Leukoreduction Decreases Alloimmunogenicity of Transfused Murine HOD RBCs.

Abstract Abstract 640 Introduction: Alloimmunization against transfused red blood cell (RBC) antigens can lead to hemolytic transfusion reactions as well as difficulties in locating compatible units of blood for future transfusions. Despite the clinical significance, factors influencing rates of RBC alloimmunization are not clearly defined. Leukoreduction decreases rates of HLA alloimmunization, because HLA antigens are carried on white blood cells (WBCs). However, most human RBC antigens are not carried on WBCs, and the effect of leukoreduction on RBC alloimmunization is not known. Many potential variables, including the large number of RBC antigenic differences between donor and recipient, make this question difficult to answer in the setting of human transfusion medicine. We hypothesized that WBCs in transfused RBC units would increase alloimmunization to an RBC specific antigen, and used a reductionist murine model to investigate this issue. Materials and Methods: Transgenic HOD donors, with RBC specific expression of the model antigen hen egg lysozyme (HEL) linked to a multi-pass human Duffy antigen (Fyb), were bled into CPDA-1 to give a final storage medium concentration of 14%. Leukoreduction was performed over a Pall neonatal leukoreduction filter, with residual WBCs quantified by propridium iodide and Trucount beads. Expression of HEL and Fyb on HOD RBCs was assessed by flow cytometry prior to transfusion. Leukoreduced or non-leukoreduced pRBCs (75 μL) were transfused via lateral tail vein, and post-transfusion survival of HOD RBCs was determined by analysis of HEL and Fybexpression at 10 and 30 minutes, 2 and 24 hours following transfusion. Alloimmunization was assessed 2 weeks later by HEL-specific ELISA and flow cytometric crossmatching (using HOD or control FVB RBCs). Statistical significance (p<0.05) was determined using PRISM software and a two-way ANOVA with a Bonferroni post-test. Results: Alloantibodies against HOD RBCs were detectable by HEL-specific ELISA in recipients following a single transfusion of non-leukoreduced or leukoreduced RBCs. In 4 of 5 experiments (n=50 mice total), non-leukoreduced HOD RBCs were statistically significantly more immunogenic than leukoreduced HOD RBCs (sera diluted 1:50). Alloantibodies against HOD RBCs were detected in a minority of mice by flow cytometric crossmatching, which is less sensitive than ELISA. By histogram overlay of sera crossmatched with HOD RBCs compared to control FVB RBCs, 6/25 recipients of non-leukoreduced RBCs and 2/25 recipients of leukoreduced RBCs had a detectable anti-HOD response. In each experiment, there was a >3 log10 decrease in leukocytes after leukoreduction. HEL and Fyb expression was comparable as determined by flow cytometry in samples pre- and post-leukoreduction. Mean 24 hour post-transfusion survival was 98.7% (95% CI 97-100%) and 97.9% (95% CI 95.4-100%) in recipients of non-leukoreduced and leukoreduced RBCs, respectively. Conclusions: Leukoreduction of murine HOD RBCs decreases alloimmunogenicity of the RBC specific HEL antigen. This decrease is subtle and is better detected by HEL-specific ELISA than by less sensitive flow cytometric crossmatching. Although one explanation for these findings is that the WBCs themselves are contributing to the increased immunogenicity by providing a danger signal, it is also possible that non-specific alloreactivity of transfused WBCs or other cells is playing a role. Furthermore, it cannot be ruled out that changes in platelets or RBCs induced by the leukoreduction filter are involved. However, the observed changes in immunogenicity cannot be explained by a decrease in HEL expression post-leukoreduction or by altered clearance of leukroreduced RBCs after transfusion, as HEL and Fyb expression, as well as 24 hour post-transfusion survival of HOD RBCs, were similar in both groups. If the immunogenicity of other RBC antigens is also decreased after leukoreduction, then patients at highest risk of RBC alloimmunization may benefit from receiving solely leukoreduced blood products. Furthermore, the development of higher efficiency and/or modified leukoreduction filters may be warranted. Disclosures: No relevant conflicts of interest to declare.

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Development of a Murine Model of Weak Kel: Similarities to Weak Rh(D)

Blood ◽

10.1182/blood.v120.21.842.842 ◽

2012 ◽

Vol 120 (21) ◽

pp. 842-842 ◽

Cited By ~ 1

Author(s):

Jeanne E. Hendrickson ◽

Nicole H. Smith ◽

Kathryn R. Girard-Pierce ◽

Christopher A Tormey ◽

Kate L. Henry ◽

...

