Formalin-resistant leukocyte differentiation antigens in dermatohistopathology

The diagnosis of cutaneous leucocytic and particularly lymphocytic infiltrates with a considerable degree of reliability, has become possible owing to the use of monoclonal antibodies against leucocyte differentiation antigens. This method of diagnosis depends heavily on the availability of frozen biopsies, however, since most antigens are destroyed by the routine tissue processing, including formol fixation, paraffin embedding and alcohol dehydration. In recent years an increasing number of antibodies has become available that allow the detection of formalin-resistant leucocyte differentiation antigens and thus can be used on conventional paraffin sections. When using such antibodies, which will be invaluable in routine diagnosis and, furthermore, will allow the investigation of old biopsies, the dermatopathologist must be familiar with the exact distribution of the detected antigen and should be aware of crossreactivities with other structures. This review presents the most important antibodies and gives hints on their rational and economical use.     

巢式PCR检测石蜡包埋组织中着色霉、孢子丝菌和马尔尼菲青霉

目的 评价巢式PCR对不同深部真菌感染组织石蜡包埋标本检测的价值。方法 收集着色芽生菌病、孢子丝菌病、马尔尼菲青霉病及其他深部真菌病石蜡组织标本共44份,行组织病理观察并提取石蜡组织标本中DNA。使用针对着色霉、孢子丝菌及马尔尼菲青霉核糖体DNA特定区域的特异性巢式PCR引物,分别对所提取的真菌DNA进行扩增。分析巢式PCR对这3种病原真菌扩增的敏感性和特异性,并与组织病理检查方法比较。结果 20例着色芽生菌病组织蜡块中8例扩增阳性,10例孢子丝菌病组织蜡块中7例扩增阳性,10例马尔尼菲青霉病组织蜡块均扩增阳性,其余对照深部真菌病组织蜡块扩增均为阴性,巢式PCR检测3种真菌的敏感性分别为40%、70%和100%,特异性均达到100%。组织病理检查3种真菌的阳性率分别为95%、70%、80%。结论 巢式PCR扩增石蜡包埋组织中的真菌DNA是诊断深部真菌病的一种方法,尤其适用于诊断马尔尼菲青霉病。     

A case of cicatricial pemphigoid with circulating IgA and IgG antibodies directed against 280 kD, 165 kD and 120-130 kD epidermal antigens

A case of subepidermal autoimmune blistering disease in an 86-year-old woman is reported. Clinical features were those of a cicatricial pemphigoid, with prominent mucosal involvement leading to conjunctival and nasal scarring. Direct immunofluorescence findings were consistent with either cicatricial pemphigoid or linear IgA dermatosis, since both IgG and IgA linear deposits were found at the basal membrane zone. Immunoelectron microscopy of perilesional skin revealed IgA deposits within the lamina lucida and immunoblotting of the patient's serum disclosed IgA and IgG antibodies directed against epidermal antigens of 280, 165 and 120-130 kD.     

Basal Cell Carcinoma with Eccrine Differentiation

We describe a light, electron microscopic, and immunohistochemical study of basal cell carcinoma with eccrine differentiation. The cytoplasm did not stain with fat stain on cryostat sections, but contained numerous gland-like structures. Immunohistochemistry on formalin fixed, paraffin embedded tissues using antibodies to human involucrin, thirteen kinds of cytokeratin, S-100 protein, and carcinoembryonic antigen (CEA) was performed. We found that tumor cells were positive for keratin, PKK1, MA-903, No 8, No 19, AE 1, AE 3, and 5+8. Tumor cells were negative for S-100 protein, CEA, and the other antigens examined. Electron microscopy demonstrated short microvilli and amorphous materials within the intracytoplasmic cavity. We concluded that this tumor is basal cell carcinoma with eccrine differentiation.     

Detection of immunoglobulin G antibodies in melanoma sera reactive with intracellular proteins

Immunoglobulin G (IgG) antibodies reactive with intracellular components of transformed cells were detected in 26/35 sera from patients with melanoma using immunofluorescence and/or Western blotting. By extracting cellular proteins with either sodium dodecyl sulphate or moderate concentrations of salt (400 mM NaCl), the protein antigens were partially characterized by immunoblotting procedures. Although considerable heterogeneity in the molecular weights of the protein antigens was observed, two common groups were delineated. The anti-Pol antibodies reacted with 30 kd cytoplasmic protein and the anti-Ca antibodies recognized acidic high molecular weight (75-95 kd) proteins. These antigens were detected in all transformed cell lines tested, but were not restricted to them. Anti-Ca and anti-Pol antibodies were not found in sera from patients with other solid tumors or in systemic lupus erythematosus.     

