Computerized microscopic analysis of prostatic fine needle aspirates. Comparison with breast aspirates

Computerized image analysis was employed to analyze fine needle aspiration smears of the prostate and breast using both high-resolution images of individual cells and medium-resolution images of scenes and clusters (contextual analysis). A linear discriminant analysis was used to demonstrate the computer's ability to discriminate between benign and malignant categories for both types of tissue. Correct classification as benign or malignant using contextual analysis was achieved in 22 of 26 prostatic aspirates and in 15 of 18 breast aspirates, as determined by comparison with histology. The addition of high-resolution single-cell analysis resulted in correct classification of 24 of 26 prostatic aspirates and all breast aspirates. For virtually all features, the distinction between benign and malignant was more subtle for prostatic than for breast tissue. The data indicate that contextual analysis may be less effective as an adjunct to high-resolution single-cell microscopy of prostatic specimens than it is for breast specimens.     

Specific tumor markers in diagnostic cytology. Immunoperoxidase studies of carcinoembryonic antigen, lysozyme and other tissue antigens in effusions, washes and aspirates

The immunoperoxidase technique was used to identify specific tumor markers in exfoliated cells in fine needle aspirates and body fluids. Carcinoembryonic antigen (CEA) and lysozyme staining was evaluated in cytocentrifuge preparations from 42 malignant effusions and aspirates and 16 benign effusions. Reactive mesothelial cells were negative for CEA and lysozyme or showed faint peripheral cytoplasmic staining. Malignant cells from 50% of the adenocarcinomas studied were positive for CEA. All tumors studied were negative for lysozyme. These staining patterns are helpful in the differential diagnosis of reactive mesothelial and adenocarcinoma cells, a frequent diagnostic dilemma. Moreover, demonstration of specific tumor antigens (e.g., prostatic acid phosphatase, calcitonin and immunoglobulin) helped define the origin of metastatic malignancy in selected cases. Estrogen receptor activity was also identified in tumor cells using this technique. Immunoperoxidase was helpful in the evaluation of malignant cytologic specimens from patients with more than one tumor. Interpretation of staining patterns is discussed, with reference to the limitations of the technique. Immunoperoxidase methods maintain cytologic detail, are readily adaptable to diagnostic cytology and increase the specificity of cytologic diagnosis.     

Chondroblastoma in fine needle aspirates.

The cytologic features of smears of fine needle aspirates from four chondroblastomas were studied. In an ideal fine needle aspiration biopsy sample of chondroblastoma, all the diagnostic details can be found: chondroid matrix surrounding individual round to oval mononuclear cells, calcifications among cells and multinucleate osteoclasts. Chondroid matrix, which we believe to be the decisive feature, and calcifications are seen better in Giemsa-than in Papanicolaou-stained smears.     

Immunoperoxidase confirmation of parathyroid origin of ultrasound-guided fine needle aspirates of the parathyroid glands

It may be difficult to differentiate between cells of parathyroid and thyroid origin in ultrasound-guided fine needle aspirations of the neck region, even in patients with a clinical history of hyperparathyroidism. A parathyroid hormone antibody was used in an immunohistochemical system to confirm a parathyroid origin in fine needle aspirate smears from nine patients with hyperparathyroidism. Immunoperoxidase positivity for parathyroid hormone confirmed a parathyroid origin in six of nine cases and was strongly suggestive, although equivocal, in the remaining three cases. Technical problems included nonuniform staining and background staining.     

Ringlike structures in fine needle aspirates from the breast

Spherical ringlike structures of various sizes resembling Liesegang rings (LRs) are described in four fine needle aspirates (FNAs) from breast lesions over a period of 13 years. A characteristic finding in these structures was a distinct double layer outer wall with striations and an amorphous central nidus. Under polarized light they were non-refractile and no birefringence was noted in Congo red. Immunohistochemical stains for calcium, iron, mucus, glycogen, amyloid, cytokeratin and epithelial membrane antigen in all the cases were found to be negative. Since LRs can be mistaken for ova or parasites, their presence in aspirates of breast should be kept in mind to avoid misdiagnosis.     

