Characteristics of Laboratory Screening Suggest That Paroxysmal Nocturnal Hemoglobinuria (PNH) Is Perceived Primarily as a Bone Marrow Failure Syndrome Rather Than as a Hemolytic Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4107-4107
Author(s):  
Hwee Yong Lim ◽  
Marjorie Farley ◽  
Carl Wittwer ◽  
Charles Parker
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Small Populations ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Bone Marrow Failure Syndromes

Abstract PNH is a hematopoietic stem cell disorder in which the predominant clinical manifestations are hemolysis, bone marrow failure and thrombophilia. PNH arises as a result of somatic mutation of PIGA, an X-linked gene required for synthesis of the glycosyl phosphatidylinositol (GPI) moiety that anchors some proteins to the cell surface; and consequently, progeny of affected stem cells are deficient in all GPI-anchored proteins (GPI-APs). The hemolysis of PNH is the result of deficiency of CD55 and CD59, GPI-APs that normally inhibit complement activation on the red cell surface, but the relationship between GPI-AP deficiency and the bone marrow failure and thrombophilia of PNH are enigmatic. The peripheral blood of patients with PNH is a mosaic of normal and abnormal cells, and the degree of mosaicism varies greatly among patients. By using fluorescently labeled antibodies, GPI-AP deficient cells (GPI-AP−) can be distinguished form GPI-AP sufficient cells (GPI-AP+) cells by flow cytometric analysis, allowing quantitation of mosaicism. Flow cytometry has been used diagnostically for more than a decade, and technical modifications have improved resolution so that very small populations of GPI-AP− peripheral blood cells can be accurately detected. The purpose of these studies was to generate insights into how PNH is perceived in the community by analyzing the results of a commercially available screening assay using data from a national clinical diagnostic laboratory (ARUP Laboratories, Salt Lake City, UT). The flow cytometric method used in these studies is a modification of the high-resolution two-color assay of Sugimori and colleagues (Blood2006, 107:1308–1314). Clients are given the choice of testing for PNH by analyzing peripheral blood RBCs or PMNs (or both). The acidified serum test (Ham’s test) and the sucrose lysis test (sugar water test) are also available for screening for PNH. For flow cytometric analysis of RBCs, a value of ≥0.005% GPI-AP− cells is considered abnormal, while for PMNs ≥0.003% is abnormal. From January 1, 2008 to June 30, 2008, 1,113 RBC assays and 133 PMN assays were performed. An abnormally large population of GPI-AP− RBCs was identified in 55 cases (5%). The percentage of GPI-AP− RBC ranged from 0.009–69.603% with a median of 1.405%. Twenty-two cases (40%) had >5% GPI-AP− RBCs, while 18 cases (33%) had >10% GPI-AP− RBCs. Of the 133 PMN assays performed, 15 (11%) were abnormal. The range of GPI-AP− PMNs was 0.004–97.727% with a median of 18.327 %. Eight samples (53%) had >10% GPI-AP− PMNs. During the 1-year period from July 1, 2007-June 30, 2008 the acidified serum lysis test (Ham’s test) was performed on 212 samples while the sucrose lysis test was performed on 148 samples. These studies suggest that screening for PNH is common (~43 RBC assays/week compared to 44 assays/week for flow cytometric screening of peripheral blood for lymphoproliferative disorders and leukemia), but the vast majority of samples tested show normal expression of GPI-APs. That so many of the test samples are negative, and that the median for abnormal RBC samples is ~1.5 % GPI-AP− cells, suggest that most of the screening is done because of the association of PNH with bone marrow failure syndromes rather than because of evidence of intravascular hemolysis. These studies underscore the need to understand the pathophysiological basis and clinical implications of small populations of GPI-AP deficient cells in patients with bone marrow failure syndromes. Nonetheless, 18 cases with >10% GPI-AP− RBCs were detected during the 6 months of observation, indicating that the prevalence of classic PNH in the US is substantial. That PNH clone size is best determined by analysis of GPI-AP expression on PMNs does not appear to be widely appreciated in the community as the PMN assays is requested 12% as often as the RBC assay. Flow cytometry has largely, but not completely, replaced Ham’s test and the sucrose lysis test as screening assays for PNH.

