Deoxyribonucleic Acid Flow Cytometry in the Assessment of Spermatogenesis

Purpose

We compared deoxyribonucleic acid (DNA) flow cytometric analysis of testicular tissue to quantitative assessment of spermatogenesis.

Materials and Methods

We studied 35 infertile men with azoospermia or oligospermia. All patients underwent incisional testicular biopsies. DNA flow cytometric analysis was performed on each specimen to evaluate the ability of the method to quantify alterations in spermatogenesis. The results were compared to quantitative histological examination. At least 100 spermatic tubules were examined on each specimen and the number of spermatids per tubule was counted. All histological specimens were examined by the same pathologist.

Results

Of the 35 specimens analyzed with DNA flow cytometry 5 were normal, while the percentage of haploid cells (spermatids and spermatozoa) was decreased (hypospermatogenesis) in 14, complete maturation arrest was noted in 2 and almost complete absence of haploid cells was found in 14. Comparing the findings on histological examination with histograms, excellent correlation was noted in cases of the Sertoli-cell-only syndrome and complete maturation arrest, while 3 of 14 histograms with hypospermatogenesis demonstrated normal spermatogenesis on histological examination. Additionally 1 of 5 histograms with normal spermatogenesis demonstrated hypospermatogenesis on histological examination.

Conclusions

DNA flow cytometry of the testicular tissue seems to be an objective and quantified method that can be used to investigate spermatogenesis in infertile men. It is also less time-consuming than any histological examination, permits management decisions within 1.5 hours after biopsy and may replace testicular histopathological study. Flow cytometric diagnoses correlated well with histopathological findings.     

Deoxyribonucleic Acid Measurements in Transitional Cell Carcinomas: Comparison of Flow and Image Cytometry Techniques

Purpose

We compared the results of deoxyribonucleic acid (DNA) ploidy determinations performed by flow cytometry and image cytometry in transitional cell carcinomas of the bladder.

Materials and Methods

In 81 cases of transitional cell carcinoma of the bladder DNA indexes were measured by flow cytometry of bladder washings and tumor tissue samples, and by image cytometry of imprints from tumor tissue samples.

Results

There was good correlation between bladder washings and tissue samples analyzed by flow cytometry in 68 cases but aneuploidy was missed with bladder washings in 13 (16 percent). There was also good correlation between flow and image cytometry in 75 cases (92.59 percent) regarding the detection of aneuploidy. There was agreement between detection of aneuploidy and DNA index in 45 cases (55.5 percent), while both methods detected an aneuploid population in 30 (37 percent) but there was disagreement regarding DNA index. Aneuploid populations were missed by flow cytometry in 6 cases (7.4 percent). Furthermore, in 10 cases peridiploid peaks were found on the image cytometry histograms, which were not visible on flow cytometry. However, it was not possible to assess accurately if these were true peridiploid populations.

Conclusions

There is good overall correlation between DNA content measured by flow and image cytometry but image cytometry has the advantage of visual discrimination, permitting preferential selection and analysis of tumor cells. However, certain problems remain with image cytometry, particularly in the case of peridiploid peaks, which cannot be classified accurately as showing true peridiploid or right shifted diploid populations.     

Clinical Evaluation of Random Biopsy of Urinary Bladder in Patients with Superficial Bladder Cancer

Background Superficial bladder cancer has a tendency to recur in the urinary bladder. One reason for recurrence is the presence of concomitant carcinoma in situ (CIS) or dysplasia. However, the usefulness of random biopsy of the urinary bladder has been unclear.
Methods Between September 1990 and March 1996, 83 patients with superficial bladder cancer underwent mucosal biopsy of 6 different sites in the urinary bladder with macroscopically normal findings (random biopsy). The relationship between a positive biopsy (CIS or dysplasia) and the tumor characteristics was examined. The disease-free survival of the patients according to the biopsy results was determined.
Results The positive biopsy rate was 24.1% (CIS, 14.5% dysplasia, 9.6%). The incidence of positive biopsy in patients with high-grade (C3), pT1 tumors, 3 or more and non-papillary wide-based tumors was significantly higher than that in patients with 1 or 2 tumors, low-grade (G1, G2), pTa tumors and papillary tumors (P< 0.05). In patients with a single papillary tumor, positive biopsy was found in 9.5%. The disease-free survival in patients with a positive biopsy did not differ from that in patients with a negative biopsy, because intravesical bacillus Calmette-Guérin was instilled in patients with a positive biopsy.
Conclusion Our results indicate that random biopsy is useful for detecting concomitant CIS or dysplasia and in the choice of drugs for intravesical instillation.     

