Image analysis and flow cytometric DNA studies of benign and malignant body cavity fluids: reappraisal of the role of current methods in the differential diagnosis of reactive versus malignant conditions.

Cytologic examination of body fluids is commonly performed in the clinical laboratory. Determination of the presence of malignancy may sometimes be difficult. In this study, we prospectively studied 60 body fluids with a panel of antibodies, including MOC-31, epithelial membrane antigen, carcinoembryonic antigen, B72.3, keratin, desmin, and CA-125. DNA and S-phase studies were performed both by flow cytometry and image analysis. Thirty-seven fluids were classified as benign and 23 were classified as malignant. The sensitivity of the antibodies for identification of carcinoma in descending order of percentage detection rate were MOC-31 (95%), epithelial membrane antigen (93%), B72.3 (84%), and carcinoembryonic antigen (80%). Desmin stained mesothelial cells in all cases. CA-125 gave similar results but was less specific. Flow cytometry detected 14 of 20 malignant fluids and image analysis 17 of 23 by identifying an aneuploid population. Benign reactive mesothelial cells were not aneuploid. Tetraploidy due to reactive mesothelial cells was found in 9 of 37 body fluids. Their S-phase fraction was low (average, 3.2%). Tetraploidy in malignant cells was distinguished from the reactive mesothelial cells by high S-phase (average, 25.95). S-phase had some use as a discriminating factor, because no benign reactive cases had more than 17%. However, 7 of 23 malignant cases had a value below 17%. DNA analysis by image was more sensitive and specific than flow. Either may be used when immunocytochemistry is nondiagnostic or cannot be performed.

The role of conventional cytology, immunocytochemistry, and flow cytometric DNA ploidy in the evaluation of body cavity fluids: a prospective study of 52 patients.

Fifty-two specimens of body cavity fluids from 52 patients were analyzed with conventional cytology, immunocytochemistry, and flow cytometric DNA ploidy methods to evaluate the most appropriate way of applying and interpreting immunocytochemistry and to evaluate the contribution of DNA ploidy analysis to conventional cytology in the diagnosis of body cavity fluids. The results suggest that conventional cytology still has an important role in the diagnosis of body cavity fluids. MOC 31 is the most sensitive monoclonal antibody for distinguishing benign mesothelial cells from malignant epithelial cells. Immunocytochemistry with the combination of cytokeratin, desmin, and MOC 31 with or without epithelial membrane antigen is suggested as a helpful ancillary method for the differential diagnosis of body cavity fluids. Flow cytometric DNA ploidy analysis also provides additional information in some difficult cases. Appropriate integration of clinical information and results of conventional cytology, immunocytochemistry, and flow cytometry are necessary to achieve the most accurate diagnosis in patients with effusion involving a body cavity.

N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome.

To evaluate the clinical significance of N-ras mutations in the myelodysplastic syndrome (MDS) archival bone marrow samples from 252 patients were studied for the presence of N-ras exon I mutations using polymerase chain reaction amplification and differential oligonucleotide hybridization. Subsequently, clinical information about these patients was obtained and analyzed. Of 220 evaluable patients, 20 (9%) had point mutation of N-ras involving codon 12. Individuals with N-ras mutation had a significantly shorter survival period than those who were N-ras negative (P = .02). An increased risk of acute myelogenous leukemia (AML) was also found in patients with N-ras mutations (P = .005). N-ras mutations were not associated with any French-American-British (FAB) subtype, with the presence of increased myeloblasts, or with chromosomal aberrations in the bone marrow. However, the presence of increased bone marrow blasts was strongly associated with poor survival rate and risk of AML (P < .001 for each). After stratifying for the percentage of blasts, N-ras mutations remained significantly associated with shorter survival period (P = .04) and increased risk of AML (P = .02). Bone marrow cytogenetic abnormalities, particularly when multiple abnormalities were present, were significantly associated with a poor prognosis (P < .001). In conclusion, N-ras mutation, although relatively infrequent in MDS, is associated with short survival period and increased probability of developing AML.

Combined histochemical stains in the differential diagnosis of Cryptococcus neoformans.

