）;应用单抗湿板进行供受者ＨＡＬ－Ⅰ类抗原分型;应用微量序列特异性引物（Ｍｉｃｒｏ－ＳＳＰ）进行ＨＡＬ－Ⅱ类基因分型。结果 １７例受者ＰＲＡ阳性率为５．１％￣８０％,平均３７．８９％;按交叉反应组（ＣＲＥＧｓ）配型原则,供受者ＨＬＡＣＲＡＥｓ０．１和     

B淋巴细胞交叉配型在致敏肾移植受者中的作用

致敏肾移植受者术前需做淋巴细胞毒交叉配型。T淋巴细胞交叉配型（TXM）的意义已得到公认，但B淋巴细胞交叉配型（BXM）的临床意义尚有争论。我们发现BXM阳性的肾移植受者术后急性排斥反应发生率显著升高，1年移植肾存活率显著降低，报告如下。     

高致敏肾移植患者血浆置换后的HLA配型处理一例

患者，女性，48岁，原发病为多囊肾所致尿毒症。2004年10月19日初次检测群体反应性抗体（PRA）为阴性。2004年12月2日患者因消化道大出血，连续输血3d，输血量分别为400ml、200ml、400ml。2005年3月10日再次检查PRA，HLA-I类抗体为53．6％、Ⅱ类抗体阴性，HLA抗体特异性为A1、A23、1360。3月16日再次复查，结果如上，确认为高致敏受者。患者分别于2005年4月1日和4月8日各进行了1次血浆置换术（PE），置换量为3000ml。     

采用流式细胞微珠法检测肾移植受者体内供者特异性抗体39例

目的 应用流式细胞微珠法检测肾移植受者供者特异性抗体(DSA),并探讨DSA阳性受者的HLA配型及移植物排斥反应发生情况.方法 检测39例亲属肾移植受者移植前、后的DSA,检测供、受者HLA错配情况,记录受者移植后排斥反应的发生情况.分析DSA阳性及阴性受者的HLA错配及排斥反应的发生情况.结果 39例共检测DSA 313次,其中移植前78次,移植后235次,移植前出现DSA阳性的均暂缓手术.5例HLA无错配的受者移植后DSA均为阴性,34例HLA错配的受者移植后12例出现DSA阳性(35.3％,P＜0.05).12例DSA阳性受者中,5例发生排斥反应(41.7％),其排斥反应发生率显著高于DSA阴性的移植受者(7.4％,P＜0.05).发生排斥反应且DSA阳性受者的单抗原微珠免疫荧光强度均值为5723.9±1030.5,高于未发生排斥反应的DSA阳性者的2355.2±609.7(P＜0.05).DSA阳性的受者治疗后,DSA免疫荧光强度有所下降.结论 采用流式细胞微珠法动态监测DSA效果较好,有利于预测和及时防治肾移植后排斥反应.     

脱敏预处理对高致敏活体肾移植受者PRA水平和疗效的影响

目的分析双重滤过血浆置换(double filtration plasmapheresis,DFPP)等脱敏处理对等待活体肾移植的高致敏患者的群体反应性抗体(panel reactive antibody,PRA)滴度以及对移植术后疗效的影响。方法 11例PRA强阳性(人类白细胞抗原Ⅱ类抗体80%)患者为研究对象,所有患者既往均有肾移植史。其中5例为实验组,于手术前隔日行DFPP(3～7次),加用免疫抑制剂。实验组患者每次DFPP前后抽取的血清及滤过弃物均采用流式细胞法测定PRA滴度变化。其余6例患者为对照组,移植前未作脱敏预处理。术后观察排斥反应发生情况及临床疗效。结果实验组早期第1例患者做了7次DFPP并加其它免疫处理,前5次抗体滴度明显下降,但后两次处理后抗体出现反弹。其余4例患者经3～5次DFPP处理后,抗体滴度不断下降。移植术后11例患者均未发生超急性排斥反应,实验组中有1例(1/5)发生细胞性排斥反应,而无体液性排斥反应,对照组中有4例(4/6)发生细胞性排斥反应,有3例(3/6)并发体液性排斥反应。结论 DFPP结合其它免疫处理方法进行脱敏预处理,能有效降低肾移植术前PRA滴度,降低移植术后体液性排斥和细胞性排斥的发生风险。     