图象分析法检测乳腺癌细胞DNA含量及其意义

目的：检测乳腺癌及其癌变过程中细胞DNA的含量，探讨DNA含量在这一过程中的变化规律和临床意义。方法：对1988年63例在我院手术治疗的浸润性乳腺癌，以及1984～1992年间手术治疗的13例导管内癌（ductalcarcinomainsitu，DCIS）、6例导管内癌伴早期浸润（DCIS＋Ca）、6例导管上皮高度增生（proliferativebreastdisease，PBD）的原发灶进行DNA图象分析（imagecytometry，ICM）,结合临床资料，分析63例浸润性乳腺癌细胞DNA含量与预后关系的观察。结果：发现浸润性乳腺癌与DCIS及PBD相比，有较高的DNA指数（DNAindex，NA）DI)、异倍体检出率、核面积（nucleararea，NA）和5C细胞阳性率（P＜0.05）。在DCIS与PBD两组病人中，DI、NA无明显差别（P＜0.05），而异倍体肿瘤,5C细胞出现在DCIS中（P＜0.05），且PBD中无5C细胞、异倍体出现。在乳腺癌病人预后的单因素分析中，发现5C细胞阳性、异倍体肿瘤等的无病生存率低（P＜0.01）。结论:异倍体细胞群的出现是细胞癌变的早期标志，DNA含量随着乳腺癌恶性程度增加而发生显著改变。     

LIN28在膀胱癌和膀胱癌细胞系中的表达及意义

目的：探讨LIN28在膀胱癌组织和细胞系中表达情况,以及与mieroRNA初级Let-7g（pri—Lev7g）之间关系,推测其可能临床意义及对肿瘤进展的影响。方法：采用常规RT-PCR、miRNA转录、免疫荧光和免疫组化方法,检测LIN28mRNA和pri-Let-7g表达,以及LIN28蛋白表达定位。结果：2例膀胱癌细胞系均表达LIN28mRNA,T24表达较强,免疫荧光显示这两个细胞系均表达LIN28蛋白,阳性部位位于细胞胞质,T24荧光强度强于5637。所选10例膀胱癌和相应癌旁组织均表达LIN28mRNA,二者并无明显不同,与临床分级也无明确关系。免疫组化显示癌组织LIN28表达阳性并定位于胞质,而癌旁正常组织LIN28表达为阴性。此外,两个细胞系pri—Let-7g表达较强,而癌和癌旁组织的pri—Let-7g表达强度无明显差异,需进一步检测其成熟Let-7g在这些组织中是否存在不同,以明确这些miRNA是否发生生物合成的转录后阻断。结论：明确T24和5637两个膀胱癌细胞系均可作为研究LIN28、Let-7与其相应靶基因关系的体外实验模型。尽管并不确定膀胱癌和癌旁组织LIN28、Let-7g表达强度与临床分级是否相关,但至少明确LIN28／LIN28在膀胱癌中表达,为探讨LIN28和Let-7在泌尿系统来源的其他恶性肿瘤中的作用提供借鉴和实验依据。     

Susceptibility of Liver Allografts to High or Low Concentrations of Preformed Antibodies as Measured by Flow Cytometry

Liver grafts are more resistant to damage by HLA antibodies than other organ allografts, but it is not clear if the antibodies are associated with graft rejection or graft loss, or if different antibody concentrations have different effects. To explore potential associations between antibody concentrations and outcome, preformed IgG antibodies against donor cells were quantified by flow cytometry in 465 consecutive liver transplant recipients. Antibody-positive patients were classified according to whether they had high or low antibody concentrations and analyzed for possible correlation with graft rejection or graft loss. The results showed that the incidence of rejection was not significantly different between antibody-positive and negative patients. However, patients with high antibody concentrations had a higher incidence of steroid-resistant rejections (31% at 1 year) than patients with low antibody (4%) or no antibody (8%, p < 0.0004). These effects were mainly due to T-cell (HLA class 1) antibodies. The overall incidence of rejection at 1 year was 69% for high antibody patients, 51% for patients with low antibodies and 53% for patients with no antibodies (p not significant). In an apparent paradox, antibody-positive patients underwent fewer early graft losses. Thus, the associations of preformed antibodies and outcome depend, on the one hand, on antibody concentrations, and on the other hand on whether the outcome measured is steroid-sensitive rejection, steroid-resistant rejection or graft survival. These complex interactions may explain the controversial results observed in previous studies.     

