肝脏上皮样血管内皮瘤穿刺标本的病理诊断与鉴别诊断

目的 提高对经皮肝穿刺活检标本中上皮样血管内皮瘤(EH)的认识.方法 收集北京肿瘤医院1999-2010年3016例肝穿刺活检标本中5例诊断EH的病例,回顾性分析5个病例的光镜下病理形态学特点、免疫组织化学(polymer二步法)检测结果,以及患者的临床表现、实验室检查和影像学结果.结果 5例均为女性,年龄23～47岁,平均39岁.4例B超显示肝多发实性肿物并考虑转移癌;实验室检查各项指标未见明显异常.光镜下肿瘤细胞上皮样、排列成短条索状或小巢状,包埋于特征性黏液玻璃样基质中,可见明显的胞质内空泡;瘤细胞形态温和,未见核分裂;免疫组织化学表达血管标志物CID31、CD34、第八因子相关抗原,偶见表达平滑肌肌动蛋白.结论肝穿刺活检标本中,如果病变具有典型的EH形态特点和免疫组织化学表达,同时又有临床及影像学支持,即可明确诊断;应用多种抗体组合有助于肝穿刺活检标本EH的诊断与鉴别诊断.

Abstract：

Objective To evaluate the pathologic diagnosis of hepatic epithelioid hemangioendothelioma (EH) in needle biopsy specimens. Methods Five cases of hepatic EH diagnosed in needle biopsies encountered during the period from 1999 to 2010 in Beijing Cancer Hospital were retrospectively reviewed. The specimens were formalin-fixed, paraffin-embedded and stained with hematoxylin and eosin. Immunohistochemical study was also carried out. Results All the 5 patients were females. The age ranged from 23 to 47 years ( mean = 39 years). The tumors in 4 patients were multiple and diagnosed as "metastasis" on ultrasound examination. The blood test results in all of the 5 patients were normal. Histologically, the tumor cells had an epithelioid appearance and were arranged in cords, solid nests or isolation, amongst a myxoid or hyaline matrix. The tumor cells contained scattered intracytoplasmic vacuoles which sometimes harbored red blood cells. There was no evidence of significant cellular pleomorphism, high mitotic activity and necrosis. Immunohistochemically, all of the 5 cases were positive for at least two endothelial markers (CD31, CD34 and factor Ⅷ-related antigen). Smooth muscle actin was expressed in 1 case. Conclusions The diagnosis of hepatic EH can be established in needle biopsy specimens. The histologic pattern, when coupled with immunohistochemical findings, is useful in arriving at the correct diagnosis.     

Detection of Hantavirus gene from peripheral blood of patients with HFRS in Heilongjiang province and the epidemiological significance

The aim of this study is to further understand the genotype of Hantavirus(HV) from peripheral blood of patients with hemorrhagic fever with renal syndrome (HFRS) and the epidemiological significance of this disease in Heilongjiang province in recent years. Thirty-one serum samples of clinically diagnosed patients with HFRS were examined by RT-PCR to decide the genetic subtype. On the basis of infection season, the serum samples were divided into two groups: winter (Nov, 2003—Feb, 2004) , spring and summer (April, 2004—Sep, 2004). Further analysis was performed in combination with clinical symptoms. It was found that among the total 31 samples, 22 were sero-positive. Among 14 serum samples in winter, 8 were sero-positive, of which 5 cases were of typeⅠ(Hantaan virus, HTNV) and 3 of typeⅡ(Seoul virus, SEOV). Among 17 samples in spring and summer, 14 were sero-positive, of which 5 cases were of typeⅠand 9 of typeⅡ. So it concludes that both of the two types of Hantavinis exist in Heilongjiang. The typeⅠis the main pathogen of HFRS in winter, and typeⅡis the main in spring and summer.     

