Effect of spot size on the result of single-particle ploidy detection / 军事医学

Objective To explore the relationship between the spot size and the result of ploidy analysis in the detection technology based on single-particles, and to fix the scope of spot thickness on the basis of the experimental result.Methods The influence of spot thickness on voltage signals produced by single cells was analyzed.The parameters of the beam shap system in the incident field were designed and optimized on ZEMAX.Finally, according to the cells′diameter, the target size of spots was set.A set of spots of different thickness and of Gaussian distribution obtained from the optical experimental platform was used to conduct ploidy detection experiments.Results Target spots were both obtained from ZEMAX simulation and the optical platform.When the spot thickness was larger than both monocytes and coenocytes, the mean fluorescence intensity ratio was 2.03,which met the demand of the index.Conclusion When the height of the pulse is used to represent the fluorescence density, the relative size of spots and cells will affect the result of ploidy detection.Only when spot thickness is larger than cells is the ploidy ratio accurate.

Estudio de ploidía de células plasmáticas por citometría de flujo en pacientes conmieloma múltiple: primeros casos estudiados en Uruguay / Flow cytometry for plasma cells ploidy analysis in patients with multiple myeloma: first cases studied in Uruguay

Introducción: la historia natural del mieloma múltiple (MM) es heterogénea con sobrevidas que van desde pocas semanas a más de 20 años. El análisis de los factores pronósticos es esencial para establecer una terapéutica adaptada al riesgo. El estudio de la ploidía de las células plasmáticas es un factor que ha demostrado tener un importante valor pronóstico. Objetivo: estandarizar una técnica, no disponible en Uruguay, para determinar la ploidía de las células plasmáticas por citometría de flujo. Material y método: el estudio de ploidía se realizó en médula ósea utilizando para la marcación de las células plasmáticas los anticuerpos monoclonales anti CD38 y CD138. Para el estudio del contenido del ácido desoxirribonucleico (ADN) se utilizó ioduro de propidio. En el análisis se calculó el índice de ADN (cociente entre la moda del pico correspondiente a la cantidad de ADN de las células plasmáticas en fase Go/G1 y la moda del pico Go/G1 de las células normales residuales). Resultados: en este trabajo mostramos la estandarización de la determinación de ploidía por citometría de flujo y los primeros casos analizados en nuestro país. Se estudiaron nueve pacientes con diagnóstico de MM, hallándose dos casos hipoploides (no hiperploide), un caso diploide (no hiperploide) y seis casos hiperploides. Conclusiones: disponemos de una técnica de determinación de ploidía de células plasmáticas que es sencilla, rápida de realizar y con importante valor pronóstico para pacientes portadores de MM.

Introduction: the natural history of multiple myeloma (MM) is heterogeneous, survival rates ranging from a few weeks to over 20 years. Analysis of prognostic factors is essential to decide on a therapy that is adapted to specific risks. Plasma cells ploidy analysis has proved to be a high prognostic value factor.Objective: to standardize a technique, unavailable in Uruguay, consisting in flow cytometry for plasma cells ploidy analysis in order to determine ploidy values in plasma cells.Method: ploidy analysis was performed in the bone marrow, and monoclonal anti-CD38 and CD-138 antibodies were used to mark plasma cells. Propidium iodide was used to study the content of deoxyribonucleic acid (DNA). The DNA was calculated in the analysis (the ratio of the peak mode corresponding to the DNA present in the plasma cells during the Go/G1 phase and the Go/G1 peak mode of residual normal cells). Results:the study presented the standardization of flow cytometry ploidy analysis and the first cases analysed in our country. Nine patients with a diagnosis of MMwere studied, having found two hypoploid cases (non-hyperploid), one diploid case (non-hyperploid),and six hyperploid cases.Conclusions: there is a technique for ploidy determination of plasma cells that is simple, fast to perform and has an important prognostic value for patients with MM.

Introdução: a historia natural do mieloma múltiplo (MM) é heterogênea com sobrevidas que variam de poucas semanas a mais de 20 anos. A análise dos fatores prognósticos é fundamental para estabelecer uma terapêutica adaptada ao risco. O estudo da ploidia das células plasmáticas é um fator que mostrou ter valor prognóstico importante. Objetivo: padronizar uma técnica, não disponível no Uruguai, para determinar a ploidia das células plasmáticas por citometria de fluxo. Material e método: o estudo da ploidia foi reão da determinação da ploidia por citometria de fluxo e os primeiros casos analisados no nosso país. Nove pacientes com diagnóstico de MM foram estudados, sendo encontrados dois casos hipoploides (não hiperploide), um caso diploide (não hiperploide) e seis casos hiperploides. Conclusões: dispomos de uma técnica de determinação de ploidía de células plasmáticas que é simples, rápida de realizar e com valor prognóstico importante para pacientes portadores de MM.