Keyword(s):

Flow Cytometry ◽

Immune Response ◽

Copy Number ◽

Conflicts Of Interest ◽

Antigen Expression ◽

Antibody Responses ◽

Poly I:C ◽

Specific Expression ◽

Flow Cytometric ◽

Pre Treatment

Abstract Abstract 842 Introduction: Aspects of RBC antigens that define their immunogenicity are not fully understood. Multiple studies in humans have demonstrated Rh(D) to be the most immunogenic RBC antigen, yet the same antigen present in lower copy number (weak Rh(D)) is, with rare exception, considered non-immunogenic. A better understanding of factors influencing RBC alloimmunogenicity would be helpful in predicting antibody responses in transfusion and pregnancy situations alike, as well as in managing donor RBC inventory. Herein, we describe the generation of transgenic mice with different levels of RBC specific expression of the clinically significant human KEL2 antigen, and test the hypothesis that antigen density impacts recipient immune response post-transfusion. Materials and Methods: Mice with RBC specific expression of KEL2 were generated utilizing constructs containing the human KEL2 sequence expressed behind a B-globin promoter, using a random integration approach. TER119+, CD45+, and CD41+ cells were evaluated by flow cytometry for KEL expression using monoclonal anti-Jsa and anti-Kpb, and RBC antigen density was estimated utilizing QIFIKIT beads. MuMT recipients were transfused with RBCs labeled with a lipophilic dye, and post-transfusion RBC recovery and antigen expression were evaluated by flow cytometry. To determine the immunogenicity of KEL2 or weak KEL2 RBCs, RBCs were transfused into C57BL/6 recipients every 2–3 weeks in the presence or absence of poly (I:C) pre-treatment. Recipient serum was analyzed by flow cytometric crossmatch with KEL2 or C57BL/6 RBC targets, using IgM or IgG secondary antibodies. To determine the effect of recipient RBC expression of weak KEL2 on the immunogenicity of KEL2 RBCs, weak KEL2 animals were transfused with KEL2 RBCs, the clearance of lipophilic labeled RBCs was tracked, and anti-KEL was evaluated on the transfused RBCs and also in the serum. Results: KEL2 RBCs have approximately 1200 antigenic sites per cell, whereas weak KEL2 RBCs have fewer than 200 sites; flow cytometric studies of TER 119+, CD45+, and CD41+ cells suggest both strains have RBC specific KEL expression. Transfusion of KEL2 or weak KEL2 RBCs into muMT animals resulted in stable post-transfusion RBC recovery and antigen expression. In 3/3 experiments (n=30 animals), all C57BL/6 recipients of KEL2 RBCs generated detectable anti-KEL IgM and IgG, which boosted with subsequent transfusions and which was enhanced in the presence of recipient inflammation with poly (I:C). However, in 2/2 experiments (n=20 animals), weak KEL RBCs led to no detectable antibody (IgM or IgG) in C57BL/6 recipients following 3 transfusions, even in the presence of recipient pre-treatment with poly (I:C). Furthermore, weak KEL2 recipients of KEL2 RBCs generated no detectable IgG and demonstrated no clearance of KEL2 RBCs, though low levels of anti-KEL IgM were detectable on the transfused RBCs and in the serum from approximately 5–12 days post-transfusion. Discussion: As hypothesized, antigen density significantly impacts the immunogenicity of KEL RBCs in this reductionist murine alloimmunization model. C57BL/6 recipients, like Rh(D) negative recipients, lack the human antigen in question (KEL2 in this case), and recipient antibody responses to weak KEL2 are, like most recipient responses to weak Rh(D), undetectable. Strengths of the KEL2 and weak KEL2 system include the fact that these animals are, to the best of our knowledge, genetically identical except for RBC KEL antigen copy number. Thus, this system allows for detailed analyses of the immune response to KEL RBCs with different antigen densities, on both the donor and recipient side of the equation, without confounding factors encountered in human studies (such as HLA presentation issues or considerations of the molecular basis of a particular type of weak Rh(D)). A better understanding of primary, secondary, and other immune responses in the KEL2 and weak KEL2 system may lay the groundwork for strategies to induce non-responsiveness to RBCs in humans, not only in the setting of transfusion medicine but also potentially in the setting of hemolytic disease of the fetus and newborn. Furthermore, translation of these and future findings in the KEL and weak KEL systems to Rh(D), weak Rh(D), and other human antigen systems may ultimately allow for creative solutions in blood inventory management. Disclosures: No relevant conflicts of interest to declare.

