Performance evaluation of a novel reticulocyte identification method that uses metachromatic nucleic acid staining based on a crossover analysis of emission DNA/RNA light (RNP Determination™) in hematology analyzer Celltac G.

INTRODUCTION: Assessing the percentage of reticulocytes (%Retic) is useful for diagnosing and treating blood diseases that present with anaemia. The Celltac G+™ hematology analyzer (HA) uses a novel reticulocyte identification method that involves metachromatic nucleic acid staining with acridine orange and crossover analysis of emission light of DNA/RNA (determination of red cells, nucleic acid-containing cells, and platelets, RNP Determination™). The red and green fluorescence generated by stained single-stranded RNA and double-stranded DNA express immaturity and morphological abnormality of erythrocytes by detecting erythrocyte RNA and DNA content. METHODS: The basic performance of the test automated analyzer (TAA) Celltac G+ was evaluated and compared with the flow cytometry reference method and the comparative automated analyzer (CAA) XN-1000/2000™. In addition, its precision, limit of quantity (LoQ), linearity, analytical measurement interval (AMI), accuracy, and comparability and the effects of interfering substances were evaluated. RESULTS: Evaluation of %Retic by the TAA demonstrated good precision and linearity. The AMI was confirmed from 0.02 to 8.23, and the LoQ in %Retic as the coefficient of variation within an 11% limit (SD, within a 0.01 limit) was 0.14. TAA correlated well with the reference method and routine HA (CAA). Some deviations were found between TAA and CAA in DNA measurements of erythrocytes from abnormal samples. CONCLUSION: Celltac G+ uses a novel measurement principle and can assess erythrocyte immaturity independent of DNA contents. It represents a new HA that provides novel, useful information on immaturity and morphological abnormality of erythrocytes.

Characterisation of antithrombin-dependent anticoagulants through clot waveform analysis to potentially distinguish them from antithrombin-independent inhibitors targeting activated coagulation factors.

AIMS: While antithrombin (AT)-independent inhibitors targeting thrombin or activated factor X have been assessed through clot waveform (CWA), there are no reports on assessment with respect to AT-dependent anticoagulants. The present study aims to characterise AT-dependent anticoagulants through CWA to distinguish them from AT-independent inhibitors. METHODS: CWA was applied to the activated partial thromboplastin time (APTT) assay of plasma samples spiked with each of AT-dependent drugs (unfractionated heparin, enoxaparin and fondaparinux) and AT-independent drugs (rivaroxaban, apixaban, edoxaban, dabigatran, argatroban, hirudin and bivalirudin), which was performed using the CS-5100 or CN-6000 (Sysmex). The APTT-CWA data were automatically gained by the analyser program. The positive mode of clotting reaction curves was defined as the direction towards fibrin generation. RESULTS: Regarding dose-response curves in AT-dependent anticoagulants, the maximum positive values of the first and secondary derivatives (Max1 and Maxp2, respectively) and the maximum negative values of the secondary derivative (Maxn2) seemed to drop to zero without making an asymptotic line, consistent with the irreversibility. Such a feature was observed also in hirudin, as reported previously. Notably, the symmetric property of Max1 peaks in the waveforms was distorted dose dependently in AT independent but not AT-dependent drugs. A plot of Maxp2 logarithm versus Maxn2 logarithm was linear. The slope was about 1 in AT-dependent drugs while that was more than 1 in AT-independent drugs. These features made it possible to distinguish AT-dependent and AT-independent drugs. CONCLUSIONS: The results aid in further understanding of the pharmacological aspects of anticoagulation and in screening of candidates for novel anticoagulants.

Detection of residual disease in chronic myeloid leukemia utilizing genomic next generation sequencing reveals persistence of differentiated Ph+ B cells but not bone marrow stem/progenitors.

Persistence of leukemic stem cells (LSCs) results in the recurrence of chronic myeloid leukemia (CML) after the administration of tyrosine kinase inhibitors (TKIs). Thus, the detection of minimal residual disease (MRD) with LSC potential can improve prognosis. Here, we analyzed 115 CML patients and found that CD25 was preferentially expressed on the phenotypic stem and progenitor cells (SPCs), and TKI therapy decreased the number of CD25-positive cells in the SPC fraction. To detect MRD harboring BCR-ABL1 fusion DNA, we developed a highly-sensitive method using patient-specific primers and next-generation sequencing. By using this method, we identified that in patients who achieved molecular remission, almost all residual CD25-positive SPCs were BCR-ABL1-negative. Moreover, in some patients BCR-ABL1 was detectable in peripheral B cells but not in SPCs. We conclude that CD25 marks LSCs at diagnosis but does not mark MRD following TKI treatment and that analysis of peripheral B cells can allow sensitive detection of MRD.

Expert consensus regarding standardization of sample preparation for clotting time assays.

