A Review of Water Miscible Embedding Media

Abstract

Water soluble embedding media can be used as dehydrating agents to avoid the use of ethanol, acetone, or propylene glycol, which could destroy, alter, or relocate enzyme activity of tissue, causing misleading final interpretations. The characteristics of water miscible media include low melting point to avoid high temperature induced enzyme loss, uniform hardening, and inability to dissolve cellular materials and to inactivate enzymes.

We compared the merits of well established water miscible embedding media: glycol methacrylate, especially useful for cultured cells and bacteria; Durcupan, used as a fixative and dehydrating agent as well as an embedding medium; Aquon, water miscible epoxy resin; gelatin, used to preserve lipids for study; resorcinol-formaldehyde, used mainly for the reduction of cholesterol loss in the study of frog sciatic nerve.     

免疫分型在单株丙球蛋白血症中的应用评估

目的探讨免疫分型、血清免疫球蛋白的定量检测在单株丙球蛋白血症诊断中的价值。方法收集118例单株丙球蛋白血症患者进行血清蛋白电泳(SPE)、免疫固定电泳(IF)、免疫球蛋白(Ig)定量及血清轻链(κ、λ)检测。针对免疫分型阳性、Ig定量阴性患者进行分析。结果免疫分型阳性、Ig定量阴性患者56例,其中游离轻链型6例(κ轻链型2例,λ轻链型4例),未分泌型1例;免疫分型阳性、Ig定量阳性的患者62例。结论 IF技术对单株丙球蛋白血症的诊断及分型具有重要意义,可以结合血清Ig及轻链的定量检测等其他实验室诊断。     

Value of multi-parameter flow cytometry immunophenotyping in T/NK-cell neoplasms in cytology specimens: A retrospective study in Chinese patients

BackgroundInnate limitations of morphological diagnosis of T/NK-cell neoplasms mean that they can be misdiagnosed or missed, especially when mixed with a variety of benign and reactive conditions. The aim of this study was to investigate the application value of multiparameter flow cytometry immunophenotyping (MFCI) in screening and diagnosing T/NK-cell neoplasms with cytology specimens.Material and methodsThe clinical and pathological characteristics of 1028 newly diagnosed cases from Fudan University Shanghai Cancer Center who provided a cytology specimen between June 2010 and January 2016 with correlated histology diagnosis and clinical confirmation were retrospectively reviewed. MFCI was used for screening, diagnosis and typing. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in diagnosis of T/NK-cell neoplasms were calculated.ResultsThere were 606 males and 422 females in 1028cases, with a mean age of 47.5 years (range 9–86 years). Specimens used for cytologic diagnosis included 996 FNAs, 2 US-FNAs, 13 EUS-FNAs and 17 effusions. Screening for types of lymphoma of MFCI, 139 (13.52 %) cases were T/NK cell lymphoma, 3 (0.29 %) cases were B cell lymphoma T-NHL and B-NHL coexist. A total of 146 suspected T/NK-cell neoplasms were screened out (sensitivity = 94.64 %, specificity = 95.63 % PPV = 72.60 %, NPV = 99.32 %) by MFCI, with 112 (76.71 %) histologically confirmed cases and 6 (4.11 %) false-negative cases identified (3 cases diagnosed as B-cell neoplasms and 1 case as T-cell neoplasm with B-cell neoplasm, which also were confirmed by gene rearrangement. 2 cases were suspicious T-cell–immunophenotypic abnormalities). When used at the diagnostic level, a total of 88 T/NK-cell neoplasms were identified (sensitivity = 68.75 %, specificity = 98.80 %, PPV = 87.50 %, NPV = 96.28 %) with 11 false-positive cases recognized, 9 of which showed typical immunophenotypic T-cell neoplasms features, and 2 exhibited aberrant T immunophenotype.ConclusionsMFCI has high sensitivity and specificity in the screening and diagnosis of T/NK-cell neoplasms and may be useful as an alternative diagnosis method in cytology specimens.     

Immunomodulatory Effects of Newcastle Disease Virus AF2240 Strain on Human Peripheral Blood Mononuclear Cells

Immunotherapy has raised the attention of many scientists because it hold promise to be an attractive therapeutic strategy to treat a number of disorders. In this study, the immunomodulatory effects of low titers of Newcastle disease virus (NDV) AF2240 on human peripheral blood mononuclear cells (PBMC) were analyzed. We evaluated cytokine secretion and PBMC activation by cell proliferation assay, immunophenotyping and enzyme linked immunosorbent assay. The proliferation of the human PBMC was measured to be 28.5% and 36.5% upon treatment with 8 hemaglutinin unit (HAU) and 2 HAU of NDV respectively. Interestingly, the percentage of cells with activating markers CD16 and CD56 were increased significantly. Furthermore, the intracellular perforin and granzyme levels were also increased upon virus infection. Human PBMC treated with NDV titer 8 HAU was found to stimulate the highest level of cytokine production including interferon-γ, interleukin-2 and interleukin-12. The release of these proteins contributes to the antitumor effect of PBMC against MCF-7 breast cancer cells. Based on the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, activated human PBMC showed high cytolytic efficiency towards human breast tumor cells. In summary, NDV was able to stimulate PBMC proliferation, cytokine secretion and cytolytic activity.     

