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P1

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NORMAL SPLENIC LYMPHOID SUBSETS MIMIC ABERRANT ANTIGEN EXPRESSION.

Nidhi Aggarwal, Jason Fischer, Fiona E. Craig
University of Pittsburgh School of Medicine, Department of Pathology

Background: Flow cytometry (FC) assists in the diagnosis of lymphoma through identification of aberrant antigen expression. However, normal lymphoid subsets with less well-recognized phenotypes can mimic lymphoma. This study characterizes lymphoid subsets in normal spleen using 8-color FC.

Design: 20 spleens removed for traumatic rupture, with cell viability >50%, were analyzed within 24 hrs with the following FC combinations: CD16&57/CD7/CD4/CD2/CD56/CD3/CD5/CD8; K//CD5/CD19/CD10/CD38/CD20/CD45;TCRab/TCRgd/CD3/CD25 and CD14/CD13&33/CD45/CD34.

Results: This study reveals the normal variation in splenic lymphoid subsets and demonstrates some subsets with phenotypes that have been associated with lymphoid neoplasms. Well recognized lymphoma-associated phenotypes identified in this study include CD5+ B-cells (20 of 20 specimens), CD7- T-cells (20 of 20), and CD3 bright gamma-delta T-cells (16 of 20). In addition, less well-recognized lymphoid subsets were identified that resemble those described in lymphoma: CD5- T-cells (20 of 20) (Figure 1a), CD2- NK-cells (20 of 20) and CD7dim+ NK-cells (20 of 20) (Figure 1b).The latter two NK-cell phenotypes were seen more often in the mature subtype (p<0.01).

Conclusion: 8-color FC analysis of normal spleen demonstrates lymphoid phenotypes that may resemble those described in subtypes of lymphoma, such as LGL leukemia, hepatosplenic lymphoma, and chronic NK-cell lymphoproliferative disorders. Familiarity with these normal phenotypes can help prevent misinterpretation. Furthermore, these findings support that these neoplastic phenotypes reflect expanded normal subsets rather than aberrant antigen expression.

P2

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A METHOD FOR STABILIZING CD69 ANTIGENIC EPITOPES ON ACTIVATED LYMPHOCYTES

Jodi R. Alt, Wayne L. Ryan
Streck

CD69, also known as activator inducer molecule (AIM), early activation antigen 1 (EA-1), Leu-23, and MLR3 is a type II integral membrane protein. Although not normally expressed on peripheral blood lymphocytes, surface expression is upregulated in response to a wide variety of stimuli and is identified as an early stage T-cell activation event. In addition to its involvement in T-cell mediated responses, it plays a pivotal role in the differentiation of the Th17 immune response via direct binding and inhibition of the Jak3/Stat5 transcription factor pathway. Because of CD69's role in immune responses, immune cell development, and potential antitumor effects, it is being evaluated as an important marker for the analysis of clinical patients in a variety of applications. Thus, the ability to measure CD69 expression for clinical or research flow cytometry is of significant value. We demonstrate the in vitro stimulation of peripheral blood mononuclear cells (PBMCs) followed by stabilization of CD69 expression for 7 days in Cyto-Chex® BCT. This is in contrast to stimulated cells maintained in K2EDTA alone as cells were unstable and CD69 percent recoveries were overtly elevated. This discovery allows more flexibility in testing protocols which can benefit clinical sample analysis by contract research organizations and hospitals.1

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Figure 1. Comparison of manual gating and ensemble clustering reseult. Population membership assignments using manual gating (a, c, e) Compared against assignments using the ensemble approach (b, d, f) in the HSCT dataset example. Each identified Population is color-coded separately.

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P3

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EUROFLOW™ LST, A VERY USEFUL 8-COLOR SCREENING TUBE FOR CHRONIC LYMPHOPROLIFERATIVE DISORDERS

Maria J. Arroz, José M. Pereira, Maria J. Acosta, Lylliane Luz, Ana P. Costa, Sílvia Amaro, M. Esmeraldina C. Júnior
CHLO, S. Francisco Xavier Hospital, Clinical Pathology Department

Aim: To evaluate the efficiency of screening CLPD using an 8-color, containing 11 monoclonal antibodies, single tube, in different types of specimens.

Material and methods: A total of 1000 fresh samples of peripheral blood (502), bone marrow aspirates (342), pleural effusions (44), peritoneal effusions (10), FNAs and lymph node biopsies (73), BAL (5), CSF (7) and other tissues (17), were processed using the LST™ tube without the gamma-delta reagent: CD20+CD4 Pacific Blue/CD45 Pacific Orange/Lambda +CD8 FITC/ Kappa+CD56 PE/ CD5 PerCP CY5.5/ CD19 PC7/ CD3 APC/ CD38 APC-H7. The majority of the requests were based on the suspicion of CLPD (75.5%) or evidence of cytopenias (20.7%). Infinicyt™ software was used for analysis, performing a sequential strategy based on the identification of T cells and their subsets, B cells with their light chain expression, and NK cells. Moreover, when applied to a bone marrow sample, it can also identify B cell maturation and plasma cells.

Results: Based on the analysis of this single tube, we were able to identify 299 abnormal samples, of which 261 were B-CLPD, 17 T and NK-CLPD and 16 plasma cell disorders. 40.5% of the abnormal samples had a final diagnosis of B-CLL, which was strongly suspected upon the analysis of this tube and confirmed by a second 8-color tube.

Conclusion: 3 years of routine experience using this single tube demonstrate that it is a very powerful screening tool for all types of samples, at a relative low cost when compared to the previous use of multiple tubes.

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ABNORMAL EXPRESSION OF CD20 ON IGG4 PLASMA CELLS

Antony C. Bakke1, Kate Grimm2, Dennis P. O'Malley2
1Clarient - GEHC 2Clarient Pathology Services

Context: IgG4-related sclerosing disease (IgG4-RD) is a newly described entity which presents as mass forming lesions in soft tissue, exocrine glands and in lymph nodes as IgG4-related lymphadenopathy (IgG4-RL).The underlying pathologic mechanism of IgG4-RD is unclear, however rituximab (an anti-CD20 monoclonal antibody) has been shown to have clinical efficacy.

Objective: To look for the presence or absence of CD20 on the IgG4 expressing plasma cells in IgG4-RL.

Design: Cases were identified through a retrospective review from the files at Clarient/GE healthcare. Patients were selected by the presence of a lymph node biopsy with increased IgG4 plasma cells by immunohistochemistry and a histologic diagnosis compatible with IgG4-RL and flow cytometry performed on a concurrent sample. Twelve cases were identified.

Results: We report dim CD20 expression on plasma cells in all cases where a plasma cell population was clearly identified by flow cytometry.These cases were from patients with lymph node biopsies that met published criteria for IgG4-RL.

Conclusions: This finding may be one potential explanation for the clinical efficacy of rituximab in IgG4-RD.

P5

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PLASMOCYTOID DENDRITIC CELLS AS BIOMARKERS FOR ACUTE CELLULAR REJECTION AFTER HEART TRANSPLANTATION

Markus J. Barten, Maja T. Dieterlen, Attila Tarnok, Stefan Dhein, Friedrich W. Mohr, Hartmuth B. Bittner
Heart Center Leipzig

Background: Over the last years studies in liver transplanted recipients have shown that plasmocytoid dendritic cells (pDCs) have the potential to be biomarkers for tolerance. Thus, we investigated the influence of tacrolimus (TAC)-based in comparison with cyclosporine A (CsA)-based immunosuppressive therapies on DCs in correlation with the incidence of rejection in heart transplant recipients (HTxR).

Methods: All HTxR received either CsA or TAC in combination of a daily fixed dose of mycophenolic acid (MPA) and steroids. Blood from all HTxR was taken to analyse for blood CsA or TAC concentration before drug administration. FACS analysis was performed to assess myeloid (m) and plasmocytoid DCs in peripheral blood. Endomyocardial biopsy was done to classify the histologic grade of rejection by a pathologist blinded to drug therapy. A moderate rejection grade (ISHLT 2004) was considered to be clinically significant.

Results: Mean time after HTx was 35.9 ± 11.8 months for the CsA-group and 45.1± 15.1 months for the TAC-group. Overall HTxR with rejection had significant (p<0.05) lower pDC values (55.1±16.2%) than HTxR without rejection (63.6 ± 10.5%). The influences of TAC and CsA on DCs were significantly (p<0.05) different: 1) mDCs were less in the TAC-group (45.2 ± 10.7%) vs. CsA-group (58.0 ± 19.1%), 2) pDCs were higher in the TAC-group (67.5 ± 8.4%) vs. CsA group (53.9±13.0%).

Conclusions: Our results showed for the first time that low pDC values in peripheral blood identify HTxR at risk for rejection. Furthermore, we found different affects of immunosuppressive drugs on pDCs. Future studies in a larger cohort of HTxR are necessary to confirm our findings. 1

Table 1. Comparison of NHL and HG phenotypes
Case NumberNHL HistoryNHL PhenotypeResidual Lymphoma IdentifiedLight chain in HGs (K/L ratio)
  • *

    Immunohistochemical phenotype

1MCLCD5−. CD 10−, Lambda+NoKappa (7.0)
2MCLCD5+, CD10−, Kappa+NoLambda (0.25)
3MCLCD5+, CD10−*NoKappa (17)
4MCLCDE+, CD10−, Kappa+NoKappa (43)
5Diffuse large B cell l ymphoma (DLBCL)CDB−, CD10− Lambda+NoLambda (0)
6DLBCLCD5+, CD10+, Kappa+NoKappa (12)
7DLBCLCD10−, Kappa+NoKappa (56)
8Follicular lymphomaCD5−, CD10+, Kappa+NoKappa (4.0)
9MCLCD5+, CD10−, Lambda+YesKappa (16)
10MCLCD5−, CD 10−, Kappa+YesKappa (7.5)
11MCLCD5+. CD 10−, Kappa+YesLambda (0)

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HIGHER AND EARLIER INCIDENCE OF CARDIAC ALLOGRAFT VASCULOPATHY IS ASSOCIATED WITH NON-HLA ANTIBODIES

Markus J. Barten, Maja T. Dieterlen, Sandy von Salisch, Stefan Dhein, Friedrich W. Mohr, Hartmuth B. Bittner
Heart Center Leipzig

Objective: Cardiac allograft vasculopathy (CAV) after heart transplantation (HTx) is a major therapeutic challenge, occuring in more than 50% of HTx recipients in the first years after transplantation. Antibodies against human leukocyte antigens (HLA) and non-HLA antigens like major histocompatibility complex class I-related chain A (MICA), angiotensin type 1 receptor (AT1R) or endothelin receptor A (ETAR) increasingly gain importance as modulators of allograft function and, therefore, recipient survival.

