Clinical significance of M-type phospholipase A2 receptor and thrombospondin Type 1 domain-containing 7A in primary membranous nephropathy.
Zailiang GongShuguang YuanXuejing ZhuYouliang WangFang YuDanyi YangXiangqing XuHong LiuJun LiLin Sun
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To evaluate the value of thrombospond in Type I domain-containing 7A (THSD7A) and M-type phospholipase A2 receptor (PLA2R) in primary membranous nephropathy (PMN) and to explore the relationship between their antibody levels and prognosis.Renal tissues in 128 patients with membranous nephropathy in the Second Xiangya Hospital of Central South University were collected from February 2015 to August 2017, including 108 patients with primary membranous nephropathy (PMN group) and 20 patients with secondary membranous nephropathy (SMN) (SMN group). Indirect immunofluorescence method was used to detect the expression of PLA2R antigen in kidney tissues,and the glomerular expression of THSD7A antigen was examined by immunohistochemistry and indirect immunofluorescence. The serum levels of anti-PLA2R antibodies and THSD7A antibodies were also detected by ELISA. According to the results of PMN examination,the patients were also divided into a PLA2R-related membranous nephropathy group and a THSD7A-related membranous nephropathy group.The positive rate of PLA2R in the renal tissues in the PMN group was higher than that in the SMN group (78% in the PMN group, 35% in the SMN group, P<0.01),while the positive rate of anti-PLA2R antibody in the PMN group was also higher than that in the SMN group (50% in the PMN group, 25% in the SMN group, P<0.05).The serum level of anti-PLA2R antibody was positively correlated with 24 h urine protein (r=0.254, P<0.05) and negatively correlated with serum albumin (r=-0.236, P<0.05). The expression of THSD7A was positive in glomeruli in 7 cases of the PMN group (6%) by immuno-histochemistry, and which was positive in 1case of the SMN group (5%).The serum levels of anti-THSD7A antibody in the PMN group were higher than those in the SMN group [(0.49±0.26) pg/mL in the PMN group,(0.34±0.27) pg/mL in the SMN group, P<0.05]. There was no difference in the clinical characteristics between the PLA2R-related membranous nephropathy group and the THSD7A-related membranous nephropathy group.PLA2R and THSD7A are the target antigen of PMN, and the associated autoantibodies are helpful for the differential diagnosis of PMN. The anti-PLA2R antibody levels can reflect the severity of the disease and evaluate the effect of treatment. The incidence of THSD7A membranous nephropathy is low, and monitoring the serum anti-THSD7A antibody levels can assess patients' condition and predict disease outcome.目的: 探讨M型磷脂酶A2受体(phospholipase A2 receptor，PLA2R)、1型血小板反应蛋白7A域(thrombospondin Type 1 domain-containing 7A，THSD7A)及相应的抗体对特发性膜性肾病(primary membranous nephropathy，PMN)的诊断价值及其血清抗体水平与预后的关系。方法: 纳入2015年2月至2017年8月中南大学湘雅二医院肾内科行肾活检确诊的膜性肾病128例，其中PMN108例(PMN组)，继发性膜性肾病(secondary membranous nephropathy，SMN)20例(SMN组)。应用间接免疫荧光法检测肾组织PLA2R抗原表达，间接免疫荧光法和免疫组织化学法检测肾组织THSD7A抗原表达，ELISA检测血清PLA2R和THSD7A的抗体水平。根据检测结果，又将患者分为PLA2R相关性膜性肾病组和THSD7A相关性膜性肾病组。结果: PMN组肾小球PLA2R阳性率明显高于SMN组(分别为72%和35%，P<0.01)，PMN组血清PLA2R抗体阳性率亦明显高于SMN组(分别为50%和25%，P<0.05)。PMN组血清PLA2R抗体与24 h尿蛋白量呈正相关关系(r=0.254，P<0.05)，与血清白蛋白呈负相关关系(r=-0.236，P<0.05)。PMN组中7例(6%)患者肾小球THSD7A呈阳性，SMN组有1例(5%)患者肾小球THSD7A呈阳性。PMN组血清THSD7A抗体水平明显高于SMN组[分别为(0.49±0.26)和(0.34±0.27) pg/mL，P<0.05]。7例THSD7A相关性膜性肾病组与86例PLA2R相关性膜性肾病组的临床病理特征差异均无明显统计学意义(均P>0.05)。结论: 肾组织PLA2R和THSD7A是PMN的靶抗原，其血清抗体有助于PMN的鉴别诊断；PLA2R抗体水平可反映病情严重程度，可用来评估治疗效果；PMN患者THSD7A相关性膜性肾病的发病率低，监测血清THSD7A抗体水平可评估患者病情，并可预测疾病转归。.Keywords:
Membranous Nephropathy
Immunofluorescence
Introduction. Peritubular capillary complement 4d staining is one of the criteria for the diagnosis of antibody-mediated rejection, and research into this is essential to kidney allograft evaluation. The immu- nofluorescence technique applied to frozen sections is the present gold-standard method for complement 4d staining and is used routinely in our laboratory. The immunohistochemistry technique applied to paraffin-embedded tissue may be used when no frozen tissue is available. Material and Methods. The aim of this study is to evaluate the sensitivity and specificity of immunohis- tochemistry compared with immunofluorescence. We describe the advantages and disadvantages of the immunohistochemistry vs. the immunofluorescence technique. For this purpose complement 4d staining was performed retrospectively by the two methods in indication biopsies (n=143) and graded using the Banff 07 classification. Results. There was total classification agreement between methods in 87.4% (125/143) of cases. How- ever, immunohistochemistry staining caused more dif- ficulties in interpretation, due to nonspecific staining in tubular cells and surrounding interstitium. All cases negative by immunofluorescence were also negative by immunohistochemistry. The biopsies were classified as positive in 44.7% (64/143) of cases performed by immunofluorescence vs. 36.4% (52/143) performed by immunohistochemistry. Fewer biopsies were classified as positive diffuse in the immunohistochemistry group (25.1% vs. 31.4%) and more as positive focal (13.2% vs. 11.1%). More cases were classified as negative by immunohistochemistry (63.6% vs. 55.2%). Study by ROC curve showed immunohistochemistry has a speci- ficity of 100% and a sensitivity of 81.2% in relation to immunofluorescence (AUC: 0.906; 95% confidence interval: 0.846-0.949; p=0.0001). Conclusions. The immunohistochemistry method presents an excellent specificity but lower sensitivity to C4d detection in allograft dysfunction. The evalu- ation is more difficult, requiring a more experienced observer than the immunofluorescence method. Based on these results, we conclude that the immu- nohistochemistry technique can safely be used when immunofluorescence is not available.
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134 liver biopsies obtained from 124 patients with various diseases of the liver were examined by the direct immunofluorescent technique for the Australia antigen. FITC-labelled IgG fractions prepared from three different sera containing antibody to the Australia antigen were used. Controls included FITC-labelled IgG fractions of sera containing antibodies against human immunoglobulins, complement factors and herpes simplex virus.
The three antibodies to the Australia antigen employed showed identical fluorescent patterns and reacted with the same biopsies. In 68 biopsies prepared and examined within 12 hr, speckled or granular fluorescence was noted in the nuclei of the hepatocytes in all cases irrespective of the clinical or histological diagnosis or the serological reaction for the Australia antigen. The antigen in the nuclei of the hepatocytes proved to be very labile.
It is concluded that IgG prepared from sera containing antibodies to the Australia antigen may have two antigenic specificities, one precipitating with the Australia antigen in serum and another reacting by immunofluorescence with a labile antigen which appears to be a constituent of normal liver cell nuclei.