The CXCR4 Antagonist AMD 3100 Induces Rapid Mobilization of Facilitating Cells and Hematopoietic Stem Cells in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4694-4694
Author(s):  
Hong Xu ◽  
Ziqiang Zhu ◽  
Yiming Huang ◽  
Suzanne T. Ildstad
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
Dose Titration ◽  
Great Promise ◽  
Hematopoietic Stem ◽  
Flow Cytometric

Abstract Abstract 4694 Bone marrow transplantation (BMT) offers great promise for treating red blood cell disorders, inherited disorders of metabolism, autoimmune diseases, and inducing donor-specific tolerance to organ transplants. However, the widespread application of this approach is dependent upon the development of less toxic strategies for BMT and avoidance of graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FC) facilitate engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex barriers without causing GVHD. We previously reported that Flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) synergistically mobilize FC and HSC into the peripheral blood (PB). Recently, AMD 3100 has been found to be a rapid mobilizing agent whose effect occurs within hours after injection. It is a macrocyclic compound and potential fusion inhibitor that antagonizes CXCR4 alpha-chemokine receptor for its effect on HSC mobilization. CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), are important in HSC homing and maintenance in the bone marrow microenvironment. Here, we investigated the effects of AMD 3100 on the mobilization of FC and HSC into PB in combination with FL and G-CSF. A dose titration of AMD 3100 was first performed. B6 mice were injected subcutaneously with AMD 3100 with the doses ranging from 1.25 mg/kg to 10 mg/kg. PB was obtained 0.5, 1, 3, and 6 hours post-injection. After individual count of peripheral blood mononuclear cells (PBMC), cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−). The numbers of PBMC significantly increased even 0.5 hour after AMD 3100 treatment and peaked at 1 h. The maximal mobilization of PBMC was noted at 1 h with 5.0 mg/kg AMD 3100. Treatment with 5.0 mg/kg AMD 3100 caused a 3.1-fold increase of WBC at 1h compared with saline treated controls. An increase of FC was detectable with all doses of AMD 3100. The numbers of FC peaked between 1 and 3 h, and declined rapidly to resemble saline-treated controls at 6 h after. A 5.9-fold increase of FC was observed at 1 h with 5.0 mg/kg AMD 3100 (P = 0.012). These data suggest that AMD 3100 is a potent cell mobilizer from bone marrow to PB. We next investigated the effect of AMD 3100 in combination with FL and G-CSF on the mobilization of FC and HSC into PB. B6 mice were injected with FL (day 1 to 10), G-CSF (day 4 to 10), and AMD 3100 (day 10). PB was obtained 1 h after injection on day 10. After performing a count of peripheral WBC, cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−) and HSC (Lin−/Sca-1+/c-kit+) mobilization. The maximal mobilization of PBMC was observed when animals were treated with AMD 3100/FL/G-CSF. The numbers of PBMC with AMD3100/FL/G-CSF treatment increased with 17.2-fold and 6.4-fold when compared with controls treated with saline or AMD 3100 alone (P < 0.00001), respectively. A maximal elevation of both FC and HSC was detected when AMD 3100 was added to FL/G-CSF treatment and reached 1.91 ± 0.42 × 103/μl (Figure 1A) and 1.89 ± 0.35 × 103/μl (Figure 1B), respectively. The increase of FC and HSC was significant. There was a 10.1-fold increase in FC and 230.8-fold increase in HSC when compared with recipients treated with AMD 3100 alone (P < 0.00001). AMD 3100/FL/G-CSF treatment resulted in a 1.7-fold of FC and 2.2-fold increase of HSC when compared with recipients treated with FL/G-CSF (P < 0.05). In summary, AMD 3100, FL, and G-CSF show a highly significant synergy on the mobilization of FC and HSC. This study may be clinically relevant in efforts to mobilize immunomodulatory FC and HSC to PB for transplantation, especially to induce tolerance for organ transplant recipients. Disclosures: Ildstad: Regenerex, LLC: Equity Ownership.

