An improved method for discrimination of cell populations in tissue sections using microscopy-based multicolor tissue cytometry.

BACKGROUND: In tissue context, researchers and pathologists lack a generally applicable standard for quantitative determination of cytological parameters. Increasing knowledge of disease-specific markers calls for an appropriate in situ tissue cytometry. METHODS: Microscopy-based multicolor tissue cytometry (MMTC) permits multicolor analysis of single cells within tissue context. RESULTS: Tissue specimens stained for CD45/CD3/CD4/CD8 were analyzed. Specificity as well as reproducibility of MMTC is demonstrated and a novel MMTC-based function to improve visual discrimination of subpopulations is introduced. CONCLUSIONS: Our data demonstrate that MMTC constitutes an important step toward automated and quantitative fluorometry of solid tissues and cell monolayers.     

Intraabdominal desmoplastic small cell tumor mimicking adenocarcinoma. A case report

BACKGROUND: Fine needle aspirates and washings from intraabdominal desmoplastic small cell tumors (IADSCTs) are rarely encountered by pathologists. Immunocytochemical examination of histologic material is usually necessary for a definitive diagnosis. CASE: A 23-year-old man presented with abdominal pain, ascites and bilateral pleural effusion. Examination of ascitic fluid suggested adenocarcinoma, but histologic and immunocytochemical examination of surgically resected tissue showed features of an IADSCT. CONCLUSION: This case is a reminder that IADSCT should be included in the differential diagnosis of intraabdominal neoplasms and that its diagnosis in cytologic preparations requires a high index of suspicion. In particular, this case appears to be the first reported example of IADSCT mimicking adenocarcinoma.     

Inducible nitric oxide synthase and apoptosis in human B cell lymphomas

Nitric oxide synthases are isoenzymes that catalyse the synthesis of nitric oxide (NO). NO plays both pathological and physiological roles depending on its rate of synthesis and concentration in cellular source and microenvironment. Apoptosis is an important biological factor in lymphomas. This study evaluates expression of inducible nitric oxide synthase (iNOS) in human lymphomas and its relation with apoptosis. This study comprised 46 cases of B-cell lymphoma. The lymphomas were classified as 3 mantle cell, 5 marginal zone, 4 follicular, 2 Burkitt, 25 diffuse large cell, 2 anaplastic large cell, 3 lymphoblastic, 2 lymphoplasmacytic according to WHO classification of lymphoid neoplasms. Hematoxylin eosin slides of the cases were reviewed and immunoperoxidase technique was performed iNOS and Caspase monoclonal antibodies to selected sections of each case. Antigen staining was carried out with iNOS and Caspase proteins and Ultravision Polyvalent, HRP-AEC kit (Neomarkers-Biogen USA). For the evaluation of iNOS and Caspase, tumor areas with a high density of expression were chosen. Positive stained cells were counted in 5 different areas at a magnification ×40 by an Olympus B × 51 microscope in each case. The iNOS and Caspase expressions were independently recorded by four pathologists and the results were averaged. All of the cases were positive for the iNOS and Caspase. But there is not a statistically important relation between lymphoma grade and iNOS activity. We could not find a correlation between iNOS and patients age. This study reveals the capacity of B-cell neoplasms to express iNOS in situ. In conclusion, our study revealed that there is a positive relation between iNOS expression and apoptosis (p $=$ 0.032 spearman correlation).     

Immunohistochemical expression of Galectin-3 and HBME-1 in granular cell tumors: a new finding

Granular cell tumor (GCT) is a relatively rare neoplasm, usually located in the upper aerodigestive tract, skin and soft tissue. Because of its uncertain histogenesis, GCT has been the object of many immunohistochemical and ultrastructural studies that have suggested a Schwann cell origin. Our recent observation of a case of GCT immunoreactive for Galectin-3 and HBME-1 led us to further investigate the immunohistochemical profile of these neoplasms. We evaluated the immunohistochemical expression of the traditional markers for GCT (S-100, CD68) along with new markers (Galectin-3, HBME-1, Calretinina and Inhibin-alpha) in 22 granular cell tumors. Our results showed, in all cases, a constant diffuse positivity for S-100 protein, CD68 and Galectin-3. HBME-1 was positive in 95% of cases. The present study gives a new immunophenotypic profile for GCT, which could help pathologists in distinguishing morphologically ambiguous granular lesions in unusual sites.     

Inverse relationship between Leydig cell density and metastatic potential of prostatic adenocarcinoma.