Significance of DNA ploidy analysis in diagnosis of ASCUS / 中华妇产科杂志

Objective To investigate the significance of DNA ploidy analysis in diagnosis of atypical squamous cell of undetermined significance ( ASCUS).Methods From Jan.2009 to Jul.2011,875 women with ASCUS confirmed by liquid based thin layer cytology technique underwent DNA ploid analysis in Hubei Maternal and Child Health Hospital.Among 294 women underwent high risk HPV detection.All subjective were examined colposcopy directed biopsy at day 3 to 10 after menstruation.Results Among 875 ASCUS cases,553 cases with histologically as chronic cervicitis (63.2％),165 cases with cervical intraepithelial neoplasia (CIN) Ⅰ (18.9％ ),45 cases with CIN Ⅱ (5.1％),79 cases with CIN Ⅲ (9.0％) and 33 cases with cervical invasive cancer (3.8％) were confirmed by colposcopy.Totally 532 cases were observed with DNA heteroploid,and 343 were not observed with DNA heteroploid.When DNA heteroploid negative and more than or equal to three ploid were used to predict CIN Ⅱ or more severe cervical diseases,the sensitivity,specificity,positive predictive values and negative predictive values were 98.7％ and 90.3％,47.5％ and 46.1％,29.1％ and 40.8％,99.4％ and 92.1％,respectively.The amount of heteroploid cells ＞2.5c and ＞ 5c among every 100 detected cells in chronic cervicitis and CIN Ⅰ,CIN Ⅱ,CIN Ⅲ and cervix cancer were respectively 2.53 ± 1.99 and 0.10 ±0.07,2.24 ± 1.69 and 0.20 ±0.11,4.10 ± 1.91 and 0.28 ±0.19,7.97 ±7.33 and 1.27 ± 1.23,8.99 ±7.33 and 0.36 ±0.33,there was no statistical difference in amount of heteropolid cells between ＞ 2.5c and ＞ 5c at group of chronic cervicitis and CIN Ⅰ,CIN Ⅲ and cervix cancer (P ＞ 0.05).However,the amount of heteroploid cells at ＞2.5c and＞ 5c at group of chronic cervicitis,CINⅠ,CIN Ⅲ and cervical were higher than that of CIN Ⅱ significantly (P ＜0.05).Among 294 cases with high risk (HR) HPV detection,216 cases were HR-HPV positive,and 78 cases were HR-HPV negative.The pathology result by colposcopy at group of negative heteroploid,heteroploid ＜ 3,or ≥ 3 showed statistical distribution (x2 =115.2775,P ＜ 0.01).ConclusionDNA ploidy analysis can be used for ASCUS diagnosis,which can avoid excessive biopsy under colposcopy,in the mean time,CIN and cervical cancer could decrease missed diagnosis.

The EGFR Protein Expression and the Gene Copy Number Changes in Renal Cell Carcinomas

BACKGROUND: The epidermal growth factor receptor (EGFR) is known to be involved in many tumor promoting activities. EGFR inhibition has been tried as a therapeutic modality in many human malignancies. METHODS: The expression of EGFR protein and the gene copy number changes were studied in 135 clear cell carcinomas and 16 papillary renal cell carcinomas (RCCs), and these tumors were diagnosed between 1995 and 1997. RESULTS: An EGFR protein expression (2+ and 3+) was found in 54.1% of the clear cell RCCs and in 43.8% of the papillary RCCs. In the clear cell RCCs, its expression was associated with male gender, the tumor size (> or =4 cm) and high T stages (T2 and T3), with statistical significance. Trisomy and polysomy of the EGFR gene were found in 27 (25.7%) and 40 (38.1%) of 105 clear cell RCCs, respectively. Trisomy and polysomy were correlated with an EGFR protein expression and a high clinical T stage, with statistical significance. Among 15 papillary RCCs, 13 tumors showed trisomy (86.7%) and one showed polysomy (6.7%). Amplification was not found in both the clear cell and papillary type RCCs. CONCLUSIONS: A considerable numbers of RCCs showed an overexpression of EGFR protein and increased EGFR gene copy numbers, yet the clinical significance of conducting a FISH study in RCC patients seems to be limited.

