Extension of useful reagent shelf life beyond manufacturers' recommendations. Cell Markers Committee of the College of American Pathologists.

OBJECTIVE: The College of American Pathologists Cell Markers Committee designed a study to evaluate the use of immunohistochemistry primary antibodies beyond manufacturers' recommended dates. METHODS: Pathologists were asked to save aliquots of primary antibodies during mid-1997 so that by spring 1998 the reagents would be "outdated" according to manufacturers' recommendations. Three tumors were immunostained both in mid-1997 and early 1998 (using outdated reagents in 1998). Two hundred twenty-one laboratories participated. PATIENT SAMPLES: Immunostained materials consisted of an angiomyolipoma immunostained for muscle-specific actin and HMB-45, a melanoma immunostained for S100 protein and HMB-45, and a large cell lymphoma immunostained for common leukocyte antigen and HMB-45. Blocks from the same tumor were used in each instance. MAIN OUTCOME MEASURE: We compared the immunostaining results as a percentage of laboratories indicating a positive or negative immunohistochemical result between the 1997 and 1998 time points. RESULTS: Only minor differences were identified for the 221 reporting laboratories in 1998 as compared with those in 1997. CONCLUSIONS: The data suggest review of the Health Care Financing Administration's ruling on extending the useful reagent shelf life beyond manufacturers recommendations. Similar studies using more inherently quantitative methodology are suggested.

Expression of p53 predicts treatment failure in aggressive non-Hodgkin's lymphomas.

Approximately 50% of patients with aggressive non-Hodgkin's lymphomas (NHL) achieve a complete remission (CR) and cure with combination chemotherapy. The International Index is a useful clinical measure that predicts the patients' tolerance of therapy and likelihood of achieving CR, but it is not a direct measure of chemosensitivity. In this study we have investigated the predictive value of the tumor suppressor gene, p53, as a biological marker for response to treatment in the aggressive NHL. A retrospective study was carried out on 50 patients with aggressive NHL who were treated with CHOP chemotherapy. Treatment outcome was correlated with the expression of p53 in the lymphoma, as measured by routine immunohistochemistry using the monoclonal antibody Do-7. Forty percent of the lymphomas had >5% of the cells staining positively for p53 and this finding correlated significantly with response to treatment. Fifty percent of patients with p53 positive tumors achieved a CR versus 77% of patients with p53 negative tumors. In addition, the relapse rate and time to relapse were significantly different in the two groups. In the p53 positive group, 60% of patients relapsed in a median time of 6 months, whereas 26% of the p53 negative group relapsed with the time to relapse being >22 months. The overall survival of the p53 positive group (17 months) was significantly shorter than that of p53 negative group (>24 months). These results suggest that the immunohistochemical assessment of p53 is a simple and important prognostic measure for patients with aggressive NHL who are treated with CHOP chemotherapy.

Use of proliferating cell nuclear antigen as a marker of liver regeneration after partial hepatectomy in rats.

In order to document and compare proliferating cell nuclear antigen (PCNA) mRNA and protein levels with more traditional parameters of hepatic regenerative activity in a rat model, adult male Sprague-Dawley rats (4 to 6 per group) were killed at various times up to 96 hours after 70% partial hepatectomy. At each time interval, tissue PCNA mRNA abundance and protein levels were documented (by Northern and Western blot analysis, respectively) and compared with the results of PCNA immunostaining and 3H-thymidine incorporation into hepatic DNA. Tissue PCNA protein levels were also documented in additional groups of rats 12, 24, 36, and 48 hours after sham or 30% partial hepatectomy. PCNA mRNA expression after partial hepatectomy was variable: almost undetectable at 24 hours, levels returned to baseline at 36 hours, then fell again to low levels at 96 hours. PCNA protein levels remained stable for 36 hours, increased to fourfold above baseline (p < 0.01) at 48 hours, then remained elevated for the duration of the 96-hour study. Changes in PCNA by immunostaining were similar but tended to occur somewhat earlier (significant increases being detectable at 24 hours), whereas 3H-thymidine incorporation detected the earliest increases in DNA synthesis at 12 hours and peaked at 36 hours. Peak PCNA protein levels correlated with the extent (0%, 30%, or 70%) of hepatic resection. The results indicate that PCNA protein level as determined by Western blot analysis, but not PCNA mRNA expression, correlates with PCNA immunostaining and 3H-thymidine incorporation in the regenerating liver. These findings support the use of PCNA protein determinations as an additional quantitative measure of hepatic regenerative activity after partial hepatectomy in rats.