PURPOSE: Evaluate the relationship between metastatic potential of prostatic adenocarcinoma (PC) and testicular Leydig cell density. MATERIALS AND METHODS: Tissue samples from 111 men, age 52-85, with PC and bilateral orchiectomy were evaluated for Leydig cell density. The patients were divided into two groups: Group A were patients with metastasis (n = 36) and Group B were patients without metastasis (n = 75). Leydig cell density was determined by direct manual microscopic cell count on the tissue sections. The means of cell counts by four pathologists, expressed as cell/0.78 mm2 were used for analysis. The normally distributed data were analyzed by two-tail Student's t-test. Thirty-eight age-compatible autopsy cases who died of unrelated causes served as normal controls. RESULTS: The mean of Leydig cell count in group A patients was 14.43 (14.43+/-1.19 SE). Mean of Group B was 47.05 (47.05+/-4.05 SE) whereas normal controls displayed a mean of 48.66 (48.66+/-2.94 SE). Group A was significantly different from control (p < 0.00001). Group A and Group B were also significant different (p < 0.001) whereas control was not significantly different from Group B (p > 0.75). CONCLUSIONS: Patients with metastatic adenocarcinoma of prostate, as a group, have a significantly lower Leydig cell density than patients without metastasis or patients without PC in compatible age groups. The hormonal relationship between this observation is however unknown. One possible explanation is that PC subpopulation with metastatic potential may require different level of endogenous androgen or are androgen-independent.     

High-risk HPV DNA detection rate in patients with atypical squamous cells and its relationship to the atypical squamous cell: squamous intraepithelial lesion ratio

OBJECTIVE: To determine if there is any relationship between the atypical squamous cell (ASC):squamous intraepithelial lesion (SIL) ratio and high-risk HPV DNA detection rate. STUDY DESIGN: Data on the frequency of various cytologic diagnoses for each pathologist were retrieved from 2002 to 2003. ASC:SIL ratio for each pathologist was calculated. In addition, data related to high-risk HPV detection rate was obtained during the study period. HPV DNA testing was performed using HC II (Digene, Gaithersburg, Maryland U.S.A). Cases with insufficient quantity were excluded. RESULTS: Five pathologists, with experience ranging from 2 to 15 years, reviewed Pap tests during the study period. For the entire laboratory, the ASC:SIL ratio was 2.0, and the high-risk HPV DNA detection rate in ASC specimens was 31%. For individual pathologists, ASC:SIL ratio ranged from 1.2 to 4.0 and high-risk HPV DNA detection rate from 31% to 38%. No significant correlation was noted between the ASC:SIL ratio and high-risk HP DNA detection rate. CONCLUSION: Our ASC:SIL ratios (except for 1 pathologist) and high-risk HPV DNA detection rates were within an acceptable range. We did not observe any association between ASC:SIL ratio and high-risk HPV detection rate.     

Training in molecular cytopathology testing

Training in molecular cytopathology testing is essential in developing and maintaining skills in modern molecular technologies as they are introduced to a universal health care system such as extant in the UK and elsewhere. We review the system in place in Northern Ireland (NI) for molecular testing of solid tumours, as an example to train staff of all grades, including pathologists, clinical scientists, biomedical scientists and equivalent technical grades. We describe training of pathologists as part of the NI Deanery medical curriculum, the NI training programme for scientists and laboratory rotation for Biomedical Scientists. Collectively, the aims of our training are two‐fold: to provide a means by which individuals may extend their experience and skills; and to provide and maintain a skilled workforce for service delivery. Through training and competency, we introduce new technologies and tests in response to personalised medicine therapies with a competent workforce. We advocate modifying programmes to suit individual needs for skill development, with formalised courses in pre‐analytical, analytical and postanalytical demands of modern molecular pathology. This is of particular relevance for cytopathology in small samples such those from formalin‐fixed paraffin‐embedded cell blocks. We finally introduce how university courses can augment training and develop a skilled workforce to benefit the delivery of services to our patients.     

Fine needle aspiration cytology of desmoplastic small round cell tumor. A case report.

BACKGROUND: Intraabdominal desmoplastic small round cell tumor (DSRCT) is a recently recognized type of primitive sarcoma characterized by a predilection for young males, a usually very aggressive course and generally unsuccessful therapy. A primitive histologic appearance with prominent desmoplasia and striking divergent multilineage differentiation are well-described morphologic features of this tumor, along with a consistent fusion of the EWS and WT1 genes at the molecular level. The cytologic literature contains only scattered references to this type of neoplasm. Detailed information on the clinical and fine needle aspiration (FNA) biopsy and the immunocytochemical and ultrastructural findings in a patient with DSRCT is presented. CASE REPORT: A 23-year-old male had a firm abdominal mass with multiple secondary lesions of the liver. An FNA biopsy was performed under ultrasonographic guidance. CONCLUSION: FNA of the liver nodules showed cohesive groups of small cells with hyperchromatic nuclei and inconspicuous nucleoli; immunocytochemically vimentin and desmin showed characteristic perinuclear globular positivity. FNA cytology is an effective means of diagnosing deeply located lesions. The cytologic features of DSRCT need to become familiar to pathologists and must be considered in the differential diagnosis of liver metastasis.     

