Wojciech Gorczyca

CD5- mantle cell lymphoma.

Mantle cell lymphoma (MCL) typically expresses B-cell antigens and CD5 and overexpresses bcl-1 protein. However, unusual cases of bcl-1+ and CD5-MCL have been observed, posing a practical challenge for correct diagnosis and management. We identified 25 cases (48 samples) of bcl-1+ and CD5- lymphoma. CD5 expression was assessed by flow cytometric analysis alone (1 case), immunohistochemical analysis alone (17 cases), or dual flow cytometric/immunohistochemical methods (7 cases). The morphologic features were consistent with MCL with centrocytic cytomorphology in 20 cases and blastic variant in 5 cases. The t(11;14) was confirmed in 8 of 11 cases by fluorescence in situ hybridization of paraffin-embedded tissue. Cytogenetic analysis revealed the t(11;14) within a complex karyotype in 2 additional cases. These data show that MCL may lack CD5 expression. Evaluation of bcl-1 expression by immunohistochemical analysis or molecular genetics may be indicated if MCL is suspected clinically or morphologically despite a lack of CD5 expression.

2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasia: Medical indications†

The clinical indications for diagnostic flow cytometry studies are an evolving consensus, as the knowledge of antigenic definition of hematolymphoid malignancies and the prognostic significance of antigen expression evolves. Additionally the standard of care is not routinely communicated to practicing clinicians and diagnostic services, especially as may relate to new technologies. Accordingly there is often uncertainty on the part of clinicians, payers of medical services, diagnostic physicians and scientists as to the appropriate use of diagnostic flow cytometry. In an attempt to communicate contemporary diagnostic utility of immunophenotypic flow cytometry in the diagnosis and follow‐up of patients with hematolymphoid malignancies, the Clinical Cytometry Society organized a two day meeting of international experts in this area to reach a consensus as to this diagnostic tool. This report summarizes the appropriate use of diagnostic flow cytometry as determined by unanimous approval of these experienced practitioners.

B-Cell Lymphomas With Coexpression of CD5 and CD10

Coexpression of CD5 and CD10 is highly unusual in B-cell lymphomas and may pose a diagnostic challenge. We report 42 cases of B-cell lymphoma with simultaneous expression of CD5 and CD10. They made up approximately 0.4% of all B-cell lymphomas seen during the study period and included the following cases: large B-cell lymphoma (LBCL), 14 (33%); follicular lymphoma (FL), 10 (24%); mantle cell lymphoma (MCL), 9 (21%); chronic lymphocytic leukemia, 4 (10%); acute precursor B-cell lymphoblastic leukemia/lymphoma, 2 (5%); and other low-grade B-cell lymphomas, 3 (7%). All MCLs had overexpression of bcl-1 or the t(11;14) and were CD43+. All FLs had typical histomorphologic features and were bcl-2+ and bcl-6+ but CD43-. Of 14 LBCLs, 5 were histologically high-grade. Six (43%) of 14 patients with LBCL died within 10 months of diagnosis of CD5+CD10+ lymphoma (median survival, 4 months), including all 3 patients with stage IV disease and 2 of 5 with histologically high-grade lymphoma. Our findings indicate that coexpression of CD5 and CD10 is rare but occurs in diverse subtypes of B-cell lymphoma. Investigation of bcl-1, bcl-6, and CD43 and morphologic evaluation may resolve the potential confusion in diagnosis and lead to the recognition of the correct lymphoma subtype.

Immunophenotypic Pattern of Myeloid Populations by Flow Cytometry Analysis

We present our experience with immunophenotypic characteristics of benign and malignant myeloid populations, with emphasis on differential diagnosis especially between eosinophils, dysplastic granulocytes, neoplastic promyelocytes, and monocytes. Eosinophils are characterized by bright CD45, high side scatter (SSC), very low forward scatter (FSC), positive CD11b, CD11c, CD13, CD15, and CD33. They are negative for CD10, CD14, CD16, CD56, CD64, and HLA-DR. Mature monocytes are positive for CD11b, CD11c, CD13, CD14, CD33, and CD64, and may express CD2 and CD4. Blasts in acute myeloid leukemias (AML) with minimal differentiation have low SSC and moderate CD45 expression and are positive for CD34, CD117, CD13, HLA-DR, and CD33 and may be positive for TdT, CD4, and CD11c. In acute promyelocytic leukemia (APL), four FC patterns can be recognized. The majority of cases represented classical (hypergranular) APL and were characterized by high SSC, positive CD117, usually negative CD34, heterogeneous CD13, and bright CD33 (pattern 1). The second most common type, corresponding to hypogranular (microgranular) variant of APL, differed from classical APL by low SSC and frequent coexpression of CD2 and CD34 (pattern 2). Rare cases of APL (pattern3) showed mixture of neoplastic cells (SSC(low)/CD2(+)/CD13(+)/CD33(+)/CD34(+)/CD117(+)) and prominent population of benign granulocytes/maturing myeloid precursors (SSC(high)/CD10(+/-)/CD16(+/()/CD117(()). One case showed two APL populations, one with hypogranular and one with hypergranular characteristics (pattern 4). Detailed phenotypic characteristics of neoplastic monocytes and dysplastic granulocytes with their differential diagnosis are also presented.

