Fluorescence microscopy and flow cytometry in measuring activated caspases in human spermatozoa

Staining of spermatozoa with the fluorescein-Val-Ala-Asp-fluoromethylketone has already been performed on ejaculated sperm samples, using fluorescence microscopy (FM) or flow cytometry (FCM) in order to score activated caspases. This assay may help in assessing apoptosis and its role in male fertility. The present study compares the above two techniques in order to adopt the most accurate method for detection in human frozen-thawed testicular, epididymal and ejaculated spermatozoa. The analyses were carried out on frozen/thawed testicular (n = 14), epididymal (n = 8) and ejaculated (n = 10) sperm samples. Activated caspases were detected in living spermatozoa using fluorescein-labelled inhibitors of poly-caspases (FLICA). For the measurements by FM, the same-observer and different-observer reliability were assessed in testicular and epididymal sperm samples. The inter-method (FM and FCM) reliability was assessed both in epididymal and ejaculated sperm samples. The reliability was evaluated by intraclass correlation coefficient (ICC) and the differences between paired measurements from the same sample were tested by Wilcoxon test for matched pairs. For the same-observer and the different-observer data, the ICC were 0.980 and 0.986. In testicular suspensions, the presence of different types of germinal and somatic cells hampers the differentiation of stained spermatozoa by FCM. For the inter-method reliability, the ICC was 0.903. A lower proportion of the viable spermatozoa stained with FLICA was observed by using FM (-6.60 +/- 7.38 %, mean +/- SD; p = 0.003) compared with FCM. To measure the proportion of spermatozoa with activated caspases by this test, FM is a highly accurate and reliable method whatever the sperm origin (ejaculate, epididymis, and testis). FCM cannot be used for testicular samples but seems to be more appropriate for analysis of epididymal and ejaculated sperm samples. The systematic lower proportion by FM in measuring the proportion of stained viable spermatozoa with FLICA involves that only the data measured by the same method (FM or FCM) may be compared.     

Experimental study of spinal cord compression by epidural tumors using electron probe X-ray microanalysis and confocal laser scanning microscopy

Changes in the nerve fibers of the spinal cord were studied in rat experimental epidural tumor models. Light microscopy showed demyelinization in all with rats paraparesis and paraplegia. Cross-sectional views of nerve fibers stained with 3,3dipentyloxacarbo-cyanine iodide, obtained by confocal laser scanning microscopy, showed distorted, shrunken fibers with a low fluorescence intensity. Changes in the electrolyte contents of nerve fibers were studied by electron probe X-ray microanalysis. The K concentration in axons and the myelin sheath was increased in the paraparesis group, but was decreased in the paraplegia group. These findings suggest that, in the paraparesis group, compression of the spinal cord damaged cell membrane channels, which subsequently caused an increase in intracellular K, a decline in the action potential, and low-intensity fluorescence of nerve fibers. On the other hand, in the paraplegia group, destruction of cell membranes caused a decrease in intracellular K until it approached the extracellular level. This reduced both the action potential and the fluorescence intensity. As Ca and Mg concentrations in both axons and the myelin sheath increased in relation to the severity of neurologic damage, it appears that these electrolytes may also play an important role in damage to nerve fibers.     

Role of FSH in regulating testicular germ cell transformations in the rat: a study using DNA flow cytometry

Summary. The role of FSH in regulating testicular germ cell transformations during initiation and maintenance of spermatogenesis in the pubertal and adult rat has been studied using DNA flow cytometry (FCM). The cell types were quantified on the basis of their DNA content using DNA specific fluorochrome DAPI (4,6-diamidino phenylindole). Pubertal (30-d old) and adult (100-d old) rats were deprived of endogenous FSH support for 10 d by daily injection (200 μl d^?1) of a characterized FSH antiserum; the control group received an equivalent volume of normal rat serum. FSH deprivation did not lead to any change in serum testosterone levels. The relative proportion of testicular germ cells in the FSH deprived pubertal rat showed a 90% reduction in 1C (round spermatids) and 260% and 90% increase in 2C (spermatogonia) and 4C (spermatocytes) cells respectively. While the overall conversion of 2C to 1C (1C:2C ratio) was reduced by 98%, the transformation of 2C to 4C (4C: 2C ratio) and 4C to 1C (meiotic division 1C: 4C ratio) was inhibited by 43% and 93% respectively. In the FSH-deprived adult rat the overall conversion of 2C to 1C was reduced by 26% (P < 0.05) only. The 2C and 4C population of cells increased by 47% and 97% respectively (P < 0.025) and the 4C:2C ratio by 47% (P < 0.05). While the meiotic division (1C:4C ratio) was reduced by 54% (P < 0.001), the post-meiotic differentiation of round spermatids to elongate-spermatids (HC:1C) was inhibited by 68% (P < 0.001). A positive correlation (r = 0.95) in per cent HC and a negative correlation (r = -0.67) in per cent 4C cells to the dose of FSH antiserum injected was observed. It is concluded from this that FSH has a role in the onset of meiotic prophase and meiotic division during the first wave of spermatogenesis occuring in the pubertal rat and during adulthood FSH appears to be involved in regulating meiotic division and post-meiotic transformation of spermatids.     

