scholarly journals Platelet-Associated Antibodies By Flow Cytometry in Adult Patients with Immune Thrombocytopenia before and after Splenectomy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-5
Author(s):  
Kseniya A. Nikiforova ◽  
Elena K. Egorova ◽  
Yuliya O. Davydova ◽  
Nikolay M. Kapranov ◽  
Elena I. Pustovaya ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Immune Thrombocytopenia ◽  
Conflicts Of Interest ◽  
Polyclonal Antibodies ◽  
Fluorescein Isothiocyanate ◽  
Platelet Glycoprotein ◽  
Rank Test ◽  
Severe Thrombocytopenia ◽  
Becton Dickinson

Introduction Immune thrombocytopenic purpura (ITP), or primary immune thrombocytopenia is an autoimmune disease characterized by isolated thrombocytopenia (number of platelets in peripheral blood is less than 100×109/L) and splenic production of antibodies against platelet glycoprotein complexes and megakaryocytes, resulting in hemorrhagic syndrome. Circulating platelets attached by autoantibodies that leads to accelerated removal of these cells by spleen macrophages. The therapy for patients with newly diagnosed ITP with hemorrhagic syndrome and/or severe thrombocytopenia (number of platelets in peripheral blood <10-20×109/L) includes corticosteroids and characterized by a low rate of remission (just around 20-30%). For cases of ITP resistant to corticosteroid therapy recommends one of the second-line method of treatment - splenectomy (SE). Approximately 60-80% of the patients achieve complete remission after splenectomy. There is a technique for assessment of platelet-associated antibodies (PAA) classes IgG, IgM and IgA on platelets by flow cytometry. This method is a commodious, easy, quick, and relatively cheap and applied to estimate autoimmunity status of patients with thrombocytopenia. However, this method characterized by low specificity. Aim The aim of the study was to determine the correlation between level of PAA of IgG, IgM, and IgA classes in the peripheral blood of adults with ITP before SE, 5-7 days and 3 months after SE. Patients and methods The study included 21 patients with ITP (4 cases of persistent ITP and 17 cases with chronic form). Median age was 36.9 years, M:F ratio was 1: 4.25 (men was older than women - 46.0 years old versus 34.7). All patients underwent from 1 to 3 lines of therapy and were recommended for SE due to resistance to treatment. The PAA level was measured at three time points (before SE, 5-7 days, and three months after SE) by flow cytometry (Becton Dickinson FACS Canto II). Goat polyclonal antibodies against human IgG, IgM, IgA labeled with fluorescein isothiocyanate (FITC) (Cedarlane) were used to determine antibodies of various classes. Anti-CD41a labeled with phycoerethrin (Becton Dickinson) was used to determined platelets. PAA level was assessed based on the mean of fluorescence intensity (MFI) of the FITC-channel. Statistic analysis was carried out using GraphPad Prism 6.01. Wilcoxon signed-rank test had been used for pair comparison. The value of 0.05 had been taken as reliable. Results MFI levels of PAA IgA (391 vs 198, p = 0.005) and IgM (275 vs 142, p<0.0001) significantly decreased in patients after SE compared with the initial level (level before SE). Level of MFI PAA IgM also remained reduced (275 vs 138, p=0.0084) three months after SE (Fig. 1). MFI levels of PAA IgG did not change. Conclusions Using of flow cytometry to determinate platelet-associated immunoglobulins for diagnostic of ITP remains controversial. Despite the fact, this test can be recommended for monitoring of PAA from patients with ITP after SE. In addition, the results confirm the fact that most cells producing antiplatelet antibodies seems to be residing in a spleen. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clinical Utility of Flow Cytometry Immunophenotyping in Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 8-8
Author(s):  
Alessandra Suelen Jardim Silva ◽  
Gustavo Henrique de Medeiros Oliveira ◽  
Lenilton Silva DA Silva Júnior ◽  
Hugo Henrique de Freitas Ferreira ◽  
Maria das Graças Pereira Araujo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Fluorescein Isothiocyanate ◽  
Chlorophyll Protein ◽  
B Lineage ◽  
B Lymphoid

