WBC image classification and generative models based on convolutional neural network.

BACKGROUND: Computer-aided methods for analyzing white blood cells (WBC) are popular due to the complexity of the manual alternatives. Recent works have shown highly accurate segmentation and detection of white blood cells from microscopic blood images. However, the classification of the observed cells is still a challenge, in part due to the distribution of the five types that affect the condition of the immune system. METHODS: (i) This work proposes W-Net, a CNN-based method for WBC classification. We evaluate W-Net on a real-world large-scale dataset that includes 6562 real images of the five WBC types. (ii) For further benefits, we generate synthetic WBC images using Generative Adversarial Network to be used for education and research purposes through sharing. RESULTS: (i) W-Net achieves an average accuracy of 97%. In comparison to state-of-the-art methods in the field of WBC classification, we show that W-Net outperforms other CNN- and RNN-based model architectures. Moreover, we show the benefits of using pre-trained W-Net in a transfer learning context when fine-tuned to specific task or accommodating another dataset. (ii) The synthetic WBC images are confirmed by experiments and a domain expert to have a high degree of similarity to the original images. The pre-trained W-Net and the generated WBC dataset are available for the community to facilitate reproducibility and follow up research work. CONCLUSION: This work proposed W-Net, a CNN-based architecture with a small number of layers, to accurately classify the five WBC types. We evaluated W-Net on a real-world large-scale dataset and addressed several challenges such as the transfer learning property and the class imbalance. W-Net achieved an average classification accuracy of 97%. We synthesized a dataset of new WBC image samples using DCGAN, which we released to the public for education and research purposes.

Delayed NK Cell Reconstitution and Reduced NK Activity Increased the Risks of CMV Disease in Allogeneic-Hematopoietic Stem Cell Transplantation.

Cytomegalovirus (CMV) infection has a significant impact in patients after allogeneic hematopoietic stem cell transplantation (HSCT). We investigated natural killer (NK) cell reconstitution and cytotoxic/cytokine production in controlling CMV infection, especially severe CMV disease in HSCT patients. Fifty-eight patients with acute myeloid leukemia (AML) who received allo-HSCT were included. We monitored NK reconstitution and NK function at baseline, 30, 60, 90, 120, 150, and 180 days after HSCT, and compared the results in recipients stratified on post-HSCT CMV reactivation (n = 23), non-reactivation (n = 24) versus CMV disease (n = 11) groups. The CMV disease group had a significantly delayed recovery of CD56dim NK cells and expansion of FcRγ-CD3ζ+NK cells started post-HSCT 150 days. Sequential results of NK cytotoxicity, NK cell-mediated antibody-dependent cellular cytotoxicity (NK-ADCC), and NK-Interferon-gamma (NK-IFNγ) production for 180 days demonstrated delayed recovery and decreased levels in the CMV disease group compared with the other groups. The results within 1 month after CMV viremia also showed a significant decrease in NK function in the CMV disease group compared to the CMV reactivation group. It suggests that NK cells' maturation and cytotoxic/IFNγ production contributes to CMV protection, thereby revealing the NK phenotype and functional NK monitoring as a biomarker for CMV risk prediction, especially CMV disease.

Electrostatic Modification for Promotion of Flavin-Mediated Oxidation of a Probe for Flavin Detection.

Electrostatic effects on the redox photochemistry of synthetic probes (1, 2, and 1-Zn) are examined by adjusting the thermodynamic driving force of their oxidation reactions. The redox photochemistry was simply controlled by introducing a zinc binding site (2,2'-dipicolylamine (DPA)) on the coumarin moiety of probe 2. Zinc complexation produced a positively charged environment on the coumarin (1-Zn), which lowered the electron density of a nearby 9 H-xanthene ring, attenuating the auto-oxidation of 1-Zn by 45 % compared with that of probe 1 at 298 K. The positive net charge of 1-Zn also provided an attractive Coulombic force toward the phosphate of flavin mononucleotide and flavin adenine dinucleotide, which lowered the reduction potential of the electron acceptor (isoalloxazine) and improved intermolecular electron transfer from the 9 H-xanthene ring to isoalloxazine. The flavin-mediated oxidation rate of 1-Zn was increased to 1.5 times that of probe 2. Probe 1-Zn showed highly selective sensing behaviour toward flavins, producing an intense brightness (ϵΦF =2.80×103 m-1  cm-1 ) in the long-wavelength regions (λmax =588 nm) upon flavin-mediated oxidation. Furthermore, probes 1-Zn and 2 were successfully applied to eosinophil imaging and the differential diagnosis of eosinophilia; this demonstrates their use as diagnostic tools.