Elementary study on the antigenicity of SARS-CoV Mc region

To test the antigenic activity of M protein (Mc protein) in the inner membrane of SARS-CoV, SARS-CoV Mc protein's bases locating inside the membrane were cloned, the His-fusion protein was expressed in E. coli and analyzed for its antigenic activity. Among those 7 clinically diagnosed patients' sera , there were 5 positive and 2 negative in reaction with His-fusion protein. All of the 20 healthy persons' sera and rabbit anti-OC43 and 229E were of negative reaction with His-fusion protein. The animals immunized with His-fusion protein have produced multi-clonal antibody. The His-fusion protein could specially react with clinically diagnosed SARS patients' sera and the animals immunized with His-fusion protein could produce specifically multi-clonal antibody, but it could not react with the sera of healthy persons and the rabbit anti-OC43 and 229E.     

半巢式PCR法在鉴定风疹病毒标本基因型中的应用

目的在无法成功分离风疹病毒的前提下鉴定风疹病毒阳性标本的基因型,从而全面完整地了解河北省风疹病毒基因型的特征.方法采用半巢式PCR法,将盲传三代风疹病毒核酸检测为阴性而咽拭子标本风疹病毒核酸检测为阳性的风疹疑似病例咽拭子标本进行核酸扩增、序列测定和分析.结果检测的11份风疹疑似病例咽拭子标本中有6份为2B基因型风疹病毒,5份为1E基因型风疹病毒,11份标本与相应的各基因型参考株同源性高,重要抗原位点未发生变异.结论使用半巢式PCR法能够成功地鉴定出盲传为阴性的风疹病毒基因型,丰富了风疹病毒基因库,并为更全面地了解河北省风疹病毒分子流行病学提供依据.     

Relatives of rubella virus in diverse mammals

Since 1814,when rubella was first described,the origins of the disease and its causative agent,rubella virus(Matonaviridae:Rubivirus),have remained unclear1.Here we describe ruhugu virus and rustrela virus in Africa and Europe,respectively,which are,to our knowledge,the first known relatives of rubella virus.Ruhugu virus,which is the closest relative of rubella virus,was found in apparently healthy cyclops leaf-nosed bats(Hipposideros cyclops)in Uganda.Rustrela virus,which is an outgroup to the clade that comprises rubella and ruhugu viruses,was found in acutely encephalitic placental and marsupial animals at a zoo in Germany and in wild yellow-necked field mice(Apodemus flavicollis)at and near the zoo.Ruhugu and rustrela viruses share an identical genomic architecture with rubella virus2,3.     

Diagnosis of fetal rubella infection by nucleic acid hybridization

The efficacy of nucleic acid hybridization for the diagnosis of rubella infection in experimental and clinical materials was compared with immunoblot and virus isolation techniques. Our results showed that nucleic acid hybridization is specific and rapid but gives false-negative results when compared with conventional virus isolation in some experimental although not in clinical materials so far examined. For this reason, a failure to demonstrate rubella virus in fetal specimens by this method alone cannot yet be taken as a sole criterion for ruling out fetal rubella infection.     

Application of molecular and serological assays to case based investigations of rubella and congenital rubella syndrome

Rubella and congenital rubella syndrome continue to be important health problems worldwide. The detection of rubella RNA directly in clinical specimens is a critical factor in early laboratory diagnosis of recent or congenital infection, in addition to detection of rubella-specific IgM. In order to comply with recent WHO recommendations for establishing uniform genetic analysis protocols for rubella virus we have developed a new block based PCR assay (PCR-E317), which extends the sequence generated by the block based PCR-E592 currently in use, to cover the minimum acceptable 739 nucleotides (nt) window at the E1 gene. In addition, a real-time PCR assay has been developed to allow rapid detection of the virus in the laboratory. The assays were applied to a number of clinical specimens collected from patients including recent rubella incidences in the UK, Ethiopia and Turkey, two prenatal and two congenital rubella syndrome cases. Rubella RNA was detected in specimens from two patients that were collected too early for IgM detection, in two amniotic fluids for prenatal diagnosis and in the follow up specimens from the two infant with congenital rubella syndrome tested for viral secretion. At least four genotypes were identified among these patients. The results showed that molecular assays are important tools in the early diagnosis of rubella and congenital rubella syndrome, in the provision of molecular epidemiological information for tracking transmission pathways and in adding to the knowledge of rubella strain distribution worldwide.     