The ASCUS : SIL ratio and the reference laboratory pathologist

The atypical squamous cells of undetermined significance (ASCUS) : squamous intraepithelial lesion (SIL) ratio was proposed to monitor laboratory use of the ASCUS diagnosis. This study addresses problems associated with comparing pathologists by this means. An intuitive example showed the ASCUS : SIL ratio depends on the prevalence of smears from patients who actually have SIL. In this study of 2000 cervical smears, each of five pathologists made 400 diagnoses. Differences among proportions of SIL diagnoses were statistically significant; differences among proportions of ASCUS diagnoses were not. Had an ASCUS : SIL ratio upper limit of 3.0 been used, two pathologists would have been misidentified as having high ASCUS diagnosis rates. Unlike the situation for laboratories, potential variability in SIL prevalence requires caution in the use of this ratio in assessing pathologists. An alternative measure that is independent of prevalence, the ASCUS : SIL odds ratio, is posited.     

Mortality study of pathologists and medical laboratory technicians.

Membership lists of professional bodies were used to establish study populations of British pathologists (1955-73) and medical laboratory technicians (1963-73). The standardised mortality ratio (SMR) for pathologists was 60 and for medical laboratory technicians 67. Twenty-seven of the 310 deaths were due to suicide. These numbers gave SMRs of 250 for pathologists and 243 for medical laboratory technicians. Suicide was the commonest cause of death in female technicians. Access to lethal chemicals at work is a possible factor explaining the high proportion of suicide by poisoning compared with the general population. Suicide rates for pathologists exceed those of all medical practitioners; similary medical laboratory have higher rates than all laboratory technicians. Excess deaths from lymphatic and haemopoietic neoplasms were noted in English male pathologists (observed 8, expected 3-3; P less than 0-01). This difference is not due to Hodgkin''s disease or leukaemia and remains unexplained. No other neoplastic diseases were noted as causing excess mortality in either occupational group but a small, possibly spurious, excess number of deaths was noted for aortic aneurysm in male pathologists (observed 4, expected 1-8).     

A comparative study in morphometric grading of transitional cell carcinoma of the urinary bladder

To overcome the considerable observer inconsistency in the histologic grading of transitional cell carcinomas, the value of four different morphometric grading methods was investigated in 61 tumors of the bladder. Only two methods showed satisfactory reproducibility. Both methods, one based on random nuclear sampling and the other on selective nuclear sampling, showed an increase in the mean and standard deviation of the nuclear area with higher tumor grades (P less than .00001). Morphometric classification of the learning set (44 cases) was in agreement with the unequivocally assessed histologic grade in 35 cases (79.5%) using random sampling and in 38 cases (86.4%) using selective sampling. By reducing the grading classes to "low" (grades 1 and 2) and "high" (grade 3) and by introducing a classification probability threshold (0.80), an accurate morphometric classification was achieved in 38 cases (86.4%) using random sampling and in 41 cases (93.2%) using selective sampling. Of the 17 cases with histologic grading discrepancies, all 10 low-grade tumors (with discrepancies of grade 1 versus grade 2) were correctly classified as low-grade carcinomas by both of the morphometric methods; in the remaining 7 cases, with low-versus-high discrepancies (grade 2 versus grade 3), the selective method yielded better correlation with the tumor stage and clinical follow-up. It is concluded that morphometric classification is an acceptable alternative for histologic grading by pathologists, provided that the reproducibility of the method is confirmed. Although both random and selective sampling yielded satisfactory classifications, the selective method gave more reliable results as confirmed by the clinical behavior.     