Clinical significance of DNA haploid analysis for malignant degree and prognosis assessment of breast carcinoma / 国外医学(肿瘤学分册)

Objective To investigate the clinical significance of DNA haploid analysis for malignant degree and prognosis assessment of breast carcinoma. Methods Highclarity Colourful Pathological Analysis System-1000( HPIAS-1000) was used to analyze the DNA haploid of 120 breast carcinoma patients who had been followed up for more than 5 years. All patients were divided into three groups according to histology. 48 advanced differentiation cases, 44 middle differentiation cases and 28 low differentiation cases. Then DNA haploid analysis was made,that is diploid(2C) ,3 - 4C,aneuploid(AN). Results Except for 3 -4C,there were significant differences betweenⅠandⅡgrade ,ⅡandⅢgrade,ⅠandⅢgrade( P

nfluences of DNA Ploidy and Pre-operative CEA Level on the 5 Year Survival Rate in Colorectal Cancer

PURPOSE: The role of DNA ploidy in colon cancer as a prognostic factor and the correlation of DNA ploidy with the established prognostic factors have been studied for the past 20 years. The purpose of this study was to look into the correlation of DNA ploidy with the prognostic factors and to assess the influence of pre-operative CEA level and DNA ploidy on survival in colorectal cancer. METHODS: A total of 319 patients with colorectal cancer received radical operations, and DNA flow cytometric analyses of DNA ploidy patterns were performed at the Department of Surgery, Inha University Hospital, from June 1996 to July 2002. The patients were divided into 2 groups according to the DNA ploidy patterns. RESULTS: The DNA ploidies of the colorectal tumors were compared to various prognostic factors, the pre-operative CEA level and lymph-node metastasis. The latter two showed correlations to the DNA ploidy. The 5-year survival rate for patients with a normal pre-operative CEA level and DNA diploidy was 85.6% compared to 47.8% for patients with both high pre-operative CEA level and DNA aneuploidy, a statistically significant correlation (P= 0.0003). CONCLUSIONS: This study suggests that DNA ploidy in patients with colon cancer has a significant correlation with pre-operative CEA level and lymph-node metastasis. Especially, the pre-operative CEA level and DNA ploidy in patients with colorectal cancer may play a role as useful prognostic factors.

Expression of CD34 and Platelet Derived Growth Factor Receptor 尾 and Pattern of DNA Ploidy in Dermatofibrosarcoma Protuberans / 中华皮肤科杂志

Objective To investigate expression of CD34 and platelet derived growth factor receptor ? (PDGFR-?) and pattern of DNA ploidy in dermatofibrosarcoma protuberans (DFSP), and to study their significance and relationship. Methods The expression of CD34 and PDGFR-? was detected by immunohistochemical SP method in paraffin-embedded specimens from 39 DFSPs and 30 dermatofibromas (DFs). The findings were quantitatively analyzed by image system. The pattern of DNA ploidy was detected by flow cytometry. Results The expression of CD34 and PDGFR-? in the DFSPs was higher than that in the DFs (P

Relationship between Expression of Anaphase-promoting Complex and Prognostic Factors in Invasive Ductal Carcinoma of Breast

BACKGROUND: The role of the anaphase-promoting complex (APC) is to promote the degradation of mitotic cyclins and other substrates involved in sister chromatid adhesions. The APC appears to be responsible for the degradation of cyclin B and may have a potential role in the loss of control concerning cell proliferation in mammalian cells. However, a direct link between the defects in the APC components and oncogenesis has not been estabilished. This study investigates the relationship between APC expression and variable prognostic factors in invasive ductal carcinoma of the breast. METHODS: We evaluated 108 cases of invasive ductal carcinoma surgically resected from January, 1996 to May, 2000 at Wonju Christian Hospital, Wonju College of Medicine, Yonsei University. Immunohistochemical stains for APC, estrogen receptor, and Ki-67 were done in paraffin sections using the avidin-biotin complex method. The results were compared with clinical and pathologic parameters and flow cytometric DNA analysis factors. RESULTS: Forty cases (37.0%) showed immunopositive reactions for APC. The APC positivity in histologic grades 1, 2, and 3 were 28 cases (84.4%), 33 cases (60.0%), and 7 cases (35.0%), respectively (p=0.0011). The APC expressions in cases with the number of mitosis of less than 10, 10-19, and more than 20 per 10 high power fields, were noted in 37 cases (75.5%), 26 cases (63.4%), and 5 cases (27.8%), respectively (p=0.0016). The mean value of the Ki-67 labeling index was 221.7 in the APC-positive group and 317.9 in the APC-negative group (p= 0.0091). DNA flow cytometric analysis revealed higher APC expressions in cases with diploid patterns (p=0.0095). The APC expression rate increased significantly with decreasing histologic grade, with decreasing mitotic activity, in cases with a low Ki-67 labeling index, and those in the diploid group (p<0.05). The APC expression was not statistically correlated with clinical stage, tumor size, and estrogen receptor status. CONCLUSIONS: These findings suggest that positive APC expression may be considered as a good prognostic factor of invasive ductal carcinoma, and loss of APC expression may be related with the progression of breast cancer.