Methods: Sera of 116 HTx recipients were screened post-transplantation by Luminex-technology for HLA and MICA antibodies and their donor specificities. Non-HLA antibodies against AT1R and ETAR were analysed by ELISA. For statistical analysis gender, age, status of CAV and CMV seropositivity were documented.

Results: Gender, age or CMV infection had no influence on the incidence of CAV. In general, HTx recipients developed antibodies against HLA class I or class II to a lower extend than against non-HLA antigens. CAV appeared more frequently in recipients with non-HLA antibodies (38.3% AT1R; 44.1% ETAR; 13.0% MICA) than in recipients with HLA antibodies (8.7% HLA class I; 8.7% HLA class II). Furthermore, recipients with non-HLA antibodies developed CAV earlier (73.7±47.4mo) than recipients without non-HLA antibodies (85.5 ± 50.6mo). Donor specific antibodies against HLA-B46, -B76, -DQ7, -DQ8, MICA19 were identified as risk factor for CAV in this context.

Conclusions: Both HLA and non-HLA antibodies had an influence on CAV after HTx. Especially, non-HLA antibodies were connected to an earlier and higher incidence of CAV. These results indicate the necessity for monitoring HLA and non-HLA antibodies after HTx.1

Table 1. Comparison of NHL and HG phenotypes
Case NumberNHL HistoryNHL PhenotypeResidual Lymphoma IdentifiedLight chain in HGs (K/L ratio)
  • *

    Immunohistochemical phenotype

1MCLCD5−. CD 10−, Lambda+NoKappa (7.0)
2MCLCD5+, CD10−, Kappa+NoLambda (0.25)
3MCLCD5+, CD10−*NoKappa (17)
4MCLCDE+, CD10−, Kappa+NoKappa (43)
5Diffuse large B cell lymphoma (DLBCL)CDB−, CD10− Lambda+NoLambda (0)
6DLBCLCD5+, CD10+, Kappa+NoKappa (12)
7DLBCLCD10−, Kappa+NoKappa (56)
8Follicular lymphomaCD5−, CD10+, Kappa+NoKappa (4.0)
9MCLCD5+, CD10−, Lambda+YesKappa (16)
10MCLCD5−, CD 10−, Kappa+YesKappa (7.5)
11MCLCD5+. CD 10−, Kappa+YesLambda (0)

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IDENTIFICATION OF NON-HLA ANTIBODIES IN VENTRICULAR ASSIST DEVICE RECIPIENTS

Markus J. Barten, Sandy von Salisch, Maja T. Dieterlen, Stefan Dhein, Friedrich W. Mohr, Hartmuth B. Bittner
Heart Center Leipzig

Background: Recipients of ventricular assist devices (VADR) bridged to heart transplantation (HTx) have a higher incidence to develop antibodies against human leukocyte antigens (HLA). Beside HLA antibodies, non-HLA antibodies like major histocompatibility complex class I-related chain A (MICA) and autoantibodies against angiotensin type 1 receptor (AT1R) and endothelin receptor A (ETAR) are implicated in the pathogenesis of acute rejection and allograft vasculopathy. Therefore, we monitored non-HLA and HLA antibodies in VADR during the first year after implantation.

Methods: For antibody screening, sera of 56 VADR were analyzed by Luminex-technology for HLA/MICA and by ELISA for AT1R/ETAR antibodies several times over one year after implantation. Blood transfusions, gender and age were reviewed.

Results: The average age of VADR was 53.6±13.4 years, including 26 men. Most of the VADR were positive for AT1R (65.5%) and ETAR (68.9%) antibodies (>17 U) with high antibody titres up to 1000 U (27.6% each) or up to >2000 U (AT1R: 24.1%; ETAR: 34.5%). Almost half of the VADR (48.2%) showed moderate titres of HLA and/or MICA antibodies within the first year (anti-HLA-class I: 27.5%, anti-HLA-class II: 24.1%, anti-MICA: 17.2%). No significant difference in the number of received blood products were observed between antibody-negative or positive VADR, but AT1R/ETAR positive VADR received a higher amount of blood transfusions (55.5 ± 77.6 vs. 16.1 ± 9.5).

Conclusions: Beside HLA and MICA antibodies, VADR show most notably high titres of autoreactive AT1R/ETAR antibodies within the first year after VAD-implantation. Our data underline the necessity for monitoring non-HLA antibodies in VADR prior HTx.

P8

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EVALUATION OF IMMUNE RECONSTITUTION IN PATIENTS WITH FANCONI ANEMIA FOLLOWING BONE MARROW ALLOGENIC HEMATOPOIETIC STEM CELL TRANSPLANTATION AND MYELOABLATIVE CONDITIONING

Miriam Perlingeiro Beltrame1, Dimas Tadeu Covas2, Rita Perlingeiro3, Mariester Malvezzi1, Carmem Bonfim1, Ana Paula Azambuja1, Maria Tadeu Rocha1, Noeli Silva1, Julie Pimentel1, Yara Schluga1, Edna Martins1, Ricardo Pasquini1
1UFPR - Hospital de Clinicas 2Faculdade de Medicina de Ribeirao Preto 3University of Minnesota

Fanconi anemia is an autosomal recessive or X-linked disorder characterized by bone marrow aplasia. Allogeneic haematopoietic cell transplantation (HCT) remains the only treatment that can correct the haematological manifestations in patients with Fanconianaemia. We studied immune recovery in 23 patients during one year pos HCT. The mean age of patients was 9,6 years old and 7.6 in the control group. By flow cytometry we evaluated T, B and NK lymphocytes in blood and cytokines (IL-2, IL-4, IL-6, IL-10, TNF and INF) in serum samples. The follow-up was on days D+30, D+60, D+100, D+180, D+270, D+360 to correlate these findings with immune response and compare with pre transplant samples and the control group. The following markers were performed;CD3,CD4,CD8, CD10, CD16, CD19, CD20, CD24, CD25, CD27, CD28, CD31, CD38, CD45, CD45RA,CD45RO,CD56, CD57, CD69, CD95, CD127 CD152, CD154, CD178, FOXP3, TCRab, TCRgd. This study may lead to the need for new clinical and therapeutic approaches based on the results obtained. The use of biomarkers as a tool to monitor this group of patients may predict response to treatment and predict the risk of chronic graft-versus-host and the same diagnosis.

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RESULTS FROM FLOWCAP2 - CRITICAL ASSESSMENT OF AUTOMATED FLOW CYTOMETRY DATA ANALYSIS TECHNIQUES ON CLINICAL DATA FOR DIAGNOSIS

Ryan Brinkman1, Nima Aghaeepour1, Greg Finak2, Holger Hoos3, Tim Mosmann4, Raphael Gottardo2, Richard Scheuermann5
1BC Cancer Agency 2Fred Hutchinson Cancer Research Center 3University of British Columbia 4University of Rochester 5J Craig Venter Institute

An important use case for FCM analysis is the discovery of biomarker patterns in FCM data for the purposes of sample classification (i.e., diagnosis). We assembled a benchmark of three datasets in which the subjects/samples were associated with an external variable that could be used as an independent measure of truth for sample classification. The benchmark consisted datasets for: (1) studying the effect of HIV exposure on 44 African infants using 6 tubes of 8 color assays (HIV-exposed emph in utero but uninfected (HEU) vs. unexposed (UE)); (2) diagnosis of acute myeloid leukemia (AML) using 8 tubes of 5 color assays on 359 subjects provided by a reference laboratory (AML vs. non-AML); (3) discriminating between two antigen stimulation groups of post-HIV vaccine T-cells using two tubes of 8 color assays on 48 subjects (Gag-stimulated vs. Env-stimulated). We received a total of 43 submissions of computational approaches for the analysis of flow cytometry data and we compared the sensitivity, specificity, accuracy and F-measure of these approaches to correctly predict patient outcome. For two of the datasets (AML and HIV Vaccine Trials Network (HVTN)) many algorithms were able to perfectly predict the external variables even under very conservative conditions (i.e., using an independent test set as large as the training set).

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EXPRESSION OF CD2 AND CD25 ON MAST CELL POPULATIONS OUTSIDE THE SETTING OF SYSTEMIC MASTOCYTOSIS

Sindhu Cherian, Vivian McCulloch, Katy Dougherty, Valerie Miller, Jonathan Fromm, Brent Wood
University of Washington

Systemic mastocytosis (SM) is a diagnosis made using a variety of clinical, laboratory, and histologic parameters. The demonstration of aberrant CD2 and/or CD25 expression on mast cells provides one minor criterion for a diagnosis of SM. In order to validate a tube (CD45/CD117/CD2/CD25) for mast cell evaluation in our laboratory, we performed flow cytometry (FC) using this tube on marrow samples submitted for routine analysis by FC. Cases included in the study (n=44) had either an unknown diagnosis or a lymphoid neoplasm while cases with a myeloid stem cell neoplasm, cases in which SM was a clinical consideration, and cases with too few mast cells for evaluation (<0.01% mast cells) were excluded. The threshold for defining a case as positive was determined using a fluorescence minus one control. 36 cases showed mast cell populations with no expression of CD2 or CD25. 8 cases showed expression of CD2 and/or CD25 on ≥10% of the mast cell population (CD25 in 5 cases, CD2 in 2 cases, and both in 1 case).Review of available clinical data revealed no history of SM in the positive cases. The percentage of mast cells showing aberrant expression of CD2 and CD25 ranged from 12.1% to 98.8% and 22.2% to 74.6% respectively. Interestingly, 0/9 (0%) cases with no prior exposure to chemotherapy were positive while 8/35 (22.9%) post-chemotherapy cases were positive. These preliminary findings raise the possibility that aberrant expression of CD2 and/or CD25 may be seen on mast cells outside of the setting of SM, in particular during active marrow regeneration.

P11

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UTILITY OF CD200 IN THE DIAGNOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA

Sindhu Cherian1, Sandra Bohling2, Megan Wilson1, Greg Levin1, Brent Wood1
1University of Washington 2PhenoPath Laboratories

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is an indolent CD5+ B cell lymphoma in which flow cytometry plays an integral role in diagnosis. Mantle cell lymphoma (MCL) is often in the differential when considering CLL/SLL, and distinguishing the two is clinically important. Typically, the two can be distinguished using evaluation of CD20, CD23, surface light chain intensity, and FMC7 expression; however, additional markers are required in some cases. In the current study, we evaluate the utility of mean fluorescent intensity (MFI) of CD200 to separate CLL/SLL and MCL and compared CD200 to other established parameters. 66 consecutive cases of CD5+ B cell lymphoma with clinical and/or morphologic data compatible with a diagnosis of CLL/SLL (n=51) or MCL (n=15) were included. A MFI ratio (defined as MFI of parameter X for CD5+ B cells/MFI of parameter X for T cells) was calculated for CD200, CD20, CD23, and FMC7.Expression of each parameter was different between MCL and CLL/SLL with a p value of <0.05; however, the difference for CD200 had the lowest p value with only 2 outliers identified. In addition, an initial assessment of monoclonal B cell lymphocytosis (n=14) and CD5+ diffuse large B cell lymphoma (n=3) found variable CD200 expression.An ROC curve analysis indicates that most cases of CLL/SLL and MCL can be separated using a cut off of 1.10 for the CD200 MFI ratio (area under the curve of 0.982). Follow up studies are in progress and will focus on characterizing outlier cases and applying the CD200 MFI ratio to a validation set.

