Sysmex Corporation Research Articles

Basic hemostatic parameters in adults with sickle cell anemia at Ahmadu Bello University Teaching Hospital, Zaria Nigeria

Background: Reports from various studies showed that patients with sickle cell anemia (SCA) have altered components of hemostasis such as platelet function, the procoagulant, anticoagulant, and the fibrinolytic pathways. These altered components may be different or the same based on regions, racial, or environment. Determining the basic hemostatic parameters in our environment is imperative because from records this may be the first time such a study is being carried out and the altered components so involved may give an insight as to the clinical phenotypes we have in our setting. The data obtained from this study may also provide reference values appropriate for therapeutic intervention. The objective of this study is to determine some basic hemostatic parameters in patients with SCA in steady state attending the hematology clinic of Ahmadu Bello University Teaching Hospital Zaria, Nigeria. Materials and Methods: We conducted a case–control study involving fifty patients with SCA (HbSS) in steady state and 25 healthy volunteers with normal hemoglobin (HbAA) as controls between the ages of 15 and 50 years, with females comprising 40 (53.0%) and 35 (47.0%) males. Steady state refers to a point in time where a patient with SCA is not experiencing an acute painful crisis or any changes due to therapy for at least four consecutive weeks after a previous painful crisis. Platelet count was determined by hematology analyzer (Sysmex XT–2000i, Sysmex Corporation, CPO Box1002 Kobe 650–8691, Japan) while bleeding time (BT) was performed using Ivy's method, using disposable Bevel lancet with the test carried out in duplicate and superficial veins were avoided. Prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were estimated using semi-automated coagulation analyzer. Results: The age ranges of both the patients and the controls were 15–50 years and 15–34 years with mean ages of both was 23.80 ± 7.46 and 24.28 ± 3.48 years (P = 0.76) respectively. Females comprised 53.0% of all the study participants. The mean values of hemostatic parameters in the SCA and controls groups were: Platelets (499.82 ± 208.23 vs. 230.36 ± 106.65 × 109/L, 95% confidence interval [CI]; 180.9881, 357.9319, P

Reference intervals for hematology test parameters from apparently healthy individuals in southwest Ethiopia.

Background:Clinical laboratory reference intervals are an important tool to identify abnormal laboratory test results. The generating of hematological parameters reference intervals for local population is very crucial to improve quality of health care, which otherwise may lead to unnecessary expenditure or denying care for the needy. There are no well-established reference intervals for hematological parameters in southwest Ethiopia.Objective:To generate hematological parameters reference intervals for apparently healthy individuals in southwest Ethiopia.Methods:A community-based cross-sectional study was conducted involving 883 individuals from March to May 2017. Four milliliter of blood sample was collected and transported to Jimma University Medical Center Laboratory for hematological analysis and screening tests. A hematological parameters were measured by Sysmex XS-500i hematology analyzer (Sysmex Corporation Kobe, Japan). The data were analyzed by SPSS version 20 statistical software. The non-parametric independent Kruskal–Wallis test and Wilcoxon rank-sum test (Mann–Whitney U test) were used to compare the parameters between age groups and genders. The 97.5 percentile and 2.5 percentile were the upper and lower reference limit for the population.Results:The reference interval of red blood cell, white blood cell, and platelet count in children were 4.99 × 1012/L (4.26–5.99 × 1012/L), 7.04 × 109/L (4.00–11.67 × 109/L), and 324.00 × 109/L (188.00–463.50 × 109/L), respectively. The reference interval of red blood cell, white blood cell, and platelet count in adults was 5.19 × 1012/L (4.08–6.33 × 1012/L), 6.35 × 109/L (3.28–11.22 × 109/L), and 282.00 × 109/L (172.50–415.25 × 109/L), respectively. The reference interval of red blood cell, white blood cell, and platelet count in geriatrics were 5.02 × 1012/L (4.21–5.87 × 1012/L), 6.21 × 109/L (3.33–10.03 × 109/L), and 265.50 × 109/L (165.53–418.80 × 109/L), respectively. Most of the hematological parameters showed significant differences across all age groups.Conclusion:Most of the hematological parameters in this study showed differences from similar studies done in the country. This study provided population-specific hematological reference interval for southwest Ethiopians. Reference intervals should also be established in the other regions of the country.

Performance evaluation of the white cell precursor channel on the Sysmex XN hematology analyzer in HIV specimens.

Automated hematology analyzers generate white blood cell (WBC) suspect flags to indicate smears requiring manual review. In a human immunodeficiency virus (HIV) patient population with a high frequency of abnormal specimens, the usefulness of these flags depends on their sensitivity and specificity. The white cell precursor (WPC) channel on the Sysmex XN-9000 analyzer (Sysmex Corporation, Kobe, Japan) has recently been introduced in order to distinguish immature cells such as blasts and abnormal lymphocytes. The efficiency of the flags generated by WPC reflex testing was compared to the white cell differential (WDF) on the Sysmex XN-9000 in 400 routine HIV samples. A flag was generated by the WDF channel in 346 (86.50%) samples. Addition of the WPC to the WDF resulted in a 20.99% reduction in the smear review rate. WPC analysis correctly identified the initial WDF flag in 59 (60.82%) samples with 'blasts?' or 'abnormal lympho?' flags. WPC showed an excellent sensitivity of 100%. However, the specificity remained poor. On investigation, samples with chronic infection/inflammation (35.00%) and lymphopenia (63.25%) were associated with a high false positive rate of 76.32% and 55.56%, respectively. WPC analysis removed 45 (51.72%) coexisting 'atypical lympho?' false positive WDF flags. The 'atypical lympho?' flag showed an increased sensitivity and specificity of 88.46% and 74.67%, respectively. White cell precursor reflex testing showed improved flagging efficiency for the 'atypical lympho?' flag, which was a good indicator of reactive lymphocytes during chronic infection/inflammation. Nevertheless, the false positive rate for 'abnormal lympho/blasts?' was high in the presence of lymphopenia and infection.