Erythroid Lineage Cells Are Found In Close Association With Bone In The Marrow Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 945-945
Author(s):  
Rialnat Adebisi Lawal ◽  
Kathleen E. McGrath ◽  
Laura M. Calvi
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Erythroid Differentiation ◽  
Enzymatic Digestion ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Osteolineage Cells

Abstract Osteolineage cells within the bone marrow microenvironment have been implicated in support and regulation of hematopoietic stem cells (HSCs). Recently, augmented hypoxia-inducible factor (HIF) signaling in osteoprogenitors has been shown to expand the HSC niche, and surprisingly these cells have also been demonstrated to express erythropoietin, the critical cytokine stimulating erythropoiesis. We therefore hypothesize that endosteal cells may represent an additional regulatory site for erythropoiesis. To further delineate the role of the osteolineage cells in the support of erythropoiesis, we isolated bone associated cells (BACs) with enzymatic digestion of adult C57bl/6 mice hind limbs after bone marrow flushing and depleted the BACs of CD45+ cells to enrich for osteogenic cells. We suspected some contribution of erythroid cells to CD45- BACs, however we were surprised to find that ter119+ cells represented a large percentage of BACs after enzymatic digestion. After CD45 depletion, ter119+ cells constituted about 30% percent compared to approximately 0.85% of CD45+ cells (33 ± 4.4vs. 0.85 ± 0.26, p= 0.0018) by flow cytometric analysis. Additionally, CD45 depleted BACs had approximately 46 fold higher osteocalcin expression than CD45+ cells (1300 ± 120 vs. 28 ± 9.5, p < 0.0001), while CD45/Ter119/CD31 depleted BACs had approximately 2000 fold higher osteocalcin expression than CD45/Ter119/CD31 (+) cells (2000 ± 520 vs. 0.98 ± 0.02, p= 0.0044) by qRT-PCR, confirming enrichment of the osteoblastic lineage by this immunophenotypic panel. These data suggest that there are a large number of erythroid lineage cells associated with the BACs along the endosteum. In the bone marrow of adult mice, ter119 + cells represented approximately 85% in the CD45- pool as compared to 5% in the CD45+ cell pool. To determine if the endosteum is an active site of erythropoiesis, we quantified erythroid progenitors and precursors in the BAC pool compared to whole bone marrow (wbm) and peripheral blood (pb) by both flow cytometric analysis and colony forming assays. Flow cytometric analysis demonstrated the presence of every phase of erythroid differentiation in the BAC pool, including the presence of phenotypic MEPs (wbm vs bac vs pb: 250 ± 30 vs 84 ± 22 vs 0), BFU-E (wbm vs bac vs pb: 300 ± 14 vs 110 ± 36 vs 0 ), CFU-E (wbm vs bac vs pb: 2900 ± 2 vs 430 ± 23 vs 1 ± 0.8) and proerythroblasts (wbm vs bac vs pb: 11000 ± 2500 vs 7600 ± 1600 vs 2300 ± 920) per million cells. The phenotypic frequency of CFU-E was particularly remarkable in the BACs (430 ± 23) as compared to peripheral blood (1 ± 0.8) , demonstrating that all stages of erythroid differentiation are found in tight association with the endosteum and are not due to contamination from circulating erythroid progenitors. Colony assays were performed for CFU-E (wbm vs. bac 108 ± 16 vs 6.3 ± 2 colonies per 20,000cells plated), BFU-E (wbm vs. bac 55 ±1.0 vs 2 ±1.0; colonies per 40,000 cells plated) and myeloid progenitors (wbm vs. bac 66 ± 28 vs 11 ± 2.5 ; colonies per 10,000 cells plated) also confirmed the presence of erythroid progenitors at endosteal sites. Together these results identify the endosteal surface as a site for erythroid differentiation. The presence of all phases of erythroid lineage differentiation in the BACs suggests a potential role for osteolineage cells for maintenance and regulation of erythropoiesis. Whether osteolineage cells contribute to erythroid lineage homeostasis and/or stress response, and whether activation or damage to osteolineage cells alters local erythroid differentiation remains to be demonstrated. However our data suggest further study of the endosteum and osteolineage cells as a potential and unexpected site of erythroid regulation, which could potentially be targeted to accelerate erythropoiesis and treat anemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CCL3 Regulates Normal Hematopoiesis but Is Not Essential for the Maintenance of a Long-Term Engrafting Hematopoietic Stem Cell

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1482-1482
Author(s):  
Rhonda J. Staversky ◽  
Lila Yang ◽  
Alexandra N. Goodman ◽  
Mary A Georger ◽  
Michael W. Becker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Hematologic Malignancies ◽  
Myelogenous Leukemia ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Flow Cytometric