Assessment by flow cytometry of intracellular cytokine production in the peripheral blood cells of renal transplant recipients

INTRODUCTION: There is accumulating evidence that non-invasive immune monitoring may be useful in the early period after renal transplant, particularly with regard to predicting the presence of acute rejection. It is less clear whether chronic allograft nephropathy (CAN) is also associated with consistent changes in peripheral blood or urine cells. We hypothesized that patients with CAN would manifest different patterns of cytokine production (compared with non-CAN controls), detectable in peripheral blood mononuclear cells (PBMCs). METHODS: Flow cytometry was used to quantify production within PBMCs of multiple cytokines. RESULTS: A pilot study showed significant differences in cytokine production between healthy controls and transplanted subjects. However, differences between transplanted patients with and without CAN were small and non-significant. DISCUSSION: Flow cytometry is a potentially useful method for quantifying cytokine production by PBMCs of renal transplant recipients. The technique is sensitive enough to detect differences between distinct test groups but could not find differences between recipients with and without CAN. This probably reflects the lack of a true difference because pathological changes within the long-term allograft may simply not be reflected or detected in the total population of PBMCs. Further studies should explore the usefulness of this technique in assaying more defined populations of PBMCs (such as those activated by donor allopeptides) and in serial monitoring of individual patients.     

Relationship between sperm quality and chromatin condensation measured by sperm DNA fluorescence using flow cytometry

DNA flow cytometry of sperm from 100 randomly chosen men undergoing fertility investigation revealed a general association between reduced sperm quality, as judged by conventional parameters, and the appearance of sperm with lower degrees of chromatin condensation in the ejaculate as measured by DNA fluorescence intensity. Chromatin hypocondensation, as measured by increased fluorescence, was manifested to different degrees in different samples. In many cases of more extreme sperm pathology, such as oligoasthenoteratozoospermia (OAT), the whole population of spermatozoa appeared to be affected. Significant numbers of hypercondensed spermatozoa were present in both normozoospermic men and men with different degrees of disturbance in sperm quality. All of the different parameters of sperm quality could be correlated significantly with certain of the flow parameters, although not one in particular could be used to predict deviations from the normal flow profile. In several asthenoteratozoospermic men and a small proportion of men with OAT, the DNA profiles were normal, implying that in these cases the disturbance may not be so fundamental. The presence of leucocytes in the ejaculate was associated with a general increase in the preponderance of hypocondensed subpopulations of spermatozoa in men with OAT as well as in normozoospermic subjects, emphasizing the effect of inflammatory conditions in the reproductive tract on sperm quality.     

流式细胞术检测精子DNA损伤的标准化与质量控制初步研究

目的:对流式细胞术检测精子DNA损伤的标准化和质量控制进行初步研究.方法:随机选取精液样本,观察酸变性时间,吖啶橙(AO)染色时间,精液样本冷藏、冷冻及反复冻融对精子DNA碎片指数(DFI)结果的影响.结果:随着酸变性时间延长,精子DFI逐渐增高,与酸变性30 s相比,酸变性至2 min时DFI即显著增加(P＜0.05...     

