Flow Cytometric Immunophenotyping Research Articles

AML-248: Clinico-Pathological Profile of Therapy-Related Acute Myeloid Leukemia

<h3>Context:</h3> Secondary acute myeloid leukemia (sAML) may arise from a previous clonal disorder of hematopoiesis, usually from myelodysplastic syndrome, chronic myeloproliferative neoplasm, or as a complication in patients receiving chemotherapy or radiotherapy for a primary malignancy, including hematological and non-hematological/solid malignancies (therapy-related AML [t-AML]). Overall, frequencies of sAML and tAML were 19.8% and 6.6%, respectively. It is associated with specific clinical features with a uniformly poor prognosis. <h3>Objective:</h3> To study patients of t-AML with respect to clinical presentation, laboratory parameters (including flow cytometry immunophenotyping), cytogenetic and molecular studies, and clinical outcome. <h3>Design:</h3> This study was a retrospective analysis of patients of t-AML from January 2011 to December 2019. <h3>Patients:</h3> The patients underwent a detailed hematological work up: peripheral blood smear, bone marrow examination along with flow cytometric immunophenotyping, and karyotyping and molecular studies (NPM1, FLT3ITD/TKD, BCR/ABL, PML/RARA, INV16, AML-ETO). Outcomes of the patients were also reviewed. <h3>Results:</h3> A total of 20 cases of therapy-related AML were diagnosed in patients treated for various malignancies of the breast (9 cases), prostate (3 cases), and pancreas (2 cases), as well as lymphomas (NHL: 4 cases and HL: 2 cases). There were 7 males and 13 females (M:F: 1:1.85) with a median age of 63 years (range 35–82 years). Median latency period to t-AML was 3.5 years (range 0.5–11.0 years). Median haemoglobin was 72.5 g/L (61–151 g/L), WBC count 3.7× 10⁹/L (1.02–169.1×10⁹/L), platelet count 40 × 10⁹/L (12–257 ×10⁹/L, peripheral blood and bone marrow blast percentage 48% (5-98%) and 76% (20–98%), respectively. AML with monocytic differentiation was the most common morphological and flow cytometric subtype (50% cases). Complex karyotype was seen in 50% cases. Molecular studies for NPM, FLT3ITD/TKD, PML/RARA, AML-ETO, and INV16 showed 8 cases with NPM1 positivity and 4 case with FLT3ITD positivity. Only 4 patients (20%) achieved complete remission but relapsed within 1 year of duration. Ten patients succumbed to their disease, the majority within 1 year of t-AML diagnosis. <h3>Conclusion:</h3> The study highlights the development of therapy-related AML following chemotherapy and/or radiotherapy as an important therapy-related complication. The latency period and clinical manifestations may differ depending on the type of cytotoxic drug, cumulative dose, and dose intensity. A close hematological follow-up should always be done for an accurate and timely diagnosis.

A Pilot Study to Determine the Utility of Automated Tissue Dissociator for Flowcytometry Based Evaluation of Hematolymphoid Tumor Tissue Biopsies.

Routine diagnostic biopsy tissue processing, conventional histology/immunohistochemistry (IHC) method is a multi-step and time consuming practice. With the advanced tissue dissociation protocols and panel designing, flow cytometric immunophenotyping (FCI) can be performed on diagnostic hematolymphoid tissue samples using single cell suspensions that economize steps and the time taken. Diagnostic tissue samples from lymph node, mediastinal mass, testicular biopsies and similar sites were dissociated using gentle MACS Octo-dissociator and FCI was performed thereafter. Oral tissue biopsy samples were also processed as a validation set for the protocol. 21 prospective tissue biopsy samples with suspected involvement by a known hematolymphoid neoplasm were processed and evaluated. These included B lymphoblastic lymphoma (n = 12), T lymphoblastic lymphomas (n = 3), Burkitts lymphoma (n = 3) and one case each of granulocytic sarcoma, Hodgkin lymphoma and granulomatous disease. Tissue FCI and IHC were found concordant with identified profile FCI obtained from blood/bone marrow analyses. FCI can produce a highly sensitive and reliable report, within hours, by processing fresh tumor tissue samples from suspected hematolymphoid malignancies. This method can be considered as an effective adjunct to IHC and can be applicable in routine clinical diagnostics, especially in cases that needs quick diagnosis and immediate clinical treatment.