P12

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FLOW CYTOMETRIC TESTING FOR PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: CD64 IS BETTER FOR GATING MONOCYTES THAN CD33

Bakul I. Dalal, Nikisha S. Khare
Flow Cytometry Service, Division of Laboratory Hematology, Vancouver General Hospital

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the absence of glycosylphosphatidyl inositol (GPI)-associated ligands in neutrophils, monocytes, and red blood cells. Monocytes can be separated by their bright expression of either CD33 or CD64. We report a comparison of CD33- vs CD64-based monocyte gating in flow cytometric testing for PNH.

Methods: 119 cases tested for PNH, following the flow cytometry technic recently recommended by the international panel (Borowitz et al, Cytometry Part B: Clinical Cytometry, August 2010), were reviewed. The number of monocytes and their GPI-deficient fraction gated with CD33 and CD64 were compared. The clustering patterns were recorded and investigated when necessary.

Results: CD64 staining showed distinct separation of the monocyte cluster from lymphocytes; while CD33 staining showed a bridge of CD33-dim events (fig- 1), likely consisting of basophils, dendritic reticulum cells, promonocytes, or granulocyte precursors in 90% of cases, making precise gating somewhat subjective. The difference between the number of monocytes gated by CD33 and CD64 ranged from -26% to +32% (average: 1.69%). Six patients had GPI-deficient monocytes by both CD33 and CD64-based gating, ranging from 0.02% to 83.23%. No patients showed GPI-deficient monocytes by one but not the other gating strategy. The presence of blasts in acute leukemias resulted in abnormal clustering patterns by both gating techniques.

Conclusions: CD64 is strongly expressed in all monocytes, does not have a bridge of CD64-dim events, and shows a distinct cluster, allowing for more objective gating.

Acknowledgements: This study was supported by an unrestricted educational grant from Alexion Pharmaceuticals.

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Figure 1. Monocycle gating by CD33 (a) and CD64 (a) in PNH testing. Note the “bridge” region in the CD-stained specimen.

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P13

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CLINICAL AND IMMUNOPHENOTYPIC PROFILE OF PATIENTS WITH GPI-DEFICIENT CELLS USING 2010 CONSENSUS TESTING METHOD: BRITISH COLUMBIA EXPERIENCE

Bakul I Dalal, Nikisha S. Khare, Steven Pi
Flow Cytometry Service, Division of Laboratory Hematology, Vancouver General Hospital

Background: Cells lacking glycosylphosphatidyl inositol (GPI)-associated antigens are seen in paroxysmal nocturnal hemoglobinuria (PNH) and certain bone marrow failure states. Precise quantitation of GPI-deficient cells and indications for testing have recently been standardized (Parker, 2005; Borowitz, 2010). We report here our experience of testing patients for GPI-deficient cells based on the recommended indications and using the Borowitz 2010 protocol.

Methods: 300 tests were done on 281 patients over a 16 month period. A 7-color panel consisting of CD45, CD15, CD33, CD64, CD14, CD24 and FLAER was used for granulocytes and monocytes, while a 2-color panel consisting of CD235 and CD59 was used for RBCs. Minimum 25 events were necessary to consider a specimen positive. Indications for testing were verified by chart review, CBC, bone marrow examination, cytogenetics, and laboratory tests for hemolysis.

Results: GPI-deficienct cells were seen in 45/300 (15%) tests in 36/281 patients. The clone size varied from 0.01% to 95.09%. The difference between the number of GPI-deficient granulocytes and monocytes ranged from -47.1% to +11.96%. In 27/45 cases, monocytes returned a higher number of GPI-deficient cells than granulocytes. Frequency of GPI-deficient cells under various indications (table-1) were: 1. known PNH (6/8), hemolysis (6/22), thrombosis (1/43), aplastic anemia (16/29), myelodysplastic state (4/16), unexplained cytopenias (13/142), and others (4/54). Among patients with cytopenias, those with pancytopenia had higher incidence of GPI-deficient cells (8/36) than those with mono- (1/62) or bi-cytopenias (4/42).

Acknowledgments: This study was supported by an unrestricted educational grant from Alexion Pharmaceuticals.

Table 1. GPI Deficient Cells in PNH and Bone Marrow Failure States
Indication for testing* Number of cases (%) Number positive (%)** Clone size (%)
  • *

    A few patients had more than one indication for testing and are included under multiple categories.

  • **

    Based on the lowest and highest clone size in granulocyte and monocyte cell lines.

Previously diagnosed PNH8 (3)6 (75)0.03−95.09
Hemolysis22 (7)6 (17)0.92−37.15
Thrombosis43 (14)1 (2)0.58
Aplastic anemia29 (10)16 (55)0.05−23.00
Myelodysplastic states16 (5)4 (25)0.22−79.10
Unexplained cytopenias142 (47)13 (9)0.01−92.31
Other54 (18)4 (7)0.06−0.08
Total 300 (100) 45 (15) 0.01−95.09

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IMMUNOGLOBULIN HEAVY CHAIN ISOTYPE EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA: LACK OF CORRELATION WITH KARYOTYPIC ABNORMALITIES

Adriana Doldan-Silvero1, Laila Mnayer1,2, Joseph A. DiGiuseppe1
1Dept. of Pathology Hartford Hospital 2Clinical Laboratory Partners

Immunoglobulin heavy chain expression in chronic lymphocytic leukemia (CLL) has been shown to correlate with a number of prognostic clinical and laboratory variables. Karyotypic abnormalities as determined by interphase fluorescence in situ hybridization (FISH) have also been shown to have prognostic value in CLL. In this study, we sought to determine whether immunoglobulin heavy chain expression correlates with karyotypic abnormalities in a series of 90 cases of CLL for which both immunophenotypic and FISH data were available. Heavy chain isotype expression was determined by 4-color flow cytometry, and karyotypic abnormalities were identified using the Vysis CLL FISH probe kit (Abbott Laboratories). Cases of CLL were categorized as IgM/IgD+ (54%), IgD+ (19%), and IgD- (10%); in 17% of cases, heavy chain expression was absent or undetectable. Karyotypic abnormalities were categorized as described previously (Döhner H, et al. N Engl J Med 2000; 343:1910-6). The distribution of karyotypic abnormalities did not differ significantly among the heavy chain isotype-defined immunophenotypic categories; neither adverse (11q or 17p) nor favorable (isolated 13q) deletions were more common among any single heavy chain category (p > 0.05, Fisher-Exact test). These data provide no evidence for an association between immunoglobulin heavy chain expression and karyotypic abnormalities in CLL.

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INKT CELLS IN NASAL POLYPOSIS: DIFFERENT METHODS, DIFFERENT RESULTS

Mohammad Fereidouni1, Farahnaz Jabari Azad2, Reza Farid Hosseini2
1Birjand University of Medical Sciences 2Mahshad University of Medical Sciences

Background: Invariant Natural killer T cells (iNKT) are asmall subset of T lymphocytes which are involved in a wide variety of immune responses. In spite of their importance in different disorders, low number of these cells and technical difficulties affects the validity of studies in this field. The aim of this study was to compare frequency of iNKT cells in nasal polyp with flow cytometry and Real time PCR.

Materials and methods: 16 Nasal polyp tissue was isolated during the polypectomy and each sample divided into two parts, in one part cell suspension was made by Medimachine and the frequency of iNKT cells among CD3+gate, was measured by flow cytometry using vα24 and Vβ11 antibodies and in the other part, iNKT cells evaluated by RT-qPCR using primers for Vα24 and Vβ11 genes as well as CD3 and GAPDH genes.

Results: In flow cytometry, the numbers of iNKT cells varied from 0.5% to 11% (mean 4.7%) in Polyp cell suspensions while we could not detect iNKT cells in most of polyp samples by real time PCR and in a few positive samples, the relative expression of iNKT genes was significantly lower than whatever detected in flow cytometry.

Conclusion: The results of this study show that the method of iNKT detection has profound influence on the results and it is necessary to set up standard protocols for preventing errors in this field.

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CONTRIBUTION OFMULTIPARAMETER FLOW CYTOMETRY IMMUNOPHENOTYPING TO THE DIAGNOSTIC SCREENING AND CLASSIFICATION OF PEDIATRIC CANCER

Cristiane S. Ferreira-Facio1, Cristiane B. Milito2, Vitor Botafogo1, Marcela Fontana1, Leandro S. Thiago3, Elen Oliveira1, Ariovaldo S. da Rocha-Filho4, Fernando Werneck4, Danielle N. Forny1, Samuel Dekermacher4, Ana Paula de Azambuja5, Sima E. Ferman3, Paulo Antonio S. Faria3, Marcelo G. P. Land1, Alberto Orfao6, Elaine S. Costa1
1Pediatrics Institute IPPMG, Universidade Federal do Rio de Janeiro - UFRJ 2Pathologic Anatomy Department, Medicine Faculty/ UFRJ 3Servidores do Estado Hospital- HSE 4National Cancer Institute of Brazil – INCA 5Clinics Hospital, Federal University of Paraná - UFPR. 6Cytometry Service, Department of Medicine and Cancer Research Center (IBMCC, University of Salamanca-CSIC and IBSAL), University of Salamanca

Pediatric cancer is a heterogeneous group of tumors which require multiple procedures, for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of solid tumors. Here we propose a FC panel of markers for the diagnostic screening and classification of pediatric cancer, with emphasis on solid tumors. The proposed strategy aims at differential diagnosis between tumoral and reactive samples, hematological and non-hematological malignancies and immunophenotypic classification of solid tumors. For this purpose, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC versus conventional histopathology + immunohistochemistry. The overall concordance rate between FC and conventional diagnostic approaches was of 96% (n=50/52), with 100% agreement for both all reactive/inflammatory and non-infiltrated samples and solid tumors, with only two false negative cases. Additionally, clear discrimination among samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. A relatively limited panel of markers is proposed which may contribute to differential diagnosis: neuroblastoma (CD56hi/GD2+/CD81hi), PNET (CD271hi/CD99+), Wilms tumors (different cell populations), rhabdomyosarcoma(NuMYOD1+/NuMyogenin+), carcinomas(CD45-/EpCAM+), GCT(CD56+/CD45-/NG2+/CD10+) and hemangiopericytoma (CD45-/CD34+). In summary, our results show that FC is a fast and useful complementary diagnostic tool to routine histopathology, for the diagnostic screening and classification of pediatric cancer.1

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EXPRESSION OF CD25 CORRELATES WITH PHILADELPHIA (BCR-ABL) CHROMOSOMAL TRANSLOCATION IN PEDIATRIC B-ACUTE LYMPHOBLASTIC LEUKEMIA.