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A New Algorithm and Panel Construction for Pediatric Leukemia Immunophenotyping Using 10-Color Flow Cytometry

Blood ◽

10.1182/blood.v120.21.4799.4799 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4799-4799

Author(s):

Bettina Keller ◽

Markus P Radsak ◽

Joerg Faber ◽

Alexandra Russo

Keyword(s):

Flow Cytometry ◽

Childhood Leukemia ◽

Conflicts Of Interest ◽

High Reliability ◽

Maturation Stage ◽

Specific Information ◽

Color Flow ◽

Pediatric Leukemia ◽

Flow Cytometric

Abstract Abstract 4799 Background: Rapid identification and quantification of abnormal cell populations in minimal specimen are crucial for diagnosis and longitudinal minimal residual disease (MRD) testing of childhood leukemia. So far, most standard immunophenotypic analyses are performed using antibody panels with up to five-colors and require high cell numbers. For infant and pediatric specimen, high-level multicolor analyses is highly desirable to gather sufficient data for initial diagnostic and follow up monitoring of pathologic populations. Objective: In this study, we aimed to establish a newly defined pediatric multicolor flow cytometric panel algorithm with high reliability yet minimal specimen requirement. Results: We defined a 10-color flow cytometric panel using the new violet laser dye “KromeOrange (KO)”. Applying CD45-KO/Side Scatter gating, combined with 2 additional backbone markers the panel is designed in two consecutive steps. In the first step, a single standardized 10-color-“screening tube” (FITC-HLA-DR, PE-CD15/CD56, ECD-CD5, PC5.5-CD33, PC7-CD13, APC-CD117, APC A700-CD34, APC A750-CD19, PB-CD3, KrO-CD45) is applied for initial orientation of specific lineage assignment. Based on results obtained with the screening tube, a specific multi-tube “classification panel” is used to complete detailed characterization of lineage specific malignancy and maturation stage. Suitable specimens include fresh blood, bone marrow and all body fluids. All samples are stained directly with monoclonal antibodies, followed by the lyses of erythrocytes and a short wash. Compared to standard five color panel previously used the application of greater numbers of informative antibodies in the screening tube and in the 2ndstep muti-tube classification panel is cost and time efficient and results in a more precise characterization of any single event. Conclusion: Our panel construction and algorithm definition for infant and pediatric leukemia immunophenotyping is one of the first 10-color flow cytometry panels described for this application. Advantages are the possibility to obtain highly specific information from minimal specimens with significantly improved laboratory efficiency. The overall performance is currently tested in a routine clinical setting. Disclosures: No relevant conflicts of interest to declare.

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Impaired Precursor B-Cell Maturation/Production In The Bone Marrow: Association With Molecular and Immunophenotypic Markers Of Myeloid Dysplasia In Suspected Low-Grade MDS

Blood ◽

10.1182/blood.v122.21.4943.4943 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4943-4943

Author(s):

Charles Repetti ◽

Hsueh-Hua Chen ◽

Yongbao Wang ◽

Vanessa A Jones ◽

Albert K Ho ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Somatic Mutation ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Sequence Variant ◽

Low Grade ◽

Precursor B Cells

Abstract Rationale Myelodysplastic syndromes (MDS) are clonal stem cell disorders that disrupt orderly maturation of multiple hematopoietic lineages. Several studies have suggested that maturation of precursor B cells (hematogones) is also abnormal in MDS. As a result, the presence of normal numbers or increased precursor B cells in bone marrow (BM) is frequently used as a diagnostic feature arguing against a diagnosis of MDS. We compared the presence of myeloid-associated gene mutations and myeloid maturation abnormalities with qualitative and quantitative precursor B cell findings in BM samples submitted for workup of cytopenias or MDS. Methods Seventeen BM aspirate samples with <5% blasts submitted for cytopenia or MDS evaluation were compared with 10 samples having 5% or more blasts and changes diagnostic of MDS or AML. Mutation analysis was performed on genomic DNA using a targeted exome sequencing assay. This assay employs a TruSeq custom amplicon design on the MiSeq platform (Illumina, San Diego, CA). The assay covers the commonly mutated areas of 19 myeloid-associated genes. Somatic mutation status was assigned based on mutation levels, previous association with myeloid neoplasia, and no prior identification in public or internal databases as a normal sequence variant. Flow cytometry using 6-color (CD19/CD34) and 8-color (CD19/10) formats was used to assess lymphoblasts; CD34/13 was used to assess myeloblasts; and CD11b, CD13, CD16, and CD38 were used to assess abnormalities in myelopoiesis. Results Among the 17 BM samples submitted for cytopenia or MDS evaluation that had <5% blasts, 7 (41%) had immunophenotypic myeloid maturation abnormalities. Ten (59%) of the 17 cases had at least one myeloid-associated somatic mutation, with TET2 and ASXL1being the most commonly mutated genes. The ratio of myeloblasts to B-lymphoblasts, calculated using either CD10 or CD19, was >10:1 in 10/17 (59%) cases. Nine of the 17 (53%) cases had virtually no precursor B cells detected. Discrete abnormalities in more mature myeloid forms were seen in 7/10 (70%) cases with low numbers of B-lymphoblasts but in none of the 7 cases with significant numbers of B-lymphoblasts. MDS-associated mutations were more common in cases with rare B-lymphoblasts (7/9) than in those with higher percentages of precursor B cells (3/8), but the difference did not reach statistical significance (P = 0.15). Genes mutated in the group with B-lymphoblasts present included ASXL1 (3 cases), DNMT3A (2), TET2 (1) and TP53 (2). Two of these mutated cases presented with isolated thrombocytopenia. By comparison, myeloblast/lymphoblast ratios were >50:1 in all 10 unequivocal MDS/AML samples (>5% blasts); 8 (80%) of these cases had MDS-associated mutations, and 4 (50%) had mutations in multiple genes. Conclusions Decreases in BM precursor B cells in cases of possible low-grade MDS were usually, but not always, associated with the presence of MDS-associated mutations. However, cases with normal or increased precursor B cell numbers also showed MDS-associated mutations although immunophenotypic evidence of myeloid maturation abnormalities was not seen in this group. The identification of a subgroup of cytopenic patients with likely pathogenic mutations in bone marrow precursors but minimal phenotypic evidence of myeloid dysplasia may indicate clonal abnormalities primarily located outside the granulocyte or common stem precursor populations, e.g. restricted to the megakaryocytic lineage. Therefore, the presence of intact precursor lymphoblast and myeloid maturation by higher-dimensional flow cytometry as a primary criterion to argue against a diagnosis of low-grade MDS needs further evaluation, especially when granulocytopenia is absent. Disclosures: No relevant conflicts of interest to declare.