A Novel Clot Waveform Analysis to Measure the Plasma Coagulation Potency in the Presence of Anti-Fixa/Fx Bispecific Antibody, Emicizumab

Emicizumab (also termed ACE910) is a humanized anti-factor (F)IXa/FX bispecific antibody with FVIIIa cofactor function. A clinical phase 3 study was initiated in 2015 for hemophilia A patients (HA-pts) with FVIII inhibitors. Since emicizumab, unlike FVIII, does not require activation by thrombin, its APTT-shortening effect is much greater than that of FVIII. Thus, APTT, a conventional assay to assess whole coagulation potency, would have limited utility in emicizumab-administered HA-pts, because emicizumab would mask the effect of residual FVIII or a FVIII agent on APTT. Clot waveform analysis (CWA) can provide multidimensional coagulation potencies by monitoring the process of plasma clot formation with an automated coagulation analyzer. We considered the possibility, therefore, that this assay system would overcome the above issue on APTT. In this study, we aimed to optimize concentrations of tissue factor (TF) and ellagic acid (Elg) in a trigger reagent for CWA as well as CWA parameters to provide precise evaluation of coagulation potency even in the presence of emicizumab with neither masking nor being masked by FVIII or bypassing agents. First, we determined an optimal concentration of TF/Elg trigger reagent. Various concentrations (10, 30, 100, and 300 μg/mL) of emicizumab were spiked into commercially available FVIII-deficient plasmas (George King) for testing. Recombinant (r)FVIII (Kogenate FS; Bayer)-spiked samples were also tested as a reference. PT reagent (under development; Sysmex) and APTT reagent (Thrombocheck APTT-SLA; Sysmex), used as a source of TF and Elg, respectively, were mixed in various ratios. The optimized mixture ratio (PT:APTT:buffer=1:15:135) was chosen to ensure that the maximum coagulation velocity (|min1|) in the presence of emicizumab would be in agreement with the animal study-based estimated conversion rate "0.2-0.4 IU/dL of equivalent FVIII per 1 μg/mL of emicizumab" (Muto. J Thromb Haemost. 2014). When evaluating several lots of FVIII-deficient plasmas, however, we observed large variations in transmittance depending on fibrinogen concentration of each plasma, which resulted in large variations of |min1| between donor plasmas. To decrease the bias due to fibrinogen concentration, % transmittance of clot waveform (CW) was adjusted to 100% and 0% at the pre- and post-coagulation phase, respectively. By using |min1| from the adjusted CW (adjusted-|min1|), we successfully reduced the inter-donor variations and chose it as a main parameter. Next, we evaluated adjusted-|min1| using plasmas from HA-pts without inhibitors (severe; n=2, moderate; n=2) and HA-pts with inhibitors (<10 BU/mL; n=2, >10 BU/mL; n=2) by adding emicizumab (30, 100, and 300 μg/mL) in vitro. After the addition of emicizumab, concentration-dependent increases in adjusted-|min1| were observed in all plasmas with rather small individual variations. Finally, we examined whether adjusted-|min1| reflected the effects of FVIII or bypassing agent that was added to plasmas containing emicizumab. Additive effects of a plasma-derived FVIII agent (CROSS EIGHT M; Japan Blood Products Organization), a rFVIII agent (ADVATE; Baxalta) and activated prothrombin complex concentrate (FEIBA; Baxalta) were confirmed by the increase of adjusted-|min1|. As for rFVIIa agent (NovoSeven; Novo Nordisk), its additive effect on adjusted-|min1| was not clear enough in this assay condition, but its additive effects were confirmed by the clot time. In conclusion, we established the Elg/TF-triggered CWA assay condition and parameters for measuring coagulation potency in plasmas from HA-pts even in the presence of emicizumab and a FVIII/bypassing agent without masking each other. DisclosuresNogami:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Matsumoto:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties, Research Funding. Tabuchi:Sysmex Corporation: Employment, Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Soeda:Sysmex Corporation: Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Employment, Patents & Royalties. Arai:Sysmex Corporation: Employment. Kitazawa:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties; Sysmex Corporation: Patents & Royalties. Takaoka:Sysmex Corporation: Employment. Hattori:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties. Shima:F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Sysmex Corporation: Patents & Royalties, Research Funding.

Usefulness of Reticulocyte Hemoglobin Equivalent for Diagnosis of Iron Deficiency

▪Background: Anemia is a significant worldwide health problem, and approximately 30% of world people suffer from anemia, the half of which is iron deficiency (ID). The diagnosis of anemia requires the confirmation of a decrease in hemoglobin (Hb) concentration. For the diagnosis of iron deficiency anemia (IDA), the determinations of serum ferritin and iron related parameters must be necessary even if microcytic hypochromic anemia is confirmed. With recent technological advances, the Hb content of reticulocytes can be quantified by flow cytometry. Reticulocytes exist for 1-2 days in the peripheral blood and its Hb levels might be a good index of ID.There are several markers for the assessment of Hb content in reticulocytes, including reticulocyte Hb equivalent (RET-He) and reticulocyte Hb content (CHr). RET-He, which can be measured in the same sample used for complete blood count tests by the latest automated hematology analyzers, is considered to reflect iron content in reticulocytes. If RET-He is capable of evaluating ID, it must be useful for immediate diagnosis of IDA. Therefore, we evaluated the usefulness of RET-He for determining of ID.Methods: This prospective study was approved by the ethics committee of Asahikawa Medical University (authorization numbers 1356, 1679, and 1356-3). Blood samples were obtained from 211 patients (63 males and 148 females) from 14 to 91 years old. RET-He levels were determined using an automated hematology analyzer (XN-3000® or XE-5000®, Sysmex, Kobe, Japan). Serum iron, total iron binding capacity (TIBC), serum ferritin, and biochemical data were measured using an automated chemical analyzer. Soluble transferrin receptor (sTfR) was measured by an enzyme-linked immunosorbent assay. Anemia was defined as Hb level of <12 g/dL. ID state was defined as serum ferritin level of <12 ng/mL. Patients were classified into four groups which are IDA, ID, control, and anemia without ID groups according to their Hb and serum ferritin levels (Table 1). Laboratory parameters were compared among four groups. The changes of RET-He during oral iron administration were also determined for 21 IDA patients.Results: There were 72 (14 males and 58 females), 28 (12 males and 16 females), 67 (23 males and 44 females), and 44 (14 males and 30 females) patients in the IDA, ID, control, and anemia without ID groups, respectively. As shown in Table 1, The median RET-He levels were 22.3 pg (15.1-35.6 pg), 29.7 pg (19.2-34.9 pg), 34.0 pg (25.9-38.0 pg), and 32.5 pg (19.1-46.3 pg) in the IDA, ID, control, and anemia without ID groups, respectively. Patients in not only IDA but ID groups had significantly lower RET-He levels than those in control group (p < 0.001) while there was no significant difference in RET-He levels between anemia without ID and control. RET-He correlated positively with serum iron (r = 0.654) and transferrin saturation (TSAT) (r = 0.666), and correlated negatively with TIBC (r = -0.617) and sTfR (r = -0.655). There was no correlation between RET-He and serum ferritin when all patients were included in the analysis (r = 0.287); however, analysis of groups according to their iron status revealed a positive correlation between RET-He and serum ferritin in the IDA and ID groups (r = 0.604). The area under the ROC curve (AUC) detecting ID for RET-He was 0.902, whereas AUC for serum iron, TIBC, TSAT, and sTfR were 0.889, 0.879, 0.922 and 0.821, respectively. The cutoff value of RET-He with maximal sensitivity and specificity was 30.9 pg, and the cutoff RET-He value of 28.5 pg had a specificity of >90% (sensitivity, 68%; specificity 91%). Among patients receiving iron treatments, the Hb levels increased in 14 patients, whereas Hb values decreased or did not change in 7 patients. Serum ferritin and RET-He values seemed to change in parallel with changes in Hb levels.Conclusions: In the present study, our data showed the efficacy of RET-He for diagnosis of IDA and the usefulness for monitoring drug iron administration. Because other parameters related to ID such as iron and ferritin should be measured biochemically in serum, it takes a longer time to measure serum iron and ferritin levels when compared with complete blood count tests. We would therefore suggest that measurement of RET-He might be useful to diagnose IDA because its assessment is rapid, fully automated, and can be measured in same sample used for complete blood count test. [Display omitted] DisclosuresToki:Sysmex Corporation: Research Funding. Ikuta:Sysmex Corporation: Research Funding. Yamamoto:Sysmex Corporation: Research Funding. Hatayama:Sysmex Corporation: Research Funding. Shindo:Sysmex Corporation: Research Funding. Fujiya:Sysmex Corporation: Research Funding. Okumura:Sysmex Corporation: Research Funding.