Amos Gaikwad1,2, Tarek Elghetany2, Christopher Threeton1,2, Michael Cubbage1,2, Tatiana Goltsova1,2, Andrea M Sheehan2, Jyotinder Punia2, Timothy Moore1, Xinyan Lu1,2, Choladda Curry1
1Texas Children's Cancer and Hematology Center, Texas Children's Hospital 2Departments of Pediatrics and Pathology, Baylor College of Medicine

Background: Nearly 75-80% of pediatric B-acute lymphoblastic leukemia (B-ALL) patients undergo complete remission and long-term survival with current chemotherapy. However, approximately 20% of patients ultimately have a poor prognosis. Philadelphia chromosome-associated translocation (Ph+) BCR-ABL t (9;22), which occurs in about 3-5% of pediatric B-ALL, confers the worst outcome. Identifying Ph+ B-ALL is therefore crucial for risk stratification. CD25 (IL2a-receptor) expression was reported to have association with Ph+ B-ALL in adult population. However, no similar study has been performed in pediatric B-ALL.

Methods and Results: Retrospective analysis of consecutive patients with a new diagnosis of B-ALL (blood and/or bone marrow) over three year period (2009-2012) for CD25 expression and the presence of Ph+ translocation by cytogenetic studies was performed. Specimens were prepared according to standard procedures and analyzed using BD FACSCanto flow cytometer. A threshold of 20% was used to identify positive cases for CD25 expression. Of the 233 patients, 127 were males and 106 were females ranging in age from 3 months to 22 years (median, 5 years). The median blast count was 76%. Eight cases of Ph+ B-ALL (3%) were identified; six of these expressed CD25. In addition, eight Ph-negative-B-ALL expressed CD25. Therefore, CD25 expression in predicting Ph+ B-ALL had a 75% sensitivity, a 96% specificity, whereas 43% and 99% of positive predictive value (PPV), and negative predictive value (NPV), respectively.

Conclusions: CD25 expression is a specific and relatively sensitive marker for the identification of Ph+ B-ALL in the pediatric population. However, its NPV far exceeds its PPV.

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INCREASED TIM3 EXPRESSION ON LEUKEMIC BLASTS

Kelly Garner, Stephen Ten Eyck, Fiona E. Craig, Christine G. Roth
University of Pittsburgh Medical Center

Background: T-cell immunoglobulin mucin-3 (TIM-3) has recently been reported to be expressed on acute myeloid leukemia (AML) stem cells but not on normal hematopoietic stem cells, and has been proposed as a novel tumor marker.TIM-3 can be expressed on monocytes, natural killer cells, and a subset of T cells, however, expression on normal myeloblasts has not been well-characterized or compared with AML. The aim of this study was to evaluate the diagnostic utility of TIM-3 expression in separating leukemic from non-leukemic myeloblasts.

Design: Peripheral blood and bone marrow samples from 23 AML and 19 non-neoplastic cases were evaluated, using the following flow cytometric 8 color antibody panel:CD14/TIM-3/CD117/CD13+CD33/CD34/CD3/CD56/CD45.FACSCanto-II and BD-FACS-DIVA were used for acquisition and analysis, respectively.TIM-3 expression was quantitated as a percentage of the CD34+/CD13+33+ myeloblasts.

Results: TIM-3 positive myeloblasts were identified in all cases, however the percentage of myeloblasts expressing TIM-3 was significantly higher in AML cases (median 71.5%, range 11.2-96.3%) as compared to the non-neoplastic cases (median 53.5%, range 26.1 – 70.7%) (Mann Whitney U test, p=0.02).Receiver Operator Curve (ROC) analysis identified a cut-off value of >70.6% as a useful discriminator between leukemic and non-leukemic myeloblasts (sensitivity 57%, specificity 95%).

Conclusions: TIM-3 expression is not restricted to AML, and may be seen on non-leukemic myeloblasts. Despite this overlap, a higher proportion of leukemic blasts expressTIM-3, and >70.6% TIM-3 positive myeloblasts distinguishes between leukemic and non-leukemic myeloblasts with high specificity. Further studies are required to validate the diagnostic utility of TIM-3 in clinical practice.

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EVALUATION OF PERIPHERAL BLOOD MEMORY B-CELLS IN A SMALL RUSSIAN COHORT (A PILOT STUDY).

Margarita Ivanchenko1, Konstantin Slobodnyuk1, Margarita Gorchakova1, Vera Golubeva1, Marina Guseva2, Yekaterina Zueva1
1State Pavlov Medical University 2State Pediatric Medical Academy

The assessment of peripheral blood memory B cells is widely used in diagnostics of inherited immunodeficiencies, namely, of Common Variable Immune Deficiency and Hyper IgM-syndrome. According to EuroClass 2009 recommendations, we used a whole blood staining-lysis method. The panel of monoclonal antibodies containing the following markers: CD45 (to identify lymphocytes), CD19 (to segregate B cells), CD27 (memory B cell marker), superficial IgM and IgD. Memory B cells are CD27 positive, and, after class switching are negative for IgD and IgM (with an exception for a small subpopulation that have chosen IgD/IgM to synthesis). Age-matched reference values for the analysis were taken from the literature. However, the immunophenotyping panel (including anti-IgM or anti-IgD independently), age intervals and data representing methods differed profoundly between the researches, and were difficult to compare. Preliminary, our results match reference values obtained by Piatosa et al. in 2011.

We found that for sparse subpopulations (for instance, memory B cells in young children), the accuracy of evaluation was higher if fewer markers were used (anti-IgD only) to achieve a phenotype of interest. With this approach we were able to identify a severe decrease in memory B cells number (relative and absolute) in six patients. Age, sex, anamnesis morbi and clinical manifestation were highly variable in accordance with heterogeneity of a disease. Overall, the severity of the course was more dependent on memory B cell count, than on serum immunoglobulin levels. In some patients we also found association with neutropenia that should be later characterized in details.

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COMPARISON OF BD™ STEM CELL ENUMERATION KIT (BD-SCE) WITH CURRENT LABORATORY ISHAGE PROTOCOL STEM CELL ENUMERATION ASSAY

Abigail S. Kelliher, Michelle E. Delelys, Laura J Dillon, Frederic I. Preffer
Massachusetts General Hospital

In February of 2012, BD released the Stem Cell Enumeration Kit (SCE) for absolute counting of CD34+ stem cells. Recently, new requirements by the Foundation for the Accreditation of Cellular Therapies (FACT) necessitate the presence of a simultaneous viability marker to determine CD34+ cell viability. BD's new single platform kit satisfies these requirements. We performed a comparison with our current dual platform method that is based upon the ISHAGE Stem Cell Enumeration guidelines. Percentages and absolute counts of viable CD34+ cells were compared. We tested staining the cells in the dark and in light, determined how long cells remained viable after staining and whether or not cells needed to be left on ice. There was excellent correlation between the two assays. In comparing viable absolute counts and percentages of CD34+ cells, the r-squared values were 0.977 and 0.895, respectively. If two samples are removed from the study that were stained the following day (all others were stained the same day), the r-squared value increased to 0.942.Since the absolute value of viable CD34+ cells ultimately determines the potential engraftment of the transplant, that value is of greatest importance. The BD™ SCE Kit and the automated FACSCanto™ software require that 7- Color Setup Beads as well as BD™ Stem Cell controls be utilized prior to the analysis of patient samples. The new BD assay is 8 times more expensive than our current assay, although it is more streamlined and because the software is automated, easier to ensure accurate gating between users.

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FLOW CYTOMETRY REVEALS IMMUNE SIGNATURES ASSOCIATED TO HUMAN AGING

Anis Larbi1, Tze Pin Ng2, Tamas Fulop3
1Singapore Immunology Network, Biopolis, A*STAR 2NUHS, Singapore 3Research Center on Aging

The proportion of elderly individuals (over 65 years old) is increasing worldwide. The problem with the elderly population is that a significant fraction is not healthy. Although the number of centenarians is increasing, the number of clinical conditions affecting mortality and morbidity is high. Most of elderly individuals display at least one chronic condition that overall affects health. Based on this, we aimed at identifying in a very well-characterized elderly population the changes at the immunological levels that may be associated to one or several conditions. For this we recruited apparently healthy home-dwelling individuals via the Singapore Longitudinal Aging Study (SLAS). Using state-of-the-art flow cytometry we tested the hypothesis that T cell subset distribution was different in healthy elderly vs. individual suffering from diabetes, frailty, dementia and compared this with various clinical parameters including inflammation-related markers, also assessed by flow cytometry. We identified specific signatures in Alzheimer patients compared to age-matched non-Alzheimer patients. The frequency of the T cell subsets identified correlated to circulating levels of inflammatory mediators. Diabetic and frail elderly also display distinct profiles assessed by flow cytometry. The role of chronic infections such as cytomegalovirus in such events will be discussed.

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FREQUENCY OF COLOR COMPENSATION IN CLINICAL USE

Kim Le, Kim Romang, Melissa Cessna
Intermountain Central Laboratory, Flow Cytometry Department, Intermountain Healthcare

Introduction: Color compensation is essential in multicolor flow cytometry. The College of American Pathologists flow cytometry checklist requires having procedures for determining color compensation settings, but does not specify how often the color compensation must be done. The color compensation procedure can be costly and time consuming especially if a cytometer uses multiple fluorochromes and some of them are tandem. Tandem fluorochromes are sensitive to increased light exposure and elevated temperature; and therefore require antibody-specific compensation. We evaluated the stability of spectral overlaps using the BD FACSCanto II instrument and its compensation software and determined the frequency of color compensation performances needed. The BD FACSCanto II has three lasers and can detect up to eight fluorochromes.

Materials and Methods: FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-H7, V450, and V500 fluorochromes were used for color compensation. PE-Cy7 and APC-H7 were tandem and required eight antibody-specific compensation tubes. Voltages and spectral overlaps were computed for mean and standard deviation (SD).

Results: This study was done from April 28 to May 23, 2012. All spectral overlaps had small SD less than 2%, even with tandem fluorochromes. The voltages of all photomultiplier detectors were very stable with SD less than 4 volts.