3095 – IMMUNE THROMBOCYTOPENIA AND BONE MARROW HEMOPHAGOCYTOSIS ASSOCIATED WITH SARS COV-2

The clinical presentation of patients infected with SARS-CoV-2 is remarkably diverse. Likewise, the underlying pathophysiological mechanisms are proving complex. Disturbances in the blood coagulation system and cytokine storm, such as seen in hemophagocytic syndrome, are among the most serious ones. We present the case of a female 79-year-old patient with marked thrombocytopenia of 4 (150-450) x10^9/L occurring in the context of a confirmed SARS-CoV-2 infection. Clinically, one episode of epistaxis and petechiae was observed, otherwise no signs of bleeding occurred. Diagnostic workup included microscopic blood smear analysis, bone marrow cytologic evaluation and flow cytometric immunophenotyping. Hematological malignancies, thrombotic microangiopathies and common infections were excluded as cause of the low platelet count. In the bone marrow, cytology, the megakaryocytic lineage presented normocellular. However, several large hemophagocytes with engulfed hematopoietic cells were detected. A further evaluation of markers frequently associated with hemophagocytic syndrome was performed. Ferritin was 2888 (0-150) ng/mL, CRP and GOT were slightly elevated. The white blood count was normal with a marked decrease of lymphocytes to 0.13 (1.10-3.60) × 10^9/L. There was no fever or organomegaly and the patient was in good clinical constitution. Thus, we did not diagnose hemophagocytic syndrome. Due to no other explanation for the clinical and laboratory findings, the patient was diagnosed with immune thrombocytopenia and concomitant bone marrow hemophagocytosis associated to SARS-CoV-2. The first-line treatment consisting of prednisolone and intravenous immunoglobulins failed to induce an increase in the platelet count. As second-line treatment therapy with Eltrombopag, a TPO-agonist, was started and a sustainable response with platelets in the normal range was achieved.

Role of Flow Cytometric Immunophenotyping for Classic Hodgkin Lymphoma in Small Biopsy and Cytology Specimens.

The diagnosis of classic Hodgkin lymphoma (CHL) traditionally requires surgical tissue biopsy because of the paucity of diagnostic Hodgkin and Reed-Sternberg cells. Diagnosis can be challenging in small core needle and cytologic biopsies, which are increasingly used because of reduced costs and minimal invasiveness. Flow cytometric (FC) identification of Hodgkin and Reed-Sternberg cells is possible, but FC test efficacy is not well studied outside of validation settings, especially in small specimens. To assess the testing efficacy of FC performed on small biopsy and cytology specimens for the diagnosis of CHL. We reviewed 131 patients with CHL and 459 patients without CHL during a 3-year period who underwent a small biopsy procedure, including core biopsy and/or cytology evaluation, with concurrent routine clinical FC testing for CHL, assessing performance of FC in small specimens. Evaluating testing efficacy, sensitivity was 95.4% and specificity was 98.2%, whereas positive and negative predictive values were 92.2% and 99.0%, respectively. Although there were more false-positive results than compared with published validation studies, expert review identified distinct diagnostic pitfalls; awareness of these may improve testing efficacy. Although FC diagnosis of CHL was historically considered unfeasible, our findings in a real-world clinical setting suggest that FC adds diagnostic value to small biopsy evaluation, reducing time to treatment, costs, and invasive excisional procedures.

Flow Cytometric Assessment of Ki-67 Expression in Lymphocytes From Physiologic Lymph Nodes, Lymphoma Cell Populations and Remnant Normal Cell Populations From Lymphomatous Lymph Nodes.

Recent literature suggests conventional flow cytometric (FCM) immunophenotyping complemented by Ki-67 FCM assessment as a reliable tool to classify canine lymphomas. Ki-67 expression assessed by FCM is rarely reported in canine lymphoma cases and reference data for normal canine lymph nodes are missing. Moreover, nothing is known about the Ki-67 expression within the occasionally observed remnant cell population within the gates of normal lymphocytes in lymphoma cases. Aim of this study was to compare flow cytometric Ki-67 expression of lymphocyte populations from normal canine lymph nodes, lymphoma cells from World-Health-Organisation (WHO) classified lymphoma patient samples and their neighboring normal remnant cell population. Cryopreserved lymphocyte cell suspensions from normal lymph nodes from eight dogs free of lymphoma served as reference material. Fourteen cases diagnosed by cytology, FCM, clonality testing, histopathology including immunohistochemistry consisting of 10 DLBCL, 1 MZL, 1 PTCL and 2 TZL showed a residual small lymphocyte population and were investigated. The Ki-67 expression in normal canine lymphoid tissue was 3.19 ± 2.17%. Mean Ki-67 expression in the malignant cell populations was 41 ± 24.36%. Ki-67 positivity was 12.34 ± 10.68% in the residual physiologic lymphocyte population, which otherwise exhibited a physiologic immunophenotype pattern. This ratio was equivalent (n = 3) or lower (n = 11) than the Ki-67 expression of the malignant cell population within the sample. This is the first report of FCM derived Ki-67 expression combined with immunophenotype patterns in normal canine lymph nodes, compared with lymphoma cell populations and residual normal cell populations of lymphoma cases diagnosed by state of the art technology.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

