Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

Flow cytometry identification of nonhemopoietic neoplasms during routine immunophenotyping.

INTRODUCTION: Nonhemopoietic neoplasms (NHNs) may be encountered during routine flow cytometry (FC) immunophenotyping. The clue of their presence mainly relies on detection of CD45-negative (CD45-) cells with altered scatter parameters. METHODS: In this study, we evaluated a monoclonal antibody combination conceived to characterize the CD45- population by FC, suspected of belonging to NHNs, when present. The panel included CD45 for leucocytes identification, CD326 (clones BerEP4 and HEA-125) to mark epithelial cells, CD33 to identify myeloid cells, CD138 to trace plasma cells and CD56 useful in the identification of neuroendocrine tumours. 7AAD vital dye was used to gate out dead cells. Results were correlated with cytomorphology and confirmed by histological data, if available. RESULTS: Among 9422 specimens submitted for routine FC investigation, 47 samples that included fine-needle aspirates, bone marrow aspirates, tissue biopsies and body fluids had a detectable CD45- population and a sufficient cell amount to be further investigated. FC revealed the presence of CD326-positive epithelial cells in 38 specimens; altered scatter parameters and variable reactivity to the other antigens tested allowed to suspect NHNs in the remaining nine samples. The presence of NHNs was confirmed in all cases by morphology. CONCLUSIONS: The current results show that when CD45- cells with altered scatter parameters were detected, cytometrists involved in leukaemia/lymphoma diagnosis may require further FC investigations to rapidly identify NHNs in different specimens, thus reducing the time of the immunohistochemical diagnostic workup to reach a final diagnosis.

Post-remissional and pre-transplant role of minimal residual disease detected by WT1 in acute myeloid leukemia: A retrospective cohort study.

In acute myeloid leukemia (AML), the detection of minimal residual disease (MRD) is still under investigation. The aim of the present retrospective study was to assess the role of Wilms tumor gene 1 (WT1) overexpression in a large monocentric cohort of AML patients. Among 255 enrolled patients, MRD was investigated in those in complete remission (CR) with an available WT1 at baseline (>250 copies) and at two further time-points: after induction (n=117) and prior allogeneic hematopoietic cell transplantation (allo-HCT), n=65. Baseline BM WT1 overexpression was not associated with response to induction (p=0.244). Median overall survival (OS) and disease-free survival (DFS) were significantly shorter in patients with >350 WT1 copies after induction compared to those with ≤350 (HR for mortality 2.13; 95% CI 1.14-3.97, p=0.018 and HR for relapse 2.81; 95% CI 1.14-6.93, p=0.025). Patients with WT1>150 copies pre allo-HCT had a significantly higher 2-year cumulative incidence of relapse (CIR) compared to those with WT1≤150 (HR 4.61; 95% CI 1.72-12.31, p=0.002). The prognostic role of WT1 overexpression resulted independent from other well-established risk factors. According to these results, WT1 overexpression might represent an additional MRD tool for risk stratification in patients classified nowadays in CR.

Single-Tube Flow Cytometry Assay for the Detection of Mature Lymphoid Neoplasms in Paucicellular Samples.

OBJECTIVES: Flow cytometry (FC) has become a useful support for cytomorphologic evaluation (CM) of fine-needle aspirates (FNA) and serous cavity effusions (SCE) in cases of suspected non-Hodgkin lymphoma (NHL). FC results may be hampered by the scarce viability and low cellularity of the specimens. STUDY DESIGN: We developed a single-tube FC assay (STA) that included 10 antibodies cocktailed in 8-color labeling, a cell viability dye, and a logical gating strategy to detect NHL in hypocellular samples. The results were correlated with CM and confirmed by histologic or molecular data when available. RESULTS: Using the STA, we detected B-type NHL in 31 out of 103 hypocellular samples (81 FNA and 22 SCE). Of these, 8 were not confirmed by CM and 2 were considered to be only suspicious. The FC-negative samples had a final diagnosis of benign/reactive process (42/72), carcinoma (27/72), or Hodgkin lymphoma (3/72). CONCLUSIONS: The STA approach allowed obtainment of maximum immunophenotyping data in specimens containing a low number of cells and a large amount of debris. The information obtained by STA can help cytomorphologists not only to recognize but also to exclude malignant lymphomas.

