Laparoscopic colorectal cancer surgery in obese patients.

AIM: The aim of this study was to assess the oncological and postoperative outcomes of laparoscopic colorectal cancer surgery in obese patients. METHOD: All obese (BMI > 30) patients who underwent laparoscopic colorectal cancer surgery from January 2005 to January 2008 were compared with nonobese patients undergoing similar surgery. We recorded patient demographics, intra-operative details and postoperative morbidity and mortality. RESULTS: Sixty-two obese and 172 nonobese patients underwent laparoscopic colorectal cancer resection. Both groups were well matched for demographic parameters. Overall mean operating times were not significantly different. Conversion to open surgery was more likely in obese patients. In particular, for rectal cancers, the conversion rate was 44% in the obese group compared with 17% in the nonobese group (P < 0.05). Postoperative morbidity was also greater in obese patients (P < 0.05). The duration of hospital stay was similar for laparoscopically completed cases (6 days obese vs 7 days nonobese), but in the obese-converted group it was 14 days (P < 0.05). The resected specimen with respect to length, resection margin and lymph node retrieval was equivalent between obese and nonobese patients. Disease-free survival and overall survival at a median follow up of 2 years were also similar. CONCLUSIONS: Laparoscopic colorectal cancer surgery in obese patients is technically feasible and oncologically safe. Despite greater postoperative morbidity, obese patients benefit from shorter length of stay. However, a higher conversion rate, particularly for rectal cancers, should be anticipated in obese male patients.

Application of laser scanning cytometry for evaluation of DNA ploidy in routine cytologic specimens.

The laser scanning cytometer (LSC) is a relatively new instrument that combines the features of both flow and static image cytometry. The purpose of this study was to examine the application of the LSC for evaluation of DNA ploidy in routine cytologic specimens. The material for this study consisted of 60 routine cytologic specimens obtained from 33 males and 27 females ranging in age from 23-87 yr (mean, 58 yr). The specimens were simultaneously stained with propidium iodide and FITC-cytokeratin, either on Thin-Prep slide (35 cases) or in a concentrated cell suspension (25 cases). In each case a minimum of 500 cells was evaluated (range, 527-17,963; mean, 3,889). All abnormal cell populations were relocated for the presence of malignant cells. The results were defined as diploid and aneuploid/tetraploid. In 10 bladder washes, the results of LSC were compared to results of flow cytometry. Out of 60 specimens, 7 (11%: 6 bladder washes and 1 renal wash) were excluded due to low cellularity. Of the remaining 53 cases, 11 (20%) were aneuploid/tetraploid, and 42 (80%) were diploid. All but one cytologically diagnosed malignancy had abnormal DNA content. Additionally, two bladder washes diagnosed as suspicious and atypical were aneuploid. All abnormal LSC results were confirmed by relocation of the cells. The concordance between flow cytometry and LSC in the 10 control bladder washes was 100%. In conclusion, LSC proved to be a suitable instrument for the evaluation of DNA ploidy in routine cytologic specimens.

Comparison of histologic and cytologic specimens of urothelial carcinoma with image analysis. Implications for grading.

OBJECTIVE: To compare DNA results obtained by each of three preparation types (cytologic smears or cytocentrifuge samples, tissue sections and nuclei extracted from paraffin-embedded tissue) on patients with primary urothelial cell carcinoma of the bladder. STUDY DESIGN: Cases were selected for study based on the histologic diagnosis and the availability of corresponding bladder washing cytologic specimens. Five-micrometer sections and nuclear extracts were prepared from biopsies. DNA analysis was accomplished by use of an image cytometer on the Feulgen-stained material. RESULTS: DNA content results correlated with both histologic and cytologic grade. Using a binary classification system, bladder washings and thin tissue section results concurred in 40/43 patients. Nuclear extracts were performed on 27 of the biopsies. The DNA content determined by nuclear extract agreed with the tissue section results for all 27 biopsies. CONCLUSION: All three specimen preparations yield comparable DNA content results using a binary DNA classification scheme (diploid or aneuploid). Histologic grading can be enhanced with DNA content results, particularly in the heterogeneous, grade 2 group of urothelial carcinomas.