Systemic Epstein-Barr Virus-positive T-Cell Lymphoproliferative Disease of Childhood With Good Response to Steroid Therapy.

Systemic Epstein-Barr virus (EBV)-positive T-cell lymphoproliferative disease of childhood is a rare disease and has a very fulminant clinical course with high mortality. A 21-month-old female patient was referred to our hospital with a 1 week history of fever and was subsequently diagnosed with systemic Epstein-Barr virus-positive T-cell lymphoproliferative disease of childhood. After starting treatment with dexamethasone, she showed early defervescence and improvement of laboratory parameters, and has remained disease-free after stopping steroid treatment, although longer follow-up is necessary. Our report underscores the possibility that this disease entity may be heterogenous in terms of prognosis.

Evaluation of a novel point-of-care test kit, ABSOGEN™ PCT, in semi-quantitative measurement of procalcitonin in whole blood.

BACKGROUND: In response to inflammation, procalcitonin plasma concentrations increase more rapidly than other acute-phase reactants and higher values are associated with severe disease. Procalcitonin measurements assist in determining whether antibiotic therapy should be used. point-of-care testing (POCT) is performed for early decision making about additional testing or therapy. The ABSOGEN™ PCT (Bumyoungbio, Inc., Suwon, Korea) is a rapid novel semi-quantitative immunochromatographic PCT assay that analyses whole blood samples. We compared the patient quantitative test results to ABSOGEN™ PCT test results. METHODS: Whole blood was loaded onto an ABSOGEN™ PCT cartridge and incubated for 10 minutes. The color intensity of the band of the cartridge was measured using an accompanying device and with the naked eye. The results were graded as negative (<0.1 ng/mL), low (0.1 to <1.0 ng/mL), middle (1.0-2.0 ng/mL), and high (>2.0 ng/mL). A total of 158 specimens with procalcitonin levels measured from 0 to 18.96 ng/mL were used for comparison study. RESULTS: The concordance rate between ABSOGEN™ PCT using the reader and quantitative assay, between ABSOGEN™ PCT using naked eyes and quantitative assay, and between ABSOGEN™ PCT using the reader and naked eyes for the same category was 83.5% (P=.040), 78.5% (P<.001), and 82.3% (P=.001), respectively. The concordance rates for the ±1 categories were all 100%. CONCLUSION: ABSOGEN™ PCT is an accurate assay. It is easy to use, simple, fast, portable. The assay has potential value in clinical applications, especially in emergency care.

Wilms tumor gene 1 expression as a predictive marker for relapse and survival after hematopoietic stem cell transplantation for myelodysplastic syndromes.

Relapse after allogeneic hematopoietic stem cell transplantation (HSCT) is a major concern in myelodysplastic syndromes (MDS), but the role of Wilms tumor gene 1 (WT1) as a predictive marker for post-HSCT relapse remains to be validated. We measured WT1 transcript levels by real-time quantitative PCR from marrow samples of 82 MDS patients who underwent transplantation between 2009 and 2013. Pre-HSCT WT1 expression weakly correlated with marrow blast counts or International Prognostic Scoring System scores and failed to predict post-transplantation relapse. Regarding post-HSCT WT1, transcript levels of relapsed patients were significantly higher in comparison to those in remission. Further analysis using receiver operating characteristics curves showed that higher (>154 copies/10(4)ABL) 1-month post-HSCT WT1 resulted in a higher 3-year relapse rate (47.2% versus 6.9%, P < .001) with poorer disease-free survival (DFS) and overall survival at 3 years (41.7% versus 79.0% and 54.3% versus 82.1%, P = .003 and P = .033, respectively). Multivariate analysis after adjusting for pre-HSCT karyotype and chronic graft-versus-host disease (GVHD) also revealed that higher 1-month post-HSCT WT1 was an independent predictive marker for subsequent relapse (P = .002) and poorer DFS (P = .010). In the higher 1-month post-HSCT WT1 subgroup, patients with chronic GVHD showed lower relapse rate and favorable survival outcome. One month post-HSCT WT1 expression was a useful marker for minimal residual disease and relapse prediction in association with chronic GVHD in the context of HSCT for MDS.