Unusual side effects due to warfarin.

Two cases demonstrating unusual side effects of warfarin are described. The first case illustrates the 'purple toes' syndrome, which occurred as a complication following the use of warfarin for the treatment of atrial fibrillation and stroke. The second case is an example of warfarin-induced vasculitis in a patient with a mechanical valve prosthesis. He was unable to tolerate warfarin but was successfully managed with nicoumalone, a related compound. These complications are rare but potentially dangerous effects of a commonly used agent, warfarin.

Comparison of salivary and serum enzyme immunoassays for the diagnosis of Helicobacter pylori infection.

Infection with Helicobacter pylori has been established as an important risk factor for the development of peptic ulcer disease, gastritis and gastric cancer. The diagnosis of H pylori infection can be established by invasive or noninvasive techniques. Two noninvasive enzyme immunoassays (EIAs) for antibody detection - HeliSal and Pylori Stat - were compared with histology. Both assays detect immunoglobulin (Ig) G directed against purified H pylori antigen. The test populations consisted of 104 consecutive patients scheduled for upper gastrointestinal endoscopy. Of these patients, 97 (93%) had symptoms compatible with peptic ulcer disease. Saliva and serum were collected simultaneously at the time of endoscopy. Salivary EIA had a sensitivity of 66%, specificity of 67%, positive predictive value of 67% and negative predictive value of 66% compared with the serum EIA, where the results were 98%, 48%, 64% and 96%, respectively. Although the salivary EIA is an appealing noninvasive test, it was not a sensitive and specific assay. The serum EIA also lacked specificity, but was highly sensitive with a good negative predictive value. Although a negative serum EIA rules out H pylori infection, a positive result must be interpreted in the clinical context and confirmed with a more specific measure.

The diagnostic yield of lower endoscopy plus biopsy in nonbloody diarrhea.

BACKGROUND: Patients presenting with diarrhea frequently undergo lower endoscopy plus biopsy as part of their diagnostic evaluation. The diagnostic yield of this approach has not been systematically evaluated. METHODS: To evaluate the diagnostic yield of endoscopy and biopsy in the investigation of nonbloody diarrhea, we performed a retrospective analysis using the endoscopy unit database of a tertiary care university hospital over a 3-year period. The database was searched for cases in which colonoscopy was performed for the single indication of diarrhea. The endoscopic findings and initial biopsy reports were extracted from a chart review, and each clinician was interviewed for the patient's current clinical diagnosis. The clinical diagnoses were compared with the endoscopy and biopsy results to determine whether the tests had contributed to making the clinical diagnoses. RESULTS: Three hundred six patients were identified. One hundred one were excluded for standardized predefined exclusion criteria, leaving 205 evaluable patients, of whom 77 had flexible sigmoidoscopy and 128 had colonoscopy. Eighteen percent had specific clinical diagnoses facilitated by endoscopy and/or biopsy. Endoscopy and biopsy results were normal in 74% of cases. In 8% of the cases either the endoscopy or biopsy findings were inconsistent with the final clinical diagnoses. CONCLUSIONS: Endoscopy and biopsy are important diagnostic tools in the evaluation of patients with nonbloody diarrhea, leading to a specific diagnosis in nearly one fifth of cases.

Immunohistochemical assay for oestrogen receptors in paraffin wax sections of breast carcinoma using a new monoclonal antibody.