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Clinical Impact of CD8+ Cells in Hodgkin Lymphoma Lymphadenopaties. Contribution of Flow Cytometry

Blood ◽

10.1182/blood.v126.23.1442.1442 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1442-1442

Author(s):

Sara Alonso ◽

Maria Belen Vidriales ◽

Maria Dolores Caballero ◽

Oscar Blanco ◽

J Galende ◽

...

Keyword(s):

Flow Cytometry ◽

Cox Regression ◽

Advanced Disease ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Definitive Diagnosis ◽

University Hospital ◽

Cd8 Cells ◽

Risk Patients ◽

Conventional Histology

Abstract CONTEXT: Microenvironment of Hodgkin and Reed Sternberg(H-RS) cells is a current focus of interest to define risk and predict evolution of Hodgkin′s Lymphoma. OBJECTIVES: To determine the role of infiltrating CD8+ cells, using flow cytometry (FCM), in the evolution of patients with HL. DESIGN: Cell suspensions obtained after mechanical disintegration of ganglion biopsies of patients with newly diagnosed HL were analyzed using FCM. Definitive diagnosis was made by conventional histology and immunohistochemistry. Clinical data were collected from medical records. Statistical analysis was performed using SPSS 20.0 software. SETTING: In the University Hospital of Salamanca, we consecutively analyzed by FCM all lymph nodes with suspected lymphoma at diagnosis. There was no selection bias when collecting patients, except for some cases with inadequate quality(insufficient cells). PATIENTS OR OTHER PARTICIPANTS: From 1996(earliest available FCM data) to 2014, 104 of that samples had a definitive diagnosis of HL. Treatment depended on stage: a)early diagnosis received ABVD(x3) & local radiotherapy(RT) (20-30 Gy), b)advanced: ABVD(x6 to 8), plus RT in selected cases. Median follow-up was 10 years. INTERVENTIONS :This was a retrospective observational study. MAIN OUTCOMES MEASURES: Primary end points were overall survival(OS) and freedom from treatment failure(FFTF), considered from diagnosis to progression or relapse. RESULTS: Most cells obtained were lymphocytes (Median17.1±86.9%) with a T/B/NK distribution of 72%/27%/1.6%(median) and predominance of CD4+ (median CD4:51%-CD8:12%). Median CD8+ cells(12%) was used to divide patients into 2 groups.FFTF was longer in patients with more than 12% of CD8+cells(93% vs. 71%, p=0.01). When analyzed separately patients with early/advanced disease, the clinical benefit remained in the group with advanced disease (p=0.006), whereas the statistical significance was lost in the group with early disease, possibly due to the excellent prognosis for those patients(FFTF 10 years > 95%). No differences were observed in the OS, because second line therapy was highly effective. In the multivariate analysis using Cox regression, advanced stage (HR=9.6 with 95% CI 1.2 to 73.9) and>12% CD8+ tumor infiltrating T-cells were independent variables(HR=0.26, 95% CI 0.07 to 0.9). CONCLUSIONS: Increased number of CD8+ in the H-RSC microenvironment of HL is associated with better FFTF, particularly in the advanced-disease group. This should be considered as a new biomarker to identify high-risk patients. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Flow Cytometry-Based Maturity Score As a Novel Prognostic Parameter in AML

Blood ◽

10.1182/blood.v124.21.1006.1006 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1006-1006

Author(s):

Anne Flörcken ◽

Schneider Tanja ◽

Anju Singh ◽

Seval Türkmen ◽

Burmeister Thomas ◽

...