Accurate clotting time assay results are vital, as the test is employed to indicate the amount of oral anticoagulant to be prescribed, while it is also used for screening the hemorrhagic and thrombotic diseases. The procedure chosen for preparation of a patient blood sample including centrifugation can contribute to significant differences in the results obtained. Thus, for the purpose of proposing a standardized method to appropriately prepare blood samples prior to assay, the Japanese Society of Laboratory Hematology organized the Working Group for Standardization of Sample Preparation for Clotting Time Assays (WG). Following reviews of previously announced guidelines and original experimental results, consensus was obtained by the WG, with the main findings as follows. (1) The recommended anticoagulant in the blood collection tube is sodium citrate solution at 0.105-0.109 M (3.13-3.2%). (2) Whole blood samples should be stored at room temperature (18-25 ˚C) within 1 h of collection from the patient. (3) For plasma preparation, centrifugation at 1500 × g should be performed for at least 15 min or at 2000 × g for at least 10 min at room temperature. (4) After the plasma sample is prepared, it should be stored at room temperature and assayed within 4 h.

Application of clot-fibrinolysis waveform analysis to assessment of in vitro effects of direct oral anticoagulants on fibrinolysis.

INTRODUCTION: Acceleration of fibrinolysis by direct oral anticoagulants (DOACs) has been reported by several groups, suggesting contribution of not only anticoagulant but also fibrinolytic effects to the therapeutic efficacy. The present study aims to evaluate the usability of clot-fibrinolysis waveform analysis (CFWA) for assessment of in vitro effects of DOACs on fibrinolysis. METHODS: The experimental conditions were optimized according to how t-PA concentrations and a time length after t-PA adjustment affect parameters of CFWA. Addition of the activated partial thromboplastin time (APTT) reagent followed by that of calcium and t-PA was done to obtain clotting and fibrinolytic reaction curves which were mathematically differentiated for CFWA (APTT-CFWA). The positive and negative modes of waveforms were defined as the direction toward fibrin generation and that toward fibrin degradation, respectively. The maximum positive and negative values (Maxp 1 and Maxn 1) correspond to the maximum coagulation velocity and the maximum fibrinolysis velocity, respectively. Plasma spiked with each of DOACs (rivaroxaban, apixaban, edoxaban, and dabigatran) was subjected to APTT-CFWA. RESULTS: Optimization of t-PA use was based on Maxn 1. Roughly biphasic effects of rivaroxaban and dabigatran but not apixaban or edoxaban on fibrinolysis were observed through Maxn 1 and the fibrinolysis peak time, which was defined as a time length from the time when Maxp 1 (Maxp 1 time) to the time when Maxn 1 appears (Maxn 1 time). CONCLUSION: The results suggest the usability of CFWA for assessment of DOAC effects and provide insights into relevance of anticoagulation to therapeutic efficacy and bleeding risk from the perspective of fibrinolysis.

Distinct features of bivalent direct thrombin inhibitors, hirudin and bivalirudin, revealed by clot waveform analysis and enzyme kinetics in coagulation assays.

AIMS: Bivalent direct thrombin inhibitors (DTIs), hirudin and bivalirudin, bind to the active site and exosite 1 of thrombin irreversibly and reversibly, respectively. The present study aims to assess in vitro effects of hirudin and bivalirudin through clot waveform analysis (CWA) and enzyme kinetics in coagulation assays. METHODS: The pooled normal plasma and its dilutions were spiked with hirudin or bivalirudin. The activated partial thromboplastin time (APTT) assay and the Clauss fibrinogen assay were performed using the CS-5100 (Sysmex). The APTT-CWA data were automatically gained by the CS-5100 programme. RESULTS: In APTT-CWA, the maximum coagulation velocity, acceleration and deceleration were decreased dependently on the drug concentrations, demonstrating evidence for the blockade of thrombin-positive feedback by hirudin or bivalirudin. The Hill plot analysis was applied to the dose-dependent curves in bivalirudin. The Hill coefficients were greater than 1, showing positive anticoagulant cooperativity. Regarding the dose-dependent curves in hirudin, all the parameters dropped to almost zero without making an asymptotic line. In the Clauss fibrinogen assay, the Lineweaver-Burk plots demonstrated that both drugs exhibit mixed inhibition mimicking uncompetitive binding. The Dixon plots in bivalirudin were linear and supported the inhibition type described above. The Dixon plots in hirudin were non-linear and inappropriate to use for determination of the inhibition type. In addition, the inverse function of the clotting time appeared to drop to zero without making an asymptotic line, suggesting complete loss of thrombin activity by irreversible binding. CONCLUSIONS: The results provide insights into anticoagulation with bivalent DTIs.

Assessment of in vitro effects of direct thrombin inhibitors and activated factor X inhibitors through clot waveform analysis.