The aim of this study was to evaluate the utility of a new monoclonal antibody (AER311) that targets the oestrogen receptor (ER) in an immunohistochemical assay (IHA) applied to breast cancers. Ninety-seven cases of invasive ductal carcinoma were studied by AER311-IHA using a pressure-cooking antigen retrieval technique applied to formaldehyde-fixed, paraffin-embedded tissue sections; immunostaining was assessed by semi-quantitative scoring (H score). There was 80 per cent concordance between the ER status measured by dextran-coated charcoal (DCC) assay and AER311-IHA, with 63/97 (65 per cent) tumours positive and 15/97 (15 per cent) tumours negative by both assays. Of the 12 DCC-positive cases that were negative by AER311-IHA, 11 were borderline positive (3-8 fmol/mg). Similarly, six of seven DCC-negative cases that scored positive by AER311-IHA had only borderline positive H scores (< 50). When AER311-IHA was compared with ID5-IHA, there was good concordance in ER status (77 per cent) and a significant correlation (r = 0.7, P < 0.001) between H scores. Nevertheless, the correlation between ER level determined by AER311-IHA and that measured by DCC (r = 0.53, P < 0.001) was higher than that for 1D5-IHA (r = 0.32, P = 0.002). AER311-IHA can therefore provide reliable information about the ER status of breast carcinoma on paraffin sections and is an acceptable alternative to other commercially available monoclonal antibodies.

The differential expression of a novel intestinal epithelial glycoprotein in various forms of inflammatory bowel disease.

A13D8 is a monoclonal IgM antibody that identifies an as yet unknown antigen that is expressed intensely and ubiquitously in enterocytes. Immunohistochemically, it was shown that A13D8 has a granular supranuclear staining pattern in columnar epithelial cells of normal small intestine and the colon. In ulcerative colitis, this staining pattern was retained. However, during active inflammation, staining also was evident in goblet cells. To test whether this feature of goblet cell staining was unique to ulcerative colitis, tissue sections from a variety of colitides were examined. Crohn's disease, infectious colitis, and ischemic colitis had similar staining patterns to that seen with ulcerative colitis. There was significantly more inflammation in the biopsies from patients with ulcerative colitis and Crohn's disease with positive goblet cell staining than in the biopsies from those patients with negative goblet cell staining. Almost all positive goblet cell staining in ulcerative colitis and Crohn's disease occurred in biopsies that were actively inflamed, whereas there was rare staining in biopsies that were noninflamed (regardless of whether or not there was active inflammation elsewhere in the colon). Ileal goblet cells stained positively with A13D8 only in cases of active ileitis. In cases of collagenous colitis, with comparable degrees of inflammation to that seen in ulcerative colitis and Crohn's disease, there was rarely goblet cell staining and in graft-versus-host disease goblet cell staining of A13D8 was not observed. The binding of A13D8 to tissue sections was completely inhibited by N-acetyl-D-galactosamine. These results, in conjunction with immunochemical studies, suggest that the antibody recognizes an N-acetyl-D galactosamine-containing epitope on a glycoprotein(s). In conclusion, these data suggest that A13D8 recognizes a glycoprotein expressed by intestinal columnar epithelial cells and during specific inflammatory states, particularly those associated with a neutrophilic infiltrate, becomes evident in goblet cells. Further work is required to establish the exact nature of this molecule and whether it is a pro- or anti-inflammatory factor.

Diagnostic potential for cancer via 1H magnetic resonance spectroscopy of colon tissue.

Specimens of colon tissue were examined by 1H MRS (360 MHz) in order to determine the usefulness of rat colon (n = 44) as a model for human colon (n = 60), particularly for the characterization of preneoplastic lesions. Human tissue was characterized by 1H MRS as a precursor to in vivo studies. For both tissues, resonances from mobile lipids were not characteristic of pure mucosa, but correlated with the presence of submucosa. The mean intensities of the resonances at 3.2 and 3.4 ppm (assigned mainly to choline-containing compounds and taurine, respectively) of rat mucosa compared to those of human mucosa, and of rat tumours compared to human tumours, were not significantly different, while both resonances were significantly more intense in rat tumours compared to rat mucosa. The spectra of premalignant lesions in rat colon have features between those due to tumours and normal tissue. We conclude that rat colon is a useful model for human colon in 1H MR spectroscopic studies. MR spectra from human colon control tissue and tumours were classified with 100% accuracy using multivariate analysis.