CD2+, CD3-, CD56/NCAM+ malignant lymphoma with TCRβ gene rearrangement: A case report

A case of CD56/NCAM+ malignant lymphoma is reported. Only a rare malignant lymphoma cell showed azurophilic granules in the cytoplasm of Giesma-stained preparations, while electron microscopic examination revealed occasional cytoplasmic granules with paracrystalline inclusions. The most common phenotype seen in NK lymphomas, CD2+, CD3-, CD56+, CD16-, CD57-, was present in the case. Cases with this phenotype have been interpreted to represent either true NK lymphoma or T-cell lymphoma with NK expression. Genotyping, where performed, has shown TCR germline configuration. Our case showed TCRβ rearrangement indicating that the above phenotype can be associated with a peripheral T-cell lymphoma.     

慢性胃炎患儿家族内幽门螺杆菌亚型感染分析

目的了解慢性胃炎患儿家族幽门螺杆菌（Hp）感染的情况以及家族内幽门螺杆菌亚型的分布。方法采集14例因反复腹痛来我院就诊的患儿及其家庭成员共73例的静脉血,应用幽门螺杆菌金标免疫斑点法和免疫印迹法对患儿及其家族成员进行Hp抗体和亚型的检测。结果慢性胃炎患儿家族中Hp抗体阳性率i00％,免疫印迹Hp亚型抗体阳性率95．9％,其中I型Hp感染占60．3％,Ⅱ型Hp感染占35．6％。I型HP及Ⅱ型Hp感染阳性率在患几与二级亲属之间差异均有统计学意义（P〈0．05）,VacA抗体检出率在有胃肠道疾病组（66．7％）及无胃肠道症状组（35．3％）之间差畀有统计学意义（P〈O．05）。家族成员患Hp感染胃肠道疾病有家庭聚集现象。结论慢性胃炎患儿家族中Hp感染阳性率高,I型Hp感染阳性率随年龄的增加而升高,Ⅱ型Hp感染阳性率随年龄的增加而降低,VacA抗体的分布与消化道症状有关。     

多发性骨髓瘤免疫表型研究进展

多发性骨髓瘤(MM)是一种克隆性的B细胞疾病,以恶性浆细胞(Pc)在骨髓中大量增殖,产生溶骨性破坏及过量单克隆免疫球蛋白为主要表现.MM的诊断主要依靠骨髓中存在浆细胞浸润,血、尿中存在一定量的单克隆蛋白以及瘤细胞浸润或单克隆蛋白引起的器官损害(高钙血症、肾功能不全、贫血、骨质破坏等).本文就流式细胞术免疫表型检测对MM诊断和预后意义的研究进展做一综述.     

脑脊液流式细胞术在检测中枢神经系统白血病中的应用

目的：探讨流式细胞术（flow cytometry,FCM）对患者脑脊液（cerebrospinal fluid,CSF）细胞免疫表型的检测,在诊断中枢神经系统白血病（central nervous system leukemia,CNS-L）中的价值。方法采用多色流式细胞术 CD45／SSC双参数散点图设门方法,对195例患者CSF细胞免疫表型进行分析,同时行细胞形态学检测,比较两种方法的差异。结果195例患者中有71例为非霍奇金淋巴瘤（Non-Hodgkin’s lymphoma,NHL）患者,FCM检测有24例发现异常表型细胞,阳性率为33.80％。经形态学分析,15例发现异常细胞,其阳性率为19.72％,差异具有统计学意义（P＝0.022）。58例急性淋巴细胞白血病（acute lymphoma leukemia,ALL）患者,FCM检测有13例发现异常表型细胞,阳性率为22.41％。经形态学分析,12例发现异常细胞,其阳性率为18.97％,差异无统计学意义（P＝1.000）。66例急性非淋巴细胞白血病（acute nonlymphocytic leukemia,ANLL）患者,经 FCM和形态学检测均有4例患者检测出异常细胞,其阳性率均为5.97％,差异无统计学意义（P＝1.000）。结论脑脊液 FCM检测敏感性高,淋巴瘤患者行 FCM分析更有助于中枢神经白血病的诊断,是细胞形态学检测方法的重要补充。     