Shift to Anti-Fibrinolysis on the Administration of L-Asparaginase (L-Asp) during Induction Therapy for Pediatric Acute Lymphoblastic Leukemia (ALL)

Introduction: Thromboembolism is a serious complication associated with ALL. The use of central venous catheter and treatment protocols involving corticosteroids and L-Asp is assumed as important thrombogenic factors at the induction phase. In particular, L-Asp has profound effects on hepatic synthesis of pro-, anti-coagulant and fibrinolytic factors. In this study, we hypothesized that change of coagulation and fibrinolytic function contributes to hyper-coagulation condition during the induction phase with L-Asp. In order to clarify this, we evaluated the dynamic change in coagulation and fibrinolysis by simultaneous measurement of both thrombin and plasmin generation assay (T/P-GA). Patients: Twenty-seven pediatric patients with newly diagnosed ALL were enrolled from Aug. 2014 to Oct. 2015 at 3 hospitals in Japan. All cases had no thrombotic predisposition. Eighteen cases (66.7%) (BCP-ALL; n=17, T-ALL; n=1) received Berlin-Frankfürt-Münster (BFM)-95 oriented induction therapy included prednisolone (and dexamethasone for T-ALL), vincristine, daunorubicin, and E.coli L-Asp (a total of 8 doses of 5,000 U/m2). The others (BCP-ALL; n=8, T-ALL; n=1) received Japan Association of childhood Leukemia Study (JACLS) ALL02 oriented induction therapy included prednisolone, dexamethasone, vincristine, daunorubicin, cyclophosphamide and E.coli L-Asp (a total of 6 doses of 6,000 U/m2). Methods: The individual hemostatic parameters were monitored by fibrinogen (Fbg), FDP, AT, TAT and PIC. Additionally, the global functions of coagulation and fibrinolysis were evaluated using T/P-GA established by our group [Matsumoto et al. TH 2013]. This assay was initiated by the addition of a mixture of optimized concentrations of tissue factor and tissue-type plasminogen activator. Thrombin and plasmin generation were monitored simultaneously using individual fluorescent substrates in separate microtiter wells. Standard curves were set using purified alpha-thrombin and plasmin. Patients' plasmas were collected at the following points, T0; pre-phase of L-Asp, T1; intermittent phase of L-Asp, T2; post-phase of L-Asp, and T3; post-induction phase. Endogenous potentials of thrombin generation (T-EP) for coagulant activity and plasmin peak levels (P-Peak) of plasmin generation for fibrinolytic activity were selected as parameters for evaluation in this study. A ratio of T-EP and P-Peak of patients' plasmas to those of control normal plasma were calculated. Results: All cases obtained first remission, and none of them developed coagulopathy. Six cases received FFP transfusion for low Fbg level, whilst 21 cases received AT supplement for low AT level. Fbg showed a median of 170, 99.0, 99.0 and 328 mg/dl at T0, T1, T2 and T3, respectively, whilst the other individual parameters showed relatively unchanged. T-EP revealed a median of 1,126, 1,059, 1,175, 1,343 and 1,132 nM, whilst P-Peak showed a median of 6.67, 4.54, 4.12, 5.50 and 5.77 nM for T0, T1, T2, T3 and control plasma, respectively, indicating the elevated T-EP ratios and reduced P-Peak ratios (Fig. 1). The most significant difference in both ratios demonstrated a median of 1.5-fold (range, 1.0 to 2.6) at T2, consistent with the lowest Fbg levels. The FFP transfusion group showed significantly lower T-EP ratios than non-transfusion group at T1 (a median of 0.87 vs. 1.01, P=0.041) and T2 (a median of 0.96 vs. 1.07, P=0.009), whilst P-Peak ratios revealed no significant changes. The AT supplement group showed no significant changes of both ratios. Conclusion: The results from decreased Fbg and unchanged FDP might reveal the hepatic synthesis disorder of Fbg, whilst the results from T/P-GA showed that their hemostatic dynamics appear likely to be thrombotic tendency, since their coagulation state was hyper-coagulation and anti-fibrinolysis at post-phase of L-Asp. These results suggest that the impaired balance of coagulation and fibrinolysis due to L-Asp therapy might play an important role of a thrombotic complication at induction phase. On the other hand both conventional FFP transfusion and AT supplement therapy might not dramatically repair this unbalance state. A further research would be required to examine the role of coagulant and fibrinolytic function using T/P-GA in the pathogenesis of coagulopathy associated with L-Asp therapy in order to establish the optimal supportive therapy. [Display omitted] DisclosuresNogami:F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Matsumoto:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties, Research Funding. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding.

Activated FVIII Released from FVIII/VWF Complex Facilitates Thrombus Development Under High Shear Flow Condition

Activated FVIII Released from FVIII/VWF Complex Facilitates Thrombus Development Under High Shear Flow Condition