DNA Flow Cytometry in Surgically Treated Lung Cancer: Prognostic Significance

Although DNA aneuploidy and high proliferative activity (S-phase fraction, SPF) of tumour cells, measured by flow cytometry, have proved to be indicators of poor prognosis in most solid tumours, there have been conflicting results in lung cancer studies. During a four-year period we studied the prognostic significance of DNA ploidy and SPF in 99 surgically treated lung cancer patients. Flow cytometric analysis was done from archival, formalin-fixed, paraffin-embedded tumour specimens. DNA index and SPF were determined, using MultiCycle software with sliced nuclear correction to compensate for debris. There were 61 DNA diploid and 38 DNA aneuploid tumours. The median SPF was 10.2%. Neither ploidy nor SPF was associated with previously known prognostic factors. Survival was poorer in patients with aneuploid tumours than in the other patients, but the difference was not statistically significant. DNA ploidy and SPF thus do not seem to be useful prognostic indicators in surgically treated lung cancer.     

流式细胞术在精子功能检测中的应用

目的:研究流式细胞术在精子功能评价中的作用。方法:对104例男性不育患者和10例正常生育男性的精液标本进行精液量、精子密度、活动率、畸形率等精液常规分析(CASA法),对精子存活率、染色质结构和线粒体膜电位等进行流式细胞术分析,并将检测结果通过SAS软件进行统计学分析。结果:成组U检验结果表明:不育患者的精子与正常生育男性精子相比除了具有密度低(U=2.51,P=0.014 3)、活动率差(U=3.44,P=0.001)、畸形率高(U=-5.88,P<0.000 1)等特点外,经流式细胞术测定精子结果表明不育男性的精子质膜完整率(U=4.72,P<0.000 1)、线粒体膜电位正常率(U=-2.53,P=0.030 9)和染色质完整率(αt:U=-3.82,P=0.000 3;SDαt:U=-3.98,P=0.000 1;COMPαt:U=-3.57,P=0.000 5)均显著低于正常生育男性。多元逐步回归分析结果表明:精子质膜完整性与精子活动率(t=1.66,P=0.101 6)、精子的线粒体膜电位与精子活动率(t=3.33,P=0.001 4)、精子染色质的完整性与精子活动率(t=3.24,P=0.001 9)均呈正相关,而精子的线粒体膜电位与精子颈、尾部的畸形率呈负相关(t=-3.44,P=0.001)。结论:流式细胞仪测定对精子质量评定意义显著,对男性不育的诊断和治疗也具有较高的参考价值。     

环氧化酶-2抑制剂塞来昔布对膀胱癌T24细胞杀伤作用的研究

目的：体外观察塞来昔布对人膀胱癌T24细胞株的抑制作用,并探讨其可能的机制。方法i采用MTT法检测塞来昔布对T24细胞的生长抑制作用;采用Hoechst 33258染色观察细胞凋亡形态;采用Western印迹法检测塞来昔布作用前后T24细胞COX-2蛋白的表达变化;采用RT—PCR法检测塞来昔布作用前后T24细胞Bcl-2及Bax基因的表达变化。结果：塞来昔布（≥100μmol／L）作用24h及48h后能明显抑制T24细胞的生长并诱导凋亡;塞来昔布100μmol／L作用,24h及48h后,T24细胞COX-2蛋白表达量下降,Bcl-2基因表达亦下调,而Bax基因在塞来昔布作用24h后变化不明显,48h后表达上调。结论：塞来昔布能抑制T24细胞增殖并诱导其凋亡,可能与下调Bcl-2、上调Bax基因表达有关。     