Keyword(s):

Clinical Outcome ◽

Conflicts Of Interest ◽

Risk Group ◽

Statistical Significance ◽

Risk Groups ◽

Outcome Analysis ◽

Intermediate Risk ◽

Logrank Test ◽

Maturity Score ◽

Flow Cytometric

Abstract Background: Based on cytogenetic and molecular phenotypes, the European LeukemiaNet (ELN) classification is widely accepted for risk stratification of patients with acute myeloid leukemia (AML). Single leukemic blast cell antigens were also shown to have prognostic value, but the influence of AML blast maturity within the different ELN risk groups is largely unknown. Methods: Our study includes 300 AML patients, who were diagnosed at our institution between 01/2003 and 04/2012 and in whom morphologic, immunophenotypic, cytogenetic and molecular data from primary diagnosis were available. A flow cytometric maturity score was developed in order to distinguish mature AML (AML-ma) from immature AML (AML-im) by means of a quantitative analysis of the expression of early progenitor cell antigens (CD34, CD117, and TdT). Clinical outcome analysis was performed both in the whole cohort and in the different ELN risk groups using Chi Square and Fisher's Exact Test (CR/CRi) and Logrank Test (RFS, OS). Results: A mature AML blast phenotype (AML-ma) was associated with a significantly longer RFS (7.5 vs. 4.3 years) and OS (5.3 vs. 2.7 years) than an immature phenotype (AML-im) (p < 0.001). Low-risk phenotypes according to ELN (NPM1, PML-RARA) significantly accumulated in the AML-ma group, whereas high-risk aberrations (monosomal and complex aberrant karyotypes, -5 or del(5q), -7) had a higher frequency in AML-im. Interestingly, the differences in RFS and OS were maintained in a subgroup analysis within the different ELN risk groups. Statistical significance was obtained in the intermediate risk group with AML-ma being superior to AML-im (RFS: 7.0 years (AML-ma) vs. 3.3 years (AML-im); p = 0.002; OS: 5.1 years (AML-ma) vs. 3.0 years (AML-im); p = 0.022). In contrast, there was no correlation between AML blast maturity and CR/CRi rates. Conclusions: Our novel flow cytometric score easily determines AML blast maturity and can predict clinical outcome, even within the different ELN risk groups. Particularly for intermediate risk patients according to ELN, the score may be useful in post remission therapy decision-making. It remains to be clarified whether these results reflect differences in genetic instability and/or a different frequency of leukemia stem cells in AML-ma and AML-im. Figure 1 Figure 1. Figure 2 Figure 2. RFS (A) and OS (B) of AML patients with mature (AML-ma) and immature (AML-im) blast phenotype within the ELN intermediate risk group. AML-ma show a significantly longer RFS and OS compared to AML-im. Disclosures No relevant conflicts of interest to declare.

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CD166+CD34+ Cells Exhibit Marked Functional Differences During Fetal and Adult Life

Blood ◽

10.1182/blood.v122.21.2435.2435 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2435-2435

Author(s):

Saloomeh Mokhtari ◽

Zanetta S. Lamar ◽

Chris Booth ◽

Frank Marini ◽

Christopher D Porada ◽

...

Keyword(s):

Flow Cytometry ◽

Growth Medium ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

Adult Life ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Cd34 Cells

Abstract ALCAM/CD166 is expressed from the onset of hematopoiesis in the yolk sac and in a variety of hematopoietic tissues throughout ontogeny. Both hematopoietic and stromal cells in the AGM region, fetal liver, and fetal and adult marrow express this molecule. CD166 double knockout mice are viable and fertile, without any major blood defects, but their microenvironment and hematopoietic stem cells (HSC) exhibit deficiencies in their ability to support and engraft long-term, respectively. In order to further study the role of CD166 in hematopoiesis, we characterized, during ontogeny, the origin, function, and sub-populations of CD166+ cells in different hematopoietic organs. To this end, we used flow cytometry, confocal microscopy, and colony-forming assays to analyze human fetal liver (FL) at 18 and 20 gestational weeks (gw), bone marrow (BM) from 10 to 20gw, and adult BM. Flow cytometric analysis of FL at 18 and 20gw demonstrated that although 3±1% of liver cells at this age were CD166+, less than 1% were endothelial CD166+CD34+ cells, and no hematopoietic CD166+CD34+CD45+ cells were detected. In fetal BM, CD166+ cells emerged after 15gw, expressed Flk-1 and CD34, and their percentage increased progressively with gestational age. Flow cytometric analysis at 20gw showed that 1.5±1% of cells were CD166+CD34+, of which 93±0.5% were CD45+. Human adult BM contained 2±0.5% of CD166+CD34+ cells, of which only 66±0.6% were CD45+. In order to functionally characterize CD166+CD34+ cells from adult and fetal BM (20gw), we plated these cells in mesenchymal cell growth medium (MSCGM), endothelial growth medium (EGM-2), and complete methylcellulose (MC). MSCGM and EGM-2 did not support growth and expansion of fetal or adult CD166+CD34+ cells. Quantification of the hematopoietic colony-forming potential of these cells demonstrated that fetal CD166+CD34+ generated/1000 cells: 8±1 Blast; 27±2 CFU-Mix; 51±10 CFU-GM; and 0 BFU-E, while adult CD166+CD34+ gave rise to 7 Blast; 17 CFU-Mix; 36 CFU-GM; and 10 BFU, demonstrating differences in the hematopoietic potential of these cells. Furthermore, at day 12 of MC culture, adherent stromal cells were detected underneath MC, but only in cultures from fetal BM. Characterization of these cells by flow cytometry showed that more than 90±2% of these cells were CD166+CD9+, and 30±5% were CD146+. Furthermore, these stromal CD166+ cells did not express CD34, CD45, CD31, CD209, or CD6. Immunostaining demonstrated that the CD166+CD146+ cells expressed osteopontin and Stro-1. A CD41+CD68+ population of cells was also found. In conclusion, we found that, during ontogeny, expression of CD166 in FL is not associated with hematopoietic cells. In the BM, expression of CD166 is associated with CD34 and Flk2, and its expression on HSC commences later in gestation, suggesting that these cells either arise in the BM, or that CD166 expression is triggered at a certain time point in gestation, probably associated with rapid proliferation of HSC during this time period. Furthermore, we demonstrated that CD34+CD166+ cells from 20gw fetal BM contain hematopoietic and stromal cell populations, while adult BM-derived CD34+ CD166+ cells are exclusively hematopoietic. Disclosures: No relevant conflicts of interest to declare.