DNA analysis by flow cytometry of paraffin embedded core biopsies of the prostate

The majority of literature concerning DNA analysis of prostate cancer involves testing formalin-fixed prostatectomy tissue, fresh or formalin-fixed transurethral resections of the prostate (TURP), or fresh core biopsies. We were interested if flow cytometry could analyze the DNA of formalin-fixed, paraffin-embedded, core biopsies separated into normal versus malignant segments. Of the 50 potentially available samples for analysis representing 11 controls of normal core tissue and 39 core biopsies from the 11 patients, one patient had no normal tissue, one core had no malignancy, and three cores had no tissue visibly remaining in the paraffin blocks for analysis. Therefore, of 45 actual samples available for processing, sometimes representing segments as small as 0.2 cm, separate segments containing malignant glands or normal glands were excised from the blocks, and processed separately by the Hedley technique. Forty-four of the 45 available samples produced interpretable DNA histograms as defined by discernible G0/G1 peaks, a calculable cell cycle analysis, and the qualitative appearance of a “smooth” histogram appearance, reflecting sufficient nuclei were analyzed. This is the first report to our knowledge where flow cytometry has successfully been used to analyze paraffin blocks of core biopsies which were, in addition, separated into malignant versus normal enriched segments. © 1994 Wiley-Liss, Inc.     

Quantitative DNA measurement by flow cytometry and image analysis of human nonseminomatous germ cell testicular tumors

Current clinical staging, which includes the use of serum tumor markers and imaging techniques, fails to identify the 30–40% of clinical stage I (CS I) nonseminomatous germ cell testicular tumor (NSGCT) patients who have occult metastatic disease. Therefore, there is a real clinical need to evaluate new biological parameters of the primary tumor that might be useful as predictors of occult metastatic disease. This study was undertaken to compare quantitative DNA measurements by flow cytometry and image analysis in CS I NSGCT, and to analyze the relevance of these parameters for predicting occult lymph node involvement. Different blocks of formalin-fixed, paraffin-embedded NSGCTs of 62 CS I patients who underwent retroperitoneal lymph node dissection between 1985 and 1989 were prepared according to the Hedley technique, and analyzed by quantitative cytometry. Thirty-six (58.1%) patients had histologically proven lymph node involvement (pathological stage II), whereas 26 (41.9%) patients (pathological stage I) had neither lymph node metastases according to retroperitoneal lymph node dissection (RPLND) specimens nor tumor recurrence during follow-up. Concordant results were found in 76.5% of the samples by both cytometric techniques. For flow cytometry, the percentages of aneuploid cells in the S- and the G2M+S-phase were the most robust predictive parameters for lymph node involvement, whereas for image analysis the 5c exceeding rate (5cER) had the most predictive significance. Based on the experience obtained in this study, both cytometric techniques provide additional information on tumor aggressiveness that might be useful in therapeutic selection of early stage NSGCT patients for either RPLND or surveillance only.     

Discrimination between benign and malignant prostate tissue using chromatin texture analysis in 3-D by confocal laser scanning microscopy

BACKGROUND: Analysis of chromatin texture may improve both the diagnosis and the assessment of the prognosis of prostate cancer. Confocal laser scanning microscopy (CLSM) allows performing measurements in nuclei reconstructed in 3-D. The aim of this study was to evaluate the clinical usefulness of 3-D texture analysis of prostate tissue. METHODS: Image stacks of eight prostate cancer sections were obtained by CLSM of both benign and malignant areas. Texture feature values were computed for individual nuclei. The discriminative power of the texture features was established by receiver operating characteristic (ROC) analysis and linear discriminant analysis (LDA). RESULTS: Texture features were identified that could discriminate between benign and malignant nuclei. LDA correctly classified 89% of the nuclei of the pooled set of benign and malignant nuclei. CONCLUSIONS: 3-D nuclear texture features allow discrimination of most benign and malignant prostate nuclei. We estimate that the classification rates can be increased by improving the image quality.     

SYBR-14/PI双染法流式细胞术检测精子质膜完整性的研究

目的:探讨应用荧光染料SYBR-14/碘化丙啶(SYBR-14/PI)双色标记法进行流式细胞术检测精子质膜完整性的可行性及其临床意义。方法:收集208例男性精液标本,按WHO精液分析标准分为正常组(n=31)与异常组(n=177)。通过计算机辅助精液分析系统进行精液常规分析。精液标本洗涤处理后经SYBR-14/PI双染后上流式细胞仪(FCM)分析,用发绿色荧光精子百分率(SYBR-14+/PI-%)表示质膜完整精子(PMI)的比例。结果:正常组与异常组SYBR-14+/PI-与SYBR-14-/PI+精子百分率均存在统计学差异(P均<0.05)。正常组精子SYBR-14+/PI-%为(55.66±20.64)%,显著高于异常组[(39.71±19.21)%,P=0.000]。208例标本中,SYBR-14+/PI-%与精子活动率呈显著正相关(r=0.408,P=0.000),与(a+b)级精子百分率呈显著正相关(r=0.398,P=0.000),与d级精子百分率呈显著负相关(r=-0.413,P=0.000);SYBR-14-/PI+%与精子活动率呈显著负相关(r=-0.380,P=0.000),与(a+b)级精子百分率呈显著负相关(r=-0.397,P=0.000),与d级精子百分率呈显著正相关(r=0.385,P=0.000);SYBR-14+/PI+%与精子活动率呈正相关(r=0.172,P=0.013),与(a+b)级精子百分率呈正相关(r=0.177,P=0.011),与d级精子百分率呈负相关(r=-0.164,P=0.018)。结论:应用流式细胞术SYBR-14/PI双染法检测精子质膜完整性具有可行性,可用于评估男性生育力。     

Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.     

三色荧光标记鉴定早孕蜕膜及外周免疫细胞组成

目的建立早孕妇女蜕膜免疫细胞高纯度的分离方法,流式细胞术多色荧光直接标记分析早孕蜕膜及外周血主要免疫细胞的构成比。方法经胶原酶消化、Percoll密度梯度离心、短期培养结合的方法分离纯化蜕膜免疫细胞,采用三色、双色及单色荧光直接标记流式细胞术分别检测早孕妇女和对照组中蜕膜、外周血CD3-CD56+CD16-和CD3-CD56+CD16+NK细胞、NKT和γδT细胞、T细胞和单核细胞的阳性率。结果与外周血相比,蜕膜免疫细胞以CD3-CD56+CD16-数量最多,约占免疫细胞总数的(67.02±18.33)%,T细胞占(11.05±7.22)%,单核细胞占(5.28±0.29)%,NKT细胞占(2.35±1.62)%;早孕妇女外周血T淋巴细胞较正常生育期妇女明显下降(P<0.05),而早孕妇女外周血中NK细胞、NKT细胞、单核细胞数量与对照组相比无显著差别。结论早孕妇女蜕膜中具有与外周血不同的免疫细胞组成,应用多色荧光标记的流式细胞术能够简单、直接地鉴定各免疫细胞亚群。     

In vitro culture of spermatogonial stem cells isolated from adult alpaca (Vicugna pacos) testes analysed with Dolichos biflorus by flow cytometry

Spermatogonial stem cell (SSC) is known for its self‐renewal capacity. We have studied the in vitro proliferation of isolated SSC from adult alpaca (Vicugna pacos) testes. A total of 107 samples were evaluated of which 31 were evaluated at baseline, 36 were cultivated in DMEM and 40 in STEMPRO media. Half of the cultivated samples was analysed after 14 days, and the rest after 21 days. Round cell subpopulations were identified with FITC‐DBA by flow cytometry: strongly positive DBA (sDBA+) as SSC, weakly positive DBA (wDBA+) as in early differentiation and negative DBA as differentiated. At the beginning, 4.16% of the cells were SSC, 37.61% wDBA+ while 54.12% were DBA?. After 14 days, 42.28% of SSC, 44.68% wDBA+ and 11.07% DBA? were found in DMEM while 47.09% of SSC, 32.57% wDBA+ and 18.48% DBA? in STEMPRO. After 21 days 38.66% were SSC, 52.78% wDBA and 7.65% DBA? in DMEM and on STEMPRO media 47.92% SSC, 44.20% wDBA+, 4.93% DBA?. There is a significant difference between the number of initial and SSC cultivated, as well as between DBA? (p < 0.05), while there is no significant difference between the wDBA+ (p > 0.05). Our results suggest that both culture media are appropriate for the in vitro proliferation of alpacas SSC.     

The use of multiparameter flow cytometry to assess tumor cell heterogeneity and grade prostate cancer

Multiparametric flow cytometry has permitted the quantitative assessment of tumor cell heterogeneity within a single tumor. This technique was applied to four tumor lines with biological activity similar to the variety observed in human prostate cancer. These tumors were derived from the Dunning R3327 transplantable rat prostatic adenocarcinoma and have permitted the development of an index to assess, quantitate, and discriminate variation in the aggressive nature of these tumors. The index is based upon measurements of the mean and the variance for perpendicular light scatter and forward light scatter. Perpendicular light scatter has been shown to correlate with nuclear shape, volume, and roundness as measured by digitized image analysis. Forward light scatter increases in quantity and range as the tumors become more undifferentiated. This technique is rapid, reproducible, and applicable to the quantitative measurement of cytometric parameters on large numbers of individual tumor cells.     