Prognostic factors in carcinoma of the vulva: a clinicopathologic and DNA flow cytometric study.

The clinical staging of carcinoma of the vulva is a predictor of patient survival; however, the significance of other prognostic factors remains somewhat controversial. Length of survival after diagnosis of invasive squamous cell carcinoma was determined for 39 clinically staged and surgically treated patients who were followed at our institution. Clinical stage, tumor type, use of radiotherapy (RT), histopathologic features (invasive pattern, depth of invasion, lymph node status, nuclear grade, adjacent dysplasia, desmoplasia, inflammation) and DNA ploidy (determined by flow cytometry from paraffin-embedded tissue) were evaluated as predictors of survival. Kaplan-Meier survival curves were generated for strata defined by each of the various predictors and compared using the log-rank test. Advanced stage (p = 0.0002), RT use (p = 0.0004), "spray" invasive pattern (p = 0.005), positive lymph node status (p = 0.001), increased positive lymph node number (p = 0.016), and greater depth of invasion (p = 0.039) were associated univariantly with decreased survival time. Spray invasive pattern (p = 0.018), positive lymph node status (p = 0.030), positive lymph node number (p = 0.040), and RT use (p = 0.045) continued to be associated with decreased survival time after controlling for stage. Of the significant factors, invasive pattern stands out as a qualitative feature that may have potential benefit in predicting survival independent of clinical stage in patients with vulvar carcinoma.

Alterations in circulating intercellular adhesion molecule-1 and L-selectin: further evidence for chronic inflammation in ischemic heart disease.

Atherosclerosis is increasingly thought to be a chronic inflammatory disease. Inflammation requires transmigration of leukocytes from the circulation to the tissues. Adhesion of leukocytes to endothelial calls is the initial event in an inflammatory response and is mediated by expression of several adhesion molecules. In this study we characterize the contribution of intercellular adhesion molecules (ICAM-1) and L-selectin in patients with different coronary artery disease syndromes. Serum concentrations of cICAM-1 and sL-selectin were measured by enzyme-linked immunosorbent assay in 31 patients with stable angina, 30 patients with unstable angina, 18 patients with acute myocardial infarction and 20 healthy subjects in a control group. All patients underwent coronary angiography. Mean (+/-SE) cICAM-1 levels were higher (p < 0.05) in patients with stable angina (249 +/- 6 ng/ml), unstable angina (260 +/- 16 ng/ml), or acute myocardial infarction (261 +/- 24 ng/ml) compared with those in subjects in the control group (171 +/- 11 ng/ml). In contrast, levels of sL-selectin were lower (p < 0.01) in patients with stable angina (1.2 +/- 0.1 microg/ml), unstable angina (1.1 +/- 0.6 microg/ml), or acute myocardial infarction (1.1 +/- 0.1 microg/ml) compared with those in subjects in the control group (1.8 +/- 0.1 microg/ml). No difference was found in cICAM-1 or sL-selectin levels among patients with stable angina, unstable angina, or acute myocardial infarction. No correlation was seen between cICAM-1 or sL-selectin levels and extent (or severity) of coronary artery disease or leukocyte count. L-selectin expression was observed to be depressed in patients with severe angina compared with that in members of the control group. To examine the mechanism of reduction in sL-selectin levels and L-selectin expression on leukocytes, leukocytes from the control group were stimulated in vitro. Stimulation of leukocytes resulted in a rapid downregulation of surface L-selectin expression, measured by flowcytometry, similar to the suppressed expression of L-selectin found on leukocytes from patients with coronary artery disease. In conclusion, altered cICAM-1 and sL-selectin levels in patients with coronary artery disease reflect the presence of a chronic inflammatory process. This inflammatory process results in downregulation of leukocyte expression of L-selectin and thus lower circulating sL-selectin levels.

Computer-assisted and visual methods of assessing cellular proliferation in tissue sections from non-Hodgkin's lymphoma a comparison.