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Marginal Zone B Cells Mediate Alloantibody Formation to a Clinically Significant Human RBC Antigen in a Murine Model

Blood ◽

10.1182/blood.v120.21.843.843 ◽

2012 ◽

Vol 120 (21) ◽

pp. 843-843 ◽

Cited By ~ 2

Author(s):

Kathryn R. Girard-Pierce ◽

Sean R. Stowell ◽

Nicole H. Smith ◽

Kate L. Henry ◽

James C. Zimring ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Marginal Zone ◽

Conflicts Of Interest ◽

Model System ◽

Single Amino Acid ◽

Specific Expression ◽

Marginal Zone B Cells ◽

Clinically Significant

Abstract Abstract 843 Introduction: Alloantibodies generated against red blood cell (RBC) antigens can lead to severe and potentially fatal outcomes such as hemolytic transfusion reactions (HTRs) or hemolytic disease of the fetus and newborn (HDFN). Several mouse models have been generated in efforts to study the alloimmune response to RBC antigens; however, few express clinically significant human RBC antigens. As a result, we developed a novel transgenic mouse model with RBC specific expression of the human KEL antigen, one of the most common non-ABO(H) antigens implicated in HTRs and HDFN. Using this model, we examined the rate, magnitude and consequence of alloimmunization following transfusion of RBCs expressing the human KEL2 antigen in moderate copy number (approximately 1200/RBC). In addition, we evaluated the potential involvement of marginal zone B (MZ B) cells, a B cell population involved in antibody-mediated immunity toward blood borne antigens, in the RBC alloimmunization process. Materials and Methods: C57BL/6 or KEL2 transgenic control recipient mice were transfused with KEL2 and C57BL/6 RBCs labeled with distinct lipophilic dyes to facilitate detection of cells following transfusion. To determine the role of MZ B cells, an additional group of C57BL/6 mice was treated with a-LFA-1 and a- CD49d blocking antibodies, four days prior to transfusion, in order to selectively deplete B cells from the marginal zone; additional animals were treated with an isotype matched control antibody or were surgically splenectomized. Post-transfusion survival and RBC bound antibody levels were determined by flow cytometry at specific timepoints after transfusion utilizing the fluorescent lypophilic dyes, anti-IgM, and anti-IgG. Serum antibodies were also analyzed by indirect immunofluorescence using flow cytometry with KEL2 and control C57BL/6 RBCs as targets. All experiments were completed at least three times with 3–5 recipients per group per experiment. Results: In C57BL/6 recipient mice, anti-KEL glycoprotein IgM was detectable within 2 days after KEL2 RBC transfusion, and peaked at day 5 post transfusion (compilation data of 3 experiments: adjusted MFI of IgM bound to KEL2 RBCs was 24.5 +/− 8.2 (mean +/− SD) on D5). Anti-KEL glycoprotein IgG was detectable on the transfused KEL2 RBCS and in the serum by day 5 and peaked between 14 and 21 days post transfusion (compilation data of 3 experiments: adjusted MFI of IgG bound to KEL2 RBCs was 19.7 +/− 16.7 on D14). Clearance of KEL2 RBCs began as early as 2 days following transfusion and continued to largely parallel the formation of anti-KEL alloantibodies. In contrast, KEL2 recipients failed to make anti-KEL alloantibodies (adjusted MFI of IgM bound to KEL2 RBCs was 2.1 +/− 2.7 on D5; adjusted MFI of IgG bound to KEL2 RBCs was 1.2 +/− 0.6 on D14, p<0.001 (IgM) and <0.05 (IgG) compared to KEL2 RBCs into C57BL/6 recipients). Furthermore, KEL2 recipients failed to clear transfused cells, strongly suggesting that KEL2 RBC clearance in C57BL/6 recipient mice was an antibody-mediated process. Splenectomy abrogated the alloantibody response to KEL2 RBCs, and selective depletion of MZ B cells greatly reduced alloimmunization (adjusted MFI of IgM bound to KEL2 RBCs was 9.3 +/− 6.1 on D5, adjusted MFI of IgG bound to KEL2 RBCs was 1.7 +/− 3.2 on D14, p<0.001 (IgM) and p<0.01 (IgG) compared to KEL2 RBCs into C57BL/6 recipients); furthermore, MZ B cell depletion also prevented KEL2 RBC clearance. Discussion: Transfusion of RBCs expressing the KEL2 antigen elicits a robust alloimmune response in recipient mice lacking expression of the human KEL glycoprotein. Given the complete lack of the human KEL glycoprotein antigen on recipient RBCs, the described system more closely models Rh(D) than KEL1/KEL2 (which involves a single amino acid polymorphism). The anti-KEL alloantibody response observed during primary exposure in the described model system leads to clearance of the transfused KEL2 RBCs, and suggests that initial alloantibody formation may reduce therapeutic efficacy when antigenic differences exist on transfused RBCs. In addition, these results suggest a key role for splenocytes in RBC alloimmunization, specifically MZ B-cells. Ongoing studies are further examining alloimmune responses and clearance patterns within this model system, with a focus on manipulation of MZ B cells as a potential method to prevent RBC alloimmunization. Disclosures: No relevant conflicts of interest to declare.