Comparison between histopathology and DNA histogram of testis

From January to July 1989, the DNA histogram of testicular open biopsies was done for 11 patients of primary infertility and 7 patients of a control group. There were 2 failures in these 36 specimens. The flow cytometric analysis revealed characteristic patterns in the relative numbers of haploid (1C), diploid (2C), and tetraploid (4C) cells. In the presence of normal spermatogenesis, the haploid compartment contained the majority of cells, followed by the diploid, and then the tetraploid (1C greater than 2C greater than 4C). The other diagnostic criteria of flow cytometry were as follows: hypospermatogenesis (2C greater than 1C greater than 4C), the maturation arrest (2C greater than 4C greater than 1C), and Sertoli-cell-only syndrome (2C, near 100%). According to the aforementioned diagnostic criteria, the results of DNA histograms were compared with the histopathology diagnosed by junior or senior pathologists. These patterns of DNA histograms correlated with the diagnoses by senior pathologists in 28 of 34 specimens (82.6%), while there were only 20 of 36 pathologic diagnoses (55.6%) which matched between junior and senior pathologists. It is shown that abundant experience is needed for testis pathologists to diagnose accurately. The DNA histograms correlate well with pathological findings, with the advantages of quantification and fewer specimens needed. In conclusion, testicular tubular cell DNA histograms appear to be a reliable modality in the evaluation of spermatogenesis. They provide the quantitative information of sperm maturation and help in making appropriate decisions in the management of male infertility.     

α-Fetoprotein-Producing Non-Germ Cell Tumors of the Urological System

Elevated serum levels of α-fetoprotein (AFP), a fetal serum protein, occur mainly in the development of hepatocellular carcinoma (HCC) or germ cell tumors, mainly yolk sac tumor. Rarely, other tumors of the urological system produce AFP. This article reviews the AFP-producing non-germ cell tumors of the urological tract reported to date. These include different types of tumors of the adrenal glands, kidney, ureter, urinary bladder, and testis. It is important for pathologists, urologists, and oncologists to be aware of such cases as the diagnosis affects the management plan for the patient.     

Classification of routinely processed anaplastic large cell tumours with a small panel of antibodies. An immunohistochemical study with clinical follow-up

A proportion of anaplastic large cell tumours is difficult to classify on sections of routinely processed, paraffin-embedded tissue. Differentiation into large cell lymphoma, carcinoma, melanoma or sarcoma is important in order to assess prognosis and proper treatment. Although the use of immunohistochemistry has been reported in the differentiation between some of these types of neoplasms, no antibody panel, which can directly differentiate all of them, has been described. In the present study we evaluated the value of a panel of 5 antibodies for the classification of 29 anaplastic large cell tumours, which could not be classified by experienced pathologists using conventional histological and histochemical techniques. The panel, which can be used on routinely fixed paraffin-embedded tissue, consisted of 5 different antibodies directed against keratin, vimentin, the human milk-fat globule membrane antigen MAM-6, a melanoma associated antigen and common leucocyte antigen. The use of this panel directly resulted in a definite diagnosis in 95% of the cases and provided valuable information for the diagnosis in the remaining cases. The diagnosis was confirmed by additional marker studies and electron microscopy. Moreover, clinical follow-up, including treatment data, was in accordance with the diagnosis based on the panel.     

Grading ovarian tumors. Evaluation of decision making by different pathologists

Although grading of ovarian tumors is widely performed, the criteria for each grade are not well defined; as a result, pathologists tend to establish their own criteria without, however, assessing the actual predictive value of the criteria. In order to investigate this relationship, four gynecologic pathologists independently reviewed and carefully graded as benign, borderline or malignant (grade I, II or III) 40 "common" epithelial tumors of the ovary, without reference to clinical, prognostic or other findings. Intermediate grades were allowed. Subsequently, a subjective grading form was completed for each case; the form contained questions regarding the histologic and cytologic features. The sets of features with the biggest correlation with the tumor grades differed among the pathologists. This may indicate that the observers use different features in their grading processes. Moreover, the pathologist with the highest number (five) of significant microscopic features in the multivariate model had the lowest coefficient of correlation between his tumor grade and his feature set. The correlation coefficients for the other pathologists were quite similar, although the features used (no more than two or three) varied. The participants in the study felt that the methodologic approach had an educational value for them. Further investigations are required to evaluate whether the differences in the underlying decision making process also result in frank disagreement in ovarian tumor grading.     

Mitotic figure recognition: agreement among pathologists and computerized detector

Despite the prognostic importance of mitotic count as one of the components of the Bloom-Richardson grade, several studies have found that pathologists' agreement on the mitotic grade is fairly modest. Collecting a set of more than 4,200 candidate mitotic figures, we evaluate pathologists' agreement on individual figures, and train a computerized system for mitosis detection, comparing its performance to the classifications of three pathologists. The system's and the pathologists' classifications are based on evaluation of digital micrographs of hematoxylin and eosin stained breast tissue. On figures where the majority of pathologists agree on a classification, we compare the performance of the trained system to that of the individual pathologists. We find that the level of agreement of the pathologists ranges from slight to moderate, with strong biases, and that the system performs competitively in rating the ground truth set. This study is a step towards automatic mitosis count to accelerate a pathologist's work and improve reproducibility.