Conclusions: Color compensations remained stable over time. Daily color compensation performance on the BD FACSCanto II was not needed due to its daily performance check to ensure the stability of photomultiplier voltages. We suggested doing color compensation weekly instead of daily (per manufacturer's recommendation) to reduce reagent cost and processing time.

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VALIDATION OF EOSIN 5 MALEIMIDE STAINING FOR THE DETECTION OF SPHEROCYTES IN HEREDITARY SPHEROCYTOSIS

Michael A. Liew1, Roberto Nussenzveig1, Archana Agarwal1,2, Carl T. Wittwer1,2
1ARUP Institute for Clinical & Experimental Pathology, 2University of Utah School of Medicine

Hereditary spherocytosis (HS) is a common inherited hemolytic anemia characterized by the presence of spherical erythrocytes or spherocytes. Spherocytes have a smaller surface area and can maintain healthy oxygen levels, but are physically fragile. HS can be diagnosed by clinical features, peripheral smear examination as well as clinical laboratory tests including osmotic fragility, ektacytometry or flow cytometry. Our aim was to validate the flow cytometry assay using eosin 5 maleimide (EMA) labeled intact red blood cells that bind to band 3. One microliter of whole blood was washed in 200 μl of phosphate buffered saline (PBS), and stained using 0.5mg/ml EMA for 60mins at room temperature. The sample was then washed 3 times using PBS+0.5% (w/v) bovine serum albumin (BSA) before being resuspended in 200 μl PBS+0.5%BSA. One hundred thousand erythrocyte events were acquired on a FACS Canto II. When data obtained by flow cytometry was correlated with visual microscopy looking for spherocytes, a correlation was evident with rare visual spherocytes (n=26) showing a mean fluorescence of 9100, occasional spherocytes (n=10) with a mean of 8700, and moderate spherocytosis (n=6) with 6800. Samples also appear to be reasonably stable with the EMA fluorescence decreasing by approximately 8% 4 days post draw. This assay will be a useful diagnostic tool for the diagnosis of HS. This work was funded by the ARUP Institute for Clinical and Experimental Pathology.

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DETECTION OF MINIMAL RESIDUAL DISEASE (MRD) IN B LYMPHOBLASTIC LEUKEMIA/LYMPHOMA (B-ALL) USING A 9 PARAMETER KALUZA RADAR PLOT

Joanne M. Luider1, Karan Paisooksantivatana1, Maria C. Gentile2, Iwona A. Auer1, Adnan Mansoor1
1Calgary Laboratory Services 2Beckman Coulter Canada

Introduction: Identification of B-ALL MRD to a sensitivity level of 0.01% is now routinely required for risk-stratified treatment protocol as well as for clinical trials. In addition to all technical issues, data analysis is the most crucial step for accurate quantification of MRD. For this purpose, sophisticated and extensive sequential gating strategies are typically utilized in many laboratories using different analysis software.

Aim: To determine if the Kaluza radar-plot (showing 9 parameters in one plot) can simplify MRD analysis for B-ALL.

Methods: Ten normal/regenerating bone marrows and fifty bone marrow samples from both adult (n=27) and paediatric (n=20) patients were stained in a single 10-colour tube with the following antibodies:CD58-FITC, CD49f-PE, CD34-ECD, CD38-PC5.5, CD81-APC, CD22-APC-AF700, CD10-APC-AF750, CD123-PB, and CD45 Kro. All CD19-positive events were gated and subsequently analyzed for MRD using both a standard sequential gating strategy and a single 9 parameter Kaluza radar plot. MRD was identified as a cell cluster (at least 10 events) falling outside the normal maturation “zone” defined by the 10 reference BM's.

Results: All fifty samples, 34 MRD-negative and 16 MRD-positive bone marrows, showed complete agreement between the sequential gating strategy and the single 9-parameter Kaluza radar plot in terms of either qualitative or quantitative results.

Conclusions: B-ALL MRD can be accurately and efficiently quantified using a simple 9-parameter Kaluza radar plot from a single 10-color tube analysis.

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COMBINED TCR GAMMA/DELTA T CELL SUBSETS IN HEPATOSPLENIC T-CELL LYMPHOMA

Neil V McNamara, Aysha Othman, Anne-Marie Watson, Lindsay Dunlop, Silvia Ling
Haematology, Liverpool Hospital

Hepatosplenic T-cell lymphoma (HSTL) is a rare and aggressive disease of cytotoxic T cells expressing T cell receptor (TCR) gamma/delta. Presentation may include, fever, abdominal pain and hepatosplenomegally without lymphadenopathy. Bone marrow involvement is common, with thrombocytopenia and occasional pancytopenia. Immunophenotyping by flow cytometry is a principle diagnostic tool for HSTL. We describe 2 cases of this rare condition, with immunophenotypes in bone marrow samples of Case (1): CD2+, CD3+, CD4-, CD5-, CD7+/- CD8-, CD16+, CD56+, TCR gamma/delta+, and Case (2): CD2+,CD3+,CD4-, CD5+, CD7+, CD8+, CD16+, CD56-, TCR gamma/delta+. Diagnostic populations were also found in blood, however additional TCR gamma/delta subsets were unexpectedly detected in both cases with unique phenotypes, possibly arising from immune responses, and appeared less sensitive to chemotherapy treatment. TCR gamma/delta markers are not always included in routine lymphoma screens. Small numbers of these cells in the blood are easily overlooked and appropriate follow up markers not always examined. It is recommended that TCR surface markers be included in all T cell lymphoma screening panels as recommended by EuroFlow. We suggest cases of HSTL may be under diagnosed due to incomplete immunophenotype testing and distinction between TCR gamma/delta subsets needs to be made during treatment monitoring.

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APPLICATION OF EIGHT-COLOUR FLOW CYTOMETRY (FC) IN THE ASSESSMENT OF PLASMA CELL MYELOMA (PCM) AT PETER MACCALLUM CANCER CENTRE (PMCC).

Vuong Van Nguyen1, Peter Gambell1, David Westerman1,2, Neil Came1,2
1Pathology Department, Peter MacCallum Cancer Centre 2University of Melbourne

Aim: The utility of our 8-colour/3-tube FC method (table 1) for differentiating normal and abnormal bone marrow (BM) plasma cells (nPC/aPC) at diagnosis and for stringent complete response assessment (sCRa) of PCM was compared to our superseded 4-colour/5-tube protocol (italics).

Methods: Consecutive BM aspirate samples were analysed on a BD-FACSCanto-II to define aPC (with or without 5% nPC/total PCs) at diagnosis, aPC during treatment (Rx), and aPC at 0.01% sensitivity for sCRa in patients with PCM. Comparison of sCRa by 4-colour BD-FACSCalibur (same gating strategy) was performed in the same number of consecutive samples analysed immediately prior to commencement of 8-colour FC. Undetectable aPC, or of any PC, defined sCR provided that >100000 events (averaged/tube) were analysed and that CD45/SSC confirmed representative BM cellularity.

Results: From October 2011-July 2012, 85 eligible BM samples were assessed by 8-colour FC; 69 for PCM (30 sCRa, 27 inter-therapy, 12 diagnostic) and 16 miscellaneous. 100% sCRa samples were suitable by 8-colour vs 22/30 (73%) by 4-colour (p=0.005, Fishers); both 100% sensitivity; 86% vs 27% specificity; median events analysed (average/tube) 463426 (100000-733955) vs 164343 (27292-500000). An aPC phenotype was defined in 67/85 (79%) cases (table 2). Table 3 highlights likelihood of each antigen dichotomizing nPC/aPC in the same sample.

Conclusion: Eight-colour FC of the BM in PCM for sCRa at PMCC is superior to previous 4-colour method. Additional markers (see CD200) and reliable analysis of more BM cells per tube provides improved specificity for defining sCR and adds clinically relevant prognostic information to guide therapy.

Table 1. 8-Colour FGFG Panal 4-Colour PGFC Panel (Italics)
V450Pac OrangeFITCPEPerCP-Cy5.5PE-Cy7APCAPC-H7
CD38CD3CD81 CD27 CD28 CD19 CD138 CD45
CD38CD3CD20CD200CD56CD19CD138CD4S
CD38CD3 Cyt Kappa Cyt LambdaCD117CD19CD138CD45
Table 2. Frequency of Antigen Expression on aPC (% of cases) N=67
CD19Light chain restrictionCD200CD56CD45CD81CD117CD38CD27CD28CD20
41008272313754100513115
Table 3. Frequency of each antigen differentiating aPC from nPC (% of cases) N=44
CD19Light chain restrictionCD200CD56CD45CD81CD117CD38 weakCD27CD39CD20
9595917566815954482714

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IMPACT OF GALLIOS™ ACQUISITION SETTINGS AND INFINICYT™ ANALYSIS SETTINGS ON DATA ANALYSIS AND INTERPRETATION (LESSONS LEARNED)

Melanie O'Donahue, Susan Schmitt
1Michael McPherson 2Esther Alvaro 3Marla Dorman

Fluorescent compensation can be tricky, especially when data is acquired and analyzed with different software platforms. A growing trend in flow is to have the technical component (staining and acquisition) and the professional component (analysis and interpretation) performed at separate locations. Our site acquires samples on a Gallios™ and the data is analyzed off site using Infinicyt™.

Compensation samples acquired on the Gallios™ using AutoSetup settings did not appear to look optimally compensated. We manually set the compensation settings by aligning the MFI of the dual negative to the positive PE and PC-5 positive populations, respectively. Both sets of data were sent to our professional site for analysis. It came to our attention that the results of the sample acquired on the Gallios™ that appeared to have acceptable fluorescent compensation did not look acceptable by the flow techs analyzing the data with Infinicyt™.

Our investigation found that data acquired with the Gallios™ Baseline Offset function “ON” displayed differently in CXP™ than in Infinicyt™ software. We also discovered that the Infinicyt™ Negative Visibility feature changes the display of data post-acquisition. Without using post-acquisition compensation, FCS Express displayed data similarly to CXP™ (it does recognize the Baseline Offset function) while Beckman Coulter's Kaluza™ Software displayed data similarly to Infinicyt™ (it does not recognize the Gallios™ Baseline Offset function).

Due to the visual nature of flow cytometry interpretation, it is critical that both the acquiring and analyzing operators have a full understanding of the effects of all acquisition and analysis software functions.

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FREQUENCY OF CD34+ HEMATOGONE SUBSET IN PERIPHERAL BLOOD STEM CELL APHERESIS PRODUCTS USING THE ISHAGE PROTOCOL

Sarah L. Ondrejka, Deborah Katanik, Betty Gay, Bruce Briggs, Florence Namiotka, Lisa McTaggart, Hien Duong, Eric D. Hsi
Cleveland Clinic

Background: CD34+ cell enumeration is a critical step in graft harvesting for hematopoietic stem cell transplantation. Using the ISHAGE protocol, we periodically find a separate population of CD34+ events, suspected to consist of hematogones (B-lymphocyte precursors) that falls within the gating guidelines for R3 and thus is not excluded from the total CD34+ value reported. We investigated the frequency with which these cells occur in peripheral blood stem cell (PBSC) apheresis products and characterized their phenotype.