Extranodal Lymphoproliferative Processes and Flow Cytometry.

OBJECTIVE: Fine-needle aspiration (FNA) cytology is a safe and cost-effective technique for the diagnosis of lymphoproliferative processes, especially when correlated with clinical and imaging studies. However, cytology alone may be unable to detect a lymphoid neoplastic process, as architectural features are less obvious than in histologic preparations and, in certain cases, reactive processes may mimic lymphoma. Flow cytometry (FC) has been recognized as an important ancillary technique in the diagnosis of lymphoid neoplasms and it can be used in conjunction with FNA in the evaluation of lymphoproliferative processes. STUDY DESIGN: We performed a review of the published literature concerning FC applied to the detection of salivary glands and thyroid lymphoproliferative processes, which are frequently related to autoimmune diseases and difficult to diagnose by cytomorphology alone. RESULTS: FC is able to detect and subtype non-Hodgkin lymphomas and may contribute to the exclusion of a neoplastic process in cytologically unclear cases. CONCLUSIONS: FC can be successfully applied in the differential diagnosis of lymphoproliferative processes in the head and neck region. The FNA-FC combined approach can reduce time to therapy and may prevent unnecessary surgical biopsies.

Utilility of flow cytometry as ancillary study to improve the cytologic diagnosis of thyroid lymphomas.

BACKGROUND: To evaluate the efficacy of the use of flow cytometry (FC) immunophenotyping together with fine-needle aspiration cytology (FNAC) in the diagnosis of thyroid lymphoma. METHODS: FC was performed in parallel with FNAC in 35 samples of suspected thyroid lymphoma over a 12 years period. Results were correlated with histological or molecular findings and follow-up, when available. RESULTS: A final diagnosis of lymphoma was given in 13 of 35 (37.1%) specimens. Among the 22 cases considered negative for lymphoma by FC, 11 were diagnosed as thyroiditis by cytology, 7 as reactive, 2 were anaplastic carcinoma, and 2 cases were considered cytologically suspicious for lymphoma but were not confirmed by further investigations. Histology on core biopsy or molecular analysis was available in 12 of 13 lymphoma cases (92.3%). Data obtained by the combination cytology/FC were confirmed in all cases on histology biopsies. Correlation with histology showed a sensitivity and a specificity of 100% for the combination cytology/FC. CONCLUSIONS: FC is an important additional test that can contribute with cytology to the identification of lymphomas of the thyroid. FC can detect the presence of small neoplastic lymphocyte populations and may contribute to the diagnosis of cases in which the lymphoid infiltrate is difficult to interpret on cytology alone.

Immunophenotyping of paucicellular samples.

Immunophenotyping of paucicellular samples may represent a diagnostic challenge in the flow cytometry (FC) laboratory routine, as the scarcity of cells limits the number of tests that can be performed. Specimens such as fine needle aspirates (FNA), human body fluids (BF), cerebrospinal fluid (CSF), or ocular fluid (OF) sent for FC investigations in the case of suspicion of lymphoma, or for lymphoma monitoring, may contain very low numbers of cells. In these cases, it is mandatory to obtain the largest amount possible of useful information from a single tube. The basic protocol described in this unit provides a method that combines the use of multiple monoclonal antibodies (MAbs) with a Boolean gating strategy to identify and quantify the main lymphocyte populations, as well as to detect lymphomatous B cells or any aberrant T cell expression, if present, in paucicellular samples.

Tissue flow cytometry immunophenotyping in the diagnosis and classification of non-Hodgkin's lymphomas: a retrospective evaluation of 1,792 cases.