Abstract Background/Rationale: Hematologic malignancies are known to remodel the bone marrow microenvironment, reducing support for normal hematopoiesis while increasing support for the malignant clone. The chemokine CCL3 has been demonstrated to play a role in microenvironmental dysfunction in multiple malignancies including myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome. In addition, CCL3 has been shown to be critical for the progression of chronic mylogenous leukemia in murine models. However, to consider anti-CCL3 therapy as an option for hematologic malignancies we must first understand its role in the regulation of normal hematopoiesis. To date the role of CCL3 in this process is poorly understood. Methods/Results: In these experiments we utilized genetically altered mice with a global loss of CCL3 (CCL3KO) on a C57bl/6 background. Peripheral blood counts revealed that monocytes, granulocytes, and red blood cells were all significantly decreased in the peripheral blood of CCL3KO mice as compared to WT controls at 12 weeks of age (9.78 ± 0.3 vs. 8.06 ± 0.2 RBCs*106/μl, WT vs. CCL3KO p≤0.001 n=8 mice/group). CCL3KO mice also demonstrate a 2-fold increase in the frequency and number of phenotypic long-term hematopoietic stem cells (LT-HSCs: Lin-sca1+ckit+flt3-CD150+CD48-) at 12 weeks of age in the bone marrow by flow cytometric analysis (0.0053 ± 0.0005 vs. 0.0106 ± 0.0007 % of cells, WT vs. CCL3KO p≤0.0001 n=8 mice/group). A significant increase was also seen in short-term HSCs (ST-HSCs), but not in multipotent progenitor (MPP) populations (data not shown), suggesting that CCL3 regulates the most immature hematopoietic cells. To quantify functional hematopoietic stem cells in the marrow of CCL3KO mice competitive transplants were performed using whole bone marrow cells. In primary transplants CCL3KO mice demonstrated a small but significant decrease in engraftment over 22 weeks when compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Decreased engraftment was seen in B cells, T cells, and myeloid cells in the peripheral blood. Upon secondary transplantation the decrease in engraftment of HSCs from CCL3KO donor mice was much more profound. At 16 weeks post-transplant HSCs from CCL3KO donors contributed to hematopoiesis at a rate 5 times lower than WT littermate controls (64.67 ± 1.967 vs. 11.97 ± 5.322 % of cells, WT vs. CCL3KO p≤0.0001 n=10 mice/group). These results were seen in both male and female mice and suggest that, although phenotypic HSCs were increased in the bone marrow of CCL3KO mice, those HSCs were defective. To test this hypothesis we sorted Lineage-Sca1+Ckit+Flt3- (Flt3-LSK) bone marrow cells enriched for LT-HSCs in order to establish stem cell activity on a per cell basis through competitive transplantation. As with the whole bone marrow transplants, primary transplant of sorted Flt3-LSK cells resulted in reduced engraftment of CCL3KO cells as compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Surprisingly, upon secondary transplantation, CCL3KO Flt3-LSK donor cells performed better than the WT littermate controls (2-way ANOVA, p<0.05 over 16 weeks, n=8 mice/group). This result suggests that a transplantable population of cells excluded by the Flt3-LSK sorting parameters is responsible for repression of long-term engraftment capacity of marrow from CCL3KO mice. In establishing the mechanism by which CCL3 regulates hematopoiesis we investigated the rate of apoptosis by quantification of caspase 3 activation, as well as cell cycle status by quantification of Ki67 positivity and DNA content by flow cytometric analysis. We found no difference in the rate of apoptosis, however there was a significant decrease in the fraction of short term HSCs (ST-HSCs) (Flt3-CD48-CD150-LSK) that were actively cycling (2.06 ± 0.43 vs. 1.23±0.44 % of ST-HSCs WT vs. CCL3KO p<0.05 n=3 mice/group). This suggests that CCL3 regulates the proliferation of hematopoietic progenitor cells downstream of the LT-HSC. Conclusions: These results highlight a role for the chemokine CCL3 in the maintenance of the hematopoietic system under benign, physiologic conditions. However, a long-term engrafting HSC population is clearly maintained even in the complete absence of CCL3 suggesting that anti-CCL3 therapy would be well tolerated by the hematopoietic system. Disclosures No relevant conflicts of interest to declare.