Wheat protein antibodies in dermatitis herpetiformis

An enzyme-linked immunosorbent assay was used to detect class-specific antibodies to wheat protein antigens. Antibodies which we detected by this technique reacted indistinguishably with antigens prepared from crude gluten, crude gliadin, alpha-gliadin, Frazer fraction III, and subfraction B and B3 of Frazer fraction III. No sera reacted with a human serum albumin control antigen. The prevalence of IgG antibodies to wheat protein antigens was significantly greater in patients with gluten sensitive enteropathy, 12 of 17, (p = .00011) and in patients with dermatitis herpetiformis, 5 of 14, (p = .046) than in normal control subjects. Strongly positive reactions for IgG antibodies were present only in patients with gluten sensitive enteropathy or dermatitis herpetiformis. IgA antibodies to wheat protein antigens were found only in gluten-sensitive enteropathy patients. We have found this to be a sensitive, precise technique for measurement of antibodies to wheat protein antigens and feel that it will prove useful in evaluation of the role of immune complexes involving wheat protein antigens and their antibodies in the pathogenesis of dermatitis herpetiformis.     

Monoclonal antibody labeling of mononuclear cell surface antigens in formaldehyde-fixed paraffin-embedded cutaneous tissue

The influence of the sequential stages of conventional formaldehyde fixation and paraffin embedding of cutaneous tissue on monoclonal antibody labeling of cell surface antigens is described. The effects of variation in fixation time, dehydration, clearing, wax embedding, and enzyme treatment of cutaneous sections were examined. By curtailing fixation time, using cold ethanol dehydration, and limited cold clearing with xylene, immunoreactivity of several important monoclonal antibodies was retained. Wax embedding could be achieved at 58 degrees C for 1 h or by using low-melting-point wax at 42 degrees C for 3 h. Thus was derived an optimal processing procedure which afforded good tissue morphology and allowed reliable reproducible labeling by monoclonal antibodies to cell surface antigens.     

原位杂交法检测尖锐湿疣中人乳头瘤病毒DNA型别

以非放射性地高辛配基标记人乳头瘤病毒（ＨＰＶ）６ｂ、１１、１６、１８型探针，用原位杂交法检测了重庆地区５０例经临床病理学诊断为尖锐湿疣的石蜡组织切片中ＨＰＶＤＮＡ型别，杂交条件分别采用严格条件（Ｔｍ－１２℃）和非严格条件（Ｔｍ－３５℃）进行。结果：尖锐湿疣中ＨＰＶＤＮＡ阳性率为８２％，其中ＨＰＶ６ｂ、１１、１６型分别为６２％、２４％、１０％，ＨＰＶ１８型未检出。杂交信号主要分布于表皮浅中层，且许多分布于空泡细胞内，但并非所有杂交信号均在空泡细胞内。     

建立巢式PCR和异源双链法检测石蜡标本麻风菌DDS耐药株

目的：为了能检出石蜡包埋组织中麻风菌氨苯砜（DDS）的耐药基因folPl，有必要建立敏感、特异的巢式PCR，为开展回顾性麻风菌DDS耐药流行病学研究服务。方法：从石蜡组织中提取麻风菌DNA，建立巢式PCR，优化PCR条件，扩增DDS耐药基因片段。用异源双链法筛选突变菌株，并经直接测序进一步证实。结果：巢式PCR将PCR检测folPl的敏感性，从6．5％（3／46）提高到76．7％（33／43）；优化后的巢式PCR，最终使敏感性更进一步提高至90．7％（39／43）。8株folPl突变菌、两种突变型被发现。结论：巢式PCR是一项快速、简便、可行的方法。通过优化多项PCR条件，提高了石蜡包埋组织中folPl检测的敏感性和特异性。异源双链法可用于筛选DDS耐药突变菌株。     