Identification of a Factor X-Interactive Site on the Factor VIII A2 Domain

We have experienced a case of acquired hemophilia A with inhibitor recognizing only a factor (F) VIII A2 epitope, and reported the inhibitory mechanism for disappearing FVIII activity (Blood, 124, 4226, 2014). In summary, the patient’s inhibitor IgG bound to FVIII A2N (residue 372-562) fragment and inhibited Arg372 cleavage in FVIII by FXa, suggesting that FX(a) bound to FVIII A2 domain. ELISA-based assay showed that FVIII A2 fragment bound to FX (Kd; 338 nM). We hypothesized that FVIII A2 residues 400-429 might be FX binding site according to the 3-D model of FVIII molecule, and prepared synthetic peptides (400-409, 409-419, and 420-429). The 400-409 peptide inhibited the FVIII A2-FX interaction, suggesting that the 400-409 region contributed to FX-interactive site. In this current study, we further performed the localization of a FX-interactive site on the 400-409 region in the A2 domain. A purified FXa generation assay demonstrated the 400-409 peptide decreased the generation of FXa in a dose-dependent manner up to 38% of 100 μM (Ki; 23 ± 9 nM). In comparison, scrambled peptide of 400-409 decreased up to 10% of 100 μM. These data demonstrated that the 400-409 peptide inhibited the generated FXa, suggesting the 400-409 region contributed to regulate the coagulation function. Covalent cross-linking was observed between the biotinylated 400-409 peptide and FX following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. This cross-linking formation was blocked by the addition of unlabeled 400-409 peptide. N-terminal sequence analysis of the peptide-FX product demonstrated that two sequential residues (Lys408 and Ser409) could not be detected, supporting that two residues participate in cross-link formation. To confirm the significance of these residues in A2 domain for FX-binding, the mutant forms of the A2 domain, converted to alanine, were expressed in BHK system and purified. Compared with wild type FVIII (Kd; 10 ± 3 nM), the binding affinity of Ser409Ala FVIII mutant for FX was no significant difference (Kd; 14 ± 1 nM) on SPR-based assay. Lys408Ala or Lys408Ala/Ser409Ala double FVIII mutant, however, decreased the binding affinity by 3.6~4.3-fold (Kd; 36 ± 7 or 43 ± 2 nM, respectively), suggesting contribution of Lys408Ala to the binding interaction. For the functional evaluation of the association with FVIII mutants to FX, a FXa generation assay was repeated. Lys408Ala, Ser409Ala, or Lys408Ala/Ser409Ala FVIII mutant reacted with varying concentrations of FX decreased by 1.2~1.6-fold (Km; 53 ± 12, 69 ± 15, or 65 ± 15 nM, respectively) compared to wild type FVIII (Km; 43 ± 9 nM), supporting a contribution of these mutants to Km and overall catalytic efficiency. Vmax values were largely unaffected by the mutations with most values within approximately 30% of the wild-type value. On the other hand, Kcat/Km value of Lys408Ala, Ser409Ala, or Lys408Ala/Ser409Ala FVIII mutant were decreased by 0.5~0.7-fold (Kcat/Km; 1.0, 1.3, or 0.9 nM-1min-1, respectively) compared to wild type FVIII (Kcat/Km; 1.8 nM-1min-1), suggesting low catalytic efficacy of Lys408Ala and Ser409Ala. These results indicate that the 400-409 region in the FVIII A2 domain, and in particular Lys408 and Ser409, may contribute to a unique FX-interactive site. DisclosuresNogami:Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding. Shima:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Mechanism of Tissue Factor (TF) Enhancing Factor (F)VIII Activity on FXa Generation in Initial Phase of Coagulation and Interaction Between TF and FVIII C2 Domain

In the cell-based coagulation model, factor (F)VIIa complex with tissue factor (TF) initiates the blood coagulation by generating FXa as the extrinsic tenase complex and activates FIX which composes the intrinsic tenase complex. We demonstrated that FVIIa/TF directly activated FVIII in an early coagulation phase (Soeda, JTH, 2010), and TF enhanced the intrinsic tenase activity via possible interaction with FVIIIa (Ogiwara, ASH, 2010). In this study, we clarified the enhancing mechanism of intrinsic tenase activity in the TF-related up-regulation of FVIII, and identified the TF-interactive region on FVIII.To explore the enhancing mechanism of TF for FVIII regulation, we performed the FXa generation assay with various amounts of FVIII or thrombin-mediated FVIIIa, constant FIXa (1 nM), FX (300 nM) and phospholipid vesicles (PL; 20 µM) in the presence of recombinant lipidated TF (rTF, Innovin®). The Km value for FVIII in the presence of rTF was ~2.4-fold lower than that its absence (Km; 5.6±0.5, 13.3±3.7 nM, respectively, p<0.05). Similarly, the Km for FVIIIa in the presence of TF was ~1.5-fold lower than that in its absence (Km; 6.3±0.6 nM, 9.7±1.6 nM, respectively, p<0.05), supporting that the presence of TF could promote the FXa-catalyzed activation of FVIII and FVIIIa-dependent generation of intrinsic FXa. To further evaluate the effect of TF on FVIII-dependent FXa generation, the FXa generation assay with FVIIa/TF-activated FVIIIa (FVIIIa-VIIa/TF) was also performed. The initial velocity on FXa generation with FVIIIa-VIIa/TF was 22.6 nM/min. However, the initial velocity on FXa generation with FVIIIa-VIIa/TF by addition of FVIIa-inhibitor (E-76), not to generate FVIIa/TF-dependent FXa, was 3.4 nM/min, and that with FVIII alone was 0.05 nM/min. In addition, the initial velocity with FVIIa/TF alone was 10.4 nM/min. These findings supported that the TF increased FXa generation greater than the additive effect of FVIII-dependent and FVIIa/TF-dependent FXa generation in early initiation phase of coagulation prior to thrombin generation. Since tissue factor pathway inhibitor (TFPI) is present in physiological circulating whole blood, a similar experiment on FXa generation assay was repeated under the presence of TFPI, estimated to be present at 0.5 nM in the pre-coagulant state or at 15 nM in the coagulant state in circulating blood. The initial velocity on FXa generation with FVIIIa-VIIa/TF was reduced by the presence of 0.5 or 15 nM TFPI (17.9 and 12.6 nM/min, respectively). By contrast, the initial velocity on FVIIIa-VIIa/TF-dependent FXa generation with addition of FVIIa inhibitor was little reduced by 0.5 nM TFPI, whilst was reduced by 15 nM TFPI (3.5 and 2.5 nM/min, respectively). These findings supported that the TFPI possibly didn’t inhibit TF on the enhanced intrinsic tenase on association with FVIII in the pre-coagulant state.We further reported that TF enabled FXa to activate FVIII, irrespective of von Willebrand factor (VWF), and the direct association of rTF and non-lipidated TF with FVIII (Furukawa, ISTH, 2015). Since TF is transmembrane protein, however, we performed a surface plasmon resonance (SPR)-based assay (BIAcore®) and solid phase-based ELISA to identify the interactive region(s) on FVIII to recombinant soluble TF (sTF; Altor BioScience), a portion of TF outside of the PL membrane. An SPR-based assay revealed the direct binding of intact FVIII, LCh (a3-A3C1C2, A3C1C2) subunit, C2 domain to immobilized sTF (Kd; 2.3±0.6, 5.8±1.0, 10.5±3.5, 11.8±0.5 nM, respectively). The intact HCh, A1 or A2 domain to sTF failed to bind, however. A non-equilibrium ELISA also revealed that sTF bound to immobilized C2 domain with moderate affinity (Kdapp; 16.9±2.2 nM), and the interaction was dependent on ionic strength and Ca2+. In addition, the presence of VWF significantly competitively inhibited the C2 and sTF binding by ~90% (IC50; 5.7 µg/ml) at the maximal concentration employed, suggesting that the C2 domain-TF interaction could activate FVIII by FXa even in the presence of VWF.We concluded that it might be possible that TF enhanced the FVIII-mediated FXa generation by not only FVIIa but also FXa, additionally this enhancing mechanism might not be suppressed by TFPI in the initiation phase of coagulation. Furthermore, TF might function to FVIII activation, irrespective of presence of VWF, by the binding to C2 domain through the competition with VWF. DisclosuresNogami:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Shima:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Identification of a Protein S-Interactive Site on the Factor VIII C2 Domain

Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL) surface-dependent conversion of FX to FXa by FIXa. On the other hand, protein S (PS) functions as a cofactor of activated protein C that inactivates FVIII(a) and FV(a). We have reported a new regulatory mechanism on coagulation that PS directly impaired the FXase complex by competing the FIXa-FVIIIa interaction (Takeyama, Br J Haematol. 2008;143:409), and identified the PS-interactive site on the FVIII A2 domain (Takeyama, Thromb Haemost. 2009;102:645). However, the contribution of FVIII light chain (LC) to PS-binding has not been determined. In this study, several approaches were employed to assess a PS-FVIII LC interaction. The binding of FVIII LC to active site-modified FIXa (EGR-FIXa) was inhibited by PS dose-dependently (Ki; 4.6 nM) on ELISA. Because FVIII C2 domain has a binding site for FIXa, we examined whether FVIII C2 domain bound to PS. PS bound to FVIII C2 domain (Kdapp; 283 nM) by the ELISA. A SPR-based assay also revealed that FVIII C2 domain bound to PS (Kd; 62 nM). We have reported that a FIXa-interactive site exits on FVIII C2 domain (residues 2228-2240) (Soeda, J Biol Chem. 2009;284:3379). Therefore, we assessed the interaction of the synthetic peptide spanning the residues (2228-2240) with PS. ELISA showed that the peptide bound to PS (Kdapp; 104 µM). SPR-based assay also revealed that the peptide bound to PS (Kd; 31.4 µM), although scrambled peptide failed to bind to PS. Covalent cross-linking was observed between the biotynated 2228-2240 peptide and PS following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) using SDS-PAGE. This cross-linking formation was blocked by the addition of the unlabeled peptide. Furthermore, N-terminal sequence analysis of the peptide-PS product showed that one basic residue (K2239) could not be detected, supporting that this residue participates in cross-link formation. These results indicate that the 2228-2240 residues in the FVIII C2 domain, in particular K2239, may contribute to a PS-interactive site. To inquire further into the interactive region of FVIII C2 domain to PS, we prepared a stably expressed recombinant B-domainless FVIII mutants (E2228A, D2233A, K2236A, and K2239A), which were polar amino acids in the 2228-2240 residues, subsequently compared interaction with PS of the mutants with B-domainless FVIII wild type. To evaluate the binding affinity to PS, we performed SPR-based assay with the wild type FVIII and the FVIII mutants. The assay showed K2239A bound to PS with ~1.9-fold higher Kd value (28.1 nM) than that of wild type (Kd; 14.7 nM), although E2228A, D2233A, or K2236A bound to PS (Kd; 12.7, 16.3, and 10.3 nM, respectively) with no significant difference compared with wild type. In addition, to evaluate the effect of mutants for FXa generation, we assessed FXa generation assay with wild type or K2239A in the presence or absence PS, because K2239 might contribute to a PS interaction of FVIII. After each FVIII (1 nM) was reacted with various concentrations of PS in the presence of PL (20 µM) for 30 minutes, FVIII was activated by thrombin (30 nM), followed by the reaction with FIXa (40 nM) and FX (300 nM) for 1 minute. Although generated FXa with both wild type and K2239A were reduced in the presence of PS dose-dependently, compared with its absence, the inhibition ratio of K2239A was less than that of wild type (18.2% and 31%, respectively in the presence of 500 nM PS). On the other hand, the Km value on FXa generation of K2239A for various concentrations of FIXa was ~1.8-fold higher than that of wild type (5.5±0.9 and 3.1±0.2 nM, respectively), suggesting that K2239 residue, involved in the FIXa-interactive site, might contribute to the inhibition of FVIII function by PS. In conclusion, FVIII C2 domain, in particular K2239, was possible to play an important role of the inhibitory mechanism to FVIII function by PS, due to the binding to PS. DisclosuresShima:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Nogami:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

A New Classification of Mild/Moderate Hemophilia Α Based on a Multi-Dimensional FVIII(a) Functions Evaluated By a Plasma-Based Factor Xa Generation

In mild/moderate hemophilia A (MHA), the real hemostatic potential of their innate factor (F) VIII(a) may not be evaluated by FVIII activity (FVIII:C) obtained in a one-stage clotting assay. We often experience the discrepancies between clinical phenotype and FVIII:C in MHA patients. In order to elucidate the hemostatic potentials of the innate FVIII in MHA patients with a simple method, we here have established a plasma-based FXa generation assay (PB-XaG) to clarify the multi-dimensional functions of FVIII(a) in MHA determined by the interaction with the components of tenase complex consisting of FIXa, FX, and phospholipid (PL). XaG was evaluated in the patients’ plasmas and in the control plasma dilutions consisting of FVIII-deficient plasma and serial dilutions of recombinant FVIII (1-100 IU/dL) by a chromogenic substrate S-2765 together with human plasma-derived (pd)FIXa (0-20 nM), pdFX (0-200 nM), and PL vesicles (0-114 µM) initiated by thrombin (0.01 nM). The interaction between FVIIIa and each component of tenase complex was evaluated by Michaelis-Menten’s kinetic parameters, Vmax and apparent Km (Kmapp). We investigated the PB-XaG in the plasmas obtained from 17 MHA patients, carrying F8 mutations and FVIII:C as shown in the table. The PB-XaG showed that the group A, consisting of the case 1, 2, 3 and 4 with mutations at the residue(s) essential to formation of the inter-domain surface in the FVIII molecule, possessed ~1.8-7.1-fold increases in Kmapp for FIXa (10±0.9, 49±17, 31±6 and 9±3 nM, respectively) compared to FVIII:C-matched control (6.5±1.3 nM), whilst Kmapp for FX and PL in this group were of little difference from that in control, indicative of the reduced affinity for FIXa. The group B, including the case 5, 6, 7, 8 with mutations at the A2 or A3 domain remote from the PL-binding region on the C2 domain (residue 2303-2332) and the case 9 with the mutation at the PL-binding region, demonstrated ~1.7-4.8-fold increases in Kmapp for PL (4.3±2.4, 5.0±1.0, 6.5±1.3, 12±4.0 and 3.0±0.6 µM, respectively) compared to control (2.0±0.4 µM), whilst Kmapp for FIXa and FX in this group were similar to that in control, indicative of the reduced affinity to PL. Noteworthy, the apparent catalytic efficiencies defined as Vmax/Kmapp were 0.033 min-1 for the case 6 and 0.019 min-1 for the case 8 with mutations at the A3 domain, 0.3 or 0.1-fold lower than that for the case 9 (0.19 min-1) with mutation at the PL-binding region. The PB-XaG showed that the group C consisting of the case 10, 11, 12, 13, 14 and 15 demonstrated ~1.6-7.5-fold increases in Kmapp for FX (22±9, 23±9, 24±9, 10±4, 4.6±1.6 and 5.8±3.0 nM, respectively) compared to control (4.5±0.8 nM), whilst Kmapp for FIXa and PL in this group were similar to that in control, indicative of the reduced affinity to FX. Interestingly, the case 16 classified in the group C’ showed enhancing affinity with FX represented by a half decrease of Kmapp for FX compared to FVIII:C-matched control (2.0±0.9 and 4.8±1.5 nM, respectively). By contrast, the case 17 with mutation at the thrombin cleavage site categorized in the group D demonstrated any little XaG initiated by thrombin for all axes of FIXa, FX and PL. Taken together, the function of FVIII(a) in MHA could be evaluated multi-dimensionally and clearly classified into the some types of interaction with FIXa, FX, PL and thrombin by the PB-XaG. Our plasma-based XaG assay would be a useful tool for functional analyses of FVIII(a) to clarify the mechanism(s) of hemostatic heterogeneity in mild/moderate HA. [Display omitted] DisclosuresNogami:Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding. Shima:F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Bleeding Events and Safety Outcomes in Patients with Hemophilia a with Inhibitors: A Prospective, Multicenter, Non-Interventional Study