Defining the Cell Surface Proteome of MLL Rearranged Leukemia

Introduction: B-acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr ) have a particularly dismal clinical outcome. Although it is known that this oncoprotein radically alters epigenetic regulation, the precise molecular mechanisms that define the cellular phenotype of these cancers remain poorly understood and new therapeutic strategies are sorely needed. Furthermore, although flow cytometric immunophenotyping has been performed for decades on B-ALL blasts, relatively little is known about the cell surface proteome of these cancers outside of canonical CD markers. Due to its role in modulating cellular behavior as well as accessibility to immunotherapy, the cell surface represents an attractive target for unbiased proteomic profiling in order to gain insight into cellular behavior as well as enabling the development of targeted therapies. Here, we aim to resolve the cell surface proteomic landscapes of MLLr and non- MLLr B-ALL cellular models. Our goal is to generate new mechanistic insights into the molecular re-wiring that occurs in these forms of cancer and identify novel candidates for targeted immunotherapy.Methods: We examined 10 B-ALL cell lines that harbor different oncogenic fusions including the MLL-AF4, BCR-ABL, and ETV6-RUNX1 translocations, respectively, using both “classical” lines as well as low-passage patient-derived lines, in addition to non-malignant B-cells as a control. We performed unbiased labeling of cell surface proteins by biotinylation of glycosylated amino acids (3 biological replicates each) followed by affinity pull-down with streptavidin. After on-bead trypsin digestion, peptides from bound glycoproteins were analyzed on a Thermo Q-Exactive Plus Mass spectrometer using 4-hour LC runs, data-dependent acquisition, and label-free quantification to identify and quantify members of the cell surfaceome for each cell line. To confirm our proteomic findings we performed flow cytometry using a BD CytoFLEX flow cytometer on select cell lines with fluorescently conjugated monoclonal antibodies, in order to assess presence and qualitative abundance of particular cell surface targets.Results: We quantified on average >900 high-confidence membrane proteins (FDR=0.05) per cell type, comprising ~40% of all proteins identified in each experiments. Principal component analysis identified unique cell surfaceome signatures that distinguish several of the leukemia subtypes and normal B-cells from each other, implying different cell-surface phenotypes associated with specific B-ALL genetic alterations (Figure 1A). As positive controls, we found several proteins differentially expressed in MLLr that were also previously identified based on transcriptomic profiling of MLLr patient samples, such as FLT3 and PROM1. In our data, the MLLr surfaceome was surprisingly enriched for Ephrin receptors, matrix metalloproteases, and other proteins that play a role in cell motility, invasion of tissues, and modulation of the tumor microenvironment (significantly enriched membrane proteins colored in red, Figure 1B). Using flow cytometry with monoclonal antibodies specific for select cell surface markers we found excellent agreement with our proteomic data. We will next move to patient-derived xenografts and tissues for further validation. These findings suggest these novel markers may be candidates for downstream immunotherapy development, potentially specific to MLLr B-ALL, where there is particularly high clinical need.Summary and Conclusions: In this work, we profiled the cell surface proteomes of multiple B-ALL cell models, identifying cell surfaceome signatures unique to different B-ALL subtypes. Overall, our work demonstrates the power of performing un-biased interrogation of the tumor surfaceome to identify new therapeutic targets for immunotherapy, as well as provide a resource to advance our phenotypic understanding of these aggressive cancer subtypes. [Display omitted] DisclosuresWiita:TeneoBio, Inc.: Research Funding; Sutro Biopharma: Research Funding.

Measurement of PD-1 Expression on Lymphoma Cells By Flow Cytometry Is a Useful Tool in Diagnosing and Monitoring Minimal Residual Disease in Angioimmunoblastic T-Cell Lymphoma