Flow cytometry in the diagnosis of mediastinal tumors with emphasis on differentiating thymocytes from precursor T-lymphoblastic lymphoma/leukemia.

Flow cytometry (FC) has become the routine technique in the evaluation of hematopoietic neoplasms. Since the anterior mediastinum is a frequent site of involvement by both primary and secondary lymphoma/leukemia, flow cytometry plays an important role in the evaluation of mediastinal masses. The present study reviews 100 flow cytometry cases from patients presenting with mediastinal lesions. In 5 cases (5%) flow cytometry was not diagnostic due to either insufficient cell yield or low viability. In the remaining cases (95/100) cell suspensions were adequate for flow cytometry evaluation. Results showed that in 31/32 (96.8%), 2/3 (66.7%), 7/9 (77.8%), 7/8 (87.5%) and 11/11 (100%) cases of B-cell lymphoma, T-cell lymphoma, carcinoma, T-ALL/LBL and thymoma/thymic hyperplasia, respectively, the diagnosis could be reached by flow cytometry alone. Excluding HL, the general sensitivity of FC in diagnosing mediastinal tumors was ∼92%. Among the 100 cases, flow cytometry gave non-informative results in 3 cases of diffuse large B-cell lymphoma, 1 case of peripheral T-cell lymphoma, and 3 cases of carcinoma. No false positive results were encountered. The phenotypic pattern, especially surface CD3 expression versus forward scatter, reliably discriminated between immature thymocytes from thymoma/thymic hyperplasia from T-ALL/LBL. Flow methodology has the advantage of rapid turn-around time as well as high sensitivity, enabling patients with large anterior mediastinal masses and/or superior vena cava syndrome to begin treatment as promptly as possible. In experienced hands, flow cytometry plays a valuable and complementary role to histology and immunohistochemistry in diagnosing mediastinal tumors.

Flow Cytometry Immunophenotypic Characteristics of Monocytic Population in Acute Monocytic Leukemia (AML-M5), Acute Myelomonocytic Leukemia (AML-M4), and Chronic Myelomonocytic Leukemia (CMML)

Publisher Summary Monocytic proliferations comprise a heterogeneous group of disorders ranging from reactive monocytosis to acute monocytic leukemia. Based on the cytomorphological and phenotypic features, differential diagnosis includes acute promyelocytic leukemia (especially microgranular variant), acute myeloid leukemia (AML) without maturation, minimally differentiated AML, chronic myelomonocytic leukemia (CMML), acute megakaryocytic leukemia, and acute myelomonocytic leukemia (AML-M4). Flow cytometric immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells. It plays important role in diagnosis, classification, and monitoring of hematopoietic neoplasms, including acute leukemias. This chapter presents the phenotypic characteristic of monocytic populations from acute monocytic leukemia (AML-M5), CMML, and AML-M4. The chapter describes the different types of leukemia such as acute monocytic leukemia, chronic myelomonocytic leukemia, and acute myelomonocytic leukemia. Flow cytometry (FC) can identify and differentiate abnormal monocytes and complements cytomorphology and cytochemical staining in diagnosis of myeloid proliferations with monocytic differentiation.

Cytochemical diagnosis of Gaucher's disease by iron stain

Gaucher’s disease, the most common lysosomal storage disorder, arises from mutations in the gene encoding glucocerebrosidase. The glucocerebrosidase enzyme, which cleaves glucose from ceramide, is markedly reduced or absent. This results in accumulation of glucocerebroside, primarily in phagocytic cells. Of the various subtypes, the most common form, accounting for up to 99% of cases, is that of type I, or the chronic non-neuronopathic form. The disease is inherited in an autosomal recessive fashion, manifests in adulthood with splenic and skeletal involvement, and occurs predominantly in the Ashkenazi Jewish population. Clinical features include pathological fractures as well as cytopenia(s) caused by hypersplenism. The diagnosis is generally made by cytomorphologic examination of the bone marrow, and confirmed by measurement of glucocerebrosidase activity in peripheral blood leucocytes or cultured skin fibroblasts. Gaucher’s cells show diffuse and avid iron staining using a Prussian blue iron stain (left, Prussian blue stain with Wright–Giemsa inset), in contrast to normal bone marrow histiocytes or pseudo-Gaucher histiocytes associated with chronic myeloproliferative disorders such as chronic myeloid leukaemia (right, Prussian blue stain with Wright–Giemsa inset). This was determined over a generation ago to be probably due to phagocytosed erythrocytes. The current generation of haematologists and pathologists need to be aware of this phenomenon. Any histiocyte with diffuse iron uptake should be considered as highly suspicious for Gaucher’s disease, and an appropriate clinical work-up should be instituted.

