A case report of cutaneous melanocytoma with CRTC1‐TRIM11 fusion: Is CMCT distinct from clear cell sarcoma of soft tissue?

Pathological diagnosis of dermal melanocytic tumors is often problematic owing to histological resemblance. Recently, cutaneous melanocytoma with CRTC1‐TRIM11 (CMCT) was added to this category. However, only six cases have been reported so far. We herein present a case of a 77‐year‐old Japanese man with CMCT. The patient presented a nodule in the right thigh and underwent surgical resection. Histological examination indicated a well‐demarcated 6 × 5 mm‐sized tumor nodule in the dermis and subcutis. The tumor was amelanotic, consisting of uniform nests and fascicles of spindled, or epithelioid cells. The melanocytic nature was evident by immunohistochemistry. The CRTC1‐TRIM11 fusion was detected by TRIM11 immunostaining, chromogenic in situ hybridization, and RT‐PCR/direct sequencing. He has been free from the tumor for 1 year after additional resection. The main differential diagnosis of CMCT includes primary and metastatic dermal malignant melanomas (MM) and dermal/subcutaneous clear cell sarcoma (CCS). Additionally, histological overlap with paraganglioma‐like dermal melanocytic tumor was considered. Although some investigators argue that CMCT is a variant of CCS, we think it should be separated from CCS, and subcutaneous/dermal CCS should be confined to tumors with EWSR1‐ATF1/ CREB1 fusion. However, longer follow‐up and more case studies are needed for revealing the true prognosis of CMCT.     

A multicenter pharmacokinetic study of the B-domain deleted recombinant factor VIII concentrate using different assays and standards

Summary. When the one‐stage clotting assay is used in comparison with the chromogenic and immunological assays, plasma levels of factor (F)VIII are underestimated by 40–50% after infusion of B‐domain deleted recombinant FVIII (BDD‐rFVIII) in patients with hemophilia. A possible way to counteract the underestimation of FVIII levels by the one‐stage assay is the adoption of a recombinant FVIII reference standard instead of a plasma standard. To evaluate the usefulness of such a standard [ReFacto^® Laboratory Standard (RLS)], the pharmacokinetic parameters of a single dose of BDD‐rFVIII (25 U kg^?1) were evaluated in a multicenter study carried out in 18 patients with severe hemophilia A. The very low in vivo recovery, obtained with the combination of the one‐stage assay and plasma reference standard, was increased up to the values obtained by the chromogenic assay when the results were expressed in terms of RLS. When the plasma standard was used, the one‐stage/chromogenic ratio was 0.82 ± 0.12 for FVIII levels above 25 U dL^?1 and 1.42 ± 0.99 for FVIII levels below 25 U dL^?1. Using the RLS, the one‐stage/chromogenic ratio increased to 1.01 ± 0.19 at FVIII levels above 25 U dL^?1, as a consequence of a complete overlap of the two decays; however, at FVIII levels below 25 U dL^?1, the one‐stage/chromogenic ratio was still 1.6 ± 0.85. After the twelfth hour, FVIII concentrations obtained by chromogenic assay were always lower than those resulting from the one‐stage clotting assay, independently of the standard used. Results obtained by chromogenic assay were not affected by the type of standard used. Compared with those obtained by the one‐stage assay, higher values of clearance, lower volume of distribution area and shorter plasma half‐life or mean residence time were obtained by chromogenic assay because of a shape change of the decay curve due to a shift to higher values in the first part (time interval 0–12 h) and to lower values in the second part of the decay curve (time interval 12–48 h). As a consequence, the slope of the decay curve obtained by means of chromogenic assay was steeper. In conclusion, the more homogeneous results of in vivo recovery and pharmacokinetic analysis, due to the decrease of discrepancy between the two methods when RLS was used, make the cheaper and more widely used one‐stage assay preferable to the more expensive chromogenic assay, on condition that the ReFacto specific standard has used.     

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

Summary.  Introduction:  In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. Methods:  Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. Results:  The chromogenic FVIII:C levels were higher (0.90 ± 0.15 and 0.47 ± 0.13 IU mL⁻¹) than the 1-st clotting ones (0.14 ± 0.05 and 0.10 ± 0.05 IU mL⁻¹) in family X and Y, respectively ( P  < 0.001). Mean endogenous thrombin potential (ETP) was 1579 ± 359 n m  min⁻¹ and 1060 ± 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 n m  min⁻¹, whereas for those of family Y they ranged from 447 to 1122 n m  min⁻¹. Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. Conclusions:  Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.     

一种用于艰难梭菌分离培养的新型显色培养基的性能评价

目的评价自主研发的艰难梭菌显色培养基(CDCA)性能。方法选用艰难梭菌及其他细菌的标准株进行CDCA显色的特异性评价;将艰难梭菌标准菌株梯度稀释后测定BD公司CDSA、生物梅里埃公司CDIF和CDCA的灵敏度。收集临床120份粪便样本分别采用3种培养基进行艰难梭菌分离培养,并对生长菌落进行质谱鉴定和tpi基因检测,以3种平板中任1种能培养出艰难梭菌的样本定义为真阳性。结果在CDCA平板上除了梭形梭菌、双酶梭菌、第三梭菌、脆弱拟杆菌等菌种能够少量生长并显色外,对其他实验菌种均有抑制作用。CDSA、CDIF和CDCA检测艰难梭菌的灵敏度分别为2.0×10~5 CFU/mL、8.0×10~1 CFU/mL和4.0×10~2 CFU/mL。120份标本中检出艰难梭菌31份(25.8%),CDSA、CDIF和CDCA培养阳性标本分别为25份(20.8%)、28份(23.3%)和26份(21.7%),3种显色培养基对临床腹泻标本检测阳性率差异无统计学意义(χ~2=0.418,P=0.811)。3种显色培养基的敏感性、特异性、阳性预测值、阴性预测值分别为:CDCA 83.8%、100%、100%、94.7%;CDIF 90.3%、98.9%、96.6%、96.7%;CDSA 80.6%、100%、100%、93.7%。结论本实验室研发的CDCA的特异性和灵敏度较高,可用于临床艰难梭菌的初步分离。     