A retrospective analysis of 1,792 solid tissues suggestive of lymphoma, submitted over a 12-year period, was carried out and flow cytometry (FC) results were compared with histologic findings. The final histologic diagnosis of cases documented in this report is as follows: 1,270 non-Hodgkin's lymphomas (NHL); 17 composite lymphomas; four NHL plus carcinomas; five post-transplant lymphoproliferative disorders; 105 Hodgkin's lymphomas (HL); eight acute leukemias; 42 tissue cancers; and 341 non-neoplastic diseases. A strong correlation between morphology and FC data was observed among hematological malignancies (1,268/1,304, 97.2%) with the exception of HL. Among B-NHL, FC detection of clonally restricted B-cell allowed the identification of lymphomas that were not histologically clear and the differential diagnosis between follicular lymphoma and reactive hyperplasia. A high correlation level (r = 0.83; P < 0.0001) was obtained in comparing proliferation results obtained by FC and immunohistochemistry. Among T-NHL, FC detection of an aberrant phenotype direct histologic diagnosis in cases having less than 20% of neoplastic cells. In nine cases, FC suggested the need to evaluate a neoplastic population, not morphologically evident. Results show that FC routinely performed on tissue samples suspected of lymphomas is a fundamental adjunct to morphology in the diagnosis of NHL and may enhance the performance of the histologic evaluation so as to achieve the final diagnosis. To the best of our knowledge, this is the first report in the literature of a wide series of tissues also studied by FC.

Final results of a multicenter trial addressing role of CSF flow cytometric analysis in NHL patients at high risk for CNS dissemination.

This prospective study compared diagnostic and prognostic value of conventional cytologic (CC) examination and flow cytometry (FCM) of baseline samples of cerebrospinal fluid (CSF) in 174 patients with newly diagnosed aggressive non-Hodgkin lymphoma (NHL). FCM detected a neoplastic population in the CSF of 18 of 174 patients (10%), CC only in 7 (4%; P < .001); 11 patients (14%) were discordant (FCM(+)/CC(-)). At a median follow-up of 46 months, there were 64 systemic progressions and 10 CNS relapses, including 2 patients with both systemic and CNS relapses. Two-year progression-free and overall survival were significantly higher in patients with FCM(-) CSF (62% and 72%) compared with those FCM(+) CSF (39% and 50%, respectively), with a 2-year CNS relapse cumulative incidence of 3% (95% confidence interval [CI], 0-7) versus 17% (95% CI, 0-34; P = .004), respectively. The risk of CNS progression was significantly higher in FMC(+)/CC(-) versus FCM(-)/CC(-) patients (hazard ratio = 8.16, 95% CI, 1.45-46). In conclusion, FCM positivity in the CSF of patients with high-risk NHL is associated with a significantly higher CNS relapse risk and poorer outcome. The combination of IV drugs with a higher CNS bioavailability and intrathecal chemotherapy is advisable to prevent CNS relapses in FCM(+) patients.

Flow cytometric detection of liposomal cytarabine in cerebrospinal fluid of patients treated with intrathecal chemotherapy.

We report the unusual flow cytometric detection of liposomes in the cerebrospinal fluid of patients with aggressive non-Hodgkin's lymphoma and acute myeloid leukemia, treated with intrathecal liposomal cytarabine.

Ten antibodies, six colors, twelve parameters: a multiparameter flow cytometric approach to evaluate leptomeningeal disease in B-cell non-Hodgkin's lymphomas.

BACKGROUND: Flow cytometry (FC) is considered a sensitive and specific technique for the detection of occult lymphoma cells in cerebrospinal fluid (CSF). METHODS: The diagnostic sensitivity of a FC approach which uses a combination of 10 antibodies, a single-tube evaluation, and a six-color instrument, was evaluated and compared to conventional cytology (CC) for the detection of lymphomatous cells in the CSF of 44 patients affected by B-cell non-Hodgkin's lymphoma (B-NHL) considered at high risk of central nervous system spread. RESULTS: The CSF obtained from 36 newly diagnosed and 8 relapsed patients affected by B-cell lymphoma was assessed by FC and CC on a total of 62 samples; 52/62 (82.6%) were considered paucicellular as they had fewer than 10 cells/µl. All cases were evaluated by both methods. FC gave 15/62 (24%) positive results, CC 10/62 (16%) positive results; none of the samples evaluated had a positive CC with a negative FC result. CONCLUSIONS: The use of a multiparameter FC approach, which collects an elevated number of monoclonal antibodies in a single tube and identify different cell populations with a selective gating strategy analysis, allows for the evaluation of lymphocyte subsets and the detection of leptomeningeal disease in B-NHL, even in the presence of paucicellularity of samples.

Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature.

AIM: To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL). METHODS AND RESULTS: CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl-6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases. CONCLUSIONS: CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion.