Diagnostic schemes for fine needle aspirates of breast masses

A comparison was made of four statistically based schemes for classifying epithelial cells from 243 fine needle aspirates of breast masses as benign or malignant. Two schemes were computer-generated decision trees and two were user generated. Eleven cytologic characteristics described in the literature as being useful in distinguishing benign from malignant breast aspirates were assessed on a scale of 1 to 10, with 1 being closest to that described as benign and 10 to that described as malignant. The original computer-generated dichotomous decision tree gave 6 false negatives and 12 false positives on the data set; another tree generated from the current data improved performance slightly, with 5 false negatives and 10 false positives. Maximum diagnostic overlap occurred at the cut-point of the original dichotomous tree. The insertion of a third node evaluating additional parameters resulted in one false negative and seven false positives. This performance was matched by summing the scores of the eight characteristics that individually were most effective in separating benign from malignant. We conclude that, while statistically designed, computer-generated dichotomous decision trees identify a starting sequence for applying cytologic characteristics to distinguish between benign and malignant breast aspirates, modifications based on human expert knowledge may result in schemes that improve diagnostic performance.     

Estrogen receptor determination in fine needle aspirates of the breast. Correlation with histologic grade and comparison with biochemical analysis

Material obtained by fine needle aspiration (FNA) of 25 surgically removed breast carcinomas was tested for the immunocytochemical localization of estrogen receptor (ER) using the peroxidase-antiperoxidase method and a monoclonal antibody developed against human breast cancer ER. The results were compared to those obtained by the conventional biochemical analysis of cytosol protein. A semiquantitative relationship between the immunoperoxidase stain and the biochemical analysis suggests that cases in which greater than 70% of the cells stain and in which intense staining is present are likely to contain ER in a concentration of greater than 250 fmol/mg of cytosol. Less than 15% stained cells and an absence of intense staining is indicative of a concentration of less than 10 fmol/mg. In only one case was there a significant difference in positivity between the two methods, possibly as a result of a functional heterogeneity of the tumor cell population. Intense staining is strongly suggestive of a tumor of low histologic grade and was never seen in tumors with a high histologic grade or nuclear grade. The immunoperoxidase method of ER detection on material obtained by FNA is a semiquantitative means of selecting patients with breast cancer who are likely to respond to hormonal therapy. The method overcomes many important disadvantages of cytosol analysis and provides clinically significant information regarding the ER content and the degree of tumor differentiation.     

Morphometric analysis of fine needle aspirates from breast lesions

In an attempt to discover the morphometric variables with the most diagnostic power in the differentiation of benign from malignant breast disease, 20 unequivocally benign and 20 unequivocally malignant and histologically confirmed breast aspirates were examined on an image analyser. It was found that standard deviation of nuclear area was the most discriminant variable. Then 23 aspirates initially diagnosed as 'suspicious of malignancy' were measured by the same technique, and standard deviation of nuclear area correctly differentiated all but three cases.     

The role of micronucleus scoring in fine needle aspirates of ductal carcinoma of the breast

S. Samanta, P. Dey and R. Nijhawan The role of micronucleus scoring in fine needle aspirates of ductal carcinoma of the breast Aims and objectives: Micronucleus (MN) scoring was carried out in benign (fibroadenoma) and malignant (infiltrating ductal carcinoma) breast lesions to evaluate the role of MN as a biomarker in breast carcinomas. We also compared MN scores among different cytological grades of breast carcinoma. Materials and methods: A total of 31 archival cases of fibroadenoma (FA) and 40 cases of infiltrating ductal carcinoma (IDC) were selected. The best May‐Grünwald–Giemsa (MGG) stained fine needle aspiration cytology (FNAC) smear of each case was selected. The MN scoring was carried out independently by two observers on 1000 epithelial cells in oil immersion magnification (100× objective). The MN scores in FA and IDC were compared. The IDC cases were graded and the MN scores in different cytological grades of IDC were compared. Results: The mean MN scores (± standard deviation) in FA and IDC were 0.6 (± 1.1) and 13.6 (± 12.8), respectively, which were significantly different (P < 0.0001). There were seven grade 1, 13 grade 2, and 20 grade 3 IDCs. The mean MN scores (± standard deviation) of grade 1, 2 and 3 IDC were 4.3 (± 2.3), 11.95 (± 9.2) and 21.1 (± 16.7), respectively. An analysis of variance (anova ) test showed a significant difference in MN score between all the grades of IDC (P < 0.05). However, there was no significant difference between fibroadenoma and grade 1 IDC. The Pearson’s correlation coefficient showed positive correlations between MN scoring in the different grades of IDC. Conclusions: MN scoring on routinely stained smears of IDCs was significantly higher than in fibroadenoma and was relatively easy, reliable and reproducible. As MN scoring of grade 1 IDC was similar to fibroadenoma, a larger study should be conducted to compare grade 1 IDC with other benign breast lesions.     

Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates

?. P. Marin?ek, N. Nolde, I. Kardum‐Skelin, R. Nizzoli, B. Önal, T. Rezanko, E. Tani, K. T. Ostovi?, P. Vielh, F. Schmitt and G. Kocjan
Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates Objectives: To collect data on the variability of immunocytochemical (ICC) procedures used to detect oestrogen/progesterone receptors (ER/PR) on cytological material; to test the reproducibility of results; and to identify the crucial points in the ICC procedures that affect the result. Methods: Ten laboratories from eight countries participated in a two‐part study. In the first part, one of the participants (the coordinator) prepared and distributed cytospins from a fine needle aspirate of a primary breast carcinoma. Laboratories performed ICC staining for ER/PR according to their own methods on the test slides and in‐house positive controls. Slides were returned to the coordinator together with information on the preparation of positive control slides and the ICC methodology used. In the second part, obligatory methods of fixation and antigen retrieval were specified. Evaluation of results included grading the number of positive cells, staining intensity, background staining, cytoplasmic staining, sample condition and cellularity. Participants evaluated their own results, which were subsequently evaluated by the coordinator. Results: There was great variability in the preparation of slides for in‐house controls and ICC methodology. The outcome of ICC staining of in‐house control slides was excellent in two laboratories, adequate in three, sub‐optimal in four and inadequate in one. Only six obtained a positive reaction on the test slides and not all were of a high quality. Results of the second run were greatly improved in terms of cellularity of in‐house positive control slides, and scores for the percentage of stained cells and staining intensity of control and test slides. Cytospins and monolayer (ThinPrep^®) preparations were superior to direct smears; methods of fixation and antigen retrieval were the key points in the staining process. Conclusions: Our experience points to the need for guidelines for hormonal receptor determination and external quality control on cytological material, in order for cytological methods to be used in routine clinical practice with a suitable degree of confidence.     

Development of a new antigen detection dot-ELISA for diagnosis of tubercular lymphadenitis in fine needle aspirates

A sandwich dot enzyme-linked immunosorbent assay (ELISA) was standardized to detect mycobacterial antigen in fine needle aspirates of patients with tubercular lymphadenitis (TBLN). The assay was performed on nitrocellulose paper by using antibodies raised in mice and rabbits against crude soluble protein (CSP) of Mycobacterium tuberculosis. The test was able to detect as low as 5 ng protein/ml. A total of 225 suspected cases of tubercular lymphadenopathy were screened, out of which 96 were cytomorphologically confirmed as cases of tubercular lymphadenitis (50 acid-fast bacilli (AFB)-positive and 46 AFB-negative). These were considered as positive controls. Only 28 cases were proven to be of nontubercular etiology and were considered as negative controls. In the remaining 101 (39 scanty) aspirates, tubercular etiology could neither be ruled out nor confirmed. Out of 50 AFB-positive confirmed cases of tubercular lymphadenitis, 46 were ELISA-positive. Out of 46 AFB-negative but cytomorphologically confirmed aspirates, antigen could be demonstrated in only 42 aspirates. Four samples from patients with nontubercular etiology were also found to be ELISA-positive. Antigen was picked up in a total of 90.3% of aspirates with suspicion of tuberculosis and 79.5% of scanty aspirates. The assay was found to be 91.6% sensitive and 85.7% specific. The assay was found to be simple and rapid, and hence, could be performed in areas where health facilities are rudimentary.     