Methods: PBSC apheresis samples were analyzed for the presence (≥0.01%) of the additional CD34+ population. The CD34+ subset was analyzed with antibodies for CD19, CD20, and CD10.The proportion of these cells relative to total CD34+ cells was compared between allogeneic and autologous sources.

Results: Between 1/4/2011 – 12/6/2011, 274 apheresis samples were evaluated.61% contained the additional CD34+ population.This population comprised a median of 7.14% of total CD34+ cells (range 1.25 – 77.97).CD19+, CD20+/-, and CD10+ expression pattern in a subset (=43) of positive samples confirmed that the population corresponded to hematogones. The CD34+ hematogone subset in allogeneic PBSC apheresis products (median 2.99% of total CD34+ cells, range 1.29 – 33.33) was not significantly different from autologous products (median 5.63%, range 1.25 – 16.67; P=0.23).

Summary: Hematogones are captured in the ISHAGE gating protocol and may comprise a substantial fraction of CD34+ cells in PBSC apheresis products. Whether inclusion may adversely affect engraftment clinical endpoints is the subject of ongoing investigation.

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PEDIATRIC REFERENCE RANGES FOR PERIPHERAL B CELL SUPOPULATIONS BASED ON THE EUROCLASS CLASSIFICATON OF OF CVID

Michele Paessler1,2, Leslie Koutafaris2, Joe McMann2
1University of Pennsylvania School of Medicine 2Children's Hospital of Philadelphia

The evaluation of pediatric patients for immunodeficiencies, including B cell immunodeficiencies and common variable immunodeficiency (CVID) are common. In recent years, flow cytometric studies have demonstrated various B cell subsets and the basis for a new classification scheme defining subgroups in common variable immunodeficiency. These specific B cell subsets correlate both clinically and prognostically with CVID and also have diagnostic value. However, the data from these studies were based largely on adults and are not applicable to the pediatric population. Previous studies of pediatric reference ranges demonstrated significant differences from adult values and changes in the B cell compartment during development, particularly in infancy and early childhood. Immunphenotyping is a useful tool in diagnosing immunologic and hematologic disorders and when values fall outside of the defined normal ranges it may indicate disease and the need for further work-up. Evaluating the pediatric patient for immunodeficiencies, including CVID, can be challenging due to the lack of reliable reference ranges. Reference ranges for B cell subsets in children have been hampered due to significant age-related changes in the developing immune system in the pediatric population and small numbers of patients in previous studies. At the Children's Hospital of Philadelphia, we are comprehensively evaluating age-related B cell subsets in a healthy cohort of children from birth to 21 years using 10-color flow cytometry (Beckman Coulter Galios). The age ranges for the study are:<6 months, 6 months-1year, 1-2, 2-4, 5-8, 8-12, 12-18 years. In this study, flow cytometry is performed on PBMCs and analyzed by gating on lymphocytes by CD45 versus side scatter. B cells are then identified by CD19 positivity. The B cell subsetsin our comprehensive B cell panel include those descrfibed in theEUROclass trial. The following immunophenotypic subtypes ofCD19+ B cells are analyzed: IgD+/CD10+; CD40+, IgD+/IgM+; CD27+/IgD+; CD27+/IgD-; CD27+/IgM+; CD27+/IgM-; CD27-/IgM+; IgG/CD20-; CD5+/CD19+;CD21+/CD38+; CD38+/IgM+, and CD38+/IgM-.The values are given as a percentage of B cells and total events. Thus far 62 patients have been analyzed and the study is in progress. To date, we have seen a decrease in the percentages of all B cells subsets compared to those published values in other pediatric cohorts and adults. The most significant differences we have seen are in naive B cells and in both switched and unswitched memory B cells, which were ∼30-40% less than those published age-related values. This finding further documents the need for age-related reference values in a large pediatric cohort.1

Table 1. Comparison of NHL and HG phenotypes
Case NumberNHL HistoryNHL PhenotypeResidual Lymphoma IdentifiedLight chain in HGs (K/L ratio)
  • *

    Imnnunohistochemical phenotype

1MCLCD5−. CD 10−, Lambda+NoKappa (7.0)
2MCLCD5+, CD10−, Kappa+NoLambda (0.25)
3MCLCD5+, CD10−*NoKappa (17)
4MCLCDE+, CD10−, Kappa+NoKappa (43)
5Diffuse large B cell lymphoma (DLBCL)CDB−, CD10− Lambda+NoLambda (0)
6DLBCLCD5+, CD10+, Kappa+NoKappa (12)
7DLBCLCD10−, Kappa+NoKappa (56)
8Follicular lymphomaCD5−, CD10+, Kappa+NoKappa (4.0)
9MCLCD5+, CD10−, Lambda+YesKappa (16)
10MCLCD5−, CD 10−, Kappa+YesKappa (7.5)
11MCLCD5+. CD 10−, Kappa+YesLambda (0)

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FLOW CYTOMETRIC AND PROTEOMIC FEATURES OF PLATELET-FREE PLASMA MICROPARTICLES IN THALASSEMIA

Kovit Pattanapanyasat1,2, Porntip Chaichompoo1,3, Panida Kumya1, Ladawan Khowawisetsut1, Wararat Chiangjong1,4, Sakdithep Chaiyarit1,4, Nutkridta Pongsakul1,4, Noppadol Sirithanaratanakul5, Suthat Fucharoen3, Visith Thongboonkerd1,4
1Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University 2Center for Emerging and Neglected Infectious Diseases, Mahidol University 3Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University 4Center for Research in Complex Systems Sciences, Mahidol University 5Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University

Thalassemias are inherited hemoglobinopathies characterized by absent or partial production of α- or β-globin chain synthesis. Several thalassemia genotypes exist, and the corresponding manifestations can vary from barely detectable abnormalities to severe even fatal anemia. Profound hemostatic changes with thromboembolic complications are common in thalassemia patients. Several etiologic factors including oxidative damage of red blood cells (RBCs), platelet activation and the high level of membrane-derived microparticles (MPs) stemming from blood cells are thought to be responsible for the associated thrombotic risk, but the mechanisms remain unclear. In this communication, we report the oxidative stress, procoagulant properties and proteomic profiles as well as their number and cellular origin of these MPs isolated from β-thalassemia/Hemoglobin E patients' blood. Flow cytometric results showed that platelet-free-plasma (PFP) MPs from thalassemia patients expressed significantly higher levels of phosphatidylserine (PS)-bearing MPs than those from normal subjects. The highlevels of these PS-bearing MPs correlated with increased platelet counts and their procoagulant activity. These PS-bearing MPs showed mostly platelet and RBC origin which both had high levels of oxidant activity. Proteome and Western blot analyses of isolated PFP MPs from thalassemia displayed several distinct protein features that involved in regulation of cell stress, i.e. peroxiredoxin 6, apolipoprotein E, heat-shock protein 90, etc. These findings suggest that elevated oxidative damage in platelets and RBCs potentially induce MP formation, and that these MPs contain proteins with oxidative features that might aggravate thrombotic events when the number is excessive as commonly seen in thalassemia patients.

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DFTD AN INFECTIOUS PARASITIC CANCER

Terry Pinfold, Greg Woods, Alexandre Kriess, Gabriella Brown
Menzies Research Institute Tasmania

Devil Facial Tumor Disease (DFTD) is a truly infectious cancer. It is not caused by a viral or bacterial infection or carcinogens in the environment. It is a parasitic clonal cell line transmitted between hosts through the natural biting behaviour of Tasmanian devils.

The disease appears limited to Tasmanian devils which is a unique species of carnivorous marsupial endemic to the island state of Tasmania, just south of mainland Australia. It is highly infectious, has a 100% mortality rate, and has decimated the population to less than 20% of previous estimates. This single disease is expected to cause the extinction of the Tasmanian devil in the wild within the next 30 years. We have developed a mouse model to study DFTD and trial immunotherapy and vaccine treatments. Current work includes “devilising” the NOD/SCID mice by partial reconstitution of their immune system with effector cells from Tasmanian devils. Flow cytometry has been used to identify antibodies against surface antigens of viable DFTD cells and quantify cytokine responses to DFTD. However, the optimization of a non-radioactive cytotoxicity assay for DFTD proved challenging. For the first time we have been able to successfully induce protective cytotoxic responses with the Tasmanian devil effector cells. We are exploiting a sensitive flow cytometry based assay to measure the cytotoxic responses being induced by Tasmanian devils lymphocytes. We are optimistic of a future immunotherapy against DFTD. Funding: Australian Research Council Grants and Dr Eric Guiler Tasmanian Devil Research Grants

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QUALIFICATION OF A HUMAN BLOOD PSTAT3 ASSAY FOR ANALYSIS OF CLINICAL SAMPLES: AN INVOLVEMENT OF ITS NUCLEAR TRANSLOCATION IN CD4+ CELLS

Susan Pleasic-Williams1, Ming Zhu2, David Wunderlich1, Jaime Masferrer2, Fei Hua2, John Cheng1
1Pfizer, Inc 2Pfizer, Inc

The JAK3 signaling pathway transmits information from IL21 receptors in lymphocytes. Measurements of JAK3 phosphoryation (pSTAT) in these cells may serve as a pharmacodynamic endpoint for assessing IL21R activation and blockage. Using the FACSCanto-II and ImageStream cytometer (ISC), we developed IL21 mediated pSTAT assays with blood CD4+T cells.Incubation of 10ng/mL of IL21 for 15min at 37°C in human blood triggered a pSTAT response measured by phospho-flow. The FACSCanto assay was used to test the effect of ATR-107, a human IgG 1lmAb against IL21R, in a Phase Ia study to supplement the receptor occupancy assay. In blood samples from healthy volunteers at day 42 after treatment with a single dose of ATR107, the pSTAT response in CD4+T cells was inhibited by >90% in treated subjects (n=4) as compared to placebo (n=3), consistent with IL21 receptor occupancy results. Separately, we also investigated a possibility of nuclear translocation of pSTAT3 using ISC. Analysis of samples by the ISC revealed a high similarity between the image pairs of pSTAT-AF647 and nuclear DAPI dye in the cell, indicating pSTAT translocation upon stimulation. Furthermore, we demonstrated an acceptable intertube precision (CV= 6.9%) and sample stability (<30% decrease post 48hr storage at 4°C) using K2EDTA Vacutainer™. Collectively, we have demonstrated that IL-21 stimulation results in rapid translocation of pSTAT3 into the nucleus ofCD4+T cells; and that the pSTAT assay could be used to detect inhibition of the IL-21 pathway in clinical samples.