Chronic RPS19 Deficiency Leads to Bone Marrow Failure In a Mouse Model for Diamond-Blackfan Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 193-193
Author(s):  
Pekka Jaako ◽  
Johan Flygare ◽  
Karin Olsson ◽  
Ronan Quere ◽  
Jonas Larsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Flow Cytometric

Abstract Abstract 193 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al, 2009 ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the Collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells, and flow cytometric analysis of bone marrow after a short-term induction reveals increased frequencies of hematopoietic stem and progenitor cells reflecting the onset of stress hematopoiesis. In the current study we have analyzed the long-term effect of RPS19 deficiency in bone marrow. In contrast to a short-term induction, flow cytometric analysis of bone marrow after 51 days revealed decreased frequencies of hematopoietic stem and progenitor cells that correlate with a severe peripheral blood phenotype. In addition, we observed a 3–6 fold increase in apoptosis in RPS19-deficient bone marrow compared to controls based on TUNEL assay. Furthermore, transplantation of whole bone marrow cells from transgenic donors into wild type lethally irradiated recipients confirms that the observed phenotype is autonomous to the blood system. To study whether long-term RPS19 deficiency functionally impairs hematopoietic stem cells, we pre-induced mice for 30 days followed by 15 days without doxycycline to restore the RPS19 expression. Mice were sacrificed and total bone marrow cells were transplanted together with wild-type competitor cells (1:1) into wild type lethally irradiated recipients without doxycycline. This experimental setting allows us to assess the functionality of pre-induced hematopoietic stem cells in absence of ribosomal stress. Flow cytometric analysis of peripheral blood one month after transplantation clearly demonstrates decreased reconstitution from pre-induced donors compared to the wild-type competitor. While this time point reflects mainly the function of transplanted progenitors, long-term analysis of hematopoietic stem cell function in these recipients is ongoing. To study the molecular mechanisms underlying the hematopoietic defect we performed comparative microarray analysis. We chose to analyze preCFU-E/CFU-E erythroid progenitors since we have previously located the erythroid defect at the CFU-E – proerythroblast transition based on flow cytometry and clonogenic proliferation cultures of prospectively isolated erythroid progenitors. Microarray analysis of preCFU-E/CFU-E progenitors reveals deregulation of several genetic pathways, including a robust upregulation of p53 pathway genes, and these targets have been confirmed by real-time PCR. Furthermore, many of p53 target genes are also upregulated in the Lineage− Sca-1+ c-Kit+ (LSK) population that contains immature hematopoietic progenitors and stem cells suggesting that the activation of p53 is not restricted to the erythroid lineage. To ask whether increased activity of p53 can solely explain the hematopoietic phenotype, we have crossed our mouse model into a p53-null background. In summary, our data suggest that RPS19-deficient mice fail to uphold stress hematopoiesis for extended periods of time, with chronic RPS19 deficiency causing bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Update on the diagnosis and management of paroxysmal nocturnal hemoglobinuria

Hematology ◽  
2016 ◽  
Vol 2016 (1) ◽  
pp. 208-216 ◽  
Author(s):  
Charles J. Parker
Keyword(s):  
Bone Marrow ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Cell Clone ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Disease Process ◽  
Hematopoietic Stem ◽  
Glycosyl Phosphatidylinositol ◽  
Flow Cytometric ◽  
Immune Mediated

Abstract Once suspected, the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) is straightforward when flow cytometric analysis of the peripheral blood reveals a population of glycosyl phosphatidylinositol anchor protein-deficient cells. But PNH is clinically heterogeneous, with some patients having a disease process characterized by florid intravascular, complement-mediated hemolysis, whereas in others, bone marrow failure dominates the clinical picture with modest or even no evidence of hemolysis observed. The clinical heterogeneity is due to the close, though incompletely understood, relationship between PNH and immune-mediated bone marrow failure, and that PNH is an acquired, nonmalignant clonal disease of the hematopoietic stem cells. Bone marrow failure complicates management of PNH because compromised erythropoiesis contributes, to a greater or lesser degree, to the anemia; in addition, the extent to which the mutant stem cell clone expands in an individual patient determines the magnitude of the hemolytic component of the disease. An understanding of the unique pathobiology of PNH in relationship both to complement physiology and immune-mediated bone marrow failure provides the basis for a systematic approach to management.