Bleeding Events and Safety Outcomes in Patients with Hemophilia a with Inhibitors: A Prospective, Multicenter, Non-Interventional Study

Quality Control of Structural Parameters (CELL POPULATION DATA) of Sysmex XN Series

INTRODUCTION:Cell population data (CPD) measured by hematology analyzers (HA) describe morphological parameters of white blood cell (WBC) subpopulations and are reported to be as useful parameters in the screening of several hematological and non-hematological diseases. The Sysmex XN is an HA that uses fluorescence flow cytometry for WBC differential, combining forward scatter (FSC), side scatter (SSC) and fluorescence intensity (SFL) to identify WBC populations. These three measures, along with their Distribution Width (DW), are used to provide CPD of Neutrophils (NE), Lymphocytes (LY) and Monocytes (MO). Recent articles have shown that neutrophils CPD parameters (NE-SSC, NE-SFL) may be useful in the diagnosis of myelodysplastic syndromes [1,2] and sepsis [3]. However, little is known on their analytical quality specification, as the comparability of CPD between instruments and laboratories. The aim of the study was to evaluate the between-analyzers consistency of CPD provided by Sysmex XN comparing the results obtained in five different XN modules installed in the same laboratory. Here we present the results for NE-SSC and NE- SFL, the CPD parameters mainly used to support the diagnosis of suspected sepsis and myelodysplasiaM&MPeripheral blood samples collected in K3EDTA tubes (Becton Dickinson, NJ) in three different days from 30 healthy blood donors (HBD, 10 samples/daily) were analyzed within 30 minutes on each XN module. Additionally, all normal samples analyzed by the lab were selected from the routine workload of one month (November 2015) consisting of a total of 35.500 samples. The inclusion criteria (that led to a selection of 6,702 normal routine samples, NRS) were as follows: no morphological flags, WBC from 4.0 to 10.0 x 109/L; Hgb from 120 to 170 g/L; MCV from 84 to 98 fL, PLT from 150 to 450 x 109/L; RDW-CV < 14.0%; NRBC=0.00 x 109/L; NE from 2.00 to 5.60 x 109/L, LY from 1.50 to 3.50 x 109/L, MO from 0.30 to 0.80 x 109/L, EOS from 0.10 to 0.60 x 109/L, BASO <0.20 x 109/L. Finally, the daily results of XN-Chex control blood (Normal level) were included in the evaluation.For each group of samples (HBD, NRS, XN-Check) we compared the median values of CPD parameter obtained on each XN modules. The between-analyzers consistency of CPD parameter was assessed calculating the percentage of Bias (Bias%) between the median value of each XN module and the overall median value of all the five analyzers.To evaluate the stability over the time of CPD parameters, the same study on 30 HBD, 6,690 NRS and XN-Chex control blood was repeated after two months (January 2016).RESULTSThe median values of NE-SSC and NE-SFL obtained in each group of samples are shown in Table 1. The results highlight a good agreement between analyzers, with a maximum Bias of -3.85%. Interestingly, the median values of NE-SSC and NE-SFL were practically the same in HBD and NRS while, as expected, they were significantly different on XN-Chex (due to the presence of stabilized cells). However, the Bias observed for each module XN on control blood was similar to that observed on fresh blood (HBS and NRS groups). Median values of NE-SSC and NE-SFL were practically the same after two months both in HBD and NRS, proving that CPD parameters are stable over the time. Similar results were obtained on all the other CPD parameters provided by Sysmex XN (data not shown)CONCLUSIONSCPD parameters are stable over the time and their inclusion on the automated QC of the analyzer based on the moving average of normal may be a reliable and inexpensive method to assess the between-analyzers consistency of CPD parameters. Providing the expected values of CPD parameters, XN-Check could be useful for the daily QC, to assess the need of calibration and to improve the inter-laboratory comparability being the Neutrophils the best WBC population to control using XN-Check control blood. The moving average can be used too as a way to monitor the calibration and to decide what to do when the QC materials give a result out of range (moving average).The results of this study show the possibility to calibrate the CPD parameters. The availability of a calibration material also for CPD parameters would guarantee a further improvement of standardization and their use as reportable diagnostic parameters.1. Le Roux G, Vlad A, Eclache V et al. Int J Lab Hematol 2010;32:237-2432. Furundarena JR, Araiz M, Uranga M et al. Int J Lab Hematol 2010;32:360-3663. Park SH, Park CJ, Lee BR et al. Int J Lab Hematol 2014;37 [Display omitted] DisclosuresSimon-Lopez:Sysmex Corporation: Consultancy, Honoraria. Buoro:Sysmex EMEA: Research Funding. Pacioni:DASIT Diagnostica SpA: Employment, Other: Scientific Manager of DASIT (Distributor of Sysmex in Italy). Seghezzi:Sysmex EMEA: Research Funding. Manenti:Sysmex EMEA: Research Funding.

First-Line Dasatinib Treatment of CML-CP Leads to Earlier Achievement of MMR and MR4.5 with High Safety