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MATRIX METALLOPROTEINASES INDUCER (EMMPRIN/CD147) EXPRESSION ON B CELLS OFHEALTHY DONORS AND CHRONIC LYMPHOID LEUKEMIA

Cecilia M. Rodriguez1, Monica B. Gilardoni2, Dario A. Sastre1, Viviana B. Heller1, Carolina Ramello2, Ana C. Donadío2
1Hospital Nacional de Clinicas 2Facultad de Ciencias Quimicas-UNC

Matrix metalloproteinases (MMPs) play a pivotal role in cell migration and invasion through extracellular matrix. CD147, a cell surface glycoprotein, stimulates production of MMPs on tumor and stromal cells. In this study, we analyzed theCD147 and MMP9 expression in B lymphocytes of healthy donors (HD B cell) and neoplastic B lymphocytes (B-CLL) of patients with Chronic Lymphocytic Leukemia (CLL) and their regulation through B-CLL-fibroblasts (Fb) contact(coculture) by Flow Cytometry. CD147 median fluorescence intensity (MFI) range in HD B cell (21,6 - 63,7) allowed us to separate CLL in low MFI (19,1 ± 3,2;n=3), middle MFI (42,9 ± 12,5; n=18) and high MFI (77,7 ± 8,3; n=7). CD147expression was significantly higher in high MFI CLL group compared with moderate (p<0.01), low (p<0.05) and HD B cells (p<0.01). Higher CD147expression was associated mainly with CD19+/CD5+highfraction. The MMP-9 concentration in culture supernatants after 72 hours of coculture was heterogeneous, exhibiting higher levels than B-CLL cultured alone in 3/5 patients. Our results showed an increase in CD147 expression and MMP-9levels in B-CLL induced by Fb contact. These findings could suggest the role ofCD147 in the promotion of B-cells movement and migration towards tissues.

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CLINICAL RELEVANCE OF THROMBOSPONDIN RECEPTOR (CD36) EXPRESSION IN EGYPTIAN DE NOVO ADULT ACUTE MYELOID LEUKEMIA

Dalia Salem1, Sherine Abd El-Aziz1, Manal Salah-Eldin2
1Clinical Pathology Department, Oncology Center, Mansoura Faculty of Medicine 2Medical Oncology Department, Oncology center, Mansoura Faculty of Medicine

Acute myeloid leukemias (AML) is a heterogeneous group of disorders which often present with different morphological, immunophenotypic and cytogenetic patterns. Identification of these characteristics may be useful for a better prognostic evaluation and for a more appropriate therapeutic approach.CD36 is a transmembrane, highly glycosylated, glycoprotein commonly expressed on blasts in acute monocytic leukemia, megakaryoblastic leukemia, and erythroleukemia. In this study, we evaluated CD36 surface expression in 97 newly diagnosed Egyptian AML patients, and results were correlated with morphology, immunophenotype, cytogenetic pattern and clinical outcome. CD36 antigen was recorded in 48 out of 97 cases (49.5%) and particularly in those with M5 and M6 FAB subtypes. Moreover, CD36 expression was significantly associated with expression of CD11b (p = 0.001) and CD14 (p = 0.0001), unfavorable cytogenetic abnormalities (p = 0.001), shorter overall survival (p > 0.0001) and leukemia free survival (p = 0.03).On the basis of the study results, it can be concluded that CD36 expression in AML patients may identify a subgroup with poor prognosis, and thus may be a valuable adjunct to be added to the current prognostic factors.1

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THE EFFECT OF DIFFERENT ANTICOAGULANTS ON SPECIMEN STABILITY AND LEUKOCYTE SUBSETS

Margo I. Santiago, Maryalice Stetler-Stevenson, Constance Yuan, Prashant Ramesh
NCI

Introduction: A single cell suspension is required for flow cytometry (FC). For this reason clotting must be prevented in blood or bone marrow via anticoagulant. Cells must also be viable or non-specific binding of antibodies will occur. Ethylene-diaminetetraacetic acid (EDTA) and sodium heparin are commonly used anticoagulants for both blood and bone marrow. Debate still remains about which anticoagulants produce the most viable results when processed immediately, and those processed in 24 hours after collection. In this study we compare FC analysis of split EDTA and sodium heparin anticoagulated blood processed immediately and 24 hours after collection

Methods: Both EDTA and sodium heparin anticoagulated blood were collected from normal donors at the same time point. Half of each was processed immediately and the remaining half held at room temperature overnight. NH4Cl red cell lysis was performed and both viability analyzed (using 7AAD staining) and T, B and NK cell subsets quantified by FC.

Results: Although viability was comparable when staining was performed same day, there was a significant decrease in viability in EDTA compared to heparin anti-coagulated blood at 24 hours.

Conclusion: Heparin anti-coagulant is preferable to EDTA in specimens not being immediately processed for FC

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FLUOROCHROME CHOICE INFLUENCES ESCAPEE FORMATION IN HIGH DIMENSIONAL FLOW CYTOMETRY

Richard D. Schretzenmair, Andrew D. Bantly, Lifeng Zhang, Amy Steinmetz, Jonni S. Moore
University of Pennsylvania

Introduction: The apparent loss of mononuclear populations during staining with fluorescent antibodies, the so-called lymphocyte escapee phenomenon, was noted as early as 1994. The effect was most evident with FITC in ammonium chloride lysed whole blood. Although there has been a large expansion of fluorescent probes, it has not been determined if the escapee phenomenon occurs with the new fluorochromes and if it is restricted to certain subsets. This information is critical to panel design and to accurate measures of individual leukocyte subsets.

Materials and Methods: Pre-lysed whole blood was stained with CD3 antibodies conjugated to more than 20 different fluorochromes and analyzed on a BD FACSCanto with 11-color upgrade or a BC Gallios. Events were analyzed using a pan-Leukocyte gate, excluding only debris and CD3 vs. SSC plots were generated. Escapees were classified as events with CD3 intensity similar to T cells (CD3+ low SSC) which had SSC properties more appropriate to that of Granulocytes or Monocytes. In addition several other pan-T cell, T cell subset and B cell markers were tested.

Results: Escapee formation is evident with multiple probes and is dependent upon the fluorochrome tag and not on the antibody clone. Large difference between donors was noted, some having virtually no escapees while others with substantial numbers.

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IMMUNOPHENOTYPICAL FEATURES OF ACUTE MYELOID LEUKEMIA CELLS IN ASSOCIATION WITH ITD MUTATION IN GENE FLT3.

Konstantin Slobodnyuk, Margarita Gorchakova, Margarita Ivanchenko, Irina Fridlin, Mikhail Zaraiski, Yekaterina Zueva
State Pavlov Medical University

Background: Immunophenotypical and genetic alterations in acute myeloid leukemia have been demonstrated to be important prognostic factors for the stratification of the patients in the different risk groups. There are some controversial data concerns on concordance immunophenotypical pattern of leukemic cells in AML patients with internal tandem duplication (ITD) in gene FLT3. Objective: By using 5-colour flow cytometry and PCR with detection in gel electrophoresis, we have studied the association of immunophenotypical features of leukemic cells in AML with the mutation FLT3-ITD.

Methods: We have studied leukemic cells of bone marrow from patients with AML (n=45) and healthy controls (n=13). For surface labeling antigens on blast cells we used monoclonal antibodies: CD 2, 3, 4, 5, 7, 11b, 13, 14, 15, 16, 19, 20, 22, 33, 34, 36, 38, 41a, 56, 64, 71, 117, 184, HLA-DR (Beckman Coulter), CD133 (Miltenyi Biotech). Cells were labeled according to the standard procedures of manufacturer instructions. For PCR amplification we used primers FLT3-ITD (forward): 5′-TCTGCAGAACTGCCTATTCCT-3′; FLT3-ITD (reverse): 5′-CTTTCAGCATTTTGACGGCAA-3′. For the gel of electrophoresis we used 3% polyacrylamide.

Results: Mutation FLT3-ITD was detected in 13 cases out of 45 (29%). We found significant difference (p<0,05) in the number of leukemic blasts in the bone marrow that were increased in the patients with the mutation FLT3-ITD at initial diagnosis. Acute myeloid cells with the mutation FLT3-ITD showed a significant increase in the surface expression of antigens CD7 and CD13 respectively. Kaplan-Meier analysis showed significant decrease in the overall survival in patients with either the mutation FLT3-ITD or the surface expression of CD7.

Conclusions: Mutation FLT3-ITD associates with the CD7 expression and both can be considered as the markers of poor prognosis in the overall survival in patients with AML. The authors declare that they have no competing interests.

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THE FREQUENCY OFLEUKEMIA-ASSOCIATED PHENOTYPE (LAP) IN PATIENTS WITH ACUTE MYELOID LEUKEMIA.

Konstantin Slobodnyuk, Margarita Ivanchenko, Margarita Gorchakova, Maria Estrina, Ekaterina Rusanova, YekaterinaZueva
State Pavlov Medical University

Background: Immunophenotyping is of paramount importance for the detection of the independent prognostic factors in acute myeloid leukemia (AML). The aim of our study was to determine the frequency of different immunophenotypes in patients with AML.

Methods: Bone marrow (BM; n=45) samples from patients with acute myeloid leukemia, and BM samples from 13 healthy donors, were stained with a five-color fluorescent monoclonal antibody (MAb) cocktails. We used following MAb panel with CD45 ECD combined in each tube with FITC, PE, PC5, and PC7 conjugated MAbs: CD5/CD10/CD34/CD19, CD15/CD33/CD34/CD117, CD34/CD33/CD7/CD117, CD14/CD13/HLA-DR/CD184, CD64/CD11b/CD16/CD2, CD36/CD71/CD33/CD61, CD4/CD8/CD3/CD56, CD22/CD133/CD25/ CD20.

For the detection of blast cells we used gating strategy CD45/SS. The data was acquired on BC FC500 cytometer and analyzed by CXP Analysis software™.

Results: We detected LAP in 41 of 45 cases (91%). Two or more LAP types were detected in 37 patients (83%). The asynchronous antigen expression was the most frequent type of abnormal phenotype profile of cells whereas the cross-lineage antigen expression was the less one (14 cases, 31%). The absence of the CD13 antigen expression was prevalent among myeloid antigens, and the most commonplace cross-lineage antigen was CD7.

Conclusions: The presented results demonstrate that leukemia-associated immunophenotype can be detected virtually in all cases (91%) prior to the diagnosis of acute myeloid leukemia. The most frequent type of abnormal phenotype is asynchronous antigen expression. The authors declare that they have no competing interests.