DNA ploidy patterns in gastric adenocarcinoma

To assess the value of DNA ploidy, flow cytometric analysis was performed on unfixed fresh materials obtained from 86 patients with gastric cancer who underwent stomach resection. We evaluated the DNA content of gastric carcinoma cells from four different sites and compared it with Ki-67 proliferating activity, and other pathologic parameters. The incidence of aneuploid and diploid was similar (48.8% vs. 51.1%). Early gastric carcinoma showed a higher rate of the diploid pattern (75%) compared to that of advanced gastric carcinoma 7.3%). DNA diploidy was noted increasingly in diffuse-type tumors according to uren, in signet ring cell type tumor according to WHO classification and in orly differentiated tumors (p<0.05). Well and moderately differentiated rcinomas revealed the aneuploid pattern more frequently than poorly fferentiated tumors. The aneuploidy was associated with high S phase fraction d high proliferative index. Aneuploidy was noted in the mucosa adjacent to the mor (26%), in the close normal-looking mucosa (7%) and in the remote rmal-looking mucosa (3%). This result suggest the possible role of field ncerization in the development of gastric adenocarcinoma.

Comparative Study of the Breast Carcinoma by the Measurement of Nucleolar Organizer Regions and the Flow Cytometric DNA Analysis

It is important to predict the prognosis of patient with cancer. Well known parameters predicting the prognosis are staged and degree of differentiation. Recently, the prognostic and diagnostic significance of silver-stained nucleolar organizer regions(Ag-NORs) and flow cytometric DNA analysis are introduced. This study aims to detect the diagnostic significance of Ag-NORs in breast lesions, and to correlate the number of Ag-NORs and cellular ploidies in breast carcinoma. In addition, the existence of heterogeneity according to the sample in the same patient is studied Sixty six paraffin blocks(two primary lesions and the two metastatic lymph nodes in 13 patients, and one primary lesion and one metastatic lymph node I 10 patients) of breast carcinoma, 30 paraffin blocks of fibroadenoma and 23 paraffin blocks of normal breast are studied. The numbe of Ag-NORs increase from normal breast(mean 1.45) through fibroadenoma(mean 1.60) to carcinoma(mean 2.43). It was significantly different between each lsions(P<0.0005). The number of Ag-NORs increased in the aneuploidy carcinoma than that in the euploidy one(P<0.05). But, there was difference between the number of Ag-NORs and DNA index according to the sample area in the same patient with carcinoma. In conclusion, the number of Ag-NORs is diagnostically significant in breast lesions and is correlated with cellular ploidies in breast carcinoma. In addition, Ag-NORs and cellular ploidies are heterogenous in the same patient.

Expressions of proliferating cell nuclear antigen, bcl-2 and c-myc proteins, and DNA ploidy in thyroid tumor cells / 中华内分泌代谢杂志

Objective To investigate the expressions of proliferating cell nuclear antigen (PCNA), bcl-2, and c-myc protein, and to explore diagnostic value of DNA ploidy in benign and malignant thyroid neoplasms. Methods The expressions of PCNA, bcl-2 and c-myc proteins in 36 cases of thyroid adenomas and 37 cases of thyroid carcinomas were examined by immunohistochemistry technique. DNA ploidy was measured by imaging analysis technique in 8 cases of thyroid adenomas and 17 cases of thyroid carcinomas. Results Among thyroid carcinomas, the positive rates of PCNA (43.02?31.16)% and c-myc protein 89.2% were significantly higher than those of thyroid adenomas (16.15?9.28)% and 50.0% respectively (both P