(INTRODUCTION) Several clinical studies have revealed that dasatinib demonstrated deep and fast responses. We report a phase II study to evaluate the efficacy and safety of dasatinib in patients with newly diagnosed chronic-phase chronic myeloid leukemia (CML-CP) in Japan (IMIDAS PART2 study).(PATIRNTS AND METHODS) Between July 2011 and June 2013, a total of 79 consecutive patients with CML-CP received 100 mg dasatinib daily as a first-line therapy. Treatment was continued until disease progression or until toxicity became unacceptable. The primary end-point was the rate of major molecular response by 12 months. Secondary end-points included the rate of complete cytogenetic response, rate of molecular responses with a 4.5 log reduction (MR4.5) by 12 months, and adverse events. The median age was 62 years, ranging from 27 to 80 years. Patients older than 65 years comprised 41.7% of all patients. Two-thirds of patients (68.4%) were male. Nearly all patients were ECOG performance status 0. Most patients (83.6%) had low and intermediate Sokal scores. The BCR-ABL1 International Scale (BCR-ABL1 IS) in the peripheral blood was measured by the central laboratory center (BML, Tokyo, Japan).(RESULTS) The median BCR-ABL1 IS before therapy was 54.0% (7.8-230.2). Seventy patients (88.6%) received dasatinib therapy for 12 months. The median BCR-ABL1 ISs were 0.25 % at 3 months (range 0.0002-52.2 %, n =77), 0.03 % at 6 months (range not detected-29.2 %, n = 75) and 0.008 % at 12 months (not detected -0.86 %, n = 70). MMR rate was 77.2% (95% CI, 67.9-86.5 %) by 12 months. The rates of CCyR and MR4.5 by 12 months were 88.6% (95% CI; 81.5-95.7 %) and 35.4% (95% CI; 24.8-46.1 %), respectively (Figure 1). Multivariate analysis of MMR or MR4.5 by 12 months showed that female sex (odds ratio 1.1, P = 0.92, odds ratio 1.7, P = 0.35, respectively), low and intermediate Sokal score (odds ratio 0.9, P = 0.90, odds ratio 3.2, P =0.23, respectively), and BCR-ABL1 IS less than 54% at diagnosis (RR =odds ratio 0.8, P = 0.74, odds ratio 0.7, P = 0.55, respectively) were not significantly correlated with MMR by 12 months nor MR4.5 by 12 months (Table 1). However, patients who were more than 62 years old were significantly correlated with MR4.0 and MR4.5 by 12 months (odds ratio 2.8, P =0.04, odds ratio 3.5, P =0.01). Treatment-related all AEs were reported in 98.7% patients (78 of 79). Grade 3/4 non-hematologic AEs were observed in only a few cases. Lymphocytosis (more than 4x 109/L) was observed in 34.1% of patients, which was within grade 2. Only 9 patients withdrew the study because of adverse events (4 patients), ineffectiveness (3 patients), and others (2 patients).(DISCUSSION AND CONCLUSION) The incidence of lymphocyte predominance may depend on a history of previous cytomegalovirus (CMV) infection in CML-CP patients (Leukemia. 2011 25(10): 1587-97). Although the frequency of CMV-positive patients was unknown in this study, that of blood donors in Japan was almost positive in the ages with 60s or older. The median age of CML-CP patients in this study was 62 years. Therefore, we assumed that some immunological effects induced by dasatinib might have improved the clinical efficacy in Japanese CML-CP patients compared to those worldwide. Our phase II study to evaluate the efficacy and safety of dasatinib in patients with newly diagnosed CML-CP in Japan revealed that the first-line dasatinib treatment of CML-CP leads to earlier achievement of MMR and MR4.5 with high safety. Elder age patients (>62 years) were significantly associated with achievement of MR4.5. [Display omitted] [Display omitted] DisclosuresShindo:Sysmex Corporation: Research Funding. Sakamoto:Yakult: Other: Remuneration; Takeda Pharmaceutical: Consultancy.

Percentage of hypochromic erythrocytes and reticulocyte hemoglobin equivalent predictors of response to intravenous iron in hemodialysis patients.

Reticulocyte hemoglobin content and percentage of hypochromic red cells are incorporated into the European best practice guidelines on anemia management in chronic kidney disease. Sysmex XN analyzer (Sysmex Corporation, Kobe, Japan) reports reticulocyte hemoglobin equivalent (Ret-He) and the hypochromic fraction of erythrocytes (%Hypo-He). Our aim was to assess the value of these parameters, in terms of the sensitivity and specificity for detecting functional iron deficiency, in hemodialysis (HD) patients. Forty HD patients in the maintenance phase of erythropoietin therapy were included. Intravenous iron supplementation was interrupted at least 3 weeks before recruitment. Two samples were analyzed for each patient: the baseline after the iron-free period and the second sample after 4 weeks of IV iron administration. Hemogram and biochemical parameters of the iron status were measured. Patients were classified as responders or nonresponders to an iron load; responders had an increase in Hb of at least 10 g/L after iron administration, compared to the baseline. To identify the efficiency of the test for predicting the response to iron administration, receiver operating characteristic analysis (ROC) was performed. According to the established criteria, 21 patients were responders and 19 nonresponders. ROC analysis results: Ret-He area under curve (AUC) was 0.84 (95% CI 0.64-0.93), at cutoff 30.8 pg, sensitivity 78.7%, and specificity 87.2%. % Hypo-He AUC was 0.78 (95% CI 0.64-0.91), at cutoff 2.4%, sensitivity 72.2%, and specificity 88.1%. % Hypo-He and Ret-He are reliable parameters for the study of erythropoiesis status in HD patients.

Particular features of the immune status in microbial eczema and erysipelas patients: a therapeutic method

Goal of the study. To study particular features of the immune status in microbial eczema and erysipelas patients in the process of a complex immune modulating therapy with Xymedone®. Materials and methods. the study involved 45 microbial eczema patients and 31 patients with a combined form of microbial eczema and erysipelas (the study group) and healthy subjects (60 and 50 subjects). All of the subjects underwent immunology examinations of their cell and humoral immunity prior to and after the pathogenetic treatment with Xymedone®. The traditional therapy of a combined course of microbial eczema and erysipelas comprised the following basic treatment: third-generation cephalosporins (ceftriaxone): 1.0 g a day for 10 days; second-generation antihistamine drugs (peroral loratadine 10 mg once a day for 10 days) and ascorbic acid 0.5 g a day for 14 days. topical treatment comprised antiseptic agents (furacilin solution 1:1000) at the acute stage, emulsions and creams (depending on the disease stage): topical mometasone furoate 0.1% twice a day for 14 days. The patients receiving Xymedone were treated in the dose of 0.25 g, one pill tree times a day for 14 days. The patients underwent an immunology study using the Sysmex Corporation XT2000 automated analyzer at the CityLab laboratory centers in the city of Kazan. Key findings. Prior to the pathogenic therapy, a high level of CD4 (T helpers) and reduced level of the immunoregulatory index (T helpers to T suppressors ratio) (IRI) was revealed in the microbial eczema patients. Ig А and Ig G concentrations were reliably increased. According to a study of immunology indices, the pathogenic therapy using Xymedone® normalized the number of peripheral blood leucocytes and reliably increased the IRI (p &lt; 0.001) in 45 microbial eczema patients. Prior to the pathogenic therapy, a reduced level of CD3 and CD4 cells was revealed in patients with a combined form of microbial eczema and erysipelas; the levels of CD8 and NK cells (CD16) were also reduced. Ig А, Ig M and IgG concentrations were reliably reduced. According to a study of immunology indices, the pathogenic therapy using Xymedone® increased the concentration of CD3 and CD4 cells. Conclusion. Xymedone®, an immune modulating drug, administered as a part of a combination therapy of microbial eczema patients and patients with a combined form of microbial eczema and erysipelas is a form of supplementary pathogenic I therapy; the treatment improved the cellular and humoral immunity indices.