术前预处理及组织配型对高度致敏患者移植肾功能的影响

目的：探讨术前预处理及组织配型对高度致敏患者移植肾功能的影响。方法：对38例高度致敏患者(高敏组)肾移植术前进行预处理及组织配型,观察患者术后移植肾功能延迟(DGF)、排斥反应的发生和血肌酐(SCr)水平的变化。结果：高敏组术后发生超急性排斥反应(HAR)2例;其加速性排斥反应(ACR)、急性排斥反应(AR)以及DGF发生率均高于非高敏组受者,1年移植肾存活率则较低。高敏组中组织配型良好的受者较配型欠佳者AR发生率及术后1年SCr水平较低;术前预防性使用赛尼哌可降低术后的AR发生率。结论：预处理降低高度致敏患者群体反应性抗体(PRA),使患者易于配型成功,良好的组织配型和使用赛尼哌可降低术后AR的发生,均有助于移植肾功能的恢复。     

交叉配型阳性受者超高供者特异性抗体基线水平提示肾移植预后差

<正>肾移植受者预存供者特异性抗体会增加体液性排斥反应的发生风险。为了明确供者特异性抗体基线水平(移植前)与抗体介导的排斥反应、移植肾肾病、移植肾长期存活的相关性,美国梅约医疗中心选择2000     

交叉反应组配型在高致敏患者肾移植中的应用

目的 探讨交叉反应组(CBEG)配型在高致敏患者肾移植中的临床意义。方法 动态监测肾移植受者体内群体反应性抗体(PRA)的水平及其特异性，按照CREG配型原则选择最匹配的供者。结果 60例受者术前PRA超过11％，均有单纯性或混合性升高；按照CREG配型，0～1个抗原错配、2个抗原错配、3～4个抗原错配者术后肌酐恢复正常的时间平均为6．5d、7．0d、12．7d，发生肾功能恢复延迟的例数分别为0、7例、3例，各组间的差异具有显著性(P＜0．05)。结论 高致敏受者在肾移植时采用CREG配型，可避开受者预存的HLA抗体特异性所对应的抗原，对于提高肾移植人／肾存活率具有重要意义。     

Clinical relevance of antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test

Recent literary data suggest that antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test may cause not only chronic, but also acute immunological complications after kidney transplantation. The aim of this study was to investigate the significance of non-cytotoxic antibodies to HLA antigens for the development of immunological complications and a worse graft prognosis after first kidney transplantation. Sera before and early after transplantation from 120 first kidney recipients were analyzed by flow cytometry (FCXM), ELISA and the standard complement-dependent cytotoxicity (CDC) test. Pre-transplant FCXM negativity was related to a lower incidence of rejection episodes in the first post-transplant year ( P<0.01). A significant association between acute rejection and the presence of antibodies to HLA class II antigens before and after transplantation was also found ( P<0.05). Our study supports the findings of other centers of the detrimental role to the kidney graft played by anti-HLA antibodies undetectable by the classical CDC test.     

Clinical relevance of antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test

Recent literary data suggest that antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test may cause not only chronic, but also acute immunological complications after kidney transplantation. The aim of this study was to investigate the significance of non-cytotoxic antibodies to HLA antigens for the development of immunological complications and a worse graft prognosis after first kidney transplantation. Sera before and early after transplantation from 120 first kidney recipients were analyzed by flow cytometry (FCXM), ELISA and the standard complement-dependent cytotoxicity (CDC) test. Pre-transplant FCXM negativity was related to a lower incidence of rejection episodes in the first post-transplant year (P<0.01). A significant association between acute rejection and the presence of antibodies to HLA class II antigens before and after transplantation was also found (P<0.05). Our study supports the findings of other centers of the detrimental role to the kidney graft played by anti-HLA antibodies undetectable by the classical CDC test.Abbreviations ACR Acute cellular rejection - AHR Acute humoral rejection - CDC Complement dependent cytotoxicity - ELISA Enzyme linked immunosorbent assay - FCXM Flow cytometry crossmatch - HLA Human leukocyte antigens - LAT Lambda antigen tray - PTC Peritubular capillary     