The acute lymphoblastic leukemia (ALL) is a malignant disease of the immune system and hematologic characterized by accumulation of neoplastic B lymphoid precursors or T (lymphoblasts) in the bone marrow and / or peripheral blood. The diagnosis of these leukemias occurs by morphological classification of French-American-British (L1, L2 or L3) associated with features of immunological profile T or B cell malignancies, based on the expression profile of monoclonal antibodies (MoAb) directed against the antigens of cell differentiation by flow cytometry (FC). Several studies have shown that blast cell immunophenotypes of cases of acute lymphoblastic leukemia does not always exhibit characteristics of lymphoid differentiation normal but exhibit aberrant immunophenotypes. Thus, blasts some cases of acute lymphoblastic leukemia of B lineage may show myeloid or T antigens. Also blasts of cases of acute lymphoblastic leukemia T cell determinants may possess B or myeloid cells.Objective:To determine the immunophenotypic profile by FC in 88 patients with ALL (B or T lineage) diagnosed in the Laboratory of Flow Cytometry Blood Center of Dalton Cunha - HEMONORTE, from State of Rio Grande do Norte, Brazil.Methods:All samples from peripheral blood and / or bone marrow were subjected to FC immunophenotyping using a panel of MoAb specific for diagnosis of acute leukemia (AL) directly conjugated to fluorochromes as fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll protein (PerCP) and allophycocyanin (APC).Results:The patients' age range was between 1 month and 84 years old, with an average of 20.3 years, with 62.5% of the patients being male. The most frequently observed strain was B and the most evident subtype was the Common Pre-B ALL. Among the cell markers evaluated, the most expressed in lineage B were CD19, CD10, HLADR and cCD79a and the antigens most frequently expressed in lineage T were cytoplasmic CD3 (cCD3) and membrane (mCD3), CD7, CD5 and CD2. A small percentage (6.8%) were doubly positive T cells.Conclusion:It is concluded that individuals with ALL in this study have demographic, clinical and immunophenotypic characteristics similar to those observed in other studies, demonstrating that CF immunophenotyping is an essential methodology in the diagnosis of follow-up of these leukemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals Efficiency and Safety of Eltrombopag for Multi-Line Failed Chinese Patients with Immune Thrombocytopenia: Cases with Decreased Megakaryocyte Response Well from Single Center Experience

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2082-2082
Author(s):  
Qi Liu ◽  
Yingying Shen ◽  
Yuzhu Li ◽  
Huijing Hu ◽  
Wenbin Liu ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Immune Thrombocytopenia ◽  
Conflicts Of Interest ◽  
Chinese Patients ◽  
Ivig Treatment ◽  
Severe Thrombocytopenia ◽  
Line Treatment ◽  
Upper Respiratory Tract ◽  
First Line