OBJECTIVE: To compare computer-assisted and visual methods of assessing cellular proliferation using tissue sections from non-Hodgkin's lymphoma (NHL). STUDY DESIGN: The study group consisted of 20 specimens of NHL (10 indolent and 10 aggressive). Three-micrometer serial sections were prepared and labeled for Ki-67 (MIB1) and argyrophilic nucleolar organizer regions (AgNORs). Labeling was assessed by classic visual inspection and quantitative image analysis. RESULTS: Computer-assisted and visual Ki-67 labeling indices were significantly higher in aggressive than indolent NHL and were linearly related (r = .850, P < .0001). Although the visual and computer-assisted AgNOR counts were significantly higher in aggressive than indolent NHL, the correlation between these two counting methods was not significant (r = .407, P < .075). Linear regression analysis demonstrated a significant correlation between the Ki-67 visual labeling index and visual AgNOR count (r = .630, P < .003); however, no such relationship could be demonstrated between the remaining methods. CONCLUSION: The results of this study suggest that visual and computer-assisted methods of immunohistochemical and AgNOR analysis may not yield comparable results. This fact may be related to the method of analysis and the computer-assisted technique.

Oxidized low-density lipoproteins facilitate leukocyte adhesion to aortic intima without affecting endothelium-dependent relaxation. Role of P-selectin.

Inflammatory cell deposition in atherosclerotic blood vessels has been thought to relate to loss of endothelium-derived nitric oxide (NO). To examine whether cell deposition correlates temporally with the loss of NO activity, rat aortic rings were incubated with buffer, native LDL (n-LDL), oxidized LDL (ox-LDL), or the endothelium-derived relaxing factor synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) for 2 hours, and vascular contractile response to norepinephrine and relaxant response to acetylcholine, thrombin, and calcium ionophore A23,187 were examined. Thereafter, the rings were exposed to biotin-fluorescein isothiocyanate-labeled fluorescent or unlabeled leukocytes for 30 minutes. Cell adhesion was quantitated by fluorescent microscopy as well as by scanning electron microscopy. Incubation with n-LDL or ox-LDL did not affect either the contractile or the relaxant response of rings. However, leukocyte adhesion increased markedly in all ox-LDL-treated rings but not in those treated with n-LDL. Thus, leukocyte adhesion occurred independent of NO activity. In keeping with this concept, pretreatment of rings with the NO precursor L-arginine failed to influence leukocyte adhesion to rings incubated with ox-LDL. Treatment of rings with L-NAME also resulted in adhesion of a large number of leukocytes. Furthermore, all rings treated with ox-LDL or L-NAME demonstrated marked expression of P-selectin leukocyte adhesion molecules, determined by immunohistochemistry. Pretreatment of rings with the P-selectin blocking antibody PB1.3 markedly decreased deposition of leukocytes in rings exposed to ox-LDL.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA tetraploidy in Feulgen-stained bladder washings assessed by image cytometry.

The prognostic utility of DNA cytometry has been demonstrated for irrigation specimens from bladder neoplasms. While the traditional method of measuring the DNA content of cells recovered by bladder irrigation is flow cytometry, image analysis has been applied increasingly, with successful results. In some cases, image analysis has been shown to detect DNA aneuploid populations missed by flow cytometry. The DNA aneuploid population most frequently missed by flow cytometry is in the DNA tetraploid range. The purpose of the present study was to review image cytometry data on bladder washings analyzed at the University of Florida Diagnostic Referral Laboratories during a one-year period, with special emphasis on the subset with DNA tetraploid histograms. Of the 205 cases reviewed, 127 (62%) were DNA diploid, 36 (18%) DNA aneuploid and 42 (20%) DNA tetraploid. Corresponding cytology was negative in 113/127 (89%) of DNA diploid, 3/36 (8%) of DNA aneuploid and 29/42 (69%) of DNA tetraploid cases. Within the DNA tetraploid group, 45% of cases had no clinical (cystoscopic) or pathologic (cytologic and histologic) evidence of neoplasia. None of these patients developed tumors during follow-up. The presence of DNA tetraploidy in cytologically negative cases should be interpreted cautiously.

Retroviral Producer Cells Cause Sarcoma in Severe Combined Immunodeficiency Mice.