Introduction: Angioimmunoblastic T-cell Lymphoma (AITL) is one of the most common types of peripheral T-cell lymphoma (PTCL), representing about 15-20% of cases of PTCL. Diagnosis of AITL is often challenging and a definitive diagnosis requires a use of ancillary testing including broad panels of immunohistochemistry and molecular studies. We evaluated the utility of evaluation of Programmed-Death-1 (PD-1) expression on lymphoma cells by flow cytometry as a diagnostic tool as well as monitoring minimal residual disease (MRD) of AITL.Methods: The study group was composed of 81 patients who were diagnosed with PTCL at Memorial Sloan Kettering Cancer Center between May 2015 and July 2017. This includes 36 patients with AITL, 6 with ALK-negative anaplastic large cell lymphoma (ALCL, ALK-), 2 with ALK-positive anaplastic large cell lymphoma (ALCL, ALK+), 9 with adult T-cell leukemia/lymphoma (ATLL), 11 with peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), 6 with nodal involvement by mycosis fungoides (MF), 5 with T-cell large granular lymphocytic leukemia (T-LGL), and 6 with T-cell prolymphocytic leukemia (T-PLL). The expression of PD-1 (CD279) on lymphoma cells was evaluated by 10-color flow cytometry using BV605-conjugated anti-CD279 antibody, and mean fluorescence intensity (MFI) was evaluated. Immunohistochemistry for PD-1, CD10, BCL6, and CXCR13 were also performed on tissue samples of AITL. Flow cytometry for variable regions of T-cell receptor beta chain (Vbeta) and genetic analysis for T-cell receptor gene rearrangement were performed on sorted abnormal T-cell population by flow cytometry in selected cases for the evaluation of clonality. Peripheral blood sample was used for the monitoring of MRD.Results: All AITL cases with tissue biopsy showed PD-1 immunoreactivity by immunohistochemistry (IHC), and PD-1 expression by flow cytometry showed excellent correlation with IHC. The abnormal CD4-positive PD-1 bright T-cell population detected by flow cytometry in AITL cases was confirmed to be clonal by restricted Vbeta fragment expression and molecular genetic analysis (Fig 1). On tissue samples, expression of other T-follicular helper cell markers was not observed by IHC in 7 cases (25%), and additional 3 cases (11%) presented with concomitant lymphoid neoplasm, which obscured morphologic and immunohistochemical evaluation for AITL. The latter 3 cases would be misdiagnosed by morphologic assessment alone. However, flow cytometry reliably detected AITL in all 10 challenging cases. Overall flow cytometry including PD-1 expression had 100% sensitivity in detection of AITL. The expression of PD-1 was significantly higher in AITL than that of other PTCL (CD279-BV605 MFI; Mean ± SEM; 6990.0 ± 934.9 vs. 547.3 ± 155.5; p<0.0001) (Fig 2A). PD-1 MFI differentiated AITL from other PTCL with high sensitivity and specificity (AUC 0.978; Fig 2B). The significant increase in PD-1 expression in AITL was a consistent finding with peripheral blood specimen. In 54 patients who had circulating lymphoma cells in peripheral blood, PD-1 expression was significantly higher in AITL than that of other PTCL (CD279-BV605 MFI; Mean ± SEM; 3079.0 ± 508.8 vs. 341.8 ± 54.17; p<0.0001: AUC 0.986) (Fig 2C, 2D). In 12 AITL patients who had tissue sample with concomitant bone marrow and peripheral blood specimens, flow cytometry detected lymphoma cells in bone marrow and peripheral blood in all patients. Among them, 9 patients were followed for monitoring of MRD. MRD in peripheral blood was not detected in 4 patients after therapy (Median 4.5 months; Range; 2-6 months); 2 patients received stem cell transplant, and 2 patients received chemotherapy only, and all 4 patients were free of disease at the time of last follow up (Median 18.5 months; Range 17-26 months). 5 patients kept having MRD after therapy (Median 13 months; Range: 3-21 months), and one of 5 patients presented with new lesion of AITL 12 months after the initial diagnosis.Conclusions: Flow cytometric immunophenotyping with anti-PD-1 antibody reliably detects neoplastic population in AITL cases, and PD-1 MFI differentiates AITL from other PTCL with high sensitivity and specificity. Bright PD-1 expression alone is a reliable indicator of TFH immunophenotype by flow cytometry. Measurement of PD-1 expression by flow cytometry is a useful tool both in diagnosing and monitoring MRD of AITL. [Display omitted] DisclosuresMoskowitz:ADC Therapeutics: Research Funding; Seattle Genetics: Honoraria, Research Funding; Bristol Myers-Squibb: Consultancy, Research Funding; Takeda: Honoraria; Incyte: Research Funding. Horwitz:Seattle Genetics: Consultancy, Research Funding; Millenium/Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; ADCT Therapeutics: Research Funding; Kyowa-Hakka-Kirin: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; Infinity/Verastem: Consultancy, Research Funding; HUYA: Consultancy; BMS: Consultancy; Mundipharma: Consultancy; Aileron Therapeutics: Research Funding. Dogan:Celgene: Consultancy; Roche Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Peer Review Institute: Consultancy.

Clinical Utility of Flow Cytometry Immunophenotyping and Hematological Profile in Chronic Lymphoproliferative Disorders

Background: Chronic lymphoproliferative disorders (CLPD) are a group of lymphatic malignancies characterized by proliferation of neoplastic mature lymphocytes, and may be sourced B, T and natural killer cells (NK). Flow cytometric (FC) immunophenotyping is a routine practice in the differential diagnosis of these tumors, contributing to greater accuracy and new therapeutic perspectives. Methods: Patients (n = 500) with CLPD were assessed by FC. In addition, we gathered patient information such as age, sex and hematologic data. Results: The results showed that 86.6% of cases were B-cell and 13.4% T/NK CLPD. B-cell CLPD distribution was as follows: 265 B-cell chronic lymphocytic leukemia, 17 B-cell prolymphocytic leukemia, 11 hairy cell leukemia, 3 follicular lymphoma in leukemic phase, 3 splenic lymphoma with villous lymphocytes, 12 mantle cell lymphoma, one Waldenstrong macroglobulinemia, 47 multiple myeloma, 12 plasma cell leukemia, 9 Burkitt lymphoma and 37 B-cell CLPD with non-specific immunoclassification. The T/NK CLPD classifications were as follows: 11 large granular lymphocyte leukemia, 14 T-cell prolymphocytic leukemia, 10 adult T-cell leukemia lymphoma, 8 Sézary syndrome and 24 T-cell peripheral lymphoma. Conclusion: The data suggest that immunophenotyping is an important method in diagnosis, monitoring and prognosis when determining the pathological mechanisms of CLPD evolution. DisclosuresNo relevant conflicts of interest to declare.

Diagnostic utility of cerebrospinal fluid cytology and flow cytometric immunophenotyping in Burkitt lymphoma

Although central nervous system involvement by lymphomas is infrequent, it is often associated with fatal outcomes, and is therefore indicative of poor prognosis. Cytologic examination of the cerebrospinal fluid (CSF) complemented by flow cytometric immunophenotyping and/or immunocytochemistry can help in establishing a prompt diagnosis. In this report, we present the cytologic features and flow cytometric immunophenotyping of Burkitt lymphoma involving the CSF.