Effects of L-acetylcarnitine (LAG) on the post-injury recovery of mouse spermatogenesis monitored by flow cytometry. 2. Recovery after hyperthermic treatment

It has been demonstrated that L-acetylcarnitine (LAC) can enhance the recovery of spermatogonial cells after X-ray damage. In the present work, the effects of LAC on the recovery processes of mouse spermatogenesis after local acute hyperthermia (42 degrees C, 1 hour) was investigated. LAC was administered i.p. (100 mg/kg body weight) every other day for four weeks after heat treatment. At intervals of 8, 14, 21, 28, 35, 40, 45, and 60 days after hyperthermic treatment, testes were weighed and their DNA content analysed by flow cytometry; the round spermatid fraction was found to be higher at 45 days in the LAC-treated animals than in controls (P less than 0.01). Correspondingly, at the same experimental point, the animals without LAC administration showed a lower testicular weight (P less than 0.05). Combined with histological analysis, these results suggest a more rapid recovery of normal spermatogenesis after physical insult with LAC treatment.     

Specific determination of endothelial cell viability in the whole cell fraction from cryopreserved canine femoral veins using flow cytometry

Abstract: An efficient method for specifically determining the viability of endothelial cells (EC) from cells dissociated from the human saphenous vein was investigated. Three different methods, trypan blue staining assay, [3H]-proline incorporation assay, and flow cytometry (FCM), combined with the fluorescein isothiocyanate conjugated with Griffonia simplicifolia agglutins (GS1-FITC)/propidium iodide (PI) double staining, were used. Both trypan blue staining and [3H] proline incorporation assays demonstrated less sensitivity to determine viability of EC differentially from the other cells. FITC-GS1 showed prominent binding to the vascular EC and could be counted by FCM including PI on dead cells. Following the cryopreservation process, the GS1-FITC/PI FCM analytical method was adopted to test simultaneously the viability of whole cells and EC from the same tissue, human saphenous veins, and mongrel dogs' femoral veins after harvesting, antibiotic solution treatment, and thawing. The viability of the whole cells from veins decreased with a significant difference (p < 0.05) from that of EC after thawing.     

Monitoring of anti-HLA class I and II antibodies by flow cytometry in patients after first cadaveric kidney transplantation

While the relevance of pre-formed anti-human leukocyte antigen (HLA) antibodies has been studied extensively, the role of anti-HLA class I and II antibodies produced after cadaveric kidney transplantation is still a matter of discussion. As it has been proposed that they are involved in a considerable number of cases, it should be investigated whether a post-transplant monitoring is a sensitive parameter for the early diagnosis of acute rejection episodes. Additionally, it has been suggested that antibodies are a major cause for chronic rejection; thus, it would be of interest to correlate antibody detection and graft survival. We retrospectively investigated 59 patients after a first cadaveric kidney transplantation without known anti-HLA antibodies (complement-dependent cytotoxicity [CDC] testing). The panel reactivity was determined with a new highly sensitive and specific flow-cytometric technique (Flow-PRA Screening Test, One Lambda, Canoga Park, USA) in sequentially collected serum samples pre- and post-transplant. In patients with acute rejection episodes during the clinical course, the last sample prior to rejection, and in patients without rejection, the last sample prior to discharge, was analyzed. Furthermore, we analyzed 3-yr graft survival and several clinical parameters such as cold ischemia time (CIT). Twenty-four of 59 patients (41%) experienced acute rejections during the clinical course. Five of 59 died with a functioning graft within the first 3 yr. Seven of 54 patients, still alive after 3 yr, lost their graft. Anti-HLA antibodies were detectable in only 7/59 patients and a correlation between antibody positivity and acute rejections (p = 0.32 and 0.54 for anti-HLA class I and II, respectively) could not be identified (sensitivity 12.5 and 8.3%). However, we found a significant correlation between the detection of anti-HLA class II and graft loss within 3 yr (p = 0.005, specificity 97.9%). Additionally, anti-HLA class II positive patients had significantly longer CIT (p = 0.003). Whether the detection of anti-HLA class II antibodies in the early post-transplant phase is of great value for the identification of patients at high risk for early graft loss needs additional investigation. However, we found that anti-HLA antibodies are detectable only in a minority of unsensitized patients and we conclude that flow-cytometric monitoring with Flow PRA is not a sensitive parameter for the early diagnosis of acute rejection episodes in patients after first cadaveric kidney transplantation.