Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classification of non-Hodgkin lymphomas

Barrena S, Almeida J, García‐Macias M D C, López A, Rasillo A, Sayagués J M, Rivas R A, Gutiérrez M L, Ciudad J, Flores T, Balanzategui A, Caballero M D & Orfao A
(2011) Histopathology  58 , 906–918
Flow cytometry immunophenotyping of fine‐needle aspiration specimens: utility in the diagnosis and classification of non‐Hodgkin lymphomas Aims: To establish the utility of flow cytometry (FCM) for screening and diagnosis of B cell non‐Hodgkin lymphoma (B‐NHL) from lymphoid tissue samples obtained by fine‐needle aspiration (FNA). Methods and results: We compared prospectively FCM versus cytology/histology analysis of FNA samples for the diagnostic screening and further World Health Organization (WHO) subclassification of B‐NHL. FCM and cytology showed a high degree of agreement (93%); however, diagnosis of reactive processes (RP), B‐NHL and T‐NHL by FCM showed higher sensitivity than cytology (92–100% versus 64–94%, respectively), without false positive NHL cases. The antibody combination used did not allow a positive diagnosis of Hodgkin lymphoma as distinct from a RP. A high concordance rate was found between FCM and histopathology (74%) in subtyping B‐NHL. In this regard, mantle‐cell lymphoma and chronic lymphocytic leukaemia/small lymphocytic lymphoma showed the highest degree of agreement (100% concordant rates). In turn, FCM showed higher sensitivity/specificity in classifying follicular lymphoma (FL) and large B cell lymphomas, while the opposite occurred for marginal‐zone and lymphoplasmacytic lymphomas. Conclusions: FCM enhances the diagnostic ability of FNA cytology, playing a crucial role in a rapid and accurate differential diagnosis between RP, B‐NHL and T‐NHL. In addition, immunophenotyping of FNA samples contributes to a more precise subclassification of B‐NHL when combined with histopathology and genetic/molecular data.     

The diagnostic approach to fine‐needle aspiration of malignant lymphoma: Using cytomorphology and immunocytochemistry with cell transfer method

Fine‐needle aspiration (FNA) cytology is generally considered to be the screening tool for lymphoproliferative lesions. The differential and decisive diagnosis, however, of malignant lymphoma from benign reactive hyperplasia by FNA cytology is sometimes challenging. The diagnostic features compatible with lymphoma as opposed to reactive hyperplasia in FNA cytology were investigated with 31 cases of lymphoma and 31 cases of reactive hyperplasia, and immunocytochemistry with cell transfer method was additionally applied to FNA cytology. The predominance of large lymphocytes, the clustering of large lymphocytes, the presence of markedly large and/or highly pleomorphic cells, the presence of apoptotic and/or necrotic cell debris were considered characteristics of lymphomas, whereas the predominance of small lymphocytes and the presence of histiocytes were considered characteristics of reactive hyperplasia. Using these cytomorphologic characteristics, the diagnostic accuracy for malignant lymphoma in FNA cytology had a sensitivity of 80.6% and a specificity of 100%. By cell transfer method, one of Papanicolaou‐stained slides could be used in immunocytochemistry for several markers. Using such methods, sensitivity of FNA cytology for lymphoma was upgraded to 100%, and decisive diagnoses of diffuse large B‐cell lymphoma, Burkitt lymphoma, low grade B‐cell lymphoma, T‐ or NK‐cell non‐Hodgkin lymphoma (NHL), or Hodgkin lymphoma was possible. Differential diagnosis of malignant lymphoma from reactive hyperplasia, and decisive diagnoses of high, and low grade B‐cell NHL, T‐ or NK‐cell NHL, and HL could be possible by FNA cytology with cytomorphology in conjunction with immunocytochemistry using cell transfer method. Diagn. Cytopathol. 2014;42:671–679. © 2014 Wiley Periodicals, Inc.     