Planimetric studies on fine needle aspirates from follicular adenoma and follicular carcinoma of the thyroid

A planimetric study was performed on fine needle aspiration biopsy smears from 21 follicular thyroid adenomas, 13 follicular thyroid carcinomas and 7 nontoxic goiters. The nuclear and the cytoplasmic projected areas were measured in each smear on 50 cells with intact nuclei. The nuclear-cytoplasmic area was calculated. Significant differences in mean nuclear area were found between benign and malignant follicular neoplasms and between neoplastic cells and cells from nontoxic goiter. Planimetry of cells aspirated from follicular neoplasms permitted differentiation between carcinomas and adenomas with a high degree of statistical probability.     

Semi-quantitative analysis of soft-tissue reactions in fine needle aspirates from tissue cysticercosis

Fine needle aspiration cytology (FNA) has a well-documented role in the diagnosis of cysticercosis. However, little is discussed about the associated inflammatory response in the host tissues. Aspirates from 182 cases of subcutaneous cysticercosis were semiquantitated for the type and degree of inflammatory response, and the amount and preservation of the parasite. Tissue sections were reviewed where available. In the FNA where no parasite was observed but a confirmatory tissue diagnosis was available, it was found that eosinophils (52%), epithelioid cell granulomas (30%), palisading histiocytes (33%) and giant cells (28%) were seen less frequently than in those where larval fragments were identified in the aspirated material in varying quantities, the response being 88-92% eosinophils, 50-70% palisading histiocytes, 68-80% epithelioid cell granulomas and 46-74% giant cells. Repair cells were maximally seen when readily identifiable larval fragments were seen in the aspirate. Bizarre cells were equally distributed in these aspirates. The tissue response in FNA from subcutaneous cysticercosis can be varied and eosinophils are found to increase with the presence of the degenerating parasite. In soft-tissue aspirates, palisading histiocytes with epithelioid cell granulomas with or without giant cells and an inflammatory exudate with predominantly eosinophils alerts one to search diligently for a parasite.     

Mean nuclear area of fine needle aspirates of primary preoperative palpable breast carcinoma using image cytometry

OBJECTIVE: Early, operable breast cancers in appropriate patients are increasingly being treated preoperatively using neoadjuvant chemotherapy. A good response rate is seen with high grade tumors. Nuclear size, which may reflect the grade of the tumor, is also of possible prognostic value in breast cancer. STUDY DESIGN: We measured the mean nuclear area (MNA) of 114 consecutive preoperative fine needle aspirates of palpable, operable breast cancers. We used computerized image cytometry to measure nuclear area to determine tumor biology and possible grade prior to treatment. RESULTS: Histologic grade distribution was as follows: low grade, 15%; moderate grade, 40%; and high grade, 45%. Mann-Whitney test for trend on tumor size and histologic grade between MNA showed a significant relationship between MNA and tumor size (P=.016) but no significance between MNA and histologic grade (P =.22). The chi2 and Fisher Exact Test between MNA and node-positive or -negative patients showed no significance. CONCLUSION: When correlating MNA with tumor size and histologic grade, high MNA is present at a higher frequency as tumor size and histologic grade increase.     

High-resolution and contextual analysis for the diagnosis of fine needle aspirates of breast.

A study was undertaken to confirm earlier work on a smaller number of patients that had suggested that medium-resolution contextual analysis complements high-resolution individual cell analysis for cytomorphometric classification of fine needle aspirate smears of breast. The objectives of this study were to improve and verify the method. Sixty-one biopsy-confirmed hematoxylin and eosin-stained aspirate smears of breast were restained using the Feulgen technique. Individual nuclei were digitized at a resolution of 0.25 micron. Features describing size, shape, density and texture were extracted from the images. Individual cell analysis correctly classified 84% of cases, contextual analysis correctly classified 70% of cases, and the combined use of both techniques resulted in 87% classification accuracy. However, if fibroadenoma cases are excluded, the combined correct classification rate is 93%. Geometric and densitometric features contributed most to correct classification in individual cell analysis, while the most important contextual feature was the number of clusters per scene. We conclude that the addition of quantitative measures of smear patterns, termed "contextual analysis," improves automated classification schemes.     

Dependence of DNA-histograms on the sampling techniques in fine needle aspirates of the breast.