The Utility of Extended White Blood Cell Differential by Flow Cytometry in Routine Hematology.

Abstract 4708Today's Hematology instruments identify white blood cells (WBC) by their physical characteristics using various measurements such as Impedance, Conductivity and Light Scatter. A complete cell identification and enumeration is performed but is limited to only six cell types: neutrophils, lymphocytes, monocytes, eosinophils, basophils and NRBCs. Automation of the WBC manual differential is possible by flow cytometry for more accurate and automated information (van Lochem et al. 2004, M. Roussel et al. 2007, J. Feuillard et al. 2007).The CytoDiff” tube is a 5-color / 6-marker panel that provides an extended cytometric differential for whole blood specimens. The CytoDiff” tube is comprised of: CD36-FITC, (CD2+CD294)-PE, CD19-ECD, CD16-PC5, and CD45-PC7 (Beckman Coulter, Inc. (BEC), For Investigational Use Only. The performance characteristics of this product have not been established).The peripheral blood (PB) specimens were lyzed using VersaLyse (BEC) to prevent selective loss of cells and to conserve light scatter characteristics. PB leukocytes were analyzed according to their immunofluorescence reactivity. At least 20,000 CD45+ events per sample were acquired on an FC500 Cytomics flow cytometer (BEC) and analyzed using CXP software (BEC). For each case with elevated numbers of leukocytes or with abnormal cells, additional analyses were performed using light microscopy and full panel extended flow cytometric immunophenotyping to identify lineages and the level of differentiation of the cells. PB was evaluated from 62 patients with different pathological conditions. Half the patients were females and half males, with adult median age 60. Four boys; median age 10.5, and two girls; median age 4.5 were also evaluated. The study was approved by The Pavlov State Medical University's Institutional Review Board, and the following results were obtained:Two cases had no immunological abnormalities in WBC subsets. Extended immunophenotyping determined 13 lymphocytosis cases with more than 50% of nucleated cells (53.1–94.5%) to be B-lymphocytosis, with 12 B-ChLL and one PLL. One specimen with normal lymphocytes had high B-cells (45%) and extended immunophenotyping specified it as hairy cell leukemia.Six cases were identified with B-blasts, nine with noTnoB (myelo) blasts and two with immature granulocytes. The analysis of these specimens using light microscopy, extended immunophenotyping and cytogenetics identified six B-cell precursors ALL, two cases of MDS transformation to AML, two relapses of AML with maturation, one new diagnosed case of AML with maturation, one case of AML with myelomonoblastic differentiation, three cases of residual disease after AML chemotherapy, one plasmoblastic leukemia (transformed after multiple myeloma) and one acute promyelocytic leukemia with t(15;17). Therefore, true positive blasts were identified in15 of 17 PB specimens using extended immunophenotyping. Two cases with immature granulocytes in PB were misclassified with the CytoDiff” tube but verified as plasmablastic and acute promyelocytic leukemia after bone marrow extended immunophenotyping and cytogenetics.Nine cases had increased numbers of monocytes (14.2–32.0%). Three cases came from the surgery department, with more than 20% of monocytes being inflammatory CD16+. Two cases with multiple myeloma after chemotherapy and cytopenia recovery had 38.8% and 43.0% of monocytes being inflammatory CD16+. Another three cases (two with craniocerebral trauma, and one with phlegmona of mandibula) showed very low inflammatory monocytes (3.5–9.3%). There was one case with very high monocytes (32%) but none were inflammatory. The analysis of this specimen using light microscopy, extended immunophenotyping and cytogenetics identified promyelocytic leukemia. ConclusionThese data demonstrate that a differential leukocyte count using Cytodiff” allows for verification of a wide spectrum of normal and pathological cells in peripheral blood. This will provide clinicians with a diagnostic tool to rapidly determine appropriate therapies for a wide variety of blood-related diseases. Disclosures:No relevant conflicts of interest to declare.

Immunophenotyping for Haematologists – Principles and Practice, by Barbara J Bain and Mike Leach, Wiley Blackwell, 2021; ISBN 9781119606116

This is a deceptively slim volume. The experienced authorship team has skilfully and succinctly distilled a comprehensive wealth of information into four well organised and extensively referenced sections. An introductory chapter outlines the principles behind the methods of flow cytometric immunophenotyping of liquid specimens and immunohistochemical investigation of tissue sections. It reviews the uses of flow cytometry beyond the diagnosis and monitoring of haematological neoplasms, including the investigation of inherited and acquired red cell, platelet and immune function/dysregulation disorders, as well as differential cell counting and enumeration. There follows a useful reference chapter listing all of the individual antibodies in routine laboratory use for diagnosis and monitoring, in the order of their cluster designation (CD) number for those that have them, followed in a logical order by those which do not. A brief description for each is given of their expression in normal and neoplastic cells. The third chapter reviews the patterns of expected cellular antigen expression in haematological neoplasms, systematically following the order of the WHO classification of tumours of haematopoietic and lymphoid tissues (2017). It includes a section on the immunophenotypic assessment of ‘minimal residual disease’ (MRD) – perhaps, as the authors suggest, more appropriately termed ‘measurable residual disease’. Finally a ‘test yourself’ chapter uses a mixture of different types of multiple-choice questions and clinic-pathological cases which covers the whole field comprehensively and enables the identification of gaps in knowledge. This book is a superb resource for medical and scientific staff in training, especially those who are taking FRCPath or equivalent examinations, and for those of us who teach them, as well as for practising haematologists involved in the interpretation of immunophenotyping results.