Diagnosis and evaluation of severity of sepsis via the use of biomarkers and profiles of 13 cytokines: a multiplex analysis.

BACKGROUND: The object of this study was to evaluate biomarkers for diagnosis of sepsis, hematologic parameters, and cytokine profiles for use in the diagnosis and evaluation of severity of sepsis. METHODS: We enrolled 127 consecutive patients with systemic inflammatory response syndrome (SIRS), 97 of whom were diagnosed with sepsis. The following biomarkers were evaluated: procalcitonin (PCT); C-reactive protein (CRP); erythrocyte sedimentation rate (ESR); white blood cell count, immature granulocyte (IG) count; and multiplex cytokines, including interleukin (IL)1-ß (IL1ß), IL2, IL4, IL5, IL6, IL9, IL10, IL12p70, IL13, IL17, IL22, tumor necrosis factor-α (TNFα), and interferon-γ (IFNγ). A cytokine bead immunoassay was used to perform simultaneous measurements. RESULTS: The disease involving urinary and respiratory tract constituted 57.5% of all patients. The severity of infection was classified as follows: SIRS patients, n=30; sepsis patients, n=81; and septic shock/severe sepsis patients, n=16. PCT, IL6, and CRP had high area under receiver operation characteristic curve (AUCs) and accuracy, which is as follows: PCT: 0.841, 80.5%; IL6: 0.811, 77.1%; CRP: 0.784, 73.8%, respectively. Severity of sepsis could be discriminated by PCT, IL6, and IL5. Unlike other cytokines, IFNγ had an inverse relation with severity of sepsis. The relationship between cytokine profiles and clinical diagnosis of sepsis was unclear. CONCLUSIONS: PCT, IL6, and CRP values could assist diagnosis, and PCT, IL6, and IL5 had discriminative properties for determination of severity of sepsis. IFNγ revealed a distinct inverse relationship with severity of sepsis. As there was no relationship between cytokine profiles and sepsis, further studies are required to develop clinical applications.

FLT3 expression and IL10 promoter polymorphism in acute myeloid leukemia with RUNX1-RUNX1T1.

We investigated the correlation between FLT3 expression and IL10 gene promoter polymorphism in acute myeloid leukemia with RUNX1-RUNX1T1 and their clinical significance. FLT3 mRNA expression was measured by real-time quantitative PCR (qPCR) and immunohistochemical staining (IHC) on bone marrow (BM) leukemic cells. IL10 gene promoter polymorphisms including rs1800896 (G-1082A), rs1800871 (C-819T), and rs1800872 (C-592T) were genotyped by direct sequencing. Among 45 enrolled patients, 32 (71.1 %) exhibited FLT3 overexpression, whose FLT3 mRNA level was higher than normal cut-off value (0.02). The IHC results also consisted with FLT3 mRNA expression data achieved by qPCR. The FLT3 mRNA level was significantly different among 3 IHC staining groups (P < 0.0001); 0.031 ± 0.041, 0.106 ± 0.097 and 0.588 ± 0.573 in IHC negative, intermediate and positive group, respectively. Interestingly, the FLT3 expression level was correlated with the percentage of BM CD34 positive cells (R = 0.360, P = 0.016). The elevated FLT3 expression at initial BM were decreased after remission and maintained lower than the cut-off level. FLT3 expression was not dependent on IL10 gene promoter polymorphisms. FLT3 overexpression itself did not demonstrate significant effects on overall survival (OS). However, it is notable that IL10 rs1800896 GA genotype tended to have a lower estimated mean OS (20.1 months) compared to GG genotype (54.6 months), but the statistical significance was not derived because of limited number of patients in this study (P = 0.072). Further studies including more type of leukemia and patients may be helpful to understand the relations between cytokine genotype and FLT3 expression and their prognostic impact.

Fragmented Red Cell as a Possible Favorable Prognostic Marker of Hematopoietic Stem Cell Transplantation Associated Thrombotic Microangiopathy.