Diagnosis of hematopoietic processes by fine-needle aspiration in conjunction with flow cytometry: A review of 127 cases

Although fine-needle aspiration (FNA) is accepted as the method of choice for the initial evaluation of lymph nodes for metastatic carcinomas, its utility as the initial diagnostic procedure for hematopoietic processes is less established. We review our experience over a 3-year period with 127 FNA cases accompanied by flow cytometric (FC) analysis from 117 patients. Fifty cases had subsequent histologic examination. A hematopoietic process was identified in 85 cases, a reactive process in 27 cases, and a nonhematopoietic process in 15 cases. All non-Hodgkin lymphomas (NHL) were B-cell processes except for one T-cell lymphoma. By FNA/FC, 44 NHL had sufficient findings to be subtyped; of these, 27 had subsequent histologic examination. The correlation between the FNA/FC and histologic classification in these cases of NHL was 100%. One case was insufficient for diagnosis by FNA and six cases were inadequate for FC. We conclude that FNA in conjunction with FC can be used as the initial diagnostic approach for both primary and recurrent hematopoietic processes.     

Value and limitations of fine-needle aspiration cytology in diagnosis and classification of lymphomas: A review.

The fine needle aspiration (FNA) cytologic diagnosis of non-Hodgkin's lymphoma (NHL) depends upon finding a relatively monotonous population of lymphoid cells in smears. Lymphomas have successfully been classified by FNA cytology following the prevalent histologic classifications. The success rate of FNA cytology ranges from 80%-90% in diagnosis of NHL and from 67.5%-86% in its subtyping. The cytodiagnosis of Hodgkin's disease (HD) depends upon demonstration of Reed-Sternberg cells or Hodgkin's cells amongst appropriate reactive cell components. The diagnostic accuracy of FNA cytology for HD has also been invariably high (>85%). Yet, the role of cytology in primary diagnosis, subclassification and management of patients with lymphoma remains controversial. The differential diagnostic problems for NHL include a group of small round cell tumors, nonlymphoid acute leukemias and HD. Reservations have been expressed regarding the efficacy of cytology in separating florid reactive hyperplasia from low-grade malignant lymphoma. The reported cytodiagnostic accuracy for follicular lymphomas and nodular sclerosis type of HD is less compared to other subtypes of NHL and HD respectively since nodular pattern and sclerosis are strict histologic criteria which can not be appreciated in cytologic preparations. Entities like atypical lymphoproliferative disorders, peripheral T-cell lymphomas and Ki-1 positive anaplastic large cell lymphomas pose diagnostic challenges to cytologists. Despite these limitations, FNA cytology remains the first line of investigations (screening test) used in cases of lymphadenopathy. Besides initial diagnosis of lymphoma, it helps in detection of residual disease, recurrences and progression of low-grade to high-grade lymphoma, and helps in staging the disease. Availability of prior FNA cytology report facilitates the histologic diagnosis and classification of NHL. Various special ancillary techniques are now being performed on lymph node aspirates to diagnose lymphoma versus other malignancies, and to decide the functional character of lymphomas and their clonal nature. Diagn. Cytopathol. 1999;21:240-249.     

Fine‐needle aspiration with flow cytometric immunophenotyping for primary diagnosis of intra‐abdominal lymphomas

Cytomorphology in conjunction with immunophenotypic characterization is becoming increasingly used for the primary diagnosis of non‐Hodgkin's lymphomas (NHL). This combination is especially advantageous for the diagnosis of intra‐abdominal and intrathoracic lymphomas, since unlike superficial lesions, open biopsy of deep‐seated tissues is more invasive and more costly, and is associated with a higher risk. We report the cytologic and immunophenotypic features of intra‐abdominal NHL obtained by fine‐needle aspiration (FNA). Twenty‐two cases of intra‐abdominal lesions obtained by image‐guided FNA where flow cytometry was also performed were reviewed. Of the 22 studied cases, 7 were classified as large‐cell lymphoma, 5 as follicular center‐cell lymphoma, 2 as small noncleaved‐cell lymphoma, 2 as lymphoplasmacytoid lymphoma, one as small lymphocytic lymphoma, and one as marginal‐zone lymphoma. In the remaining 4 cases where the immunophenotypic pattern was not definitive, the cytomorphologic features were of small cleaved cells in 3 cases and of mixed small cleaved and large cells in one case. We successfully classified 9 of the 10 patients on whom histologic confirmation was obtained. The successful primary classification of most intra‐abdominal non‐Hodgkin's lymphomas can be done with a combination of cytology and flow cytometry, and this can be the initial approach in patients with deep‐seated lesions. Diagn. Cytopathol. 1999;21:98–104. © 1999 Wiley‐Liss, Inc.     