48 fine needle aspiration biopsy (FNAB) samples from 25 breast cancer cases, originally used for cytodiagnosis were subjected to DNA cytometry. There were air dried smears stained with the MGG method, and samples stained with HE or PAP stain after 50% ethanol fixation and cytocentrifugation. Different sampling strategies were applied. Four methods were tested: method 1: cell groups measured, method 2: all cells measured, method 3: free cells measured, and method 4: atypical free cells measured. Method 4 showed most often DNA aneuploid histogram patterns, sampling method 1 had the highest number of DNA diploid histogram patterns. Diagnostic approaches may benefit from a sampling method detecting the hiding aneuploid cell population. Grading of neoplasm could potentially benefit from other approaches.     

Some cytologic features of hepatocellular carcinoma as seen in fine needle aspirates

Cytologic study of fine needle aspirates in 15 cases of hepatocellular carcinoma revealed the following cellular patterns in order of decreasing frequency: trabecular, acinar and anaplastic. The most useful cytologic criteria for the identification of hepatocellular carcinoma were the similarity of tumor cells to liver cells, the prominence of nucleoli and the frequent presence of a trabecular pattern. Our study showed that the cellular changes seen in aspirated material faithfully reflect the histologic alterations seen in tissue sections. Hence, familiarity with the histology of hepatocellular carcinoma is of tremendous value in the interpretation of cytologic material.     

Carcinoembryonic antigen content in fine needle aspirates of the lung. A diagnostic adjunct to cytology.

Carcinoembryonic Antigen (CEA) was measured in 59 consecutive fine needle aspirates (FNAs) of the lung from 58 patients to determine if the CEA content would enhance the sensitivity of the cytologic diagnosis. Twenty-eight males and 30 females with tumors 1-40 cm in diameter were studied. Final diagnoses were correlated with the clinical history, radiologic studies, tissue (when available) and follow-up. Image-guided FNAs were performed by radiologists using a 22-gauge Chiba needle and 20-mL syringe with one to four passes per specimen. Cytologic examination included rapid assessment in the radiology suite and a final diagnosis in 24 hours. CEA was measured by enzyme immunoassay using monoclonal antibody. Nine benign aspirates and 50 malignant aspirates were diagnosed. The sensitivity of cytology was 86% and specificity, 100%. Using 5 ng/mL as the cutoff, the sensitivity of CEA for malignant aspirates was 50% and specificity, 90%. The combined sensitivity of CEA and cytology was 95%. The mean CEA in malignant aspirates was 131 ng/mL and in benign aspirates, 2.41. The highest mean CEA was seen in adenocarcinoma, 402.6 ng/mL. Lower CEA content was seen in epidermoid carcinoma (58.6 ng/mL), large cell carcinoma (8.09), oat cell carcinoma, metastatic carcinoma of the kidney and breast, thymoma and lymphoma (each less than 1 ng/mL). Elevated CEA alone was diagnostic in two aspirates of bronchioloalveolar carcinoma; carcinoma with an unknown primary source, three; and large cell carcinoma, one. The adjunctive use of CEA in FNAs of the lung enhances the sensitivity of the cytologic diagnosis.     

Immunocytochemistry of fine needle aspirates. A tactical approach

To maximize the potential of immunocytodiagnosis for fine needle aspiration (FNA) samples, it is necessary to be aware of the pitfalls and limitations of these techniques and to formulate a strategy to deal with the many variables involved. Five cases are presented to illustrate some of these variables, which include determining the adequacy of the FNA specimen, selecting tactics for cytologic and immunocytochemical studies, selecting methods for processing the FNA sample, preparing smears to enrich and preserve cells of interest, selecting enzyme labeling methods to optimize sensitivity and specificity, selecting monoclonal antibodies to make the study efficient and pertinent and interpreting the study results. The adequacy of the FNA specimen could be determined by an immediate cytologic assessment of the aspirate as it was obtained. Alcohol-fixed smears and formalin-fixed tissue sections prepared from the aspirate were used for diagnosis; the immunocytochemical studies were used as a diagnostic adjunct for accurate cell identification. Immunocytochemical studies were done on air-dried cytocentrifuge smears of pre-washed cells. While both immunoperoxidase and immunoalkaline phosphatase methods were suitable, we recommend the immunoperoxidase method for the study of aspirates from nonhemopoietic tissues and the immunoalkaline phosphatase method for the study of aspirates with many blood cells present. The proper selection of monoclonal antibodies and the interpretation of the results are best made in the context of the cytologic characteristics of the FNA sample and the clinical features of the patient.