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Does Expression Of CD200 Provide a Target For LIC In Childhood ALL?

Blood ◽

10.1182/blood.v122.21.2894.2894 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2894-2894

Author(s):

Charlotte Victoria Cox ◽

Paraskevi Diamanti ◽

Allison Blair

Keyword(s):

Flow Cytometry ◽

Functional Ability ◽

Conflicts Of Interest ◽

Residual Disease ◽

Treatment Strategies ◽

Whole Genome ◽

Childhood All ◽

Nsg Mice ◽

Flow Cytometric

Abstract Although the survival of children with acute lymphoblastic leukaemia (ALL) has improved considerably, there are still around 20% of cases that go on to relapse. These patients do not respond well to current therapies and there is a need to develop treatment strategies to directly target the cells that initiate the leukaemia to allow eradication of the disease. Leukaemia initiating cells (LIC) have been shown to be present in several immunophenotypic subpopulations evaluated in NSG assays. Identifying a marker that is expressed by all LIC populations may allow their discrimination from normal haemopoietic stem cells and more specific targeting of these cells. Whole genome expression studies have recently highlighted genes, including CD58, CD97 and CD99 that were over expressed in B-ALL cases when compared to normal CD19+/CD10+ cells. The addition of these markers to the minimal residual disease (MRD) flow cytometric panel increased the sensitivity of detection by 10-fold to one leukaemia cell in 105 bone marrow (BM) cells. We have previously investigated the functional capacity of ALL cells sorted for CD58, CD97 and CD99 in combination with CD34 in vivo. However, none of these antigens were specific for LIC. CD200 has been described as another marker that improved MRD sensitivity by flow cytometry and consequently we investigated its expression in LIC populations and the functional ability of these cells in vivo. Expression of CD34, CD19 and CD200 was assessed in 12 B-ALL cases (6 pre B-ALL, 6 c-ALL) and 6 cord blood samples (CB) by flow cytometry. Whole genome microarray analysis was also performed on unsorted cells and sorted CD34+/CD19+, CD34+/CD19-, CD34-/CD19+ and CD34-/CD19- subpopulations to compare expression of genes between these LIC and the bulk leukaemia. Cells from 5 of these patients were also sorted on the basis of expression of CD34 and CD200 and the functional ability of the sorted subpopulations was assessed in NSG mice. The microarray analyses demonstrated that CD200 was over expressed in all the subpopulations and was especially high in the CD34+/CD19+ and the CD34-/CD19+ subpopulations. CD200 was significantly over expressed in the CD34+/CD19+ subpopulation when compared to its expression in the CD34+/CD19- and CD34-/CD19- subpopulations (p=0.009, F=6.3, Fcritical=4.5). Flow cytometric analyses confirmed that CD200 was over expressed in unsorted B-ALL cells compared to unsorted CB (54.6%±8.1% vs 0.07±0.09% respectively, p=0.0002) and expression was higher in CD34+/CD19+ (68.4%±10.8% p=0.0006), CD34+/CD19- (39%±9.2% p=0.01), CD34-/CD19+ (48.4%±8.6% p=0.002) and CD34-/CD19- (5%±1.8% p=0.3) subpopulations compared to CD34+/CD38- CB cells (2%±1.1%). Over expression of CD200 on these ALL subpopulations indicates that it may be useful in characterisation of LIC. When cells from 5 of these cases were sorted, the CD34+/CD200+ subpopulation accounted for the greatest proportion of cells (60.5%±16.1%). Expression of CD19 was also high in this subpopulation (65±11%). CD34+/CD200- cells represented the smallest subpopulation (0.5%±0.1%) and these cells had low expression of CD19 (4±7%). The CD34-/CD200+ and CD34-/CD200- subpopulations represented 18%±11.4% and 21%±120.6% of the bulk cells, respectively. CD19 expression was higher in the CD34-/CD200+ subpopulation (30±14%) than in CD34-/CD200- cells (7±5%). Results from 4 cases to date indicate that engraftment was achieved with the CD34+/CD200+ subpopulation in every case (4-92% human leukaemia). In 3 cases, comparable engraftment levels were also achieved using CD34-/CD200+ cells (10.4-86.1%). No engraftment was detected using the CD34+/CD200- or CD34-/CD200- subpopulations, with the exception of 1 case where engraftment levels of 18-48% were achieved. These levels were lower than those achieved with CD200+ cells from the same patient. Interestingly when BM from mice engrafted with CD200- cells from this patient were analysed they now expressed CD200 (12-35%), indicating cell differentiation had occurred in vivo. Assessment of self-renewal capability of these LIC populations is ongoing. These data suggest that CD200 may be a useful marker for discriminating LIC cells from normal haemopoietic cells, irrespective of expression of CD34 and CD19 in these cases. Disclosures: No relevant conflicts of interest to declare.