Prognostic relevance of cytometric quantitative assessment in patients with myelodysplastic syndromes.

OBJECTIVES: Morphology and cytogenetics are currently used to define prognosis in myelodysplastic syndromes (MDS). However, these parameters have some limits. Flow cytometry has been recently included in the diagnostic panel for MDS, and its prognostic significance is under evaluation. METHODS: Marrow aspirates from 424 MDS patients were analyzed by flow cytometry to evaluate the impact of bone marrow cell immunophenotype on overall survival (OS) and leukemia-free survival (LFS). The immature compartment of myeloblasts was analyzed by the quantitative expression of CD34 (<3% vs. ≥3%), CD117, and CD11b(-) /CD66b(-) (<5% vs. ≥5%); myeloid maturation was analyzed by the expression of CD11b(+) /CD66b(++) (<15% vs. ≥15%) and CD11b(+) /CD66b(+) (<25% vs. ≥25%). RESULTS: In univariate analysis, the expression of immaturity markers (CD34(+) , CD117(+) , and CD11b(-) /CD66b(-) ) was associated with shorter LFS and OS (P < 0.0001); higher expression of differentiation markers (CD11b(+) /CD66b(++) and CD11b(+) /CD66b(+) ) was associated with longer LFS (P < 0.0001 and P = 0.0002, respectively) and OS (P < 0.0001). In multivariate analysis, expression of CD34(+) (P = 0.007), CD117(+) (P = 0.013), and CD11b(+) /CD66b(++) (P = 0.023) retained independent prognostic value for OS, while only the expression of CD34(+) was a prognostic factor for LFS (P = 0.0003). Two different risk groups were defined according to the presence of 0-1 or ≥2 of these factors with significant different LFS and OS (P < 0.0001). This score showed prognostic value in predicting survival even in subanalysis according to IPSS and WHO subgroups. CONCLUSIONS: Flow cytometric analysis in MDS may provide meaningful prognostic information. Blast percentage expressed as CD117(+) or CD34(+) cells and the quantitative assessment of myeloid maturation showed prognostic value for survival.

Usefulness of multiparametric flow cytometry in detecting composite lymphoma: study of 17 cases in a 12-year period.

Composite lymphoma (CL) is a rare occurrence of 2 or more morphologically and immunophenotypically distinct lymphoma clones in a single anatomic site. A retrospective analysis of 1,722 solid tissue samples clinically suggestive of lymphoma was carried out in our institute during a 12-year period to evaluate the efficacy of flow cytometry (FC) in identifying CL. We report 17 CL cases. A strong correlation between morphologic findings and FC was observed in 13 cases (76%). In the 4 cases diagnosed as non-Hodgkin lymphoma plus Hodgkin lymphoma, although FC did not detect Reed-Sternberg cells, it accurately identified the neoplastic B- or T-cell component. In 3 cases, FC indicated the need to evaluate an additional neoplastic component that was not morphologically evident. Our data demonstrate that FC immunophenotyping of tissues may enhance the performance of the diagnostic morphologic evaluation of CL. To the best of our knowledge, this is the first report in the literature of a wide series of CL studied also by FC.

Flow cytometric detection of degranulated basophils in chronic myeloid leukemia in accelerated phase.

We report a rare case of chronic myeloid leukemia in accelerated phase with basophilic transformation, in which basophils exceeding 70%, were detectable only by flow cytometry because of their morphologic atypicality and degranulation.

CD4-positive diffuse large B cell lymphoma identified by flow cytometry: two case reports.

We report two cases of diffuse large B cell lymphoma (DLBCL), both occurring in the small bowel, which coexpress PAX5, weak or no CD20 and the CD4 antigen. The CD4 was initially identified by flow cytometry and then confirmed by immunohistochemistry. CD4 is a representative marker for helper T-lymphocytes and is present on a subset of thymocytes, peripheral T cells and monocytes or macrophages. Unlike CD2 and CD5, no B cell fractions are known to express CD4. It might be hypothesized that the deregulated control of gene expression in malignant B cells, in particular PAX5, leads to the activation of some silent or repressed genes of T cell differentiation. Although lineage infidelity is described in some B lymphomas, it remains as an uncommon phenomenon; to our knowledge, cases reported here are the first two cases of DLBCL of the gastrointestinal tract coexpressing the CD4 antigen to be described to date.