The value of flow cytometric crossmatch in cardiac transplantation

Abstract One of the major clinical problems in cardiac transplantation is that of moderate rejection of the graft, and over the past few years there is increasing evidence that humoral antibody may be important in graft prognosis. The sensitivity of the conventional cytotoxic crossmatch has been questioned, and an increased significance of there of the flow cytometric cross-match (FCXM) to detect the presence of antibodies before transplantation has been reported. In this study we have examined the sera of 138 cardiac transplants (1988–1992) for the presence of donor-directed IgG and IgM antibodies using FCXM. Sera were collected immediately before transplantation and before the institution of immunosuppressive therapy. All pretransplant cytotoxic crossmatches were negative. After a minimum follow-up period of 3 months, the performance of the transplants was graded by endo-myocardial biopsy: 1, no or mild evidence of rejection; 2, patients showing moderate rejection requiring increased immunosuppression. Of the 138 patients studied, 10 patients were excluded as they died within the first week of transplantation. Eight children were excluded since they were given prophylactic ATG (Merieux). A positive FCXM result was defined as showing values in excess of that found for the AB control sera. A significant association was found between the presence of both IgG to T and B cells and IgM to T cells and graft performance (P = 0.02 and 0.93, respectively). Indeed, IgM-directed T-cell antibodies were only found in patients with moderate rejecton. These two groups were mutually exclusive, so that the FCXM was able to identify the presence of moderate rejection in 55% of the patients. In conclusion, results show that pretransplant FCXM in ***carchac transplantation provides a more sensitive assay of antibody status in recipients and has proved to be of prognostic value.     

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.     

The use of pronase-digested human leukocytes to improve specificity of the flow cytometric crossmatch

Two-color fluorescence cytometry (FCXM) has recently been introduced to improve the detection of anti-HLA antibodies that react to donor cells, especially in recipients receiving kidney allografts. Although this assay system is highly sensitive, it lacks specificity. Between 70% and 90% of potential kidney recipients with a positive FCXM would have been denied transplant if such an assay had been used alone to detect antidonor antibodies. Lack of specificity is principally due to normal or irrelevant IgG in aggregates or immune complexes binding to Fc R receptors on lymphocytes including B cells and a significant subset of T cells. To circumvent this problem, we digested Fc R receptors on lymphocytes with pronase. We present data demonstrating that pronase digestion of lymphocytes does not alter HLA antigenicity. In addition, pronased lymphocytes allow one to use either single- or two-color FCXM. With single-color FCXM, one can quantitate antibody reactivity to lymphocytes via a cursor (on the fluorescence histogram) that separates lymphocytes that do not bind to antibodies. We present data demonstrating that this modification renders FCXM highly sensitive and specific. In addition, one can discriminate between IgG and IgM antibodies that react to lymphocytes.     

Flow cytometry evaluation of urinary sediment in renal transplantation

The value of exfoliative urinary cytology for the diagnosis of different pathological conditions in renal transplantation is widely recognized. The method, however, has not yet gained full acceptance, mainly because identification of the different cells is not always possible by means of standard staining techniques. In view of its characteristics, flow cytometry (FC) seems to represent a consistently reliable, rapid and innovative approach for differentiating the various cells present in the urinary sediment and assessing their number. This study gives the examination result of 223 urinary specimens from 127 transplanted patients selected according to pathology. Sediment cells, collected from fresh urine samples, were washed, treated with a lysing solution, resuspended in saline solution and directly analysed in a FACSCAN cytometer. Morphological evaluation showed: a small number of cells in patients with stable renal function; a larger number of cells, with predominance of lymphocytes, during acute rejection episodes; an absolute predominance of neutrophils during bacterial infection; large-sized cellular debris in cases of post-transplant tubular necrosis; and small cell debris in cases of cyclosporine cytotoxicity. Lymphocyte surface-marker evaluation made it possible to differentiate lymphocyte populations observed during acute rejection episodes (cytotoxic T-cell, CD8 and HLA class II and NK cells) from those detected during bacterial infection (T-cell CD4 positive). These results suggest that urinary FC may be a reliable diagnostic tool in clinical renal transplantation.     