Direct in situ introduction of retroviral producer cells might provide a form of treatment for localized tumors. A possible undesirable consequence of this treatment could be uncontrolled proliferation of the injected producer cells. To test this possibility, severe combined immunodeficiencies (SCID) mice were reconstituted with human peripheral blood lymphocytes which were marked with a retroviral vector using a coculture method. Although specific measures were taken to remove the possible contaminating producer cells, a high percentage of mice developed fibrosarcoma 2-6 weeks after reconstitution. We hypothesized that tumors arose from a small number of contaminating producer cells in the inoculum. Tumor cells were consistently DNA tetraploid, a characteristic of the producer cell line. DNA extracted from tumor tissue was found to contain the gene (neomycin phosphotransferase) used to mark the producer cell line. Furthermore, SCID mice injected with 1 x 10(4) producer cells developed tumors with analogous characteristics. This report indicates that the retroviral producer cell line is tumorigenic in immune-deficient animals. Copyright 1995 S. Karger AG, Basel

Role of the pathologist in biomarker studies.

Cancer chemoprevention is defined as intervention by chemical agents prior to invasion to inhibit or slow the carcinogenic process. Using surrogate endpoint biomarkers in chemoprevention studies may reduce the size, length and cost of clinical prospective randomized trials in high-risk populations. Intermediate biomarkers are measurable alterations in the tissues at risk and include differentiation, genetic composition, biochemical expression, and proliferation. Assessment is possible because invasive epithelial neoplasms are known to begin as intraepithelial proliferations with a spectrum of cellular abnormalities extending to carcinoma in situ. Genetic heterogeneity begins in the intraepithelial phase; a stochastic accumulation of genetic errors characterizes the progression of clonal evolution within the tumor through the process of invasion and metastasis. Pathologic features associated with this process include tumor classification as well as whether it is intraepithelial or invasive. If the process is intraepithelial, the grade and extent of the intraepithelial lesion are reported. If the neoplasm is invasive, tumor size, extent, degree of differentiation (histologic and nuclear grade), mitotic rate, vascular invasion, and lymph node involvement are evaluated. In assessing biomarkers relevant chemoprevention, and without complete regression of the neoplasm with the chemopreventive agent or agents, measurable parameters along with histopathologic features are applicable. Three methods readily applicable for this purpose that can be applied to paraffin-embedded, formalin-fixed tissue include quantitative pathology, immunohistochemistry, and molecular biologic applications. These methods require some consistency in handling and processing the tissues under study; results may deteriorate due to a number of processing variables, including time to fixation, time in fixative, and fixative type. Quantitative pathology, including static image analysis and flow cytometry, can determine total DNA content. Using static image analysis, very small tumors can be studied. In addition, adjacent intraepithelial and invasive components of a tumor may be studied from a single slide. Steroid receptors, oncogenes, and other proteins detectable through immunohistochemical or molecular biologic methods can be quantitated by this technique as well. Cell cycle synthetic function is assayable by both methods. Flow cytometry can calculate the total percentage of cells in S-phase, or the tumor cell S-phase fraction based on the percentage of cells detected between the G0, G1 peak and the G2 + M peak. A similar approach is generally not applicable with current image analysis equipment; however, cell cycle related proteins such as MIB-1 (Ki-67 associated) can be quantified. Immunohistochemical methods can employ a wide variety of monoclonal antibodies to detect oncogene related proteins, including HER-2/neu (c-erbB-2) and p53. Molecular biologic methods, including in situ hybridization, polymerase chain reaction, and in situ PCR, can have many applications when applied to paraffin-embedded tissues, including detection of viral DNA, identification and measurement of apoptosis, and defining gene deletions.

Keratoacanthoma: a deficient squamous cell carcinoma? Study of bcl-2 expression.

Ten keratoacanthomas with both proliferative and regressive histologic features along with 10 well-differentiated squamous cell carcinomas were examined using immunohistochemistry for the expression of bcl-2, a protooncogene recently recognized to be involved in protecting cells from undergoing apoptosis. The squamous cell carcinomas had a modest but diffuse staining pattern, while the proliferative keratoacanthomas stained only at the basal cells and only rare cells stained positively in the regressive keratoacanthomas. The degree and pattern of staining suggest a loss of bcl-2 expression with tumor maturity in keratoacanthoma and a possible role in their ultimate involution.