Ciprofloxacin prevents the inhibitory effects of acute ethanol exposure on hepatic regeneration in the rat.

To determine whether the inhibitory effects of ethanol on hepatic regeneration could be prevented by ciprofloxacin, a fluroquinolone antibiotic with gamma-aminobutyric acid (GABAA), receptor antagonist properties, adult, male Sprague-Dawley rats (n = 6-8/group) received intraperitoneal injections of saline, putrescine (a hepatic growth promoter, 50 mg/kg), or ciprofloxacin (100 mg/kg), followed 1 hour later by gastric gavage with saline or ethanol (5 g/kg). One hour post-gavage, all rats underwent a 70% partial hepatectomy (PHx). Hepatic regenerative activity was documented 24 hours post-PHx by 3H-thymidine incorporation into hepatic DNA (DNA synthesis), proliferating cell nuclear antigen staining, and hepatic tissue putrescine levels. Compared with healthy controls, DNA synthesis rates were significantly lower in ethanol-gavaged/saline-treated rats (22.7 +/- 4.4 x 10(3) vs. 12.3 +/- 6.9 x 10(3) DPM/mg DNA, respectively, P < .001) but unaltered in putrescine-(18.8 +/- 3.4 x 10(3) DPM/mg DNA) and ciprofloxacin-treated (18.3 +/- 2.6 x 10(3) DPM/mg DNA) rats. Hepatic proliferating cell nuclear antigen staining supported these findings. Hepatic putrescine levels also correlated with DNA synthesis data, being decreased in ethanol-gavaged/saline-treated rats (86 +/- 14 pmoles/mg tissue) compared with healthy controls (120 +/- 12 pmoles/mg, P < .01), ethanol-gavaged/putrescine-treated (112 +/- 14 pmoles/mg, P < .05) and ethanol-gavaged/ciprofloxacin-treated (125 +/- 17 pmoles/mg, P < .05) rats. To determine whether these effects resulted from GABAA receptor-mediated changes in liver membrane potentials, intracellular membrane potentials were recorded before and 1 hour after PHx in healthy control, ethanol-gavaged/saline-treated and ethanol-gavaged/ciprofloxacin-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Familial nephropathic systemic amyloidosis caused by apolipoprotein AI variant Arg26.

A point mutation in the apolipoprotein AI (apoAI) gene causing autosomal dominant non-neuropathic systemic amyloidosis is described in a previously unreported Canadian family of British origin with five affected individuals in three generations. Amyloid deposits in the renal biopsy from the proband, a 31-year-old female presenting with hypertension and renal failure, stained immunospecifically with antiserum to apoAI. The plasma of all family members with amyloidosis contained both wild-type apoAI and a variant bearing one additional positive charge. Sequencing of the apoAI gene demonstrated that the proband was a heterozygote for a single base substitution in exon 3, changing codon 26 from GGC(Gly) to CGC(Arg). Concordance of the mutant allele with the presence of variant plasma apoAI and clinical features of amyloidosis was demonstrated. This is the third family in which this amyloidotic mutation has been described, but the distribution of amyloid deposits and their clinical effects are clearly determined by other genetic and/or environmental factors.

Hepatic fibrosis as a predictor of hepatic regenerative activity after partial hepatectomy in the rat.