Flow cytometric detection of B-clonal excess in fine needle aspirates for enhanced diagnostic accuracy in non-Hodgkin''s lymphoma in adults

Fine needle aspiration (FNA) cytology is a valuable aid to diagnosis and tumour staging in patients with non-Hodgkin's lymphoma. These tumours are often multicentric and involve sites such as the liver or the spleen which are not easily accessible to surgical biopsy. Particularly with splenic involvement, there is a diagnostic problem of morphologically distinguishing the lymphoma cells in an admixture of normal lymphocytes. Since most lymphomas in adults are of B-cell origin, we studied the diagnostic value of adding a surface immunoglobulin (sIg) light chain analysis to the cytological evaluation of FNAs. B-clonal excess was determined by flow cytometric analysis of the sIg light chain distribution and a monoclonal finding was considered diagnostic of lymphoma. In primary diagnostic procedures the light chain analysis established a diagnosis of lymphoma in 5/14 (36%) aspirates from patients with poorly differentiated tumours. Fine needle aspirates performed as part of staging procedures were morphologically normal or inconclusive in 19 cases; in seven of these (37%) lymphoma involvement was diagnosed by the light chain analysis. Diagnostic precision was enhanced by combining morphological and immunological evaluation of fine needles aspirates in patients with established or suspected non-Hodgkin's lymphoma.     

Primary diagnosis and REAL/WHO classification of non-Hodgkin's lymphoma by fine-needle aspiration: cytomorphologic and immunophenotypic approach

The Revised European American lymphoma (REAL) and World Health Organization (WHO) classification of non-Hodgkin's lymphoma (NHL) relies on the constellation of cytologic, phenotypic, genotypic, and clinical characteristics of NHL. For the most part, the classification does not rely on architectural pattern for classification of neoplasms. This classification makes it possible to diagnose and classify lymphomas by fine-needle aspiration (FNA). In this study, we attempted to evaluate the accuracy of FNA in diagnosing and classifying NHL within the context of the REAL/WHO classifications. Cases included only those in which FNA was the primary diagnosis, followed by a surgical biopsy for confirmation. Flow cytometry (FCM) for phenotyping was carried out whenever material was available. Two groups of pathologists were identified. Group A consisted of pathologists with background training in cytopathology and/or hematopathology (three pathologists). Group B consisted of experienced surgical pathologists with no training in cytopathology and/or hematopathology (four pathologists). Seventy-four cases were included in the study. FCM phenotyping was performed in 53 cases (71%). Large cell lymphoma constituted 63% of the cases. The remaining lymphomas included Burkitt's, small lymphocytic, lymphoblastic, follicle center cell, Ki-1, mantle cell, marginal zone, and natural killer cell lymphoma. The diagnosis of lymphoma was rendered for all cases. The correct classification was seen in 63% of the cases. Classification was more accurate in immunophenotyped than in nonimmunophenotyped cases (84% vs 33%; P = 0.00004). Group A pathologists showed higher incidence of proper classification than group B (80% vs 56%; P = 0.046). The diagnosis and classification of NHL can be achieved in a large number of cases on FNA material. This accuracy can be increased if cytomorphologic criteria are established for different entities of NHL aided by FCM for phenotyping.     

T-cell-rich B-cell lymphoma (TCRBCL): limitations in fine-needle aspiration cytodiagnosis

Exclusive reports on fine needle aspiration (FNA) cytodiagnosis of T-cell-rich B-cell lymphoma (TCRBCL) are scarce in literature. This report reflects the diagnostic difficulties associated with cytodiagnosis of this rare variant of diffuse large B-cell lymphoma. The study is based on 11 cases with age ranging from 16 to 63 years and a median of 50 years. Male to female ratio was 6:5. Ten cases presented with lymphadenopathy and one had lymphadenopathy as well as extranodal solid tumor. The initial cytodiagnosis was suggestive of TCRBCL in one case, TCRBCL/Hodgkin's lymphoma (HL) in three cases, TCRBCL/HL/anaplastic large cell lymphoma (ALCL) in two cases, TCRBCL/ALCL in one case, and TCRBCL/non-Hodgkin lymphoma (NHL) T-cell/ALCL in one case. There was also a cytologically diagnosed HL case, which on review turned out to be HL/TCRBCL. Histopathological diagnosis was HL in all these nine cases. There were two histologically diagnosed TCRBCL cases during this period, with cytodiagnoses of NHL other than TCRBCL in one and HL in the other. While highlighting the difficulties associated with the cytodiagnosis of TCRBCL, this study conveys a word of caution that adequate immunocytochemical studies should be performed before diagnosing this rare neoplasm with a varied cytomorphology.     