Monoclonal antibody to P-selectin (PB1.3) protects against myocardial reperfusion injury in the dog.

OBJECTIVE: The aim was to determine whether a monoclonal antibody directed at P-selectin (PB1.3) would diminish neutrophil accumulation and protect against decrease in coronary flow reserve and myocardial function after coronary occlusion-reperfusion. METHODS: Sixteen open chest anaesthetised dogs were randomly given PB1.3 (2 mg.kg-1) or buffer intravenously after 50 min of total left anterior descending coronary artery occlusion. Ten minutes later, the artery was reperfused for 1 h. Coronary flow reserve was measured as peak reactive hyperaemic flow and as increase in coronary flow in response to acetylcholine and glyceryl trinitrate. Myocardial contractile fraction was measured by ultrasonic crystals. Neutrophil infiltration and oxidative burst in the reperfused area were also measured. RESULTS: Coronary flow reserve and myocardial contractile function were markedly impaired in the supply region following left anterior descending coronary artery occlusion-reperfusion in the buffer treated dogs. In contrast, both coronary flow reserve and contractile fraction were preserved in PB1.3 treated dogs despite coronary occlusion-reperfusion. Myeloperoxidase, an index of neutrophil infiltration, was increased in the reperfused region in buffer treated dogs, but not in the PB1.3 treated dogs. Myocardial histology confirmed the reduction in neutrophil accumulation in the reperfused regions in PB1.3 treated dogs. Flow cytometry of the regions supplied by the left anterior descending coronary artery showed a marked decrease in neutrophil oxidative burst in the reperfused region in these dogs. CONCLUSIONS: Antibody to P-selectin (PB1.3) protects against attenuation of coronary flow reserve and myocardial contractile function after coronary occlusion-reperfusion, and decreases neutrophil deposition and activation in the reperfused region.

Mesenchymal hamartoma of the liver. DNA flow cytometric analysis of eight cases.

BACKGROUND: Mesenchymal hamartoma of the liver is a rare lesion seen predominantly in childhood, which is believed to be either a developmental anomaly or reactive process. Because of recent reports of specific translocations involving chromosome 19 in mesenchymal hamartomas and certain ultrastructural and histologic features suggesting a relationship between mesenchymal hamartoma and undifferentiated (embryonal) sarcoma of the liver, some have speculated that mesenchymal hamartoma may be a neoplastic lesion with uncertain malignant potential. METHODS: Because DNA aneuploidy can be useful as a marker for neoplasia, the authors decided to assess ploidy in paraffin embedded mesenchymal hamartomas using flow cytometry. The authors retrospectively examined mesenchymal hamartomas from eight children and evaluated the clinicopathologic features and the ploidy of the lesions. RESULTS: Boys and girls were equally affected, and the mean age at presentation was 11 months. Lesions involved predominantly the right lobe of the liver, with a range of greatest dimension of 7-25 cm, and a mean weight of 651 g (though weights for three of the largest lesions were not recorded). Flow cytometric analysis of nuclei extracted from paraffin embedded tissue revealed that six of the eight lesions were DNA diploid, whereas two were DNA aneuploid (with DNA indices of 1.13 and 1.25). All of the lesions had a low S phase fraction. CONCLUSIONS: The authors concluded that although most mesenchymal hamartomas are diploid, a subset of mesenchymal hamartomas is aneuploid. The finding of aneuploidy in mesenchymal hamartoma, in conjunction with the reported cytogenetic abnormalities, suggests that mesenchymal hamartoma may be a true neoplasm and not a developmental anomaly or reactive process.

Ki-67 expression in vulvar carcinoma.