CCL18在恶性黑素瘤中的表达及其与血管内皮生长因子、Ki67表达的相关性研究

目的：探讨趋化因子配体18（CCL18）在恶性黑素瘤（恶黑）组织中的表达和临床意义,与血管内皮生长因子（VEGF）、Ki67表达的相关性。方法用免疫组化方法检测58例恶黑石蜡标本中CCL18、VEGF及细胞增殖核抗原Ki67的表达,同时检测20例色素痣石蜡标本中CCL18的表达水平。对CCL18的表达与恶黑临床病理及VEGF、Ki67的表达进行相关性分析。用免疫荧光方法验证CCL18在恶黑组织中的表达。结果 CCL18在恶黑组阳性率为84.48%（49/58）,而在色素痣组均无表达,差异有统计学意义（χ2=45.46,P<0.01）。CCL18在恶黑的表达与肿瘤Clark分级、Breslow厚度呈正相关（rs值分别为0.609、0.644,均P<0.01）;在有无溃疡以及有无淋巴结转移组间差异有统计学意义（均P<0.05）。CCL18的表达在不同性别、年龄、肢端/非肢端恶黑患者组间差异无统计学意义（均P>0.05）。恶黑中CCL18阳性表达水平与VEGF表达水平呈正相关（rs=0.727,P<0.05）,与Ki67表达水平无相关（P>0.05）。免疫荧光显示,恶黑组织中肿瘤细胞胞质表达CCL18。结论 CCL18在恶黑组织中高表达,与肿瘤侵袭、转移有一定关系。     

Linear IgA disease: the IgA and IgG response to the epidermal antigens demonstrates that intermolecular epitope spreading is associated with IgA rather than IgG antibodies, and is more common in adults

BACKGROUND: Linear IgA disease (LAD; adult and childhood) is a dapsone-responsive, acquired immunobullous disorder mediated by IgA antibodies directed at target antigens within the epithelial basement membrane. These antigens have not been completely characterized. OBJECTIVES: To identify the target antigens in LAD, and to correlate these with the antibody isotype. METHODS: We used 101 LAD sera without IgG antibodies detected by indirect immunofluorescence. The sera were analysed by immunoblotting for IgA (65 adults and 36 children) and IgG (61 adults and 34 children) autoantibodies, on salt-split, urea-extracted epidermal tissue extracts. RESULTS: Antigens were targeted in LAD by IgA antibodies (54 adults and 23 children), IgG antibodies (34 adults and 19 children), and both isotypes (30 adults and 16 children). Three major antigens were recognized by IgA antibodies: LAD285 (22 adults and three children), BP230 (30 adults and eight children) and BP180 (collagen XVII), including the 97-kDa ectodomain (52 adults and 20 children). Seven 'minor' antigens were occasionally detected (18 adults and 13 children). IgA antibodies bound multiple antigens (33 adults and nine children) more frequently than single antigens (21 adults and 14 children), but the binding to multiple antigens was more restricted in children than in adults. IgG antibodies mainly bound a single antigen (29 adults and 16 children), predominantly BP180. CONCLUSIONS: There was variation in the autoantibody response within the disease and the patient, with regard to target molecules and autoantibody class. The finding that IgG as well as IgA autoantibodies predominantly target BP180 supports a pivotal role for collagen XVII in adult and childhood LAD. The IgG response was very restricted compared with IgA autoantibodies (P < 0.01). Autoantibodies from children had a more restricted antigen repertoire than from adults (P < 0.05). Epitope spreading is common in LAD and is affected by the class of autoantibody and age of the patient.     

Granulomatous slack skin: a clinicopathological and immunohistochemical study of three cases

Three cases of granulomatous slack skin (GSS), a rare variant of T-cell lymphoma, are reported. Immunohistochemical studies using a panel of 16 antibodies were carried out on both frozen tissue and tissue embedded in paraffin wax to characterize the infiltrate. A routine immunoperoxidase technique was used to identify T cells (UCHL1, CD45R0), B cells (L26, 4KB5 [CD45R]), S100 protein-positive cells, monocytes/macrophages (Mac-387, KP1 [CD68]), and dermal dendrocytes (factor XIIIa) in paraffin sections. A close association was found between UCHL1-positive T cells and KP1-positive giant cells. A number of S100-positive cells and factor XIIIa-positive cells were present in the infiltrate from all three patients. The lymphocytes in two of the patients were predominantly of the helper T-cell phenotype. Giant cells from all three patients stained with KP1 (CD68) and Leu M3 (CD14). These studies confirm that the infiltrate in GSS is predominantly a T-cell disorder associated with monocyte-derived cells rather than with resident dendritic macrophages.     