The potential role of flow cytometry in the diagnosis of small cell carcinoma

CONTEXT Virtually no information exists in the medical literature on the immunophenotyping of small cell carcinoma by flow cytometry. CD56, or neural cell adhesion molecule, is widely expressed by small cell carcinoma and easily measured by flow cytometry. OBJECTIVE To determine the potential usefulness of flow cytometry in the diagnosis of small cell carcinoma. DESIGN AND SETTING Retrospective data and archival material on 27 patients were obtained from community hospitals. Specimens (needle aspirations and tissue biopsies) from all patients demonstrated cytomorphologic and flow cytometric features consistent with small cell carcinoma. All measurements were performed at a large reference laboratory. Routine 3- and 4-color flow cytometry using a lymphoma antibody panel, including anti-CD56, was performed. Anti-cytokeratin antibody was also used in the last 12 cases. Immunohistochemical staining with a panel of conventional markers for neuroendocrine neoplasms was performed on available tissue for purposes of confirmation of small cell carcinoma. PATIENTS Twenty-seven patients whose tissue specimens showed a clearly defined population of CD45-CD56+ cells by flow cytometry and cytomorphologic features consistent with small cell carcinoma. INTERVENTIONS Needle aspiration (n = 3) and tissue biopsy (n = 24) from a variety of sites. RESULTS CD56 positivity by flow cytometry was 100 to 1000 times that of the matched isotype control in 25 cases and 10 to 100 times that of the control in 2 cases. Cytokeratin positivity by flow cytometry was found in 12 of 12 cases. Immunohistochemical staining showed positivity for at least 1 cytokeratin and 1 or more neuroendocrine markers in 26 of 27 cases and confirmed the diagnosis of small cell carcinoma. CONCLUSIONS Routine flow cytometry can identify a neuroendocrine phenotype that shows a strong correlation with confirmatory immunohistochemical markers in cases exhibiting cytomorphologic features of small cell carcinoma. Flow cytometry appears to complement and may possibly be a satisfactory alternative to immunohistochemical staining when small cell carcinoma is suspected.

CD56-positive large B-cell lymphoma.

CD56 (NCAM), a neural adhesion molecule, is normally expressed on natural killer cells and subsets of T cells and is commonly seen on hematolymphoid neoplasms such as plasma cell myeloma and acute myelogenous leukemia. It is uncommon in B-cell lymphoma. From 2001 to 2003 a cohort of 20 cases of CD56+ B-cell lymphomas was identified by flow cytometry (<0.5% of all B-cell lymphomas studied) during a 2-year period. Most (90%) expressed CD10 and 5/5 tested cases were BCL6+, suggesting a follicular origin. An extranodal disease presentation was seen in 45% and may be related to CD56 expression. These CD56+ B-cell lymphomas may represent a new subset of large B-cell lymphoma. The relationship of cells with this antigenic profile to normal B-cell differentiation is explored.

Differential diagnosis of T-cell lymphoproliferative disorders by flow cytometry multicolor immunophenotyping. Correlation with morphology

Publisher Summary T-cell lymphoproliferative disorders are a heterogeneous and relatively rare group of lymphoid tumors. Morphologically and clinically, they can mimic both benign conditions and nonhematopoietic neoplasms. The diagnosis often requires a multitechnology approach including morphology, flow cytometry (FC), immunohistochemistry, cytogenetics/fluorescence in situ hybridization (FISH), and molecular studies (for example polymerase chain reaction [PCR] and Southern blot analysis). Although there is no single phenotypic marker specific for T-cell lymphoma/leukemia, FC plays an important role in the diagnosis and subclassification of both mature (peripheral/post-thymic) and immature T-cell disorders. Thus, the studies related to T-cell disorders can be performed using the flow cytometry analysis and immunohistochemical analysis. The chapter also presents mature T-Cell lymphoproliferative disorders. FC plays an important role in diagnosis, classification, and monitoring of the T-cell lymphoproliferations. Aberrant expression of pan-T antigens; T-cell receptors (TCR), and CD45; abnormal CD4/CD8 ratio (including dual expression or dual negativity); and presence of additional phenotypic markers helps to identify abnormal T-cell populations.