Abstract Immune thrombocytopenia (ITP) is an autoimmune disease characterized by decreased platelet (PLT) count in peripheral blood and marrow megakaryocyte dysfunction, which was induced by antiplatelet antibodies or cytotoxic T cells. Many ITP patient were previously full response to first-line treatment such as corticoids (CIS) or immunoglobulin (IVIG), but were intolerant or relapse during the maintenance treatment, and turn to receive recombinant human thrombopoietin (rhTPO), rituximab (RTX), splenectomy as well as immunosuppressive therapy (IST). Unfortunately, there still some patients failed to multiple treatments are faced with high bleeding risk. Eltrombopag, the first oral non-peptide thrombopoietin receptor agonist (TPO-RA) that approved by US FDA in 2008, is the second-line treatment option for chronic ITP based on guideline recommendation. As reported, the effective rate of eltrombopag in ITP ranges from 59% to 89.7%, and limited predictive factors were available to assess the efficiency for individual. The characteristic of marrow megakaryocyte (MK) in ITP was reported as normal or hyper megakaryocytosis on the initiation (before treatment), however there are also ITP patients with decreased marrow MK, and the response of eltrombopag to these patients have not been fully reported. Bone marrow aspiration is a routine examination in China for the diagnosis of ITP with severe thrombocytopenia. Therefore, we conducted a research tried to assess the efficiency of eltrombopag to patients with multi-line failed ITP, and analyzing the possible factors may contribute to the differences based on personal characteristic. Thirty-five multi-line failed ITP patients with PLT count bellow 30× 10 9/L who received eltrombopag treatment were enrolled, and divided into three groups, the hyper-MK (MK count>35, n=17), normal-MK (8-35, n=10) and hypo-MK (≤7, n=8) groups. The average PLT count was 10.63±6.30×10 9/L before eltrombopag treatment, and it increased significantly to 12 (2-65)×10 9/L, 39 (2-212)×10 9/L, 68 (2-453)×10 9/L, 60 (3-358)×10 9/L, and 75 (3-308)×10 9/L in the 1st, 2nd, 4th, 6th , and 8th weeks after treatment (all P=0.000, except 1st week). The median intervals time required for platelet to reach 30×10 9/L and 100×10 9/L for the first time were 11 (4-40) and 21 (10-65) days, respectively (Fig. A-C). We found the overall, complete (PLT count ≥100×10 9/L) and partial response (PLT count ≥30×10 9/L or at least twice the baseline value) rates were 54.3% (n=19), 48.6% (n=17), and 5.7% (n=2) respectively to eltrombopag in our center. The overall response rate of patient with decreased MK was 75%, which was unexpectedly higher than the patient with increased or normal MK count (52.9% and 40%, respectively) (Fig. D). To explain the underling mechanism, we detected the peripheral T lymphocyte, B lymphocytes and NK cells before eltrombopag. Results showed that the patient with decreased MK were characteristic with higher T helper (Th) cells (39.62±2.01%) and regulatory T (Tregs) cells (7.93±1.63%) when compared to the hyper-MK (30.44±10.95%, 4.23±1.67%) and normal-MK group (25.67±5.72%, 4.81±2.12%). For patient with poorer eltrombopag response group, more percentage and absolute number of NK cells (15.48±7.12%, 234.4±91.80×10 6/L) were found in peripheral blood (Fig. E-F). We also discovered that previous first-line (CIS or IVIG) treatment, rhTPO effectiveness, RTX splenectomy and IST had no influence on eltrombopag response (Fig. G). As for safety, nine eltrombopag related adverse events were reported, and most commonly were upper respiratory tract infection (8.6%), elevated ALT (5.7%), and venous thrombosis (5.7%). All the side effect was cured by symptomatic treatment, eltrombopag dose reduction or discontinue. In summary, ITP patients with decreased megakaryocyte respond well to eltrombopag, and the abnormality of NK cells may play a role in patients exert a poor response. In further clinical practice, we should considering the megakaryocytes count as well as peripheral NK population to better predicting eltrombopag response in ITP patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Dissecting Macrophage-Derived Microvesicles' Content and Function.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3785-3785
Author(s):  
Noura Ismail ◽  
Kara Batte ◽  
Leni Moldovan ◽  
Clay Marsh ◽  
Melissa Piper
Keyword(s):  
Flow Cytometry ◽  
Surface Antigen ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Antigen Expression ◽  
Mononuclear Phagocytes ◽  
Gm Csf ◽  
Wide Range ◽  
Surface Antigen Expression ◽  
And Function