scholarly journals Screening for Paroxysmal Nocturnal Hemoglobinurea with Cytodiff Analysis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4962-4962
Author(s):  
Svetlana Lugovskaya ◽  
Fyodor Dyukov ◽  
Elena Naumova ◽  
Margarita Pochtar ◽  
Olga Plehanova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Wide Spectrum ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
The United States ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Cd16 Expression ◽  
Pnh Clone

Abstract Introduction. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidylinositolglycan complementation Class A (PIG-A) gene. PIGA mutations in PNH patients lead to a glycosylphosphatidylinositol (GPI)-linked membrane proteins expression deficiency.In PNH, there is a partial or absolute inability to make GPI-anchored proteins including complement-defense structures such as CD55 and CD59 on RBCs and WBCs. Clinical features of PNH include intravascular hemolysis, bone marrow failure, and thrombosis, all major causes of morbidity and mortality. Flow cytometry (FCM) plays a key role in the laboratory investigation of PNH, and rapid diagnosis of this condition is highly desirable. A definitive diagnosis of PNH can be established by demonstrating the absence of cell membraneGPI-anchored proteins from granulocytes or red blood cells (RBC) according to ICCS Guidelines for the diagnosis and monitoring of PNH by flow cytometry. It has been also described that the expression of CD16 can be decreased on PNH-affected granulocytes. Recently new method for extended flow WBC differential was introduced by Beckman Coulter. This method uses flow cytometric analysis with CytoDiff™** reagents which is a 5-color/6-marker reagent that provides a 10-part cytometric differential from whole blood specimens and comprises CD36-FITC, (CD2+CD294)-PE, CD19-ECD, CD16-PC5, and CD45-PC7 (Beckman Coulter). The protocol allows detection of mature neutrophils, total lymphocytes, total monocytes, eosinophils, basophils, immature granulocytes, B lymphocytes, CD16-negative T/NK lymphocytes, CD16-positive T/NK lymphocytes, CD16 positive and CD16 negative monocytes, and blasts cells with lineage orientation. This method also allows the detection of the abnormal antigen expression on WBC, for example low CD16 expression on neutrophils. In case of abnormal low CD16 expression on segmented neutrophils they will be classified as Immature Granulocytes (Imm Gran). The aim of the study was to evaluate the efficacy of CytoDiff** analysis of peripheral blood for PNH screening detecting low CD16 expression on neutrophils. Methods: EDTA-anticoagulated blood samples from 53 patients with PHN suspicion were prospectively included in the study. Analysis of the PNH clones was conducted in according with international protocol, using CD235a for RBC gating, CD15-PE/CD45-PC7 for granulocyte gating, CD64-PC5/CD45-PC7 for monocyte gating and GPI-anchored proteins CD59-PE, FLAER-FITC/CD24-PC5 and FLAER-FITC/CD14-PE for RBCs, granulocytes and monocytes accordingly. For extended flow WBC differential analysis the blood samples were stained with the CytoDiff** panel, lyzed with Versalyse (Beckman Coulter) and 20 000 leucocytes were analyzed on a FC500 Flow Cytometer (Beckman Coulter) using CytoDiff** CXP software. Results:Totally 53 patients with PHN suspicion were prospectively included in the study. All these patients were characterized by anemia, thrombocytopenia and/or leucopenia. PNH diagnosis was confirmed in 6 patients and in other 7 patients the final diagnosis was aplastic anemia with PNH clone. For all 13 patients (4 males, 9 females, with median age of 41.5 years) with confirmed presence of PNH clone CytoDiff** reported increased number of Imm Gran (range 3-45%). Microscopy analysis did not detect the presence of Imm Grans in the slide, so we concluded that falsely reported increased Imm Gran count was due to the decreased expression of CD16 on Neutrophils. Good correlation (r=0.9257) was observed between Imm Gran count and the size of granulocytic PNH clone. Conclusion: Our data demonstrate that CytoDiff** analysis, which is able to detect a wide spectrum of normal and pathological cells in peripheral blood allows determination of CD16-low expression on neutrophils and thus provide efficient screening for suspicious of PNH in patients with anemic syndrome. ** Not available in the United States and other geographies. Disclosures Lugovskaya: Beckman Coulter: Research Funding.