FNA cytodiagnosis of non-Hodgkin's lymphoma and its subtyping under working formulation of 175 cases

One hundred thirty-two cases diagnosed as non-Hodgkin's lymphoma (NHL) by fine-needle aspiration cytology (FNAC) and histology, and 43 cases in which there were minor or major discrepancies between cytology and histology for diagnosis of NHL, were reviewed. The diagnostic accuracy of FNAC for NHL was 86.3% at the initial diagnosis. Following review, all the 132 cases initially diagnosed as NHL by cytology and histology remained so with minor changes in subtypes in a few cases. Of the 43 discrepant cases, 28 turned out to be NHL and 6 as Hodgkin's disease (HD); 3 were anaplastic carcinoma; and in 6 cases the discrepancy still persisted. Diagnostic accuracy of FNA for NHL improved to 98.0% following review. Categorization of histologically diagnosed NHL cases under working formulation showed that 10.4%, 21.5%, and 57.7%, respectively, were low, intermediate, and high-grade lymphomas. The corresponding figures were 16.6%, 18.4%, and 60.1%, respectively, in cytology. The diagnostic accuracy of cytology for subtyping was found to be 67.5%.     

Fine-needle aspiration cytology and flow cytometric immunophenotyping in diagnosis and classification of non-Hodgkin lymphoma in comparison to histopathology

This prospective study aimed to compare the value of fine needle aspiration (FNA) cytology (FNAC) and flow cytometric immunophenotyping (FCI) with histopatopathology (HP) in the diagnosis and classification of non-Hodgkin lymphoma (NHL). Twenty-nine excised lymph nodes suspected of NHL were evaluated using FNAC, FCI, and HP. Specimens were divided into two equal parts; one for HP and the other for FNAC and FCI. Results were compared in terms of diagnosis (malignant, benign or reactive, and metastatic) and NHL class. With combined FNAC/FCI, 11 (37.9%) cases were diagnosed as NHL, 11 cases (37.9%) as reactive lymph node, six cases (20.6%) as Hodgkin's lymphoma, and one case (3.4%) as metastasis. HP revealed nine cases (31%) of NHL, five cases (17.2%) of reactive lymph nodes and all the diagnosed metastatic and Hodgkin's lymphoma. Considering histology as a gold standard method in diagnosis, the sensitivity, specificity, PPV and NPV of FNAC/FCI in differentiate malignant and benign lesion were 73.9%, 83.3%, 94.4%, and 45.5%, respectively and in differentiate NHL from others were 75%, 93.8%, 90%, and 83.3%, respectively. Cytology and HP in addition to FCI and HP are significantly different from determination of NHL lesions point of view (P = 0.001 and P < 0.0001, respectively). However, FCI can be considered as an adjunctive method for Cytology especially because Cytology is not competent enough to differentiate between benign lesions and Lymphoma. Additionally, FCI is shown to be an accurate method in classifying NHL.     

Fine-needle aspiration of a primary mediastinal large B-cell lymphoma: a case report with cytologic, histologic, and flow cytometric considerations