B超测定BPH体积参数评价膀胱出口梗阻的意义

目的 :探讨经直肠超声 (TRUS)测定良性前列腺增生 (BPH)各项体积参数对诊断膀胱出口梗阻 (BOO)的意义。　方法 :BPH病人 116例 ,年龄 5 9～ 75 (6 8.6± 5 .1)岁。应用TRUS测定前列腺各径数值 ,应用公式V =0 .5 2R1R2 R3 计算出前列腺体积各项参数 ,同时行尿动力学检查 ,并计算AG值。将前列腺体积 (PV)、移行带体积 (TZV)、移行带指数(TZI)与病人年龄、国际前列腺症状评分 (IPSS)、前列腺特异性抗原 (PSA)及AG值进行相关性分析。　结果 :PV、TZV及TZI分别为 (6 9.7± 4 5 .9)ml、(43.5± 2 5 .6 )ml和 0 .5 7± 0 .14。最大尿流率 (Qmax)、最大尿流率时逼尿肌压力 (Pdet.Qmax)及AG值分别为 (8.31± 5 .12 )ml/s、(82 .34± 33.4 7)cmH2 O和 6 6 .72± 30 .4 6。IPSS为 2 5 .3± 4 .7,PSA为 (4.12±3.6 4 )ng/ml。相关分析提示TZI(r=0 .74 2 ,P =0 .0 17)、TZV(r =0 .6 74 ,P =0 .0 31)与AG值有显著的相关性。IPSS与TZI、TZV呈正相关 ,PSA浓度与PV、TZV、TZI呈正相关。　结论 :通过TRUS测定BPH的各项体积参数和尿动力学检查一样 ,能够作为判断BOO的参考指标     

Detection of Early Bladder Cancer by 5-Aminolevulinic Acid Induced Porphyrin Fluorescence

Purpose

We determined whether the sensitivity of detecting dysplasia or early bladder cancer can be improved by 5-aminolevulinic acid induced porphyrin fluorescence.

Materials and Methods

A 3 percent 5-aminolevulinic acid solution was instilled intravesically before cystoscopy in 104 patients. The 5-aminolevulinic acid induced porphyrin fluorescence was excited by violet light from a krypton ion laser (wavelength 406.7 nm.).

Results

The sensitivity of the fluorescence cystoscopy (96.9 percent) was significantly (p less than 0.0001) greater than that of white light cystoscopy (72.7 percent). There was no impact on specificity.

Conclusions

Due to the high sensitivity of the procedure fluorescence guided biopsies are recommended instead of random biopsies.     

Role of Flow Cytometry in the Diagnosis and Prognosis of Plasma Cell Myeloma

This article provides an overview of the role of flow cytometry in the diagnosis and follow-up of plasma cell myeloma. A brief introduction to the general immunophenotypic features of normal and myeloma plasma cells is provided, followed by a discussion of technical issues as they relate to the application of flow cytometry in this entity. The prognostic and therapeutic utility of flow cytometric immunophenotyping in myeloma is also analyzed, with an emphasis on the growing role of minimal residual analysis as potential biomarker for evaluating treatment efficacy and for tailoring risk-adapted treatment, in prospective clinical trials.     

JC-1单标法流式细胞术检测精子线粒体膜电位的研究

目的:探讨应用荧光染料JC-1单色标记法进行流式细胞术检测精子线粒体膜电位(MMP)的可行性及其临床意义。方法:收集63例男性精液标本,分为生育组(n=31)和不育组(n=32)。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用JC-1染色后上流式细胞仪分析,用发橙红色荧光精子百分率(JC-1+%)表示MMP正常精子的比例。结果:生育组精子JC-1+%为(75.89±15.69)%,显著高于不育组[(54.04±22.21)%,P=0.000]。63例标本中,JC-1+%与精子活动率呈显著正相关(r=0.610,P=0.000),与(a+b)级精子百分率呈显著正相关(r=0.614,P=0.000),与d级精子百分率呈显著负相关(r=-0.504,P=0.000)。JC-1+%与已建立的罗丹明/碘化吡啶双染法检测结果(Rh123+/PI-%)呈显著正相关(r=0.938,P=0.000)。结论:应用流式细胞术JC-1单标法检测精子MMP具有可行性,精子JC-1+%可作为男性不育的辅助诊断指标。     

从膀胱癌患者分离并培养人膀胱平滑肌细胞

目的：建立从膀胱癌患者的分离并培养膀胱平滑肌细胞的实验技术.方法：取一小块无明显肿瘤生长的膀胱组织,分离并培养膀胱平滑肌细胞;动态观察细胞形态变化、生长增殖情况以及平滑肌肌动蛋白（SMA）、结蛋白（Desmin）和广谱细胞角蛋白（AE1/AE3）的表达.结果：接种24 h后即有长梭形细胞贴壁生长,10天后长至80%融合,呈典型的＂峰谷＂样形态;传代后1天为潜伏期,2～6天为指数生长期,然后进入融合平台期,需再次传代.第2代细胞的SMA和Desmin表达阳性率分别高达（99.0±0.8）%和（97.0±2.1）%,不表达AE1/AE3.随着传代次数的增加,细胞去分化,细胞形态变成短梭状或椭圆形;SMA和Desmin的表达开始下降,传至第5代时,SMA和Desmin阳性率分别降至（78.0±3.3）%和（74.0±2.6）%;至第7代时,SMA和Desmin阳性率降至（51.0±3.0）%和（49.0±2.6）%.第7代细胞经血清饥饿培养48 h后,细胞又能再分化,形态转变成长梭状,SMA和Desmin阳性率可分别升至（90.0±3.5）%和（88.0±2.5）%,具有显著性差异.结论：本研究所培养的人膀胱平滑肌细胞具有较高的纯度,血清饥饿能促进去分化的细胞再分化,能为构建组织工程膀胱提供种子细胞.     