Three combinations of histochemical stains were used to study 69 routinely processed tissues containing various "yeast-like" fungal organisms (Cryptococcus neoformans, Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, Paracoccidioides brasiliensis, and Candida albicans). Fontana-Masson stain combined either with mucicarmine or Alcian blue produced distinctive staining of C. neoformans (Fontana-Masson-positive wall and mucin-positive capsule) that was not identified in other fungi. The Alcian blue stain combined with periodic acid-Schiff reaction also was useful for fungal classification, because the Alcian blue component reacted with only C. neoformans (capsule) and B. dermatitidis (wall) and the periodic acid-Schiff reaction reacted with the cell wall and body of each fungus. We concluded that the combined histochemical stains used in conjunction with morphologic study of hematoxylin and eosin- and Gomori methenamine silver-stained sections are helpful in the differential diagnosis of yeast-like fungal organisms.

Clinical utility of the alizarin red S stain on permanent preparations to detect calcium-containing compounds in synovial fluid.

The alizarin red S stain for permanent cytologic preparations is a valuable test that is complementary to compensated polarized light microscopic examination to detect calcium crystals. Alizarin red S has the greatest sensitivity for detection of calcium pyrophosphate crystals because crystals are stained regardless of how weakly or strongly birefringent they may be. Alizarin red S stain does not distinguish between amorphous types of calcium compounds; therefore, the different types of calcium compounds can be distinguished only when typical crystal morphologic features are present. Diagnostic importance can be attached to intracellular material that is stainable. In contrast, the diagnostic value of stainable, amorphous, extracellular material is unreliable because it is difficult to distinguish this extracellular material from contaminants frequently found in clinical specimens. Alizarin red S does not stain monosodium urate or corticosteroid crystals. Air-dried cytospin smears are helpful because they may frequently demonstrate more crystals than the wet-mount preparation. Furthermore, special stains can be performed subsequently on air-dried cytospin smears if necessary.

Permanent stained preparations of synovial fluid for detection of calcium compounds using alizarin red S.

Permanent preparations of air dried synovial fluids were prepared by staining calcium compounds with alizarin red S stain; each slide was coverslipped with Permount. Variables studied were: (a) concentration of the solution of alizarin red S, (b) pH of staining solution, (c) time of incubation in staining solution and aqueous and ethanolic content of staining solution. The staining effect of each solution was tested on calcium pyrophosphate dihydrate, calcium oxalate, apatite and monosodium urate (MSU). Of all the solutions, best results were obtained with 0.25% alizarin red S in 50% ethanol at pH 7.0 for 30 min. With this solution, the calcium-containing compounds were well stained. MSU did not stain and still preserved negative birefringence on polarization. Fixation of smears with ethanol served a double purpose: It fixed the slides without dissolving or removing MSU or the calcium compounds, yet it did dissolve five corticosteroids commonly used for intra-articular injection which may interfere with interpretation of compensated polarized light microscopy of synovial fluids.

Combined Fontana-Masson-mucin staining of Cryptococcus neoformans.

Cryptococci react positively with various histochemical stains, including the Fontana-Masson (FM), which stains the cell wall, and mucin stains, such as alcian blue and mucicarmine, which stain the capsule. Combinations of the FM stain with both the alcian blue and mucicarmine stains were performed on paraffin-embedded tissue specimens that were obtained from 15 patients who had culture-proved cryptococcosis. Combined FM-mucicarmine and FM-alcian blue stains were compared with other individual fungal stains. The FM stain, followed by either the mucicarmine or alcian blue stain, distinctively demonstrated both the cell wall and capsule of most organisms. More organisms were recognized in the combined stains than with either stain done individually. No interference between the stains was noted. Combining the FM stain with either of these two mucin stains appears to be helpful for identifying cryptococci.

Apudoma of the pancreas: benign or malignant?

A middle-aged, obese, black woman complained of abdominal pain and tenderness in the left upper quadrant. An abdominal computerized tomographic (CT) scan revealed an encapsulated cystic mass in the tail region of the pancreas. Selective angiography confirmed mass and hypervascularity, definite encapsulation, and lack of capsular invasion. The diagnostic value of the CT scan, angiography, and special staining in classifying the excised pancreatic mass as an apudoma is discussed. Malignancy was excluded by the lack of capsular-vascular invasion and the absence of metastases. Nonfunctional status was determined by lack of hormone hypersecretion.