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The needle biopsies from 60 transplanted and native kidneys have been processed and a prospective analysis of pattern, intensity and distribution of immunoglobulin deposits (IgA, IgG and IgM) and complement components (C3c and C1q) identified in these lesions has been carried out by immunohistochemistry with three step immunoperoxidase, in the period from 2000 to 2004. Those deposits were previously detected and analyzed by immunofluorescence. The samples consisted of 30 renal biopsies, previously diagnosed with glomerulonephritis and positive immunofluorescence and 30 renal biopsies without morphologic changes and deposits on immunofluorescence. 78,7% of the analyzed samples showed the identical results of the deposits of immunoglobulin and components of the complement with both, immunohistochemistry and immunofluorescence method. Sensitivity of the immunohistochemistry method with three step immunoperoxidase for all analyzed immunoglobulin and complement components is high (0,93), while specificity for the same method is 0,79. Standardized method of the three step immunoperoxidase on the paraffin embedded, formalin fixed needle renal biopsies could successfully replace the immunofluorescence method in diagnostic of GN, with the emphasis on a follow up and control of each single step in the procedure of the method.
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Immunohistochemistry is a widely used technique for research and diagnostic purposes that relies on the recognition by antibodies of antigens expressed in tissues. However, tissue processing and particularly formalin fixation affect the conformation of these antigens through the formation of methylene bridges. Although antigen retrieval techniques can partially restore antigen immunoreactivity, it is difficult to identify antibodies that can recognize their target especially in formalin-fixed paraffin-embedded tissues. Most of the antibodies currently used in immunohistochemistry have been obtained by animal immunization; however, in vitro display techniques represent alternative strategies that have not been fully explored yet. This review provides an overview of phage display-based antibody selections using naïve antibody libraries on various supports (fixed cells, dissociated tissues, tissue fragments, and tissue sections) that have led to the identification of antibodies suitable for immunohistochemistry.
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The fluorescence activated cell sorter (FACS) was used for detecting circulating antibodies to the surface antigens on isolated colon epithelial cells (anti-colon antibodies) by indirect immunofluorescence. Anti-colon antibodies were found in the serum of 30 of 41 (73%) patients with ulcerative colitis. This incidence is much higher than one established in earlier reports by application of indirect immunofluorescence to colon tissue using the fluorescence microscope. The results suggest that FACS analysis is very useful for detecting antibodies to colon specific antigen.
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Double-label fluorescent immunohistochemistry (IHC) is frequently used to identify cellular and subcellular co-localization of independent antigens. In general, primary antibodies for double labeling should be derived from independent species. However, such convenient pairs of antibodies are not always available. To overcome this problem, several methods for double labeling with primary antibodies from identical species have been proposed. Among them are methods using monovalent secondary antibodies, such as Fab fragments. Soluble immune complexes consisting of primary and monovalent secondary antibodies are first formed. After absorption of the excess secondary antibody with nonspecific immunoglobulin, the immune complexes are applied to sections. By this procedure, unwanted cross-reaction between false pairs of antibodies is avoidable. However, soluble immune complexes often show reduced or no immunoreactivity to antigens on sections. I noted that antigen retrieval (AR) of tissues by heating often but not always showed improved immunoreactivity for soluble immune complexes. Here I demonstrate the examination of conditions for this soluble immune complex method using AR-treated sections and show examples of double-label fluorescent IHC with identical species-derived primary antibodies.
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Immunohistochemistry on mouse tissue utilizing mouse monoclonal antibodies presents a challenge. Secondary antibodies directed against the mouse monoclonal primary antibody of interest will also detect endogenous mouse immunoglobulin in the tissue. This can lead to significant spurious staining. Therefore, a “mouse-on-mouse” staining strategy is needed to yield credible data. This paper presents a method that is easy to use and highly flexible to accommodate both an avidin-biotin detection system as well as a biotin-free polymer detection system. The mouse primary antibody is first combined with an Fab fragment of an anti-mouse antibody in a tube and allowed sufficient time to form an antibody complex. Any non-complexed secondary antibody is bound up with mouse serum. The mixture is then applied to the tissue. The flexibility of this method is confirmed with the use of different anti-mouse antibodies followed by a variety of detection reagents. These techniques can be used for immunohistochemistry (IHC), immunofluorescence (IF), as well as staining with multiple primary antibodies. This method has also been adapted to other models, such as using human antibodies on human tissue and using multiple rabbit antibodies in dual immunofluorescence.
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