Utility of flow cytometry immunophenotyping in fine-needle aspirate cytologic diagnosis of non-Hodgkin lymphoma: A series of 252 cases and review of the literature.

Flow cytometry (FC) immunophenotyping of fine-needle aspiration (FNA) has been reported to be useful in the diagnosis of non-Hodgkin lymphomas (NHL). The authors reviewed their 5-year experience to assess the ability that FC has in improving the diagnostic capacity of cytomorphology in the diagnosis and subclassification of NHL according to the World Health Organization's classification. FC was performed on 252 FNA specimens. These included 123 cases of NHL (89 primary and 34 recurrent lymphomas). The FC immunophenotyping included CD3, CD4, CD8, CD10, CD19, CD20, CD45, and kappa/lambda antibodies combinations in the screening panel and additional panels for B or T lineage in the presence of positivity for lymphoma after the screening. An immunologic diagnosis was obtained by FC in 90% (111/123) of cases identified as NHL. FC was able to improve the total number of NHL detected in 8 cases where cytomorphology had failed to do so. In 7% (9/123) of cases, FC failed to formulate a diagnostic hypothesis owing to the sample inadequacy; 2 cases (2%) were not identified as lymphomas by FC (1 of them considered only "suggestive" also by cytomorphology); 1 case was not identified neither by FC, nor by cytomorphology. In cases having a histologic follow-up, levels of diagnostic sensitivity and specificity of the combination cytomorphology/FC were 97% and 94%, respectively. FC applied to FNA enhanced the diagnostic potential of cytologic diagnosis and subclassification of NHL, thus avoiding the need for invasive surgical biopsies in many cases.

Aberrant expression of CD8 in B-cell non-Hodgkin lymphoma: a multicenter study of 951 bone marrow samples with lymphomatous infiltration.

T-cell antigen expression can be observed in B-cell non-Hodgkin lymphoma (B-NHL). Although CD5 is expressed in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, the presence of other T-cell antigens is less common. This article reports a retrospective multicenter analysis in which flow cytometry was used to evaluate aberrant CD8 expression on the pathologic B cells of 951 bone marrow samples from patients with various types of B-NHL. In a total of 18 patients, CD8 was coexpressed: 10 had B-CLL; 1, small lymphocytic lymphoma (SLL); 1, marginal zone lymphoma; 1, lymphoplasmacytic lymphoma; 2, diffuse large B-cell lymphoma; and 3, follicular lymphoma. There was a 1.89% overall frequency of CD8 coexpression in which B-CLL/SLL had a higher frequency (3.03%) than did the other B-cell neoplasms (1.18%). Most cases were characterized by a favorable outcome.

The usefulness of flow cytometric CD10 detection in the differential diagnosis of peripheral T-cell lymphomas.

We studied the histologic and multiparameter flow cytometry (MFC) features of 12 cases of angioimmunoblastic T-cell lymphoma (AITL), 13 of mature T-cell lymphoma, and 25 control cases of reactive lymphoid hyperplasia to evaluate the role of CD10 in the differential diagnosis of peripheral T-cell lymphomas (PTCLs). A characteristic immunophenotypic profile (CD2+/CD4+) with recurrent phenotypic aberrancies (eg, CD3 and CD7 loss) was identified in most AITL cases; MFC documented CD10 coexpression on T cells in 10 (83%). Mature T-cell lymphoma showed a more heterogeneous altered immunophenotypic pattern, and 2 cases of PTCL, unspecified, had clear evidence of aberrant CD10 expression on T cells. A small physiologic CD3+/CD4+/CD10+ T-cell population was detected by MFC in all control cases tested (range, 0.28%-4.71%), suggesting that a normal subset of peripheral CD10+ T cells exists. CD10 was a highly sensitive but incompletely specific phenotypic marker for diagnosing AITL; the differential diagnosis of PTCL, unspecified, must be related with traditional histologic features. A small number of CD10+ T cells in reactive lymph nodes suggests that this subpopulation may be the normal counterpart of neoplastic T cells in AITL. The biologic role of CD10+ T cells should be studied further.