Fine-needle aspiration (FNA) cytology and immunophenotyping by flow cytometry (FCM) are increasingly being used for diagnosing and subclassifying lymphoma in the REAL/WHO classification. Herein, we report a case of primary mediastinal large B-cell lymphoma (PMBL), a subtype of diffuse large B-cell lymphoma in the WHO classification, diagnosed by FNA cytology in conjunction with FCM. This, to our knowledge, has not previously been reported. A 57-yr-old woman presented with bilateral axillary lymphadenopathy and intermittent shortness of breath. CT scan revealed a 5-cm anterior mediastinal mass and mediastinal lymphadenopathy. Endoscopic ultrasound-guided FNA of a 4.5-cm subcarinal lymph node showed medium to large atypical lymphocytes with scant to moderate finely vacuolated cytoplasm. Nuclei were enlarged, cleaved, noncleaved, lobulated, and hyperchromatic. The background showed lymphoglandular bodies. Malignant large cell lymphoma was cytologically diagnosed. FCM, performed on a portion of the FNA specimen, demonstrated large B cells devoid of surface immunoglobulin expression, the characteristic immunophenotype of PMBL. The histologic diagnosis was PMBL. Touch-imprint cytology of the histologic specimen showed large cells with a narrow rim of clear cytoplasm and prominent outer cell border. Nuclear features were similar to the FNA specimen. In the presence of a mediastinal mass, FNA cytology in conjunction with FCM can effectively diagnose PMBL in the appropriate clinical setting.     

FNA in diagnosis of orbital lesions causing proptosis in adults

The orbit is affected by a wide range of pathologic lesions, for which a morphologic diagnosis is needed to allow adequate therapy. With increasing use of fine needle aspiration (FNA) in diagnostic pathology, the procedure has been applied for the diagnosis of space occupying lesions of the orbit. We present the cytomorphological diagnosis on orbital FNA in adult patients presenting with proptosis. Records of seven adult patients who had presented with proptosis and had undergone orbital FNA were retrieved and analysed. FNA was performed from the palpable mass in six cases and under ultrasound guidance in one case. No complication during or after FNA was reported in any of the cases. Histopathological diagnosis was available in 5 cases. Out of the 7 cases, 3 were non-neoplastic (2 inflammatory lesions, 1 reactive lymphoid hyperplasia) and 4 were neoplastic (2 primary tumors and 2 secondary involvement). Both the primary tumors were non Hodgkin's lymphoma (NHL) B-cell type. Metastases included one case of uterine sarcomatoid carcinoma and one case of secondary involvement by extension of olfactory neuroblastoma. In all the neoplastic cases, cytological diagnosis corresponded with the histopathological diagnosis. It can be concluded that cause of proptosis among adults are different from those among children and include secondary malignancies. FNA is a simple and important preliminary diagnostic modality in the assessment of adult proptosis prior to any surgical intervention.     

Cytodiagnosis of primary lymphoma of bone on fine-needle aspiration cytology specimens: review of 25 cases

Diagnosis of nodal lymphomas on fine-needle aspiration (FNA) cytologic specimens has been well established. However, cytodiagnosis of primary lymphoma of bone has not been well documented because of its rarity. We undertook a retrospective study of 25 cases of FNA cytologic specimens of primary lymphoma of bone. The slides were available for review in 20 cases; each case was evaluated with 15 cytologic features in conjunction with immunophenotyping and available surgical materials. Three diagnostic categories were assigned, including nondiagnostic (4/16%), suspicious (3/12%), and malignant (18/72%). Among the 18 malignant lymphoma, all were diagnosed on the basis of cytologic materials together with immunocytochemistry, except that two cases also relied on the cell blocks. The nondiagnostic and suspicious cases were subsequently confirmed to be malignant lymphoma on the surgical core biopsies. Of the 25 cases, 23 cases were large B-cell lymphoma, one follicular lymphoma large cell type, and one small lymphocytic lymphoma. False-positive or false-negative cases were not present in this study series. In conclusion, the vast majority of primary lymphoma of bone can be accurately diagnosed and classified on FNA cytologic specimens in conjunction with immunocytochemistry. The nondiagnostic and suspicious categories can be further reduced or eliminated by improving FNA techniques or by recommendation of surgical core biopsies together with other techniques such as flow cytometry and molecular analysis.     

Fine‐needle aspiration of fibrous dysplasia of bone: A worthwhile endeavor or not?