Abstract Abstract 3785 Microvesicles (MVs) are small membrane-bound vesicles released under normal homeostatic and stimulatory conditions by a wide variety of cell types. Microvesicles are collectively referred to as exosomes and microparticles which vary in size due to different cellular mechanisms responsible for their production. These microvesicles have a wide range of functions from facilitating communication to regulating cellular growth and differentiation. During their production; microvesicles become enriched in various molecules including proteins and nucleic acids. Previously, we have shown that plasma microvesicles derived from many cell lineages contain microRNAs (miRNAs). We also found that the majority of the peripheral blood microvesicles are derived from platelets while those originating from monocytic cells including macrophages represent the second largest population. Since microvesicles derived from mononuclear phagocytes are a large subpopulation in the plasma; we were interested in understanding their content and function. We hypothesized that the content and/or quantity of macrophage-derived microvesicles could induce the maturation of monocytes. To address our hypothesis, peripheral blood monocytes were treated in vitro for 4hr with GM-CSF; washed and cultured in media devoid of cytokines for 24 h then microvesicles were collected. Flow cytometry and electron)confocal microscropy were used to quantify and visualize microvesicles production. To examine the function of the microvesicles on macrophage maturation, the purified microvesicles were then cultured with freshly isolated monocytes. Macrophage differentiation was determined by cellular adherence using a crystal violet uptake assay and changes in surface antigen expression by flow cytometry. We also examined the genetic changes induced in monocytes incubated with the microvesicles compared to GM-CSF-treated cells. We found that freshly isolated monocytes treated with microvesicles from macrophages acquired phenotypic characteristics of a macrophage such as cellular adherence and surface antigen expression. We also found that treatment of naïve monocytes with the microvesicles induced molecular changes similar to GM-CSF treated monocytes. We found more than 7985 mRNAs that were similarly expressed between the two culture conditions. Notably, we observed the unique expression of 1324 and 1079 genes in the GM-CSF-treated compared to the microvesicle-treated cells, respectively. To begin dissecting the molecules contained in the microvesicles responsible for these changes, we performed mass-spectrometry and miRNA profiling. We observed the expression of miRs-223, -222,-191, -484, -016, -026a, and -155 in GM-CSF-derived microvesicles. Notably, these miRNAs were also expressed in the cells from which the microvesicles were released. We have begun bioinformatics analyses to predict whether the expression of the miRNAs may account for the decrease expression of specific genes in cells treated with the microvesicles that undergo differentiation. Many of the proteins found in the vesicles are important in facilitating protein:protein interactions and nucleic acid binding. Based on our observations; we postulate that microvesicles in areas of inflammation may contribute to the inflammatory response through the maturation of immune cells and activation of cells responsible for tissue repair. Thus, defining key components of this response may identify targets to regulate inflammation. Disclosures: No relevant conflicts of interest to declare.

A Case of Clopidogrel Induced TTP Recurring After Four Years

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4678-4678
Author(s):  
Nanda K. Methuku ◽  
Abhinav B. Chandra ◽  
Anuradha Belur ◽  
Lech Dabrowski
Keyword(s):  
Platelet Count ◽  
Renal Insufficiency ◽  
Peripheral Blood ◽  
Blood Smear ◽  
Reticulocyte Count ◽  
Conflicts Of Interest ◽  
Peripheral Blood Smear ◽  
Severe Thrombocytopenia ◽  
Platelet Counts ◽  
Multiple Organs

Abstract Abstract 4678 Case description - A 61 year old woman was started on clopidogrel after having PTCA with stent placement in February 2006. Four weeks after starting clopidogrel she developed thrombocytopenia with platelet nadir of 17,000. Her LDH was 700 IU/L and she was anemic with hemoglobin of 7.4 gm/dl with elevated reticulocyte count. Peripheral blood smear showed schistocytes and diagnosis of TTP secondary to clopidogrel was made. She did not have renal insufficiency. Clopidogrel was discontinued and patient was started on plasmapheresis with recovery of platelet counts. Early attempts in weaning plasmapheresis resulted in drop in platelet count and Rituximab was given to the patient weekly for four weeks. Subsequently, patient was weaned off plasmapheresis. For four years patient was followed periodically with CBC showing platelet counts greater than 250,000. In May 2010, four years after initial event patient was admitted to hospital for abdominal pain and found to have splenic infarcts. Subsequently, she also developed bilateral cerebral infarcts. Platelet count had decreased to less than 100,000. Her LDH was elevated at 419 IU/L. Reticulocyte count was 2.3%. Peripheral blood smear revealed significant number of schistocytes. There was no renal insufficiency or fever. Trans-esophageal echocardiogram (TEE) was done that did not reveal any vegetations. Patient was diagnosed as having recurrence of TTP and started on plasmapheresis with recovery in platelet counts. Pt was also treated with Rituximab. Discussion- We describe a case of TTP initially occurring within weeks of starting clopidogrel. Patient was treated with plasmapheresis and Rituximab and clopidogrel was discontinued. Patient had recurrence after four years as manifested by infarcts in multiple organs, with mild thrombocytopenia, elevated LDH and significant number of schistocytes on peripheral blood smear. It is very uncommon for clopidogrel associated TTP to recur after such a prolonged period of 4 years. Most cases of clopidogrel associated TTP have mild thrombocytopenia. This patient had severe thrombocytopenia on first presentation of TTP but had mild thrombocytopenia on recurrence. This case illustrates the importance of extended follow up and high index of suspicion for TTP as delays in initiation of plasmapheresis has a poor clinical outcome. Disclosures: No relevant conflicts of interest to declare.