scholarly journals Sf3b1 K700E Mutation Impairs Pre-mRNA Splicing and Definitive Hematopoiesis in a Conditional Knock-in Mouse Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 140-140 ◽  
Author(s):  
Annalisa Mupo ◽  
Vijitha Sathiaseelan ◽  
Michael Seiler ◽  
David Kent ◽  
Shouyong Peng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Wild Type ◽  
Aberrant Splicing ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Stem And Progenitor Cells

Abstract Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by dysplastic hematopoiesis and peripheral blood cytopenias. Recently, somatic mutations affecting components of the spliceosomal machinery have been discovered in the majority of MDS patients. SF3B1 mutations are most frequent and strongly correlate with the presence of bone marrow ring sideroblasts and a favorable prognosis. SF3B1 mutations, including the K700E substitution which accounts for more than 50% of all mutations, are missense, heterozygous and cluster in a hotspot within the heat domain of the protein suggesting that they are gain-of-function variants. The molecular effects of SF3B1 mutations and the mechanisms through which they drive clonal expansion and dyserythropoiesis remain obscure. Therefore, to assess their molecular and phenotypic consequences, we generated a mouse model carrying a conditional floxed knock-in allele (Sf3b1flox-K700E/+) by homologous recombination of JM8 murine embryonic stem cells. To induce expression of Sf3b1 K700E in adult hematopoietic stem and progenitor cells, Sf3b1flox-K700E/+/Mx1-Cre+ were injected with pIpC from 4-8 weeks of age. Here we report the initial characterization of these animals. Monthly peripheral blood counts from mutants and wild-type (WT) littermates starting one month post-pIpC injection showed a reduction in hemoglobin levels (at 8 weeks WT=17g/dl mut=14.5g/dl, p<0.03). Additionally, flow cytometric analysis of bone marrow samples demonstrated a modest but consistent decrease in late erythroid progenitor cells (Ter119+ and CD71-/low). The myeloid compartment showed relative expansion of Gr1+/Mac1+ and Mac1+ cells whereas analysis of hematopoietic stem and progenitor cells (HSPCs) revealed a decrease in HSCs (% of total events WT=0.04%; Sf3b1flox-K700E/+=0.01%) in mutant mice. In competitive transplantation experiments into sub-lethally irradiated syngeneic recipients we observed a lower engraftment potential of Sf3b1flox-K700E Lin-ve HSPCs (CD45.2) compared to wild-type cells (CD45.1). Flow cytometric analysis of peripheral blood of recipient animals showed that Sf3b1flox-K700E cells contributed more to the myeloid lineage than wild-type cells (Sf3b1flox-K700E Mac1+/Gr1+ 8.95%; Mac1+ 15% vs WT Mac1+/Gr1+ 4.08%; Mac1+ 5.57%). At a median follow-up of 56 weeks, mutant animals did not show decreased survival or signs of illness as compared to WT controls. Finally, as Sfb31 mutations are predicted to affect splicing of pre-mRNA and consequently alter the gene expression, we performed RNAseq analysis in unselected and Lin-ve bone-marrow cells from mutant and controls animals. Comparison between wt and mutant samples showed deregulated expression of genes implicated in human MDS (Mmp9, Puma, Bcl2l1). We then looked at the pattern of aberrant splicing promoted by Sf3b1flox-K700E, and found that mutant animals have an increased use of cryptic 3'' splice sites (ss) throughout their genome. We showed that the majority of these alternative 3' ss are novel and we characterized them as being located 15 to 24 nucleotides upstream from the canonical 3' ss and associated with sequence features including a shorter polypyrimidine tract and an enrichment of adenines -8 to -18 bases upstream of the cryptic 3' ss. Interestingly, similar features have been reported in human cancers with SF3B1 hotspot mutations. We predict that ~33% of the mRNAs affected by aberrant splicing will include an aberrant premature termination codon, promoting RNA degradation through nonsense-mediated decay. In conclusion, our conditional Sf3b1K700E knock-in mouse is a faithful molecular model of the consequences of these mutations in the mouse hematopoietic system. The mild phenotype we observe in comparison to SF3B1-mutant human MDS may be explained by the requirement for additional mutations to progress to overt MDS and is more reminiscent of SF3B1-associated clonal hemopoiesis, relatively common phenomenon in elderly humans without overt hematological abnormalities. Additionally, our initial characterization of novel splice sites preferentially recognised by the mutant Sf3b1 protein suggests that transcriptional consequences of the mutation may differ between species, dependant on the degree of conservation of the relevant intronic regions. Disclosures Seiler: H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Campbell:14M genomics: Other: Co-founder and consultant.