An overview of turoctocog alfa pegol (N8‐GP; ESPEROCT®) assay performance: Implications for postadministration monitoring

Factor replacement therapy with factor VIII (FVIII) concentrates is the current standard of care for patients with haemophilia A. Postadministration monitoring of FVIII activity during on‐demand or prophylactic treatment is important, for example to guide a suitable dosing regimen. While the use of two‐stage chromogenic substrate (CS) assays is increasing, activated partial thromboplastin time (APTT)‐based one‐stage clotting (OSC) assays are most commonly used to measure FVIII activity in clinical laboratories. Substantial variations in activity measurements have been observed in association with some OSC assay reagents when assessing extended half‐life FVIII molecules. Certain silica‐based APTT reagents have previously been shown to underestimate FVIII activity with the polyethylene glycol (PEG)‐conjugated product turoctocog alfa pegol (N8‐GP [ESPEROCT^®]; Novo Nordisk A/S). As a wide range of assay reagents are used in clinical laboratories worldwide, it is essential to establish which can be used to accurately measure activity with modified FVIII concentrates. Here, we describe the approach taken by Novo Nordisk to determine the suitability and accuracy of assays and reagents to measure FVIII activity in samples that contain N8‐GP. While accurate activity measurements were possible with all tested CS assays and most of the OSC APTT reagents tested, three APTT reagents that contain silica as a contact activator were found to underestimate N8‐GP recovery (APTT‐SP, TriniCLOT?, STA^® PTT‐Automate). The data demonstrate the importance of characterizing the accuracy of each FVIII activity assay. Any limitations should be communicated to treating physicians and the clinical laboratories that test samples containing N8‐GP.     

植绒转运拭子联合显色培养基对鼻腔金黄色葡萄球菌定植的快速筛查

目的采用植绒转运拭子联合显色培养基对鼻腔金黄色葡萄球菌(SA)定植进行快速筛查,了解耳鼻喉头颈外科患者SA定植情况,为预防SA感染和医院感染防控提供依据。方法采用eSwab植绒转运拭子采集某院耳鼻喉头颈外科门诊患者鼻前庭标本,以WASPLab微生物自动化系统接种于羊血琼脂培养基和MRSA/SA显色培养基,孵育培养16、40 h,自动拍摄、观察菌落,通过质谱(MALDI-TOF MS)、药敏试验和mecA基因检测对SA进行验证。结果共采集200份鼻前庭标本,检出SA菌株48株,其中MSSA23株(占47.9%),MRSA25株(占比52.1%),鼻腔SA定植率为24.0%,MRSA定植率为12.5%。SA筛查阳性报告平均时间为(17.6±6.1)h。培养与质谱鉴定、耐药表型检测的符合率为100.0%,与mecA基因检测的符合率为97.9%。结论基于植绒转运拭子联合显色培养基的快速检测方法准确度较高,报告时间较短,可以用于SA定植快速筛查。     

The use of ecarin chromogenic assay and prothrombinase induced clotting time in the monitoring of lepirudin for the treatment of heparin-induced thrombocytopenia

Lepirudin (r-hirudin) is one of the two alternative anticoagulants licensed to treat patients with heparin-induced thrombocytopenia (HIT). Manufacturer's guidelines state that lepirudin should be monitored using the activated partial thromboplastin time (APTT) ratio. However, several studies have demonstrated a plateau effect of higher concentrations of lepirudin on APTT ratios and variable results when comparing different APTT reagents. This study compares APTT ratios (using two different APTT reagents) with two other commercially available methods for directly quantifying plasma lepirudin levels: ecarin chromogenic assay and prothrombinase-induced clotting time in 95 samples from five patients receiving lepirudin anticoagulation for HIT.     

发色底物法在酶促反应初速度内测定α1抗胰蛋白酶的活性

目的：优化发色底物法,使其在酶促反应初速度内测定α1抗胰蛋白酶（ AAT）的活性并用于血浆蛋白纯化过程中各样品活性的检测。方法采用酶标仪动态监测模式观察酶浓度和反应时间对酶促反应的影响;计算初速度并确定被底物饱和的最大酶浓度。将AAT与胰蛋白酶孵育,剩余靶酶和底物作用产生的光密度可反映AAT的活性。通过△D与AAT标准品活性进行拟合建立标准曲线,测定相关样品的活性,进行精密度和准确度验证。结果胰蛋白酶浓度为0．0625 mg／ml,20 min内酶促反应处于初速度内。 AAT标准曲线范围为200～1200 IU／ml, r＞0．99。该法测定Cohn Ⅳ、前处理液、洗脱峰样品中AAT的活性分别为（720．59±18．63）、（601．84±19．18）、（568．09±24．83）IU／ml。每个样品之间的RSD＜10％,加样回收率均在90％～110％。结论发色底物法经优化后,准确度和精密度大幅度提高,更适于实验室制备AAT或不同生产阶段样品的活性检测。