Dynamic Changes in Platelet Responsiveness to Thrombin and Coated Platelet Potential May Inform Risk of Hemorrhage and/or Thrombosis in a Novel Canine Mode of Immune Thrombocytopenia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2193-2193
Author(s):  
Marshall A. Mazepa ◽  
Dana N LeVine ◽  
Adam J Birkenheuer ◽  
Marjory B Brooks ◽  
Shila K Nordone ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Exploratory Study ◽  
Immune Thrombocytopenia ◽  
Treated Group ◽  
Canine Model ◽  
Severe Thrombocytopenia ◽  
Dynamic Changes ◽  
Platelet Recovery ◽  
Time Zero

Abstract Abstract 2193 In both canine and human patients with Immune Thrombocytopenia (ITP), bleeding risk is challenging to predict, and potentially leads to over-treatment of patients at low risk. Conversely, recent studies have highlighted the risk of thrombosis in ITP during platelet recovery. Given these clinical observations, we hypothesized that in ITP, changes in platelet response to agonists may occur in addition to changes in platelet numbers. In response to dual agonist activation (thrombin and convulxin), a subpopulation of platelets in both humans and dogs develops enhanced procoagulant activity. This subpopulation is termed coated platelets, and differences in individuals' potential to form coated platelets have been correlated with both hemorrhagic and thrombotic outcomes. In this exploratory study, we serially evaluated ex vivo platelet responsiveness to both thrombin and dual agonists (termed coated platelet potential) in a novel canine model of ITP. Dogs (n=4) were infused with a murine monoclonal anti-GPIIb antibody (2F9) in order to model ITP and generate predictable severe thrombocytopenia. Control dogs (n=3) were infused with a control antibody. Platelet count, thrombin responsiveness, and coated platelet potential were measured at baseline, time zero, 6 hours, 24 hours, and every 24hrs thereafter until the platelet count was ≥ baseline for at least two consecutive measures (recovery). Time zero was defined as the time when platelet count first fell to ≤ 30,000/μl following 2F9 infusion, or 1 hour following control antibody infusion. For platelet thrombin responsiveness, a monoclonal antibody to P-selectin was used to determine platelet P-selectin surface expression by flow cytometry after stimulation with graded doses of thrombin. The ED50 Thrombin was defined as the concentration of thrombin required for half-maximal P-selectin expression. Coated platelet potential was defined as the percent of platelets activated to the highly procoagulant state after dual stimulation with thrombin and convulxin, as determined by binding of biotinylated fibrinogen by platelets by flow cytometry. All dogs in the treated group developed severe thrombocytopenia (median=6×103, range=4–11×103 platelets/uL); no dogs in the control group developed thrombocytopenia. All treated dogs had platelet recovery by 240 hours (median=132 hours, range 120–240hours). Of interest, at 6 hours, ED50 Thrombin in the treated group increased nearly twofold (fig 1A) (ratio of median ED50 Thrombin treated/baseline=1.6, range 1.3–2.3), which correlated with a decline in coated platelet potential by nearly half of baseline (fig 1B) (median 52.4% of baseline, range 19.6–61.5%); minimal change from baseline was observed in controls. In both groups, ED50 Thrombin was lower at recovery than baseline (fig 1A) (treated median ED50 Thrombin=71.5% of baseline; control median ED50 Thrombin=67% of baseline). A trend of rising coated platelet potential was also noted as platelets recovered in the treated group. In conclusion, in this exploratory study of a canine model of ITP, we observed dynamic changes in platelet responsiveness. During severe thrombocytopenia, we observed a rise in ED50, indicating a decline in response to thrombin, which correlated with a fall in coated platelet potential. We speculate that this early fall in platelet thrombin response and coated platelet potential could contribute to hemorrhage risk in ITP. As a complement to this finding, in the treated group, there was a rise in coated platelet potential as platelets rebounded and coated platelet potential was slightly greater than baseline at recovery. This is consistent with others' observation that younger platelets are more likely to have coated platelet potential. We also observed a decline in ED50 Thrombin at recovery, not only in the treated dogs, but also control dogs. Thus, at recovery, the decline in ED50 Thrombin was independent of treatment group. However, this may be an artifact of our small sample size. Our observed increase in coated platelet potential during platelet recovery could potentially contribute to the thrombotic tendency of some ITP patients. Future studies are planned to explore the relationship of hemorrhagic and thrombotic risk with platelet thrombin responsiveness and coated platelet potential in this model of ITP and clinical studies of canine and human ITP. Disclosures: No relevant conflicts of interest to declare.

Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2030-2030
Author(s):  
Nishant Tageja ◽  
Neha Korde ◽  
Constance Yuan ◽  
Kristen Cole ◽  
Jennifer Hsu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
High Risk ◽  
Tumor Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cell ◽  
Myeloma Cells ◽  
Cd34 Cells ◽  
The Mean

Abstract Background Regimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. Methods Thirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient’s weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. Results The median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. Conclusions Modern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures: No relevant conflicts of interest to declare.

Value of Platelet Esoteric Testing in Laboratory Diagnosis of Platelet Disorders: A Single Center Experience

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1061-1061 ◽  
Author(s):  
Juliana Perez Botero ◽  
Deepti M. Warad ◽  
Rong He ◽  
Rajiv K. Pruthi ◽  
Dong Chen
Keyword(s):  
Flow Cytometry ◽  
Assessment Tool ◽  
Light Transmission ◽  
Conflicts Of Interest ◽  
Postoperative Bleeding ◽  
Retrospective Chart Review ◽  
Dense Granule ◽  
Platelet Glycoprotein ◽  
Molecular Testing ◽  
Platelet Disorders

Introduction Patients with inherited and acquired platelet disorders (PD) frequently present with thrombocytopenia and/or mucocutaneous bleeding. Diagnosis of PDs relies on both clinical and laboratory investigation, which encompasses both routine and esoteric platelet testing. The former includes platelet indices, light transmission aggregation (LTA) and platelet function analyzer (PFA-100). The latter includes platelet glycoprotein (GP) assessment by flow cytometry and platelet transmission electron microscopy (PTEM). Several recent studies have demonstrated limited sensitivities of routine platelet testing. It is uncertain if a combination of both routine and esoteric testing can vastly improve the sensitivity. Our goal is to assess the value of platelet esoteric testing and the ultimate sensitivity of laboratory platelet testing in diagnosing PDs. Methods Patients (between year 2012 and 2015) with a known or suspected inherited or acquired platelet disorders were recruited for platelet routine and esoteric testing. Patients' clinical features including ISTH bleeding assessment tool (BAT) scores, platelet count, LTA, PFA-100 ADP (CADP) and epinephrine (CEPI) cartridge closure times, GP expression by flow cytometry and PTEM were performed. All clinical and laboratory variables were collected and statistically analyzed. Results A total of 69 patients (51 females; median age 40 years; range: 8 months to 76 years) were enrolled to this cohort study. By retrospective chart review, 34 patients (49%) had a positive ISTH-BAT score (range 0 to 16). The most common bleeding manifestations (on any degree of severity) were cutaneous bleeding present in 53% of patients (n=37), menorrhagia in 47% of females (n=24), epistaxis in 36% (n=25) and postoperative bleeding in 35% (n=24). Joint, muscle and CNS hemorrhage were the least frequent with 7%, 6% and 1% of patients reporting bleeding in these sites. A total of 22 patients (32%) had thrombocytopenia at the time of their evaluation. Fifty-eight patients (84%) had platelet aggregation studies and of those 15 (26%) had an abnormal study. PFA-100 was performed in 57 patients (83%) of whom 42% (n=24) had prolonged closure times with either ADP or epinephrine. PTEM was performed on all samples. Twenty-seven patients (39%) had abnormal platelet structure including 17 patients who had dense granule deficiency. GP by flow cytometry was performed in 41 patients (59%) and abnormal glycoprotein expression was detected in 6 patients (15%). Of the 55 patients who had all tests performed, 22 patients (40%) had completely normal results. Of the routine tests, only PFA-CEPI or -CADP prolongation is associated with the likelihood of a PTEM abnormality (P<0.005). With the assistance of molecular testing, there were 2 cases of MYH-9 mutation-related platelet disorders, one of RUNX1 mutation-associated thrombocytopenia, one of York platelet syndrome, two of Bernard Soulier variants, one of Quebec syndrome and three of gray platelet syndrome or variants. Bleeding scores showed no statistically significant association with severity of any laboratory abnormalities. Conclusion In this cohort, the sensitivity of routine platelet testing in clinically suspected PDs is about 30-40%. Addition of platelet esoteric testing improves the sensitivity to 60%. However, the degree of platelet laboratory abnormalities do not predict severity of bleeding. The findings of this cohort underscore the importance of clinical assessment and both platelet routine and esoteric testing in investigation of patients with suspected PDs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Intracranial Hemorrhage in Egyptian Children with Primary Immune Thrombocytopenia (ITP): Report of 24 Cases in 20 Years