Fasting-induced FOXO4 blunts human CD4+ T helper cell responsiveness

Abstract Intermittent fasting blunts inflammation in asthma and rheumatoid arthritis, suggesting that fasting may be exploited as an immune-modulatory intervention. However, mechanisms underpinning anti-inflammatory effects of fasting remain poorly characterized. Here, we show that fasting in humans is sufficient to blunt CD4+ T helper cell responsiveness. RNA-seq and flow cytometric immunophenotyping of peripheral blood mononuclear cells (PBMCs) from volunteers subjected to overnight or 24-hour fasting, and 3-hours of refeeding implicate that fasting blunts CD4+ T helper cell activation and differentiation. Transcriptomic analysis reveal that the longer fast-duration has a more robust effect on CD4+ T cell biology. Through bioinformatic analyses, we identify the transcription factor FOXO4 and its canonical target FKBP5 as a potential fasting-responsive regulatory axis. Genetic gain- or loss-of-function of FOXO4 and FKBP5 is sufficient to modulate Th1 and Th17 cytokine production. Moreover, we find that fasting-induced or genetic overexpression of FOXO4 and FKBP5 is sufficient to downregulate mTORC1 signaling and suppress STAT1/3 activation. Our results identify FOXO4-FKBP5 as a novel fasting-induced, STAT-mediated, regulatory pathway to blunt human CD4+ T helper cell responsiveness.

Composition of lymphocyte subpopulations in normal and mildly reactive peripheral lymph nodes in cats.

Flow cytometric (FCM) immunophenotyping of lymphoid tissue aspirates is an available adjunct for feline lymphoma diagnostics. Reference data have only been established for feline peripheral blood. Studies investigating the composition of normal and mildly reactive feline lymph nodes (LNs) are lacking. The aim of this prospective study was to establish reference data for lymphocyte subpopulations in normal and mildly reactive feline peripheral LNs using a standardised multicolour panel of antibodies. Macroscopically inconspicuous mandibular and/or popliteal LNs from 31 adult cats, which were euthanased for reasons other than haematological diseases, were excised and processed within 5 h after death. Multicolour flow cytometry using eight different feline-specific, anti-canine and human cross-reactive monoclonal antibodies used in current diagnostic marker panels was performed after cytological exclusion of pathological states and complemented by lymphocyte clonality testing, histopathology and immunohistochemistry (IHC) to ensure the absence of lymphoid disease. Of 31 cats, the immunophenotyping data of 24 individuals could be included as histopathology and clonality testing excluded a pathological condition. Lymphocyte populations showed the following positive antibody reactions: CD18+ 86.3% ± 13.86%, CD3+ 54.81% ± 11.10%, CD5+ 57.39% ± 12.66%, CD21+ 40.42% ± 12.40%, CD79alphacy+ (CD79αcy) 30.41% ± 13.49% and CD14+ 0.75% ± 1.35%. There were 30.88% ± 13.48% CD4+ and 12.91% ± 6.68% CD8+ cells. Cytology revealed a mixed population of mostly lymphoid cells in all samples. The absence of a monoclonal/oligoclonal neoplastic population was confirmed by lymphocyte clonality testing. Histopathology and IHC showed a normal or mildly reactive pattern in all cases. This study establishes FCM immunophenotyping data of lymphocyte populations of normal and mildly reactive feline peripheral LNs. For the first time, anti-CD5, CD4, CD8 and CD21 reference data in normal and mildly reactive feline peripheral LNs are presented. CD18, CD3, CD14 and CD79αcy have been used to establish reference data for the first time in any feline material.

Comprehensive evaluation of bronchoalveolar lavage from patients with severe COVID-19 and correlation with clinical outcomes

Information on bronchoalveolar lavage (BAL) in patients with COVID-19 is limited, and clinical correlation has not been reported. This study investigated the key features of BAL fluids from COVID-19 patients and assessed their clinical significance. A total of 320 BAL samples from 83 COVID-19 patients and 70 non-COVID-19 patients (27 patients with other respiratory viral infections) were evaluated, including cell count/differential, morphology, flow cytometric immunophenotyping, and immunohistochemistry. The findings were correlated with clinical outcomes. Compared to non-COVID-19 patients, BAL from COVID-19 patients was characterized by significant lymphocytosis (p < 0.001), in contrast to peripheral blood lymphopenia commonly observed in COVID-19 patients and the presence of atypical lymphocytes with plasmacytoid/plasmablastic features (p < 0.001). Flow cytometry and immunohistochemistry demonstrated that BAL lymphocytes, including plasmacytoid and plasmablastic cells, were composed predominantly of T cells with a mixture of CD4+ and CD8+ cells. Both populations had increased expression of T-cell activation markers, suggesting important roles of helper and cytotoxic T-cells in the immune response to SARS-CoV-2 infection in the lung. More importantly, BAL lymphocytosis was significantly associated with longer hospital stay (p < 0.05) and longer requirement for mechanical ventilation (p < 0.05), whereas the median atypical (activated) lymphocyte count was associated with shorter hospital stay (p < 0.05), shorter time on mechanical ventilation (p < 0.05) and improved survival. Our results indicate that BAL cellular analysis and morphologic findings provide additional important information for diagnostic and prognostic work-up, and potential new therapeutic strategies for patients with severe COVID-19.