Laboratory confirmation of congenital rubella syndrome in infants: an eye hospital based investigation

A total of 190 specimens from South Indian children aged 0-59 months with ocular anomalies consistent with suspected congenital rubella syndrome (CRS) were investigated. Twenty-six of the 65 infants (40%) were confirmed as CRS by detection of rubella specific IgM. Rubella RNA was detected in 41 samples from 26 infants by both real-time and block based PCR. The PCR results correlated well with the presence of anti-rubella IgM/IgG (23/27 cases with rubella IgM were PCR positive). Whereas, only 17 of 26 infants met the WHO CRS case definition. Amongst the various specimens tested from the sero-confirmed cases (n = 27), a high percentage of positives were detected in lens (92%) and oral fluid (60%) specimens, when compared to other samples. The quantification of viral load by real-time PCR demonstrated higher copy number of virus in lens samples of 0-11 months infants. The rubella viruses were characterized and revealed the circulation of genotype 2B in three South Indian states. The integrated analysis of clinical manifestations, serological and molecular data in the study has generated baseline information of rubella infection and CRS in infants with ocular anomalies.     

Automated nucleic acid isolation in viral molecular diagnostics: evaluation of the QIAsymphony SP

The Qiagen QIAsymphony SP is a high-throughput (up to 96 samples per run), fully-automated nucleic acid isolation system. It was implemented in the authors' laboratory to cope with the high demand for pandemic H1N1 influenza testing in 2009. This study evaluated the QIAsymphony SP for viral nucleic acid isolation from quality control materials, pure cultures and various clinical specimens. The effect of varying sample volume on detection sensitivity was investigated using serial 10-fold dilutions of pure viral specimens and target nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Little variability in detection sensitivity was observed for all the viral targets tested, although variation in cycle threshold values was apparent in some cases. Importantly, pathogens were detectable over a broad concentration range and from diverse clinical specimens. Removal of PCR inhibitors was generally effective, as demonstrated by detection of viral nucleic acids and/or internal controls. The results demonstrate that the QIAsymphony SP is suitable for use in routine virology molecular diagnostics, and provides a high-throughput capacity, which is needed in peak seasons of infection or in centralised laboratories.     

Detection of measles,mumps, and rubella antibodies in saliva using antibody capture radioimmunoassay

Antibody capture radioimmunoassays were developed for detecting virus specific IgM (MAC-RIA) and IgG (GACRIA) to measles, mumps, and rubella and used to investigate saliva as an alternative specimen to serum for diagnosis. Saliva was collected from 63 patients with measles, 19 with mumps, and 150 with rubella, which were all clinically diagnosed and serologically confirmed. Virus specific IgM was detected in 92% of measles, 75% of mumps, and 100% of rubella saliva samples collected during the first week of illness. Between 1 and 5 weeks after onset virus specific IgM was detected in 100% of saliva specimens. After the 5th week the proportion of reactive specimens declined. The specificity of the MACRIA tests was established by testing saliva samples collected from blood donors for measles (88), mumps (88), and rubella IgM (91). All of the saliva specimens tested for measles and rubella specific IgM were unreactive, 1/88 specimens tested for mumps specific IgM contained significant reactivity. Saliva specimens collected from acute cases of MMR were tested in all 3 MACRIAs. A small proportion of saliva samples contained detectable IgM of more than one virus infection. Rubella and measles specific IgG was detected in the saliva of all cases from the 4th or 5th day of illness, respectively. Detection of mumps specific IgG was less successful. We have demonstrated that virus specific IgM can be reliably detected in saliva samples collected from acute cases of measles, mumps, and rubella and identified 1–5 weeks after onset of illness as the optimum time for collection of samples. © 1993 Wiley-Liss, Inc.     