Liver regeneration is an essential component of the recovery period after partial hepatectomy. Unfortunately, tests that accurately predict regenerative activity in the postoperative period have yet to be described. This study was designed to determine whether the extent of hepatic fibrosis correlates with liver regeneration activity after partial hepatectomy in rats with carbon tetrachloride-induced liver disease. Two groups of adult male Sprague-Dawley rats (12 to 30/group) were treated for 20 to 22 wk with weekly intragastric doses of carbon tetrachloride or vehicle. All rats then underwent 70% hepatectomy while under ether anesthesia. Liver regeneration activity was determined at 24 and 48 hr by [3H]thymidine incorporation into DNA. Hepatic fibrosis was calculated at the time of partial hepatectomy by automated image analysis on Van Gieson-stained liver tissue. Although a significant inverse correlation was found between the extent of hepatic fibrosis and DNA synthesis when all rats were considered (carbon tetrachloride-treated and vehicle-treated) at 24 and 48 hr after partial hepatectomy (r = -0.4943 and -0.7396, respectively; p < 0.05), no such correlation existed when carbon tetrachloride-treated rats were considered independently (r = -0.3231 and -0.0910 at 24 and 48 hr, respectively). In conclusion, we believe that in diseased livers, preoperative quantitation of hepatic fibrosis on automated image analysis does not serve as a useful predictor of liver regeneration activity.

Transglutaminase levels in Dupuytren's disease.

By use of immunoperoxidase staining, tissue transglutaminase and aminopropeptide of collagen type III were localized to fibroblasts running within cords of Dupuytren's fascia. Quantitative analysis of transglutaminase revealed that activity levels were significantly higher in acutely contracting fascia than in chronic contractures. The results show that contractures in Dupuytren's fascia may be reflected by the level of transglutaminase activity in the tissue. Furthermore, it is suggested that isopeptide bond formation, involving collagen type III aminopropeptide moieties, may be the biochemical mechanism by which transglutaminase maintains the contracted state.

Mechanisms of invasion and lymphatic penetration in human colorectal cancer.

The invasive areas in 24 unselected human colorectal cancers were examined by light and electron microscopy and it was shown that the invasive process involves tubes of cells rather than single cells, that degenerative changes take place in specialized cells ahead of the invasive cancer cells and that the endothelium of the lymphatic vessels disintegrates, leaving gaps in the endothelial lining.

Techniques in immunocytochemistry. Application to diagnostic pathology.

The techniques used in the localization of antigens in histologic tissues are reviewed. The principles and conditions for successful antibody-antigen binding are presented. The importance of fixation is stressed. The advantages and disadvantages of monoclonal antibodies over polyclonal antisera are described. Applications of immunocytochemical techniques in diagnostic surgical pathology and cytology are discussed.

Expression of the c-myc protooncogene in human prostatic carcinoma and benign prostatic hyperplasia.

We have examined the level of c-myc transcripts in prostate tissue obtained from patients with both benign prostatic hyperplasia and adenocarcinoma of the prostate. A significantly higher level of c-myc transcripts is observed in patients with adenocarcinoma (P less than 0.05). In addition, a subset of patients with adenocarcinoma had levels of c-myc transcripts 2-fold higher than the mean level for this group. These preliminary results indicate that the investigation of c-myc levels as a prognostic indicator in prostatic carcinoma is warranted.

Secretory meningioma. A distinct subtype of meningioma.

Six meningiomas with abundant hyaline inclusions (pseudopsammoma bodies) were studied. As seen by light and electron microscopy, hyaline inclusions are composed of material of varying structures located in intracellular lumina lined by microvilli. A remarkable pericytic proliferation within the vessel walls was found in five cases. In all six cases, immunohistochemical examination for multiple antigens showed positive staining for carcinoembryonic antigen and epithelial membrane antigen in inclusions and surrounding cells. Weak positivity was found for keratin and secretory component in five cases and for alpha-1-antitrypsin and IgM in four cases. It is concluded that secretory meningioma is a distinct type of meningioma, usually meningothelial in type. It shows characteristic light-microscopic, ultrastructural, and immunohistochemical features of epithelial and secretory differentiation with accumulation of secretory material in the form of hyaline inclusions; marked vascular pericytic proliferation is also frequently present.