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1080-1080
Author(s):  
Mohsen Saleh Elalfy ◽  
Mohamed Ahmed Badr ◽  
Ahmed Mansour ◽  
Tamer Hassan ◽  
Mohamed Meabed ◽  
...  
Keyword(s):  
High Risk ◽  
Head Trauma ◽  
Intracranial Hemorrhage ◽  
Immune Thrombocytopenia ◽  
Conflicts Of Interest ◽  
Complete Recovery ◽  
Published Data ◽  
Severe Thrombocytopenia ◽  
Egyptian Children ◽  
Neurosurgical Intervention

Background: 35 million children < 18 years; approximately 1800 new cases of ITP were diagnosed annually in Egypt. Intracranial hemorrhage (ICH) is a rare devastating complication of childhood immune thrombocytopenia (ITP). Incidence of ICH among children with ITP varies markedly in different studies from 0.2 up to 1.0 %. Intracranial hemorrhage after head trauma in children with ITP leads to significant morbidity and mortality. We published data during 1997 - 2007 (10 ICH) in children with primary ITP; risk factors and outcome. Aim & Methodology: A follow-up study to assess any change in outcome of ICH from last decade; whether new therapies might change the landscape of ICH. Centers treating > 25 child with prim ITP /year and offered a complete data of >150 children with ITP over 2 decades were enrolled. We compared 2008 - 2018 with the decade before it; variation of ICH reporting from center to center and outcome in relation to therapy. All children with ITP and ICH during study period had been treated, within < 24 hours and referred to neurosurgical hospital complex facility for consultation and intervention. Time elapsed till receiving platelet enhancing therapy and neurosurgical intervention was assessed, Outcome whether a complete recovery, permanent sequalae or death was reported. Results: Four thousand, three-hundred and forty primary ITP were evaluated, ( 380 were excluded due to incomplete data ) Twenty-four (0.6%) ICH were reported over 2 decades (14 in this decade with 20% increase incidence) and 48 matched ITP control subjects were evaluated. Platelet counts were less than 10 x 10(9)/L in 90% of children with ICH. Four (16.2%) children developed ICH within 14 days of diagnosis of ITP; one of these, was the presenting feature of ITP. four were from 3-12 months and sixteen (66.6%) of children had chronic ITP. Centers treating > 50 case/year had a higher frequency of reporting 0.8 % compared to 0.2 % in centers < 50 cases/year. Outcome is better on early intervention as well as aggressive platelet enhancing therapy with 70% complete recovery compared with 30% complete recovery on delayed intervention. Head trauma and hematuria and PC < 10 were the mostly associated with ICH, identified in 33%, 25% and 90% respectively of the patients with ICH and in 1, none and 50% of the controls (P < .001). Bleeding beyond petechiae and ecchymoses was also linked to ICH. Mortality was 25%; a further 25% had neurologic sequelae. Neurosurgical intervention was done in 25% of cases with good outcome. Reporting was more in this decade with better outcome in bigger centers. Conclusion: A rise in the incidence of ICH in Children with severe thrombocytopenia over last decade; high risk for ICH among those with PC< 10,000 plus head trauma and/or hematuria. Platelet enhancing agents whether HDMP or IVIG or TPO-RAs could not prevent ICH. However they had a good impact on survival and lessen sequalae if used in combination. Strategies by which high-risk children could be identified and well managed in small centers. Disclosures No relevant conflicts of interest to declare.