Pigmented neuroectodermal tumor of infancy. A light microscopic and immunohistochemical study

We studied two cases of pigmented neuroectodermal tumor of infancy (PNTI) by routine light microscopy and immunohistochemistry on formalin fixed, paraffin embedded tissues using antibodies to HMB-45 "melanoma associated" antigen, S-100 protein, neuron specific enolase (NSE), Leu-7 antigen, chromogranin, epithelial membrane antigen, collagen Type IV, alpha-fetoprotein and muscle-specific actin and to the intermediate filaments cytokeratin (CK), vimentin, desmin and neural filaments. We found that the large epithelioid cells, many of which contained melanin pigment, were strongly positive for CK and HMB-45, and less intensively positive for vimentin and NSE. The small neuroblast-like cells revealed only focal, weak NSE positivity. Both cell types were negative for S-100 protein and for the other antigens examined. Our results suggest that: (1) the large and small cell populations in PNTI have different immunophenotypes; (2) the expression of CK and HMB-45, together with the S-100 negativity, appears unique for the pigmented cells; and (3) this profile may be helpful in the exclusion of melanoma and peripheral neuroblastoma from the differential diagnosis.     

Immunoperoxidase techniques applied to dermatopathology

Immunoperoxidase techniques provide the pathologist with the capability for staining a wide range of antigens in tissue sections. More than 100 different antigens have been successfully demonstrated in fixed paraffin sections; other antigens can only be visualized in frozen sections. This latter group particularly includes lymphocyte surface antigens detectable by monoclonal antibodies. This review describes the current state of the art and provides several illustrations of the use of monoclonal antibodies for the identification of T-lymphocyte phenotypes in frozen section from cases of leprosy, mycosis fungoides, halo nevus, Kaposi's sarcoma, lichen planus and atopic dermatitis. Technical details and potential applications are discussed. The growing availability of commercial immunostaining kits makes these techniques more accessible to the surgical pathologist; indeed a whole new range of truly specific, special stains are available, as pathologists we must simply learn to use them.     

皮肤鳞状细胞癌中FHIT蛋白的检测及其临床意义

目的探讨皮肤鳞状细胞癌(SCC)组织中脆性组氨酸三联体(FHIT)蛋白的表达及临床意义。方法应用免疫组化SP法,检测32例SCC及12例正常皮肤组织标本中FHIT蛋白的表达。结果SCC及正常皮肤组织中FHIT蛋白的阳性表达率分别为43.75%(14/32),100%(12/12),两者差异有显著性意义(P<0.001)。淋巴结转移组和无淋巴结转移组FHIT的阳性表达率分别为30.4%(7/23)和77.78%(7/9),两组差异有显著性意义(P<0.05)。结论FHIT在SCC的发生、发展中起重要作用,检测SCC中FHIT蛋白的表达有助于了解其发生、发展及转移的机制,为临床治疗提供理论依据。     

胎儿头皮毛囊的黑素细胞定位及精细结构观察

目的 探讨胎儿毛囊黑素细胞的定位及精细结构。方法 6个月胎儿因宫内发育畸形而引产、死亡后，取其带毛头皮，一部分常规包埋切片，分别用NKI/beteb、HMB-45、酪氨酸酶、酪氨酸酶相关蛋白1（TRP1）单抗染色。另一部分无菌处理后，0.1 g/L的胶原酶Ⅱ和胰酶消化获得毛囊细胞，培养并传代后，透射电镜和原子力显微镜观察。结果 胎儿头皮毛囊NKI/beteb阳性细胞位于外根鞘，而在毛球内许多细胞HMB-45、酪氨酸酶、TRP1单抗染色阳性。在毛囊细胞的体外培养中，除去成纤维细胞和角质形成细胞后，可见两种黑素细胞，一种数目极少，色素很多，传代后消失；另一种数目较多，开始无色素，但增殖很快。传第3代后，几乎所有细胞NKI/beteb染色阳性。扫描电镜和原子力显微镜下，多数细胞为双极梭形，偶尔有3个树突。细胞体呈圆形或卵圆形，极突上无明显的分支，其内有少数散在的黑素体。结论 胎儿头皮毛囊外根鞘的黑素细胞推测为成黑素细胞和（或）其子代细胞。在早期的体外培养中，细胞增殖很快，但形态及功能上不成熟。     

Detection of high-risk human papillomavirus type 16/18 in cutaneous warts in immunocompetent patients, using polymerase chain reaction

Cutaneous warts are caused by human papillomavirus (HPV). Prevalence studies of the types of HPV present in cutaneous warts have been carried out more frequently in immunosuppressed patients. The present study was designed to study the association of high-risk HPV in cutaneous warts of immunocompetent patients. A total of 45 cases of cutaneous warts from various sites in immunocompetent subjects were analyzed for HPV. Samples included both archival material i.e., paraffin embedded and fresh tissue. Highly sensitive and comprehensive polymerase chain reaction (PCR) methodology for detection of HPV of high oncogenic potential, HPV 16/18, was employed. Human papillomavirus 16 was detected in 3 (6.6%) patients. None of the lesions demonstrated HPV 18. None of the cutaneous warts demonstrated histopathological features associated with dysplasia or neoplasia. The identification of HPV 16 in cutaneous warts, which are benign proliferations of the skin, further expands the spectrum of HPV-linked lesions. It remains of critical interest to determine whether these types are specifically associated with the development of malignant lesions analogous to those seen in anogenital cancer.     

HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin-embedded tissue specimens

AIM: This study was undertaken to assess whether heat-shock protein (HSP)47 is a useful cell marker for skin fibroblasts in formalin-fixed, paraffin-embedded skin specimens. BACKGROUND: HSP47, a 47-kDa HSP, is a collagen-specific molecular chaperone localized in the endoplasmic reticulum. HSP47 plays an essential role in collagen biosynthesis in skin fibroblasts. METHODS: Immunohistochemistry was performed to detect HSP47 in skin fibroblast cultures and skin tissue sections. RESULTS: Immunostaining for HSP47 clearly detected skin fibroblasts in paraffin tissue sections as well as in fibroblast cultures and frozen tissue sections. HSP47 staining on paraffin sections from diseased skin specimens revealed that skin ulcer, keloid, nodular fascitis, spindle cell lipoma, and dermatofibroma had strong signals for HSP47 compared with the signals obtained from normal skin. Dermatofibrosarcoma protuberans had many HSP47 positive cells, but signals on individual cells were not as strong as those seen from the above benign proliferative disease samples. In neurofibroma, a small number of faintly positive cells were detected. Our double-immunostaining studies also demonstrated that HSP47 staining distinguished skin fibroblasts from CD68-positive histiocytes/macrophages, factor VIII-related antigen-positive endothelial cells, or factor XIIIa-positive dermal dendritic cells. CD34-positive interstitial cells coexpressed HSP47 in spindle cell lipoma. CONCLUSIONS: These findings indicate that HSP47 staining can detect skin fibroblasts in routine, paraffin-embedded specimens. A panel approach using HSP47 and other cell markers on paraffin sections may help the identification of the cell type involved with mesenchymal proliferative disorders.     

Bullous pemphigoid: correlation of mucosal involvement and mucosal expression of autoantigens studied by indirect immunofluorescence and immunoblotting

Twenty patients with bullous pemphigoid were studied prospectively: sequential sera, in different phases of the disease, were collected over a period of approximately 2 years. The sera were tested using standard immunofluorescence techniques with salt-split and intact human tissue from different sites of the body (thigh, breast, oral mucosa, vagina); an early serum of each patient was tested by Western blotting. The concentration of circulating antibodies detected by the intact skin and intact mucous membranes was similar; split tissue was more sensitive than intact tissue. For eight of 19 patients, split vagina and occasionally split oral mucosa (in the same patients) were much less sensitive than all other tissues. Furthermore, there was a correlation between autoantibody reactivity with split mucous membrane tissues and clinical mucosal involvement. These results strongly suggest heterogeneity of antigens or epitopes expressed between tissues. In both split skin and mucosa all sera consistently detected an antigen on the epidermal side of the split regardless of the stage of the disease. Immunoblotting studies showed no correlation between specific antigens and mucosal expression or skin involvement.     

Use of monoclonal antibodies (UCHL1, Ki-B3) against T and B cell antigens in routine paraffin-embedded skin biopsy specimens

Diagnosis and classification of cutaneous lymphoid infiltrates are often hampered by lack of frozen tissue to allow detailed immunophenotyping. Therefore antibodies that recognize antigens resistant to routine tissue processing would be of value. In this study we present a detailed analysis of two such antibodies against CD45R antigens, UCHL1 and Ki-B3, that detect predominantly T or B cells, respectively. Tumor cells in 125 cases of various nonlymphatic skin tumors did not react with either UCHL1 or Ki-B3. Examination of 85 biopsy specimens from 13 different inflammatory skin diseases demonstrates that most reactive T cells in the skin carry the UCHL1 antigen, as controlled by simultaneous immunophenotyping in frozen tissue. With few exceptions, UCHL1 also detected the tumor cells in 28 cases of cutaneous malignant T cell lymphomas. In contrast, Ki-B3 failed to detect tumor cells in seven of eight cases of malignant B cell lymphomas of the skin.