In Vitro Regulation of Cell Growth and Angiogenesis by Inositol Hexaphosphate in Bladder Cancer

Background

Inositol Hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that is found in food sources high in fiber content. We hypothesized that IP6 would inhibit the cell growth rate of bladder cancer in vitro.

Methods

T24 and TCCSUP bladder cancer cell lines were treated with titrating doses of IP6 (0.3, 0.6 and 0.9 mM/well). Cell viability and vascular endothelial growth factor levels were measured.

Results

Significant reductions (p < 0.001) in cellular growth were noted in both cell lines at all doses and time points tested, with the exception of 0.3 mM IP6 at 24 hours in the T24 cell line. The percent inhibition of vascular endothelial growth factor was significantly higher than that observed in the TCCSUP cell line at 48 and 72 hours with 0.3 mM IP6 (p < 0.001). The T24 cells exhibited the same level of inhibition at 24 and 48 hours with 0.6 mM dose of IP6 and at 72 hours with the 0.3 mM dose (p < 0.001).

Conclusions

In vitro treatment of bladder cancer with the common dietary polyphosphorylated carbohydrate IP6 significantly decreased cellular growth by anti-angiogenic mechanisms. We feel that this data warrants further investigation and consideration for initiation of clinical trials to evaluate the safety and clinical utility of this agent.Key Words: Cellular anti-proliferation, Bladder cancer, Angiogenesis, Inositol hexaphosphate, Vascular endothelial growth factor     

Current Clinical Trials in Non-muscle Invasive Bladder Cancer

Purpose of Review

As our molecular understanding of bladder cancer continues to advance, more and more novel agents are entering clinical trials across the spectrum of bladder cancer stages. The clinical trial activity for non-muscle invasive bladder cancer (NMIBC) has been boosted further by the evolution of specific disease states that set more uniform inclusion criteria for clinical trial design. Here, we aimed to review the current clinical trials landscape in non-muscle invasive bladder cancer with respect to these disease states.

Recent Findings

Most active clinical trials focus on high-risk NMIBC in either the BCG-naïve or BCG-unresponsive setting. Strict criteria to define the disease state and a clear pathway to drug registration have encouraged trials for patients with BCG-unresponsive NMIBC. The most promising potential breakthroughs for BCG-naïve patients include alternative BCG strains, immune-priming with intradermal BCG vaccination, and systemic immune checkpoint blockade. The latter therapy is also being actively investigated in multiple trials in BCG-unresponsive NMIBC, along with novel viral agents such as INSTILADRIN (nadofaragene firadenovec) and targeted agents such as oportuzumab monatox.

Summary

After many years of relative stagnation, multiple new therapies currently under investigation in well-designed clinical trials appear poised for routine clinical implementation in the near future. These therapies should dramatically improve the outcome of patients with NMIBC. We can look forward to the challenges of biomarker-driven drug selection, optimal drug sequencing, and rational combination therapies.

     

光动力学疗法诱导人膀胱癌细胞凋亡的实验研究

目的:联合应用流式细胞仪和激光共聚焦显微镜研究叶绿素衍生物光动力学疗法诱导人膀胱癌细胞(T-24 and SCaBER)凋亡的现象.方法:光动力疗法体外作用于人膀胱癌T-24and SCaBER细胞,细胞凋亡指数采用双免疫荧光染色(Annexin V/PI)和流式细胞仪技术测定,凋亡细胞的形态学研究采用AO/EB双染色和激光共聚焦显微镜技术.结果:经光动力学作用后的膀胱癌细胞通过诱导凋亡而被抑制了细胞的活性.细胞被阻滞于G0～G1期.结论:叶绿素衍生物光动力学疗法通过诱导凋亡而抑制膀胱肿瘤的生长,激光共聚焦显微镜能提供更可靠的细胞早期凋亡的影像学资料.