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O-GlcNAcylation in early stages of chronic lymphocytic leukemia; protocol development for flow cytometry

Cancer Biomarkers ◽

10.3233/cbm-203049 ◽

2021 ◽

pp. 1-10

Author(s):

Viktória Temesfői ◽

Kinga Molnár ◽

Péter Kaltenecker ◽

Barbara Réger ◽

Árpád Szomor ◽

...

Keyword(s):

Flow Cytometry ◽

Lymphocyte Count ◽

Blood Parameters ◽

Cell Number ◽

Metabolic Changes ◽

Protein Glycosylation ◽

Lymphocytic Leukemia ◽

Tween 20 ◽

Protocol Development ◽

Flow Cytometric

BACKGROUND: Recent studies proved that metabolic changes in malignant disorders have an impact on protein glycosylation, however, only a few attempts have been made so far to use O-GlcNAc analysis as a prognostic tool. Glucose metabolism is reported to be altered in hematological malignancies thus, we hypothesized that monitoring intracellular O-GlcNAc levels in Rai stage 0-I (Binet A) CLL patients could give deeper insights regarding subtle metabolic changes of progression which are not completely detected by the routine follow-up procedures. OBJECTIVE: In this proof of concept study we established a flow cytometric detection method for the assessment of O-GlcNAcylation as a possible prognostic marker in CLL malignancy which was supported by fluorescence microscopy. METHODS: Healthy volunteers and CLL patients were recruited for this study. Lymphocytes were isolated, fixed and permeabilised by various methods to find the optimal experimental condition for O-GlcNAc detection by flow cytometry. O-GlcNAc levels were measured and compared to lymphocyte count and various blood parameters including plasma glucose level. RESULTS: The protocol we developed includes red blood cell lysis, formalin fixation, 0.1% Tween 20 permeabilisation and employs standardized cell number per sample and unstained controls. We have found significant correlation between O-GlcNAc levels and WBC (R2= 0.8535, p< 0.0029) and lymphocyte count (R2= 0.9225, p< 0.0006) in CLL patients. Interestingly, there was no such correlation in healthy individuals (R2= 0.05664 for O-GlcNAc vs WBC and R2= 0.04379 for O-GlcNAc vs lymphocytes). CONCLUSION: Analyzing O-GlcNAc changes in malignant disorders, specifically in malignant hematologic diseases such as CLL, could be a useful tool to monitor the progression of the disease.

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Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results

Diagnostics ◽

10.3390/diagnostics11081320 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1320

Author(s):

Kristýna Pekárková ◽

Jakub Soukup ◽

Marie Kostelanská ◽

Jan Širc ◽

Zbyněk Straňák ◽

...

Keyword(s):

Flow Cytometry ◽

Cord Blood ◽

Extracellular Vesicles ◽

Correlation Coefficients ◽

Flow Cytometer ◽

Liquid Biopsies ◽

Physiological Conditions ◽

Flow Cytometric ◽

Term Births ◽

And Control

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

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