Automated Nucleated RBC Measurement Using the Sysmex XE-5000 Hematology Analyzer: Frequency and Clinical Significance of the Nucleated RBCs.

We validated the automatic nucleated RBC (nRBC) count on a Sysmex XE-5000 hematology analyzer (Sysmex Corporation, Kobe, Japan) and then evaluated the frequency of nRBCs in our patient population. We correlated automated nRBC enumeration by the Sysmex XE-5000 hematology analyzer on 463 peripheral blood (PB) samples with the manual nRBC count. Results from 360,504 consecutive blood samples were reviewed to determine the frequency of nRBCs in various patient populations in our hospital. There was a strong correlation between the automated and manual nRBC count (Pearson's r = 0.97). Frequency of nRBCs varied in different patient populations and was significantly higher in the presence of other morphology flags or abnormal CBC parameters. Low-level nRBCs (0.2%-1.3%) were detected in 0.5% of samples with otherwise normal parameters. The automated method offers many advantages for high-throughput laboratories, including faster turnaround time, labor savings, and high reliability. Automated nRBC measurement allowed us to recognize a group of individuals who have low-level circulating nRBCs with otherwise normal CBC parameters. Nucleated RBC levels below 1.5% as detected by the automated count may be present in patients without increased erythropoiesis or a pathologic bone marrow process.

A comparison study of routine coagulation screening tests (PT and APTT) by three automated coagulation analyzers

Background: In recent times, growing coagulation test volume and constricted personnel budgets have enhanced interest in automated coagulation analyzers. Objective: (i) To compare the reliability of routine coagulation test [prothrombin time (PT) and activated partial thromboplastin time (APTT)] using mechanical, photo-optical, and nephelemetric methods by three automated coagulation analyzers. (ii) To evaluate the performance of a newly installed fully automatic coagulation analyzer [STA Compact Max (Stago)] and compare the consistency of its testing results with the confirmed clinical automatic coagulation analyzer at our department (Sysmex and ACL Top). Materials and Methods: Trisodium citrated (3.2%) 60 blood samples, which came to special (coagulation) laboratory with request of PT and APTT (with or without anticoagulant, abnormal, and normal/controls), Transfusion Medicine and Immunohematology Department, Christian Medical College, Vellore, Tamil Nadu, India, were included into study randomly. Sample were run on Sysmex CS2000i (Sysmex Corporation, Japan), photo-optical clot detection; ACL Top (Instrumentation Laboratory, USA), nephelometry clot detection method; and STA Compact Max (Stago, USA), viscosity-based (mechanical) clot detection. Result: Correlation was determined using Bland and Altman analysis, which demonstrated a good agreement between Sysmex CS2000i, ACL Top, and STA Compact Max for PT and APTT. A total of 10 samples (16.7%) with visually observed interferences were identified and tested on all three analyzers and showed good agreement with optical and mechanical methods. Conclusion: This study showed good agreement between newly installed STA Compact Max based on mechanical endpoint detection method with already standardized Sysmex CS 2000i and ACL Top, which work on photo-optical endpoint detection method, for evaluation of screening coagulation tests such as PT and APTT even in case of variables such as partially lysed/icteric samples. Three automated analyzers can be used interchangeably.

Enumeration of Peripheral Blood Hematopoietic Stem Cells Using Automated Hematology Analyzer, Sysmex-XN: Final Resutls of Multicenter Prospective Study

We conducted a multicenter study to evaluate a new enumeration method of hematopoietic progenitor cells (HPC) using an automated hematology analyzer, the XN-series model (Sysmex Corporation, Japan). This method appeared to be very promising in a pilot study in a single institution [Tanosaki R, et al. Int J Lab Hematol 2013]. We herein discuss the final results of our study.PB or PBSC samples were taken from G-CSF- and/or chemo-mobilized subjects. The samples were divided into two tubes, one for an in-house assay where HPC were counted in each institution and one was shipped to a central laboratory (SRL, Inc., Japan) where the CD34+ cell count was examined. Firstly, we examined the frequency of invalid data. Secondly, the correlation between HPC and CD34+ cells was investigated. Thirdly, cut-off HPC values were determined to predict certain CD34+ cell counts, i.e. 20 cells/µL for PB and 1,000 cells/µL for PBSC. Finally, for the clinical application, we estimated values which predicted more than certain CD34+ cell counts with 95% probability by constructing a two-dimensional normal distribution model. This study was approved by the IRB of each institution, and informed consent was obtained from each participant before apheresis.One hundred and nine subjects (46 healthy donors and 63 patients undergoing PBSCT) were registered in 5 institutions, and 954 HPC measurements from 614 samples were performed. Thirteen (4.4%) HPC values in 297 PBSC assays were estimated to be invalid, which was identified by an asterisk automatically applied to the corresponding result. Next, the data of the first assay from each sample where both the HPC and the central laboratory-based CD34+ count were available were extracted, and 470 HPC-CD34+ pairs from 102 subjects (43 healthy donors and 59 patients) were used for further analyses. The median age was 50.5 years (range, 3-78), 60 males and 42 females, and diagnoses were malignant lymphoma 28 patients, multiple myeloma 27, and others. It was revealed that there was one mismatched result in the PBSC samples (arrow in Fig 1B; HPC 35,768/µL, CD34+ 399/µL). When this value was removed, strong correlations between the HPC and CD34+ cell counts were observed in both the PB (r=0.94) and PBSC (r=0.97) samples (Fig 1). Strong correlations were observed also at each institution. The HPC cut-off values to distinguish CD34+ cell counts below or above 20/µL in PB and 1,000/µL in PBSC samples were determined to be 25 (AUC 0.931, Youden Index 0.713) and 921 (AUC 0.946, Youden Index 0.778), respectively, by a ROC analysis (Fig 2). Finally, HPC of 56/µL was determined to be the optimal value for predicting more than 20/µL of CD34+ cells in the PB samples with 95% probability.This study confirmed that there was a strong correlation between the HPC and CD34+ cell count in a larger number of patients with different diagnosis at different institutions. For the PBSC collection, HPC ≥ 25/µL suggested an appropriate timing, and HPC 56/µL indicated a more secure threshold. HPC may also be a useful marker to rapidly estimate the quantity of CD34+ cells in the apheresis product. In cases where the HPC value is invalid, this may be overcome by serially evaluating HPC in the clinical course. In conclusion, HPC may be a good surrogate or an alternative for the CD34+ cell count. Prospective studies are warranted to verify the usefulness of HPC in clinical practice. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immature Platelet Fraction: A Platelet Parameter With Significant Clinical Utility

Mean platelet volume (MPV), platelet distribution width (PDW) and the calculated plateletcrit (PCT) are reported to be useful in thrombocytopenia to assess platelet regeneration in the marrow. Our hypothesis was that the immature platelet fraction (IPF) is the most significant platelet parameter in patients with thrombocytopenia. Seven hundred twenty-two consecutive CBCs were analyzed on the Sysmex XN1000 (Sysmex Corporation, Kobe, Japan) using the PLT-F channel. …