The proliferative activity of invasive squamous cell carcinoma of the vulva was examined using a Ki-67 equivalent monoclonal antibody (MIB1), which gives a strong immunoreaction in paraffin-embedded tissue. Quantitation of Ki-67 immunostaining was accomplished by image analysis. Ki-67 immunostaining revealed two general patterns of reactivity in vulvar tumors: (a) a diffuse distribution of Ki-67 positive nuclei within the tumor mass and (b) a localized distribution of Ki-67 positive nuclei staining predominantly basilar components of tumor aggregates. The distribution of localized and diffuse patterns did not differ significantly between various clinicopathologic categories (age, histologic type and grade, FIGO stage, and lymph node status). However, the survival times for patients with a diffuse Ki-67 labelling pattern tended to be shorter than those for patients with a localized pattern. Survival curves based on the median positive nuclear area (PNA) calculated by image analysis did not differ significantly. Thus, the pattern of Ki-67 immunostaining, rather than the percentage of PNA, may have prognostic significance in vulvar squamous cell carcinoma.

A multilabeling technique for simultaneous demonstration and quantitation of Ki-67 and nucleolar organizer regions (AgNORs) in paraffin-embedded tissue.

Although many investigators have demonstrated a relationship between argyrophilic nucleolar organizer regions (AgNORs) and Ki-67 expression in solid tumors, no previous studies have simultaneously assessed the relationship between AgNOR and Ki-67 expression in paraffin-embedded tissue. We describe a method for simultaneous demonstration and quantitation of Ki-67 and AgNORs in routinely processed tissue. The Ki-67 equivalent monoclonal antibody MIB1, which can detect proliferative activity in routinely processed tissue with microwave heating, was employed. Fresh human tonsil tissue was fixed in formalin and embedded in paraffin for Ki-67/AgNOR dual staining. Image analysis was employed for quantitation of AgNOR staining in Ki-67-positive and Ki-67-negative nuclei. The double-staining procedure had no measurable effect on the individual parameters: Ki-67 labeling index, mean AgNOR number (NN), and NOR percentage nuclear area (NPNA). However, microwave processing for Ki-67 immunostaining significantly increased nuclear area (NA) and AgNOR area (AA). A significant difference was found between Ki-67-positive and Ki-67-negative cells for NN (p < 0.001), NA (p < 0.001), AA (p < 0.001), and NPNA (p < 0.001). These results suggest a direct relationship between AgNOR and Ki-67 in paraffin-embedded tissue.

Quality control considerations for Ki-67 detection and quantitation in paraffin-embedded tissue.

Proliferative activity has prognostic significance in many solid tumors. Immunohistochemical analysis of tumor proliferation may be accomplished with the Ki-67 monoclonal antibody which recognizes a nuclear antigen expressed throughout the cell cycle. This antibody, however, cannot be used with formalin-fixed, paraffin-embedded tissue. Recently, a Ki-67 equivalent murine monoclonal antibody (MIB-a; AMAC, Inc., Westbrook, ME) was generated which can detect tumor proliferative activity in routinely processed tissue with microwave oven heating. Using quantiative image analysis, we assessed the effect of delay in fixation, total time of formalin fixation, and microwave heating time on the immunoreactivity of this antibody. The effect of time to fixation (0, 2, 4, 8, or 24 hours) on MIB-1 immunostaining was determined in various tumor tissues using image analysis. No significant difference in positive nuclear area was observed for tissues in which fixation was delayed for as long as 8 hours relative to controls. A 24-hour fixation delay resulted in a small decrease in positive nuclear area was observed for tissues in which fixation was delayed for as long as 8 hours relative to controls. A 24-hour fixation delay resulted in a small decrease in positive nuclear area relative to controls. The effect of fixation time (4, 24, or 48 hours) and microwave oven heating time on MIB-1 immunostaining was studied in tonsil tissue, and quantitated by image analysis. Good MIB-1 immunostaining was observed for all microwave oven heating times in tissue fixed for 4 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Ploidy results in prostatic carcinoma vary with sampling method and with cytometric technique.

To evaluate the effect of sampling method and cytometric method on DNA ploidy results, a comparison study was performed on 20 whole prostate glands removed at prostatectomy. Fresh sampling was by sampling fine-needle aspiration (FNA). Paraffin sampling was by microdissection and re-embedding of 3 to 13 (average 6) 5-mm foci of microscopically proven tumor. Analysis was by flow cytometry and by image cytometry (microscopically guided). In tumor negative cases, flow and image cytometry of the FNA was diploid in each case, and flow cytometry of paraffin-embedded tissue was diploid in 5/6 cases. In tumor positive cases, non-diploid tumor was detected by image cytometry of the FNA in 70%, by flow cytometry of the FNA in 29%, and by flow cytometry of paraffin-extracted nuclei in 21%. The most effective combination was sampling fine-needle aspiration and image analysis.