Identification of WU polyomavirus from pediatric patients with acute respiratory infections in Beijing, China

A novel polyomavirus (WU virus) has been identified in pediatric patients with acute respiratory tract infections (ARI), but its role as a respiratory pathogen has not yet been demonstrated. To investigate if WU virus is related to acute respiratory infections in infants and children in Beijing, specimens collected from 674 pediatric patients with ARI from April 2007 to May 2008 and from 202 children without ARI were used for this investigation. Common respiratory viruses were tested by virus isolation and/or antigen detection by indirect immunofluorescent assay followed by RT-PCR or PCR for other viruses associated with respiratory infections in specimens collected from patients with ARI before WU virus DNA was detected. WU virus DNA was detected by initial screening and secondary confirmation PCR for all specimens. The region encoding the VP2 gene of the virus was amplified from 17 WU-virus-positive clinical specimens, and sequence analysis was performed. Thirty-eight of 674 (5.6%) specimens from patients with ARI and 3 of 202 (1.5%) specimens from children without ARI yielded PCR products with the predicted molecular weight, using either screening or confirmation primer sets, indicating that these specimens were WU virus positive. However, more than 60% of the 38 WU-virus-positive specimens from patients with ARI were also positive for one or more respiratory viruses. The nucleotide and deduced amino acid sequences of the region encoding the VP2 gene from 17 Beijing WU viruses shared high homology (>98.5%) with sequences from GenBank and among themselves. The data indicated that WU virus in Beijing occurred 3.7 times more frequently in pediatric patients with ARI than in those without ARI (p < 0.05).     

Development of polymerase chain reaction for specific identification of epizootic hemorrhagic disease virus serotype 1

Summary The diagnostic potential of the polymerase chain reaction (PCR) for specific identification of epizootic hemorrhagic disease virus serotype 1 (EHDV-1) in cell culture and clinical specimens was evaluated. Using oligonucleotide primers, selected from genome segment 2 of EHDV-1 (New Jersey strain), the PCR-based assay resulted in a 862 base pair (bp) PCR product. EHDV-1 RNA from United States prototype serotype 1 and a number of EHDV-1 field isolates, propagated in cell cultures, were detected by this PCR based assay. The specific 862 bp PCR products were visualized on ethidium bromide-stained agarose gel. Identity of the PCR product was confirmed by chemiluminescent hybridization with non radiolabelled internal probe. Using chemiluminescent hybridization, the sensitivity of the PCR assay was 1.0 fg of virus RNA (equivalent to 60 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from EHDV serotype 2 (EHDV-2); the United States bluetongue virus (BLU) prototypes serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cell; or blood cells from calves and deer that were EHDV-seronegative and virus isolation negative. Application of this EHDV-1 PCR-based assay to clinical samples resulted in detection of EHDV-1 RNA from blood samples, collected from a calf experimentally infected with EHDV-1. The described PCR-based assay provides a simple, rapid, sensitive, specific and inexpensive method for specific identification of EHDV-1 infection in susceptible ruminants.     

Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation.

BACKGROUND: Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) cause a wide range of signs and symptoms, varying from trivial mucocutaneous lesions to life-threatening infections, especially in immuno-suppressed patients. Since antiviral drugs are available, rapid and sensitive laboratory diagnosis of these virus infections is important. OBJECTIVE: To set up and evaluate HSV-1, HSV-2 and VZV qualitative real-time PCR on the Lightcycler system and to compare the results with those of the 'in-house' nested PCR and virus isolation. STUDY DESIGN: 110 consecutive samples from dermal or genital lesions from patients suspected of having HSV infections and another 110 samples from patients with suspected VZV infections were tested with real-time PCR, nested PCR and virus isolation. RESULTS: 24 samples (22%) were positive for HSV-1 by virus isolation and nested PCR, whereas 26 (24%) were positive by real-time PCR. HSV-2 was detected in 28 samples (25%) by virus isolation, in 41 (37%) by nested PCR and in 40 (36%) by real-time PCR. VZV was isolated in 15 samples (14%) and VZV DNA was detected in 51 samples (46%) by nested PCR as well as by real-time PCR. Nucleic acid amplification increased the detection rate of HSV-2 and VZV DNA in particular compared to virus isolation. No significant difference in sensitivity was found between real-time PCR and nested PCR. CONCLUSION: Real-time PCR has the advantage of rapid amplification, a reduced risk for contamination and it is a suitable method for diagnosis of VZV and HSV in specimens from skin lesions.