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QUANTIFICATION OF EXPRESSION OF ANTIGENS TARGETED BY ANTIBODY BASED THERAPY IN CHRONIC LYMPHOCYTIC LEUKEMIA

Prashant R. Tembhare1, Gerald Marti2, Adrian Wiestner3, Heba Degheidy2, Mohammed Farooqui3, Robert J. Kreitman4, Gregory A. Jasper1, Constance M. Yuan1, David Liewehr5, David Venzon5, Maryalice Stetler-Stevenson1
1Flow Cytometry Unit, Laboratory of Pathology Center for Cancer Research, NCI, NIH 2Center for Biologics Evaluation and Research, FDA, 3NHLBI, NIH 4Laboratory of Molecular Biology Center for Cancer Research, NCI, NIH 5Biostatistics and Data Management Section, Center for Cancer Research, NCI, NIH

Introduction: Chronic lymphocytic leukemia (CLL)is an indolent chronic lymphoproliferative disorder, with the majority of patients eventually requiring therapeutic intervention. Anti-CD20 (Rituximab), anti-CD52 (Alemtuzumab), anti-CD22 (BL22, HA22) and anti-CD25 (Oncotac) are mainstay therapeutic options or pre-clinical options under development for the treatment of CLL. Previous studies suggest that the level of cell surface antigen expression may affect response to monoclonal antibody-based therapy. In this study, we determine the antigen densities of these therapeutically targeted antigens in CLL cells.

Methods: Using the flow cytometric “Quantibrite” method (BD) to determine antibody binding capacity per cell (ABC), we quantified the surface antigen densities of CD20, CD22, CD25 and CD52 in CLL cells from 28 untreated CLL patients.

Results: The CLL cells in all cases expressed CD20, CD22 andCD52 but 4 cases (14%) were negative for CD25.The level of antigen density, in decreasing order, was CD52 > CD20 > CD22 > CD25. However, the highest CD20,CD22, CD25 and CD52 ABC values were observed in different cases, indicating that the level of targeted therapeutic antigen expression cannot be accurately predicted.

Conclusion: Response to therapy with these agents is highly variable among CLL patients; therefore, quantitation of potential therapeutically targeted antigens may provide a systematic approach in providing optimal individualized therapy in CLL. Antigen quantitation may also be informative in evaluating new antigens as potential therapeutic targets.

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ALVEOLAR RHABDOMYOSARCOMA MIMICKING AN ACUTE LEUKEMIA: A CASE REPORT.

Lucía Veiga, Adriana Arocena, MaríaBrasesco, Noelia García, Ana I. Landoni, Claudia Sejumil, Rossana Bonomi, Carina Di Matteo, Hugo Giordano
Specialized Techniques Laboratory, Asociación Española

Rhabdomyosarcoma (RMS) is the most common pediatric malignant soft tissue tumor. It usually presents as solid mass and the most common locations are head and neck, trunk, genitourinary tract and extremities. RMS represents around 5% of all malignancies among children and adolescents. There are two main types of rhabdomyosarcomas: embryonal rhabdomyosarcoma (ERMS), the most common, and alveolar rhabdomyosarcoma (ARMS). It is known that bone marrow infiltration may occur. We report a case of a 20 year-old man, with a 3 months history of asthenia, anemia and low back pain. Peripheral blood count showed hemoglobin concentration 6.5 g/dL, platelet count 39.000/mm3 and white blood cell count 8.900/ mm3. Bone marrow (BM) smears showed an infiltration of small round/oval cells containing one or two nucleus and scant cytoplasm which led to the suspicion of acute leukemia. Flow cytometric analysis of BM and peripheral blood (PB) showed abnormal cells CD56+ CD45-, 38.6% in BM and 3.2% in PB, without other hematopoietic markers. These results excluded the diagnosis of leukemia and suggested an extra hematologic neoplasm. The morphology and immunostaining patterns of BM biopsy revealed the diagnosis of ARMS metastasis. Cytogenetic analysis in BM showed t(2;13) (q35q14), that is present in around 60% of ARMS cases. It is important to do the differential diagnostic between acute leukemias and solid tumors with BM infiltration. Flow cytometry (FCM) had a key role in excluding important diagnostics, quantifying the grade of infiltration and orienting therapeutic steps.

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DETECTION OF EXTRAMEDULLARY (EXCEPT CSF) INVOLVEMENT OF HEMATOPOIETIC NEOPLASMS USING FLOW CYTOMETRY

Hui Wang1, Chunrong Tong1, Ping Wu1, Rui Kang2, Junyi Zhen1
1Ludaopei Hematology & Oncology Center 2Beijing Daopei Hospital

Objective: To study the value of flow cytometry in identifying extramedullary [except Cerebro-Spinal Fluid(CSF)] involvement of acute leukemia. Methods: In the patients who were diagnosed and treated in our hospital from Oct. 2008 to Oct. 2011, 52 cases were found extramedullary lump or effusion. We took their extramedullary samples to do 4 color flow cytometry detection. They included 8 lymph node samples, 31 other tissue samples, 13 effusion samples. There were 7 B cell acute lymphoblastic leukemia(ALL) cases,12 T cell ALL cases, 33 Acute Myeloid Leukemia(AML) cases. Results: Malignant cells were found in 38 samples, and 14 samples were negative. The median percentage of tumor cells were 35.95%(0.38%∼97.87%). After median following up 21 months(3 months to 3 years), all the cases could be evaluated in negative group. Except one continuously had leukemic cells in BM, others kept complete remission. 27 cases could be evaluated in positive group, of which 16 cases were refractory or relapsed of BM. 11 cases were in event-free survival (EFS). Comparing with negative group, positive group had a poor prognosis(P<0.01). CD56 positive AML was prone to extramedullary involvement (P<0.02), and might have a poor prognosis. Conclusion: Flow cytometry could effectively detect extramedullary (except CSF) involvement of hematopoietic neoplasms. The detection result had a significant prognosis value.

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STANDARDISING LEUCOCYTE PNH CLONE DETECTION: AN INTERNATIONAL STUDY

Liam Whitby1, Matthew Fletcher1, Stephen Richards2, Michael Borowitz3, Michael Keeney4, Andrea Illingworth5, Erica Acton6, Robert Sutherland6, David Barnett1
1UK NEQAS for Leucocyte Immunophenotyping 2HMDS, St James University Hospital 3Department of Pathology and Oncology, Johns Hopkins Medical Institution 4Hematology/Flow Cytometry, London Health Sciences Centre 5Flow Cytometry Laboratory, Dahl-Chase Diagnostic Services 6Dept of Lab Hematology, University Health Network. International PNH Flow Cytometry Study Group

PNH, a rare acquired stem cell disorder, is characterized by loss of GPI-linked surface structures and consequent increased sensitivity of red blood cells to complement-mediated lysis. A high risk of thrombosis means accurate diagnosis is essential for appropriate treatment. Flow cytometry is the method-of-choice to detect/quantify PNH RBC and WBC clone size. The UK NEQAS Quality Assessment Programme has identified large variance in testing protocols and reagent selection used in PNH leucocyte clone detection. To study the effect of a standardised protocol and reagent choice compared to 'in-house' methods for PNH leucocyte clone detection, 3 stabilised blood samples manufactured to contain zero, ∼8% and ∼0.1% PNH leucocyte clone populations were distributed to 19 international centres experienced in PNH analysis, together with a standardised protocol and standardised antibody cocktails: FLAER/CD24/CD15/CD45 for neutrophils; and FLAER/CD14/CD64/CD45 for monocytes (optimized for both Beckman Coulter and BD Biosciences platforms).Whilst overall medians between the two approaches for leucocyte PNH clone detection were similar, the standardised approach had lower variation around the median compared to “in-house” protocols (Table 1). Our results highlight the importance of reagent choice and a standardised approach in performing PNH analysis even among experienced laboratories; additional studies are planned in which the standardised method will be compared to in house methods among labs with less experience to evaluate further the potential benefits of standardization. This study was supported by Alexion Pharmaceuticals, BD Biosciences, Beckman Coulter and eBioscience.

Table 1. 
 Classical PNH SampleHigh Resolution PNH Sample
 Neutrophils Monocytes NeutrophilsMonocytes  
 In-houseStandardisedIn-houseStandardisedIn-houseStandardisedIn-houseStandardised
Median8.18.058.88.570.10.090.110.13
Range2.531.694.573.950.370.150.260.20
Interquartile Range1.290.481.530.910.030.020.080.08

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PRECURSOR B-CELL LYMPHOBLASTIC LEUKEMIA WITH SURFACE IMMUNOGLOBULIN LIGHT CHAIN RESTRICTION: 2 CASES REPORT

Ya-ping Zhai1, Yan-hui Liu1, Yin Zhang1, Wei Cheng1, Yu-long Li1
1The Institute of Hematology, Henan Provincial People's Hospital 2The Institute of Hematology, Henan Provincial People's Hospital 3The Institute of Hematology, Henan Provincial People's Hospital 4The Institute of Hematology,Henan Provincial People's Hospital 5The Institute of Hematology, Henan Provincial People's Hospital

Introduction: B-cell acute lymphoblastic leukemia (ALL) can be classified into two major subtypes: precursor B (pB)-ALL and mature B-cell ALL. Mature B ALLs are typically TdT negative and sIg positive, while pB-ALL characteristically express TdT and are sIg negative. Therefore, pB- ALL with sIg light chain restriction is extremely rare.

Methods: Immunophenotic and cytogenetic features of the leukemic blast cells were reviewed.

Results: The 2 patients (1 male adult aged 22 years and 1infant aged 18 months) presented with non-specific symptoms. The leukemic blast cells in both cases showed characteristic of FAB L2 lymphoblasts and co-expressed CD34/CD19/CD10/CD22/cCD79a/CD9/HLA-DR/CD38/ TdT / CD123/CD13 (partial) and unexpected single surface immunoglobulin lambda (sIg lambda) light chains restriction by flow cytometry. As expected, they were negative for surface immunoglobulin kappa (sIg kappa) light chains, CD7, CD5, CD117, CD33, CD56, CD23, FMC7, MPO. In addition, the leukemic blast cells from the infant patient were positive for CD20 while the lymphoblasts from the adult patient were negative for CD20. Cytogenetic analysis revealed the adult with t(9;22)(q34;q11) and the infant with normal karyotype. Karyotypic analysis in both cases was negative for 8q24(myc) translocation. Both cases were diagnosed and managed as pB ALL and the patients had good response to therapy for pB ALL, the patient with t(9;22)(q34;q11) also received imatinib therapy.

Conclusion: sIg light chain restriction does not necessarily represent a mature B-cell ALL phenotype. As different treatment regimens are used separately for pB ALL and mature B ALL, attention must be paid to such a rare ALL for avoiding inadequate therapy or overtreatment.