A method for detecting variability arising from errors in sample processing of paraffin-embedded tissue for DNA content analysis.

We present a method for controlling variability that may arise from inconsistencies in sample preparation for DNA content analysis of paraffin-embedded tissue. Human tonsil tissue obtained from routine surgical specimens was embedded in paraffin according to standard protocols. Fifty-micrometer sections were cut from the block and analyzed each day for 20 days to establish control ranges. One tonsil tissue section was processed in parallel with each run of clinical specimens. In this context, a run was defined as the simultaneous processing of 50-microns tissue sections for extraction of cell nuclei (dewaxing and rehydrating). If the tonsil G0/G1 peak coefficient of variation (CV) exceeded 2 SDs of the established mean, and optimum instrument performance and staining were verified, all samples prepared with the tonsil control were reprocessed. Instrument performance and staining were assessed by using the appropriate external controls. By using this rejection rule (12s), the frequency of sample reprocessing in our laboratory was approximately 6%. When the run was repeated and the tonsil control CV was within acceptable range, the G0/G1 peak CV of the corresponding clinical specimens improved 25% of the time. Because most investigators are willing to accept higher CVs for paraffin-embedded tissue than for fresh tissue, it is desirable to have a control to detect decreased peak resolution, resulting from errors in sample processing.

Comparison of two antibodies for evaluation of estrogen receptors in paraffin-embedded tumors.

Estrogen receptor (ER) content in breast cancer specimens is correlated with a prolonged disease free survival and increased likelihood of response to hormone therapy. Relatively few anti-ER antibodies are currently available for use in formalin-fixed, paraffin-embedded tissue. Recently, a new anti-ER monoclonal antibody (ERID5; AMAC, Westbrook, Maine) was generated which requires antigen retrieval by microwave oven heating for detection in routinely processed tissue. The specific aim of this study was to compare the ERID5 antibody with the commercially available rat monoclonal (ER-ICA; Abbott, Chicago, IL) which requires proteolytic enzyme digestion for detection in paraffin-embedded tissue. Sections from 20 cases of primary breast carcinoma previously assayed by dextran-coated charcoal (DCC) analysis were examined. Quantitation of ER antibody staining was performed without knowledge of the DCC values. Specimens containing > or = 20% specifically stained malignant cells were considered ER positive. The sensitivity and specificity of visual ER-ICA immunostaining were 57% and 83%, respectively. The sensitivity and specificity of visual ERID5 immunostaining were 93% and 50%, respectively. The predictive value of positive staining was 89% for the ER-ICA antibody and 81% for the ERID5 antibody. The predictive value of negative staining was 45% for the ER-ICA antibody and 75% for the ERID5 antibody. Previous studies have demonstrated a linear correlation between DCC values and the positive nuclear area (PNA) generated by image analysis for ER-ICA immunostaining. In the present study, a similar correlation between DCC value and ERID5 percentage PNA was observed (R = 0.670; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Morphometric analysis of AgNORs in imprints and sections from non-Hodgkin's lymphomas. An approach to standardization.

The argyrophilic nucleolar organizer region (AgNOR) technique was applied to tissue sections and touch imprints from 20 non-Hodgkin's lymphomas (NHL). The mean nuclear area (NA), mean AgNOR area/nucleus (AA) and NOR percentage nuclear area (NPNA) were determined using image analysis. The AgNOR count, NA and AA were significantly higher in touch imprints than tissue sections within tumors of the same histologic grade. However, no significant difference was observed for NPNA between imprints and sections within tumors of the same grade. Both the mean AgNOR number and the NPNA were significantly higher in aggressive NHL (n = 10) than indolent NHL (n = 10), regardless of sampling method. It is suggested that the NPNA is invariant to the sampling technique; NPNA values from touch imprints and tissue sections can be compared directly. Furthermore, because NPNA is calculated independent of AgNOR number, it is less subjective than manual counting.