Fine-needle aspiration (FNA) is a minimally invasive technique which is enjoying ever-increasing popularity in the initial diagnosis of many pathologic processes. However, FNA diagnosis of neoplasms occurring within bones is less commonly employed and is not the preferred method in some types of bony lesions. Fibrous dysplasia is a primary neoplasm of bone for which it is not yet clear whether FNA can reliably yield adequate diagnostic material.Review of data from 82 cases of fibrous dysplasia diagnosed between 1990 and 2006 yielded six cases, in which diagnosis was initially attempted by FNA prior to open biopsy and surgical resection. Corresponding cytologic, histologic, and imaging characteristics of the cases were reviewed.Of the six cases in which initial diagnosis was attempted by FNA, only two of six (33%) yielded adequate diagnostic material. Smears of aspirated material in all cases contained nonspecific elements, including fragments of benign host bone and cartilage, bland stromal cells, adipocytes, blood, and debris. Importantly, the two positive FNA cases were dependent on the concurrent core needle biopsy (all smears of aspirate material were nondiagnostic).Even with image guidance, FNA is insufficient to obtain diagnostic material for cases of suspected fibrous dysplasia of bone. Core needle biopsy at least is recommended to obtain adequate material, and to reduce the risk of misdiagnosis due to sampling error.     

EUS‐guided 22‐gauge fine‐needle aspiration versus core biopsy needle in the evaluation of solid pancreatic neoplasms

Endoscopic ultrasound‐guided fine‐needle aspiration (EUS‐FNA) is widely used for diagnosis of pancreatic lesions. The Echotip Procore Needle (Wilson‐Cook Medical) is a new 22G fine biopsy needle (FNB) for obtaining core biopsy material at time of EUS. This study aimed to compare the technical and diagnostic performance of conventional FNA and FNB. Thirty‐two patients met the design criteria for this prospective paired cohort study. All lesions sampled were solid (non‐cystic) pancreatic masses by EUS appearance. Patients were randomized to receive FNA or FNB by first attempt. A cytopathologist performed on‐site evaluations. Samples were assessed for accuracy of diagnosis, cellularity, contamination, and sufficiency for ancillary studies. Technical and diagnostic performances were compared. Compared to FNA, there was a statistically significant decreased ability of FNB to achieve a diagnosis (FNA 93.8%, FNB 28.1%, P < 0.001). FNB was diagnostically superior to FNA in 1 of 32 cases. Technical failures were observed in five cases due to resistance to advancement of the FNB needle. Regarding operator perceived ease‐of‐use, FNA outperformed FNB (P < 0.001). Eight cases had insufficient FNB material to survive tissue processing. There was no significant difference in mean specimen cellularity between devices. FNA samples showed an increased amount of contaminant (P = 0.036) but were more sufficient for ancillary studies (P = 0.502). Although deemed comparable to FNA when providing material for cytology, the pledged advantage of FNB acting like a core biopsy needle was not apparent in our series. Additional studies are needed before routine adoption of 22G FNB can be recommended. Diagn. Cytopathol. 2014;42:751–758. © 2014 Wiley Periodicals, Inc.     

Fine-needle aspiration in non-Hodgkin lymphoma: evaluation of cell size by cytomorphology and flow cytometry

We studied 48 non-Hodgkin lymphoma (NHL) fine-needle aspiration (FNA) specimens with initial cytomorphology (CM) and flow cytometry (FC) and subsequent surgical biopsy of the same lesion to determine whether a reliable diagnosis of large cell lymphoma or large cell transformation could be made. CM was evaluated by examining 200 lymphocytes in each specimen. FC was performed by analyzing monoclonal or abnormal B-cell populations. Percentages of large cells were evaluated by CM and FC and results correlated with the histologic diagnosis. All small cell NHLs showed fewer than 40% large cells by CM and FC; 100% (9/9; FC) and 67% (6/9; CM) of diffuse large B-cell lymphomas demonstrated greater than 40% large cells. Variable numbers of large cells were detected in grade III follicular lymphoma, low-grade lymphoma with partial large cell transformation, and large B-cell lymphoma containing fewer than 10% neoplastic cells. By using combined CM and FC, large cell lymphoma and large cell transformation can be diagnosed reliably by FNA if greater than 40% large cells are present. Surgical biopsy is necessary when there is necrosis, fewer than 10% neoplastic cells by FC, or fewer than 40% large cells with clinical signs of transformation.     