The clinical significance of conversion of complement-dependent cytotoxic T cell crossmatch test after renal transplantation

The purpose of this study was to investigate the clinical relevance of conversion of post-transplant T cell crossmatch between kidney donor and recipient. This study comprises 892 cadaveric renal transplantations performed on 874 adult patients between August 1991 and December 1997. Recipient selection was based on a negative complement-dependent cytotoxic T cell crossmatch test with current ( ≤ 2 months old) serum. For this study, on day 0 and day 14 after transplantation, serum samples were collected for later crossmatching. On day 14 after transplantation, the crossmatch had converted to positive in 76 transplantations (8.5 %). Acute rejection occurred in 50 % of the converters and 22 % of the non-converters (P < 0.005), and graft survival was significantly poorer (P < 0.025), being 85 vs 94 % at 1 and 68 vs 83 % at 5 years, respectively. In patients with delayed graft function, 1-year graft survival was 77 % in the converters and 91 % in the non-converters (P < 0.05). Conversion of T cell crossmatch, especially in connection with delayed graft function, identifies a subgroup of patients at high risk of severe rejection and poor graft survival. Received: 18 February 1999 Received after revision: 18 August 1999 Accepted: 16 September 1999     

尿流式细胞学在诊断移植肾急性排斥反应中的应用价值

目的：探讨尿流式细胞学在诊断移植肾急性排斥反应中的临床应用价值。方法：对43例肾移植受者的116份尿样本进行尿流式细胞学分析，并将急性排斥组和肾功能稳定组的分析结果进行比较。结果：急性排斥反应组尿淋巴细胞总数以及HLA－DR^ 淋巴细胞数显著增多，与肾功能稳定组比较，P＜0．01，CD8^ 细胞亦增多（P＜0．05），而CD3^ ,CD4^ ,CD19^ 细胞数变化两组差异不显著（P＞0．05），在诊断移植肾急性排斥反应上，HLA－DR阳性样本和淋巴细胞数阳性样本的诊断敏感性和特异性分别达95．2％，90．5％，和92．6％，87．4％，结论：尿流式细胞学分析可反映移植肾内的免疫状态，尿淋巴细胞数的显著增多和尿HLA－DR^ 淋巴细胞增多可以作为诊断急性排斥反应的有意义指标。     

Relevance of a positive crossmatch in liver transplantation

We studied 27 liver transplants in 24 patients performed between November 1984 and January 1988. We investigated retrospectively the importance of donor reactive HLA class I and class II and of non-HLA antibodies for graft survival in these patients. In order to determine the specificity and class of the antibodies, we used monoclonal antibodies to HLA-A,-B,-C and DR and DQ antigens to block cytotoxicity of sera and the reagent dithiothreitol to characterize the immunoglobulin class. We found that humoral immunity to HLA antigens in liver-grafted patients, demonstrable as the presence of cytotoxic antibodies reactive with donor splenic T and/or B cells in the pretransplantation period, is associated with significantly lower graft survival as compared with patients without demonstrable preformed HLA antibodies (P=0.01). In addition we found that a substantial proportion of patients had donor-reactive cytotoxic antibodies which were not HLA specific. Thus, our study shows that HLA immunity can influence liver allograft survival, and that it is useful to have patient cytotoxic antibodies characterized with regard to HLA reactivity prior to transplantation.     

Lack of correlation between IgG T-lymphocyte flow cytometric crossmatches with primary renal allograft outcome

The flow cytometric crossmatch (FCXM) has been reported to be more sensitive and capable of detecting very low levels of antibodies than the normally used complement dependent cytotoxicity test. We studied both the two colour IgG T cell FCXM and CDC-XM in 146 renal allograft recipients, 111 primary and 35 regrafts, of which 26 % (29/111) of 1st and 20 % (7/35) of regrafts had a positive FCXM. There was no overall correlation between the FCXM results and early graft outcome in primary renal allografts. The FCXM did not appear to have any advantage over the CDC-XM in predicting graft outcome in unsensitized first grafts. In the small number of regrafts studied, a positive FCXM was associated with a higher degree of graft failure. FCXM can exhibit false negative results if sera are used solely neat although these prozone phenomena do not influence subsequent graft outcome.