Investigation of the distribution of lymphocyte subsets and zinc levels in multitransfused β-thalassemia major patients

Homozygous β-thalassemia is a common genetic disorder in the Arabian Peninsula and an important cause of morbidity in Kuwait. The anemia is so severe that chronic blood transfusions, and the resulting iron overload, cause a shift in immunoregulatory balances and a deficiency in zinc. It was reported that individual immunological profile of CD8+ T-lymphocytes may have a modifying effect on the severity of iron overload in HFE homozygous hemochromatosis patients, with low numbers being negatively correlated with the total amount of body iron stores. This has not been tested in thalassemia major patients. This study was designed to utilize flow cytometric immunophenotyping to characterize effects of regular blood transfusion, and high serum ferritin levels because of irregular use of iron chelation therapy on T lymphocytes (CD2, CD3, CD4 and CD8), B lymphocytes (CD19) and natural killer cells (CD56) and zinc levels in the blood of patients with thalassemia major (n = 49) and healthy normal controls (n = 60) in Kuwait. None of the patients had active infections. T-cell markers’ percentage levels were comparable between patients and controls (P > 0.05), while B cell marker (CD19) was significantly higher in patients (P = 0.007). Patients had lower percentage levels of CD56 cells (P = 0.007) and normal serum zinc. All patients had high serum ferritin levels with no significant correlation to CD8+ T lymphocytes (P > 0.05). High iron stores did not have an effect on T lymphocytes’ profile, with normal zinc levels perhaps related to non compliance with chelation therapy. The high B cell marker may be indicative of stimulation of antibody producing cells as a result of regular blood transfusions.     

High expression of urokinase plasminogen activator receptor (UPA-R) in acute myeloid leukemia (AML) is associated with worse prognosis

Urokinase-type plasminogen activator receptor (UPA-R; CD87) is a membrane protein responsible for plasmin expression on cells facilitating cellular extravasations and tissue invasions. We studied the expression of the UPA-R on bone marrow (BM) cells of 93 patients with acute myeloid leukemia at first diagnosis and 8 healthy probands as controls by FACS analysis using phycoerythrin (PE)-conjugated antibodies. A case was defined as UPA-R-positive (UPA-R+) if >20% of the gated cells expressed UPA-R. Whereas none of the 8 healthy BM samples was positive for the UPA-R, 32 (34%) of the 93 AML samples were UPA-R+. Expression of UPA-R was heterogeneous in different FAB types, however, with the highest expression rates in monocytic subtypes (FAB M4/M5): 18%/19%/30% of UPA-R+ cases were found in M1/M2 or M3, and 58%/80% of cases with M4 or M5 were UPA-R+. Proportions of UPA-R+ cells varied between 1% and 98% of the mononuclear cell fractions, with the highest proportions in M4/M5 subtypes (on average 27%/40% UPA-R+ cells) and the lowest expression in AML M2 (11% UPA-R+ cells). The density of expressed UPA-R, estimated as mean channel fluorescence activity, was highest in cases with AML M1 (mFI: 124) followed by M4 and M5 (mFI: 78/77) and lowest in AML M2 (mFI: 43). In sAML, higher proportions of UPA-R+ cases (8 of 18; 44%) compared to pAML (24 of 75; 32%) were found as well as higher proportions of UPA-R+ cells (27% vs. 19%). Separating our patients' cohort in cytogenetic risk groups, we could not detect significant differences in the UPA-R expression profiles. For evaluations of the clinical course of AML, only patients treated by the AML-CG protocol (n = 65) were included. In the group of patients who did not respond to AML-CG therapy, significantly higher proportions of UPA-R+ cells (31% vs. 14%, P = 0.0015, t-test) were found. By evaluating a cut-off value for the percentage of positive cells that allows the most significant separation and differentiation between cases with shorter or longer relapse-free survival times, we could show that patients with >26.5% UPA-R-positive cells were characterized by a significantly higher risk for relapse compared to cases with <26.5% positive cells (P = 0.05). In summary, our data show a high expression of the UPA-R in AML, especially in (myelo)monocytoid subtypes. Cases with higher proportions of UPA-R+ cells were characterized by a significant lower remission rate after AML-CG therapy and a higher risk for relapse. Although prospective trials are still lacking, UPA-R is a prognostically relevant factor independent from the karyotype. UPA-R positivity may identify subtypes of AML associated with a more aggressive clinical course. Thus due to lower remission probabilities in UPA-R+ cases, a more intensive induction therapy regimen could be considered.