Stelzer Barnett Even though flow cytometric (FC) analysis of bone marrow aspirates is often performed in hematolymphoid disorders at diagnosis and during disease monitoring, its role has not been defined during the staging of B‐non–Hodgkin's lymphoma (B‐NHL) and B‐cell lymphoproliferative diseases. Philadelphia, PA: Saunders Elsevier: 2011. R At morphologic examination, the case of lymphoplasmacytic lymphoma showed an excess of plasma cells, which were not evaluated for cytoplasmic light‐chain expression by FC. It may be that no disruption of these small aggregates takes place during aspiration, which may in part explain the high level of FC failure reported in this study. Bone Marrow Core Biopsy: a. This is sometimes delayed due to difficulties in obtaining a bone marrow aspirate due to a "dry tap". Applied Immunohistochemistry & Molecular Morphology. Bone marrow biopsy, (BMB) is essential to detect bone marrow (BM) infiltration in B-cell non-Hodgkin lymphomas (NHLs). A minimum of 30,000 events was collected for each sample, and a minimum of 3,000 B lymphocytes (CD19+) was collected for κ and λ light‐chain analyses. Follicular center lymphomas were the most represented group, confirming previous observations that evidences the limited role of FC in detecting marrow infiltration in this B‐NHL subtype, in which neoplastic cells are frequently tightly bound to bony trabeculae (20). We also did not address separately the issue of follow-up bone marrow examinations in monitoring disease relapse or progression; doing so may also lead to a further reduction in the number of markers used for this patient subpopulation. Flow cytometry is a laboratory method used to detect, identify, and count specific cells from blood, bone marrow, body fluids such as cerebrospinal fluid (CSF), or tumors. Disease incidence among cases evaluated by the multimarker strategy or limited strategy. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. CD45 and side scatter (SSC) gating on all lymphocytes shows a normal κ:λ ratio. Upon reviewing the aspirate smears, if the blast percentage appears to be less than 5% of nucleated cells, then a full panel is not performed. The five BM−/FC+ cases belonged to different lymphoma subtypes (Table 3); no morphologic or immunohistochemical evidence of lymphoma was seen in these biopsy specimens. Introduction to Flow Cytometry for Microbiology 542, Immunology Laboratory [30 paragraphs]. Hemodiluted aspirates were not treated differently from other specimens; specimens with aparticulate smears were not analyzed by flow cytometry in the limited strategy arm. Examination of the bone marrow by aspiration and core biopsy is used for diagnosing and staging both hematologic and many non-hematologic disorders. The morphologic bone marrow inspection was performed without prior knowledge of the FC results and classified as follows: positive (BM+) when an unequivocal infiltration pattern was observed, negative (BM−) when morphologically normal, undetermined or nondiagnostic (BMu) for bone marrow involvement when smaller biopsy (<1 cm) or adipose specimens were obtained. Cannizzo Enter your email address below and we will send you your username, If the address matches an existing account you will receive an email with instructions to retrieve your username, By continuing to browse this site, you agree to its use of cookies as described in our, I have read and accept the Wiley Online Library Terms and Conditions of Use, The Non Hodgkin's Lymphoma Classification Project, A clinical evaluation of the International Lymphoma Study Group classification of non–Hodgkin's lymphoma, Classification, staging and management of non Hodgkin's lymphomas, Value of bilateral bone marrow biopsy specimens in non–Hodgkin's lymphoma, Lack of expression of surface immunoglobulin light‐chain in B‐cell non–Hodgkin lymphomas, Lack of surface light chain expression by flow cytometric immunophenotyping can help diagnose peripheral B‐cell lymphoma, Immunophenotypic analysis of peripheral blood and bone marrow in the staging of B‐cell malignant lymphoma. Moreover, the absence of gating strategies aimed at the identification of B cells may mean that the neoplastic B‐cell population hidden within the normal B cells was not evident. We demonstrated that unfixed bone marrow trephine provides an adequate and representative cell suspension for flow cytometry and it is a powerful tool when no other material (bone marrow aspirate or peripheral blood) is available to make a rapid diagnosis. Negative biopsies were considered adequate when they included at least 1 cm of subcortical bone marrow. They are converted to digital read-outs displayed on a computer screen or in a printout. The 13 BM+/FC− cases included patients with a diagnosis of follicular lymphoma, seven with diffuse large B‐cell lymphoma, two with extranodal marginal zone lymphoma, two with lymphoplasmacytic lymphoma, and one with small lymphocytic lymphoma. Among FC+ cases, 56 displayed κ and 26 displayed λ Ig light‐chain restrictions, with the clonal B‐cell population ranging from 0.5% to 97% (median 38%) of the total lymphoid gated population. Gating on CD45bright/SSC lymphocytes (green) shows neoplastic B cells displaying low levels of CD10 and κ light‐chain restriction. Advanced Imaging Techniques in Clinical Pathology. NHL, non-Hodgkin lymphoma; PCD, plasma cell dyscrasia. DM Prognostic value of bone marrow involvement by clonal immunoglobulin gene rearrangements in follicular lymphoma. Is flow cytometry a useful test? Appropriate gating strategies were applied from time to time to detect aberrant lymphoid B cells, based on their specific immunophenotypic (i.e., coexpression of CD5 and CD10 antigens with bright or dim expression) or physical (cell size) characteristics (22). In fact, considering the three assays together as gold standard, BMB alone has a sensibility of 82.1%, a specificity of 96,3%, with 1.6% of false positive but 10.4% of false negative. Flow Cytometry Tube