Abstract S03-03: Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection

Abstract Cancer patients display immunomodulation related to malignancy and anti-cancer therapies, but how these factors impact COVID-19 remains unknown. To investigate immune responses in cancer patients with COVID-19, we undertook a prospective case-control study, enrolling hospitalized solid tumor patients with acute COVID-19, as well as age-, gender-, and comorbidity-matched COVID-19 patients without cancer as controls. Using biospecimens collected during hospitalization, we performed virologic measurements as well as in-depth immunophenotyping of cellular, antibody and cytokine responses. We enrolled 17 cancer patients (cases) admitted to Yale-New Haven Hospital between March 15 and June 30, 2020 with COVID-19, as well as 17 matched non-cancer patients (controls) admitted with COVID-19. No significant differences were observed between cases and controls based on patient characteristics (age, gender, race, co-morbidities, smoking history, days from symptom onset to COVID-19 diagnosis) or outcomes (COVID-19 severity, length of hospital stay, rate of intubation or mortality). The most common primary tumor sites were lung (4/17) and gastrointestinal (4/17); all cases had received cancer-directed therapy within 6 months of COVID-19 diagnosis, with 13/17 receiving treatment less than 1 month prior to hospitalization. Three of 17 cases had received immune checkpoint inhibitor therapies. Despite having similar SARS-CoV-2 viral RNA loads at the time of COVID-19 diagnosis when compared with controls, cancer cases had increased viral RNA abundance during hospitalization, suggesting slower clearance. Antibody responses against SARS-CoV-2 were preserved in cancer cases, with cases displaying similar levels of IgM and IgG antibodies directed against SARS-CoV-2 epitopes compared to controls. Cytokine profiling revealed higher plasma levels of CCL3, IL1A and CXCL12 in cancer cases compared to controls. Using flow cytometric immunophenotyping, we found that innate immune and non-T cell adaptive immune parameters were similar between cases and controls hospitalized with COVID-19. However, among cancer cases on conventional therapies, T cell lymphopenia was more profound, and these cases demonstrated higher levels of CD8+ exhausted (CD8+CD45RA−PD1+TIM3+), CD8+GranzymeB+ and CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ activated T cells when compared with controls; interestingly, these differences were not observed in patients who had received immune checkpoint inhibition. Thus, we found reduced viral RNA clearance and specific alterations in T cell and cytokine responses in cancer patients hospitalized with COVID-19 compared with matched controls with COVID-19. This dysregulated T cell response in cancer patients, which may reflect immune modulation due to chronic antigen stimulation as well as cancer therapies, may lead to altered virologic and clinical outcomes in this population. Citation Format: Michael Chiorazzi, Erin Silva, Kristina Brower, Patrick Wong, Carolina Lucas, Jon Klein, Feimei Liu, Maura Nakahata, Jun Zhao, Nur-Taz Rahman, Camila Odio, Santos Bermejo, Shelli F. Farhadian, Charles Dela Cruz, Arnau Casanovas-Massana, John Fournier, Catherine Muenker, Anne L. Wyllie, Chantal B.F. Vogels, Chaney C. Kalinich, Mary E. Petrone, Isabel M. Ott, Anne E. Watkins, Adam J. Moore, Tara Alpert, Yuval Kluger, Aaron Ring, Nathan D. Grubaugh, Akiko Iwasaki, Albert I. Ko, Roy S. Herbst. Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr S03-03.

Cytological analysis of lymphoblastic lymphoma/acute lymphoblastic leukemia in serous effusion

Objective: To study the diagnostic clues and significance in serous effusion cytology associated with lymphoblatic lymphoma/acute lymphoblastic leukemia (LBL/ALL). Methods: Forty-five serous effusion specimens with final diagnosis of LBL/ALL were collected from August 2011 to December 2019 at the First Affiliated Hospital of Zhengzhou University. All cases were reviewed for their clinical profiles, cytomorphologic features and ancillary studies. Cell blocks and immunocytochemistry were prepared in 22 cases; flow cytometric immunophenotyping was performed in three cases and gene rearrangement analysis (T-cell recepter, TCR and immunoglobulin, Ig) was performed in five cases. Results: Among the 45 cases, there were 35 males and 10 females with male to female ratio of 3.5∶1.0. The median age was 15 years. Mediastinal mass was the initial presentation in 39 patients (86.7%) and high LDL level were observed in 34 patients (75.6%). Microscopically, the majority of the specimens (86.7%) were hypercellular. The smears demonstrated dispersed lymphoblasts that were predominantly small to intermediate in size with scanty basophilic cytoplasm and irregular or convoluted nuclei with fine chromatin condensation and inconspicuous nucleoli. Mitoses were frequently observed. Karyorrhexis and apoptosis were seen in all cases. By immunophenotyping, TdT was expressed in 19 cases (86.4%) and CD99 in 20 cases (90.9%). Ki-67 expression varied from 65% to 95%. Flow cytometry in three cases demonstrated positivity for TdT, CD2, CD3 and CD7. Monoclonal TCR gene rearrangement was found in 4 of 5 cases, and both monoclonal TCR and Igκ gene were found in 1 case. Conclusions: In LBL/ALL, primary diagnosis could be made basing on clinical features (younger male patients with a mediastinum mass) and cytomorphology (monotonous, small to medium sized lymphoid cells with prominent irregular nuclei, fine chromatin and frequent mitoses, karyorrhexis and apoptosis). If immunocytochemistry and other ancillary studies are performed, the accuracy and reliability of the results could be improved.