Lymphoid neoplasms in HIV-positive individuals in India

The HIV epidemic in the Asian subcontinent has a significant impact on India. Patients with AIDS have an increased risk of developing non-Hodgkin lymphoma (NHL). In this study, we have investigated the pattern of distribution of lymphoid neoplasms and also studied the Epstein-Barr virus (EBV)-association and p53 expression in 35 HIV-positive patients from India. The biopsy samples were studied for histology and for expression of CD20, CD3, CD15, CD30, light chains, CD138, bcl-6, epithelial membrane antigen, EBV-latent membrane protein-1, and p53 protein. In situ hybridization was performed with digoxigenin-labeled anti-sense EBV-encoded nuclear RNA-1 (EBER-1) probe. Polymerase chain reaction (PCR) was performed on DNA extracted from paraffin sections for EBV-subtype analysis. The 35 cases included 7 cases of Hodgkin disease (HD), 4 cases of plasmacytoma (PL), and 24 cases of NHL. Among the cases of NHL, 3 were Burkitt lymphoma (BL), 4 were diffuse large B-cell lymphoma (DLBL) of centroblastic type (CBL), 10 were DLBL of immunoblastic type (IBL), 4 were high-grade B-cell lymphoma (unspecified) and the rest were other subtypes. EBV-association was noted in all cases of HD, 2 of 3 BL, and 3 of 10 IBL. PCR analysis of the EBNA-3C gene revealed amplimers corresponding to type A. A p53 protein overexpression was noted in 6 of 10 IBLs, 1 of 3 BLs, 2 of 3 CBLs, and 5 of 7 cases of HD. This is the first reported study of lymphoid malignancies in HIV-positive individuals from India.     

The role of flow cytometric immunophenotyping in improving the diagnostic accuracy in referred fine-needle aspiration specimens

Flow cytometric (FCM) immunophenotyping has an important role in the diagnostic work up of fine-needle aspiration (FNA) specimens obtained from lymphoid lesions. The objective of the present study was to evaluate the feasibility of this method with respect to referred FNA specimens. One hundred and two FNA specimens referred to our laboratory for FCM analysis during the last 3 years were studied. Specimens were sent, accompanied by cytological smears, from 11 distant hospitals by ordinary mail. The evaluation of potential B-cell monoclonality, the main diagnostic issue to be resolved using FCM, was possible in 86 of these 102 cases. The remaining 16 samples could not be analyzed or adequately interpreted because of sparse or necrotic material. A monoclonal B-cell population was found in 17/86 satisfactory cases, of which 16 were histologically confirmed. Eight cases contained cells positive for the epithelial marker Ber-EP4 and were diagnosed accordingly as carcinomas. FCM analysis of specimens obtained with a clinical question of Hodgkin lymphoma or T-cell lymphomas did not yield definitive data. The time lapse between sampling and analysis (12-84 hr) did not affect the results. This probably was due to the fact that all aspirates were taken in Roswell Park Memorial Institute (RPMI) cell medium, supplemented with 50% fetal calf serum. In conclusion, this retrospective study establishes that it is possible, in the majority of cases, to refer FNA material for FCM immunophenotyping by mail, and that results regarding B-cell clonality in the case of small-cell lymphomas are reliable also after a transportation period of 3-4 days. Carcinoma may be similarly diagnosed and a diagnosis of lymphoma may be excluded in reactive proliferations. Cases with only a few atypical cells or specimens from patients suspected of having Hodgkin lymphoma or T-cell lymphomas are not suitable for analysis by FCM.     

Bilateral primary lymphoma of the breast: a case report initially diagnosed by FNAC

Primary lymphoma of the breast (PLB) is a rare disease, representing 0.04-0.5% of all malignant breast neoplasms. We present a patient with bilateral breast involvement by a high-grade diffuse large B-cell lymphoma, which was diagnosed initially by fine-needle aspiration cytology (FNAC). Mammography revealed a diffuse increase in density of the right breast and a large solitary mass on the left breast, suggestive of an inflammatory carcinoma. The patient underwent FNAC and the diagnosis of a non-Hodgkin's lymphoma (NHL) was suggested. Physical examination revealed palpable bilateral axillary lymph nodes but no evidence of concurrent widespread disease. The patient underwent complete staging evaluation. The only positive findings were an elevated lactate dehydrogenase (LDH) and evidence of axillary lymphadenopathy on CT. Excisional biopsy was performed on the left breast. The morphological and immunohistochemical analysis confirmed the diagnosis of a high-grade diffuse large B-cell lymphoma with an immunophenotype suggestive of a germinal center cell origin.