Composition for the Multimarker and Limited Strategiesa, Demographics, Mean (Range) of Complete Blood Count Indices, and Marker Utilization of Cases Evaluated by the Multimarker Strategy or Limited Strategy. et al. (8) concluded that FC has a limited role in the routine staging and follow‐up of patients with malignant lymphoma. The single-cell suspension creates unique light-scattering events that occur when each cell passes through the laser light. Four cases were excluded from the study due to suboptimal biopsy samples that could not be sufficiently correlated with other studies, yielding 1,242 cases for study. In the instrument, the fluid in which the cells are suspended passes through very narrow channels so that the cells are organized in a single file as they pass the detector(s). In cases where both morphology and flow cytometry were unrevealing, only 7 of 251 (2.8%) cases demonstrated cytogenetic abnormalities in the limited strategy arm and only 2 of 305 (0.66%) cases in the multimarker strategy arm. Wood This is accomplished at a high rate of speed (hundreds to thousands of cells per second.). One of the most common applications is in the diagnosis of leukemia and lymphoma. Cases were considered abnormal/positive if they demonstrated abnormal cellularity for age (such as hypoplastic marrows, which may be aplastic anemia or hypocellular myelodysplasia), myelodysplastic changes in any lineage, metastatic disease, lymphomatous infiltrates, leukemia, plasma cell dyscrasia, or a myeloproliferative disorder. Fifty‐four samples were evaluated as part of initial disease presentation requiring a diagnostic biopsy procedure (staging); 156 samples were sent for routine restaging in case of partial or complete remission, suspected transformation of disease, control after chemotherapy, bone marrow transplantation, or pre‐protocol study. SPECIMEN TEST MENU d. If t. he specimen is a dry tap, consider obtaining additional core biopsy specimens to obtain adequate mate-rial for diagnosis and any special studies needed. Swerdlow Chap 25. The cells of interest can then be physically collected into separate vessels for further testing. Our study did not evaluate limited flow cytometry panels in the evaluation of peripheral blood samples. Do not draw more than 2 mls /tube.Keep the sample at room temperature and use Flow Cytometry shipping package for transport. Cases of Hodgkin's disease were excluded from this study because there are no FC criteria for the diagnosis of this disease. Cut tissue specimen in thin slices (no more than 1.0 mm thick). flow cytometry… Before the biopsy, between 1 and 1.5 ml of bone marrow aspirate was collected in sodium‐citrate anticoagulant, mixed, sent to the FC laboratory for immunophenotyping, and processed immediately. Examples of gating strategies applied to a patient with small lymphocytic lymphoma. Thus we carried out a retrospective study about … It also helps tell lymphomas from non-cancer diseases in the lymph nodes. In our experience, however, the need for additional markers following an initially selected limited panel based on smear findings is a rare event. Five morphologically negative and FC+ cases were observed, belonging to different B‐NHL subtypes. To determine the clinical and financial impact and predictive value of a limited flow cytometry strategy in the evaluation of bone marrow specimens. Bray D Limited flow cytometry strategy. Please check your email for instructions on resetting your password. M 2. If an abnormal or clonal cell population was detected using the limited panel, additional markers were added, if necessary, to further classify the abnormal/clonal population. b. Key to the limited strategy is taking into account not only the bone marrow smear findings but also the patient’s clinical history and clinical findings. Flow cytometry (FC) and PCR for clonal IgH rearrangement are considered as ancillary methods, but there is increasing evidence for their clinical usefulness. In our laboratory, timely access to the smear would not be a factor in most cases since, as discussed above, the need for additional markers after a limited panel is performed is a rare event. Neither morphologic examination nor FC alone is adequate for the detection of all cases of B‐lymphoid neoplasm bone marrow involvement. The reported clinical history, laboratory data, and smear findings were used to guide the pathway taken in the limited flow cytometry strategy. The limited flow cytometry strategy, without review of aspirate or biopsy specimen slides, detected the abnormal/clonal cell population in 95 of 103 (92.2%) cases that were positive using the multimarker strategy. A limited flow cytometry strategy did not translate into a lower disease detection rate; in fact, overall, disease detection was higher in cases evaluated by a limited strategy (36.3% vs 30.7%) and, for some disease classes, was statistically significant. Applicability to the clinical laboratory: We described a method to obtain a cell suspension from core biopsies that can easily be implemented routinely in a laboratory that performs diagnostic flow cytometry immunophenotyping. Some previous studies have indicated a high degree of correlation between FC and morphologic data and reported a limited number of cases showing discrepant results, suggesting that immunophenotypic analysis is unnecessary and redundant in the management of patients affected by B‐cell disorders (7, 8). 1-3 ml of bone marrow, 1 freshly prepared smear (preferred), Green Top (Sodium Heparin) (Preferred), or Purple Top (EDTA), or Yellow Top (ACD). Wittwer., St. Louis, Missouri: Elsevier Saunders; 2018. Observations dealing with combined use of the three methods still are lacking. When necessary, analysis also was performed on B cells selected by using CD19. Either a multimarker strategy (n = 486) or a limited marker strategy (n = 756) was prospectively employed Figure 1, Table 1, and Table 2.

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