Lymphoplasmacytic lymphoma and a CD5+ lymphoproliferative process presenting as composite disease in the bone marrow: a report of two cases

Correct subclassification of B cell lymphoma requires integration of histologic and immunohistochemical findings in addition to results of flow cytometric immunophenotyping, cytogenetic analysis, and molecular studies. Evaluation for the MYD88 L265P mutation and immunohistochemical staining for LEF1 can be helpful in the workup of small B cell lymphomas. In this case report of two diagnostically difficult cases, we present two patients with lymphoplasmacytic lymphoma (LPL) and a CD5+ lymphoproliferative process presenting as composite disease within the bone marrow. In both cases, integration of ancillary studies and results of biopsy at other sites of disease in addition to a careful histologic review and an expanded immunohistochemistry panel were important for the correct final classification.

Immunophenotypic aberrancy in acute leukemia – a retrospective single institute study

Introduction: In 2016, WHO gave a comprehensive classification system that included morphology, immunophenotyping, molecular and genetic findings. Flow cytometric immunophenotyping is the backbone of WHO classification, helping to make a more accurate and faster diagnosis. Aim: This study has been conducted to find immunophenotyping aberrancy of acute leukemia (AL) and its association with gender and age group. Material and methods: This is a descriptive retrospective study of 1012 AL patients diagnosed between January 2011 and January 2019, including all new patients of all ages and both sexes, who had available immunophenotyping data. Results and discussion: The most common aberrant antigen on acute myeloid leukemia (AML) was CD7, on precursor B lymphoblastic leukemia (B-ALL) was CD33 and on precursor T lymphoblastic leukemia (T-ALL) was CD13. There was no association between sex/age and antigen aberrancy except a significant increase in CD34 loss on pre B acute lymphoblastic leukemia (pre B-ALL) and HLA-DR expression on acute promyelocytic leukemia (APL) in males compared to female patients and an increase of CD19 expression on non-APL-AML and an elevation of CD34 loss on T-ALL in adult compared to pediatric patients. Not only detection of aberrant expression of CD markers in leukemic cells helps to estimate molecular abnormalities and has a prognostic effect, but also a specification of neoplastic cell markers at the time of diagnosis is essential for monitoring patients after treatment. Conclusions: Although in this study, no association between sex/age and aberrancy of antigens was detected (except in certain AL subtypes), AL specific immunophenotype is essential for minimal residual disease detection.

Two Atypical Cases of Classical Hairy Cell Leukaemia and Hairy Cell Leukaemia Variant: A Case Study

Introduction: Classical Hairy Cell Leukaemia (cHCL) and Hairy Cell Leukaemia variant (HCL-v) are both rare and slow-growing mature B cell neoplasms. According to flowcytometry data, they fall into the group classified as CD5- CD10- B cell lymphoproliferative disorders. Methods: Two cases with features atypical to two neoplasms at the time of diagnosis were studied. Results: Case 1 was a 15 year old male with right cervical lymph nodes (1x1 cm) in the posterior triangle, a few ecchymotic patches on the arm and a massive splenomegaly. C-reactive protein (CRP) level was 53 mg/dL. Erythrocyte Sedimentation Rate (ESR) was 98 mm/1 st hour. Full Blood Count (FBC) revealed typical features of pancytopenia with monocytopenia. The liver and renal profiles were normal. Morphology of bone marrow was suggestive of cHCL. Flowcytometry and BRAF V600E mutation was positive confirming the diagnosis of cHCL. Case 2 was a 55 year old male presenting with moderate splenomegaly and absolute lymphocytosis. The FBC revealed leukocytosis which is commonly seen with monocytopenia. Blood pictures revealed many hairy cells with moderately basophilic cytoplasm and visible nucleoli suggesting HCL-v. Flowcytometry findings and negative BRAF V600E mutation confirmed HCL-v. Conclusions: Clinical findings, blood images, morphology of bone marrow, flowcytometric findings and positive BRAF V600E mutation confirmed the diagnosis of cHCL in case 1 (15 year old boy) making it as a very rare case. The morphological findings on blood, the presence of characteristic CD markers on flowcytometry and negativity of BRAF V600E confirmed the case 2 as HCL-v, despite having CD10 positivity and monocytopenia.Keywords: Flowcytometric immunophenotyping, Hairy cell leukaemia, Hairy cell leukaemia variant