Baseline Assessment Of GPI-Anchored Protein Deficient Blood Cells In Patients With Bone Marrow Failure (The OPTIMA study)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1241-1241 ◽  
Author(s):  
Naoshi Obara ◽  
Shigeru Chiba ◽  
Kohei Hosokawa ◽  
Chiharu Sugimori ◽  
Masaki Yamamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
High Resolution ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Treatment Plan ◽  
Low Frequencies ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Patient Groups

Abstract Background Paroxysmal nocturnal hemoglobinuria (PNH) is a disease derived from an acquired mutation of the phosphatidylinositol glycan class A (PIGA) gene in the hematopoietic stem cells. In some cases with aplastic anemia (AA) or low-risk types of myelodysplastic syndromes (MDS), it is known that glycosylphosphatidylinositol-anchored protein deficient (PNH-type) cells can be often detected at low frequencies (about 0.01%) through the high-resolution flow cytometry-based methFod. Because these patient groups are reported to have a good reactivity towards immunosuppressive therapies as opposed to the other patient groups lacking PNH-type cells, detection of these cells is potentially useful in determining a treatment plan for the patients with bone marrow failure syndromes. To confirm this preliminary information, a large cohort study is needed. Method A nationwide multi-center prospective observational study (OPTIMA) was started in July 2011 to determine the prevalence of patients with bone marrow failure syndromes who carried PNH-type cells and to clarify the significance of the presence and quantitative changes of these cells with regard to the clinical features. Each of the six laboratories in different universities was assigned as a regional analyzing center. The percentage of PNH-type cells was measured by the high-resolution flow cytometry-based method, originally established in Kanazawa University. At six individual laboratories, cross validations were conducted to minimize the inter-laboratory variations in the detection sensitivities, cutoff values, etc. The liquid FLAER method (≥0.003%) and cocktail method (≥0.005%) with CD55 and CD59 antibodies were used for the detection of PNH-type granulocytes and erythrocytes, respectively. Results Quality of the assay was managed in all the laboratories by periodic blind validation tests using standard blood samples containing 0.01% PNH-type cells. Until July 2013, 1214 cases were examined; 461 (38%) were positive for PNH-type cells and 141 (11.6%) had ≥1% PNH-type cells. Out of 1214, 783 patients were diagnosed to have AA (n=386), MDS (n=341), and PNH (n=56) based on the case report forms. PNH-type cells were detected in 56.2%, 19.1% and 100% of patients with AA, MDS and PNH, respectively. In a half of patients having ≥1% PNH-type cells, lactate dehydrogenase levels exceeded the ≥1.5×upper limits of normal. Conclusion Our study has successfully established the high-resolution flow cytometry-based method that enables the detection of minimal PNH-type cells (below 0.01%). Also, by implementing a uniform protocol to six individual laboratories across the country, a system has been established for the patients to undergo the detection test with equal accuracy in all of these laboratories. Further accumulation of case studies and prolonged observations are required to determine the clinical significance of the minimal PNH-type cells, especially in terms of its relation to response to immunosuppressive therapy. Disclosures: Obara: Alexion Pharmaceuticals: Honoraria. Chiba:Alexion Pharmaceuticals, In: Research Funding. Sugimori:Alexion Pharma: Honoraria. Noji:Alexion Pharmaceuticals: Honoraria. Yonemura:Alexion Pharma: Research Funding. Ando:Alexion Pharma: Research Funding. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Shichishima:Alexion Pharmaceuticals, In: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Ninomiya:Alexion Pharma: Honoraria. Nishimura:Alexion Pharma: Research Funding, Speakers Bureau. Kanakura:Alexion Pharmaceuticals: Research Funding, Speakers Bureau. Nakao:Alexion Pharmaceuticals, In: Research Funding.

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

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