Family-Associated Monoclonal B Lymphocytosis Shows Differences From CLL That Suggest An Indolent Biology.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1241-1241
Author(s):  
Mark C Lanasa ◽  
Sallie D Allgood ◽  
Susan L Slager ◽  
Nicola J Camp ◽  
Neil E. Kay ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Surface Expression ◽  
Cellular Activation ◽  
Immunoglobulin Isotype ◽  
Cell Counts ◽  
Intracellular Expression ◽  
Trisomy 12

Abstract Abstract 1241 Poster Board I-263 Background and Significance Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts >1.9 × 109 / L, progress to CLL, “low count” MBL (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL. Methods Individuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL. Results Fifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IGVH genes were 3-07 (3 MBL clones), 3-15 (3), and 4-34 (3). No VH1 family gene rearrangements were observed. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12. Conclusions Our data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL. Disclosures No relevant conflicts of interest to declare.

Neutrophil Platelet Satellitism Revisited: Sidedness and Domain of Neutrophil Associated Platelet Aggregates.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4469-4469
Author(s):  
Cannon Milani ◽  
Fred J. Schiffman ◽  
Peter J. Quesenberry
Keyword(s):  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Incidental Finding ◽  
Natural Antibodies ◽  
Polymorphonuclear Neutrophils ◽  
Platelet Glycoprotein ◽  
Clear Correlation ◽  
Platelet Aggregates ◽  
Platelet Satellitism

Abstract Abstract 4469 Platelet Satellitism surrounding polymorphonuclear neutrophils has been observed almost exclusively in EDTA-treated blood at room temperature. The mechanism underlying this phenomenon is not fully understood. In a PubMed search of the English language medical literature, there are only 44 reported cases involving the phenomenon of Platelet Satellitism. We report a case of platelet rosetting around neutrophils in a 78-year old woman with incidental thrombocytopenia. Her isolated thrombocytopenia was not mediated by any form of immunosuppression, medications, hypersplenism, intravascular consumption, or diminished platelet production. A diagnosis of Pseudothrombocytopenia was made based upon her peripheral blood smear revealing platelet aggregates displaying sidedness in relation to her neutrophils. Platelet Satellitism is postulated to represent an immunologic phenomenon caused by the presence of natural antibodies in which the platelets aggregate around polymorphonuclear neutrophils. The reasons behind why certain individuals possess agglutinating antibodies that lead to platelet clumping, and others have antibodies that cause platelet satellitism is unknown. A proposed mechanism is natural antibodies directed against different epitopes on the platelet GPIIb-IIIa complex. The reported frequency of platelet Satellitism is much lower than that of EDTA platelet clumping (approximately 1:30,000 blood counts) according to the reviewed literature. Platelet Satellitism to polymorphonuclear neutrophils was initially documented by Field and MacLeod in 1963, and has since been reported as an incidental finding in peripheral blood smears when EDTA was used as an anticoagulant. The process by which platelets bind and form rosettes around polymorphonuclear leukocytes is due to activation of EDTA-dependent antiplatelet and antineutrophil IgG autoantibodies directed against the platelet glycoprotein IIb/IIIa complex and Fc receptors of neutrophils. Further, it is theorized that a non-immunologic cause may play in role in which adherence is induced by thrombospondin or the alpha-granule protein of other platelets. In rare instances, platelets may aggregate around monocytes or basophils. Our retrospective review underscores the importance of recognizing the principle of Pseudothrombocytopenia due to EDTA-induced Platelet Satellitism. This entity is in vitro phenomena which has no clinical bearing in terms of a predisposition to increased mucous membrane bleeding. As in other literature cases, a clear correlation between the presence of IgG antibodies and a specific clinical situation, disease, or use of drugs could not be demonstrated. Therefore, these antibodies, which are present in some normal individuals, might occur naturally. Due to the exposure of certain antigenic structures present on EDTA-modified platelets and neutrophils, they may manifest themselves by triggering the Platelet Satellitism phenomenon. Disclosures: No relevant conflicts of interest to declare.

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