AIMS: Clot waveform analysis (CWA) has been reported to extend the interpretation of clotting time measurement. The parameters obtained from successive derivatives of the clotting reaction curves reflect the rates of activation of individual coagulation factors, theoretically dissecting the cascade pathway. This study aims to assess the in vitro effects of direct thrombin inhibitors (DTIs) and activated factor X (FXa) inhibitors. METHODS: CWA was applied to the activated partial thromboplastin time (APTT) assay of plasma samples spiked with each drug. For CWA of APTT measurement curves (APTT-CWA), the positive mode of clotting reaction curves was defined as the direction towards fibrin generation. RESULTS: All the maximum positive values in the successive derivatives were decreased dependently on the concentrations of each drug. Moreover, the negative values in the second and third derivatives appeared putatively due to consumption of thrombin and factor FXa, respectively, to form complexes with plasma serine protease inhibitors. The decrease of the maximum negative values observed dependently on the concentrations of each drug appeared to be consistent with the decreased generation of thrombin and factor FXa. The analysis of Hill coefficients of each drug in the dose-response of changes in the APTT-CWA parameters revealed a difference in anticoagulant cooperativity between DTIs versus FXa inhibitors. CONCLUSIONS: The APTT-CWA demonstrated evidence for the blockade of thrombin-positive feedback by DTIs and FXa inhibitors and that for the differences in anticoagulant cooperativity between them. The results demonstrate the usability of CWA for assessment of anticoagulation and provide insights into direct anticoagulants.

[Education and Training of Personnel in Morphology].

We introduce our efforts to utilize education, training, competence assessment, and quality control of personnel engaged in urinary sediment and blood cell morphology examinations in our laboratory. There are no standard samples for these morphological examinations, and standardization has not been completed for all types of blood cells or urinary sediment components. We had been carrying out simultaneous microscopic examination involving trainee staff and senior laboratory technologists as a means of education and evaluation, but acceptance criteria were unclear. Moreover, we had continued our operation without assessment of the level of achievement of routine works or the competence of individual staff members. Taking the opportunity of receiving ISO 15189 certification, we have been able to establish clear standards for evaluating personnel education and training in morphological examinations. We will continuously make efforts to maintain and manage this system.

[Potential Application of Fibrinogen Measurement Based on the Clauss Assay to Monitoring of Dabigatran].

A direct thrombin inhibitor (DTI), dabigatran was expected to be available for therapeutic use without mon- itoring. However, a number of severe bleedings occurring in patients on medication with dabigatran have been reported. The impact of dabigatran concentrations on major bleeding risk has also been revealed. Therefore, the significance of monitoring of dabigatran is of considerable interest. Hemoclot thrombin inhib- itor assay enables quantification of dabigatran concentrations but is not yet routinely available for clinical la- boratories in Japan. Based on spiking experiments with another DTI, argatroban, we previously demon- strated that the discrepancy in resulting quantification of fibrinogen concentrations between two different thrombin concentrations used in the Clauss assay may enable monitoring of DTIs. In the present study, analogous experiments using dabigatran were carried out, providing similar findings. The measured values of fibrinogen in the presence of dabigatran were similar to those in the absence of dabigatran when assayed using the high thrombin concentration (high-thrombin). The measured values of fibrinogen decreased in parallel with the increase in dabigatran concentrations when assayed using the low thrombin concentration (low-thrombin). Fibrinogen ratio, which is calculated by dividing the fibrinogen value measured with high- thrombin by that measured with low-thrombin, increased more sensitively at the high range of dabigatran concentrations than at the low range. Our observations suggest that the fibrinogen measurement based on the Clauss assay is practically applicable to monitoring of dabigatran especially for prediction of the bleeding risk. [Original].

[Measurement of plasma von Willebrand factor cleaving protease in patients with varied thrombotic microangiopathy].

Thrombotic thrombocytopenic purpura(TTP) is a multisystem disorders characterized by thrombocytopenia, microangiopathic hemolytic anemia associated with red cell fragmentation, and neurological and renal symptoms. Plasma of patients with TTP has been shown to contain unusually large von Willebrand factor(vWF) multimers that may cause platelet agglutination in vivo. Recently, a metalloprotease responsible for cleavage of vWF multimers has been isolated from normal human plasma and was found to be deficient in some patients with TTP. We examined the activity of the vWF-cleaving protease(vWF-CP), by modified Furlan's method, in plasma from patients with a familial TTP, 3 acquired TTP, 4 thrombotic microangiopathy(TMA) and 2 veno-occlusive disease(VOD) associated after allo-BMT. Diluted plasma samples of patients were incubated with protease-free vWF purified from normal human plasma, in the presence of urea and barium ions. The extent of vWF degradation was assayed by electrophoresis in SDS-agarose gels and immunoblotting. Activity of vWF-CP from 12 normal plasma have been shown as 77-180%(average 115%), whereas, no vWF-CP(below 5%) was observed in plasma from familial TTP, before and after plasma exchange, although FFP infusion therapy has been effective for this patient to recover thrombocytopenia. In 3 acquired TTP, 2 patients showed lack of vWF-CP activity in plasma, and inhibitors against vWF-CP have been elucidated by plasma cross-mixing test. After extensive plasma exchange and FFP infusion followed by corticosteroid therapy, normal vWF-CP was recovered in plasma from 2 acquired TTP patients. Among BMT patients, plasma from 4 BMT-TMA showed normal vWF-CP activities as 55-111%, whereas plasma from 2 BMT-VOD revealed low vWF-CP activity, as 24% and 37%, respectively. Thus, measurement of vWF-CP is crucial to predict differentiation of primary forms of TMA to establish the pathogenesis in varied endothelial dysfunction.