BACKGROUND: Fragmented red cell (FRC) by automated hematologic analyzer is known to detect schistocyte. In this study, it is noted that FRC might be a favorable prognostic marker of hematopoietic stem cell transplantation associated thrombotic microangiopathy (TA-TMA). METHODS: The peripheral blood samples and clinical data of 89 patients were collected. The diagnosis of TA-TMA was defined by the Blood and Marrow Transplant Clinical Trials Network's criteria and schistocyte or both schistocyte- and FRC-positive cases and other parameters fulfilled are regarded as TA-TMA. RESULTS: Schistocyte and FRC displayed a correlation coefficient of 0.461 (P < 0.001) by Spearman's method. The diagnostic concordance of TA-TMA using schistocyte and FRC was 92.1% with kappa index of 0.531 (P < 0.001). The number of diagnosed patients and mean survival month were as follows: TA-TMA by schistocyte, 8 (8.9%), 13.5 month; TA-TMA by schistocyte and FRC, 7 (7.8%), 40.4 month; No TMA, 74 (83.1%), 38.3 month, respectively. Kaplan-Meier survival analysis by log-rank method of the patient with TA-TMA by schistocyte and rest of the group showed statistical significance (P < 0.01). CONCLUSION: As evidenced by the data, FRC might be a favorable prognostic marker for TA-TMA, but additional studies with larger patients groups are required for validation of clinical applications.

Validation of Western common recurrent chromosomal aberrations in Korean chronic lymphocytic leukaemia patients with very low incidence.

In Asia, the incidence of chronic lymphocytic leukaemia (CLL) is lower than in Western countries. Only a few studies of CLL have been conducted in Korea, and no long-term clinical outcome data are available. We assessed the frequency of common chromosomal aberrations in Korean CLL patients using interphase fluorescence in situ hybridization (FISH) and investigated their relationship to clinical outcomes. Between 2000 and 2011, conventional cytogenetic studies were performed in 58 patients, and FISH results were available in 48 patients. We used six DNA probes for the detection of del(13q14), trisomy 12, del(11q22), del(17p13), IGH rearrangement and del(6q23). Chromosomal aberrations were identified in 15 of 58 patients (26%) with conventional cytogenetic studies and in 25 of 48 patients (52%) with interphase FISH, including six patients with complex karyotypes. In contrast with the results of Western studies, trisomy 12 was the most common aberration, followed by IGH rearrangement, del(13q14), del(11q22) and del(17p13). Deletion of 6q23 was not observed, and isolated del(13q14) was less frequent than in Western studies. Compared with the other types of chromosomal aberrations, patients with del(11q22) and del(17p13) were more likely to be Rai stage 3-4 and Binet stage C, resulting in poor responses to chemotherapy and worse outcomes. In contrast, patient with trisomy 12 and isolated del(13q14) showed better responses and superior survival outcomes. The incidence of CLL is lower in Korea than in Western countries, and the frequency of chromosomal aberrations differs, perhaps reflecting differences in the pathogenic mechanism between ethnicities. Large prospective studies are needed to further assess the prognostic value of these results in Korean CLL patients.

Quantitative detection of target cells using unghosted cells (UGCs) of DxH 800 (Beckman Coulter).

BACKGROUND: In the Retic channel of DxH 800 (Beckman Coulter), the red blood cells (RBCs) resistant to hemoglobin clearing are counted as unghosted cells (UGCs). The aim of this study was to evaluate that the UGC is a surrogate marker for both the detection and counting of target cells. METHODS: In total, 1181 samples including 22 from iron deficiency anemia (IDA) patients, 95 from jaundice, 2 from sickle cell anemia, 3 from thalassemia, 1 cord blood, and 269 from normal controls were analyzed. Slides were prepared from all samples except normal controls and target cells were counted for correlation analysis of target cell counts to UGCs. RESULTS: The normal control samples showed 0.01% (0%-0.01%) UGCs, and the reference range was set at ≤0.02%. The IDA samples showed 0.015% (0.01%-0.03%) UGC count and 0.05% (0%-0.2%) target cell count. The jaundice samples showed 0.98% (0.1%-5.36%) UGC count, and 1.4% (0.1%-7.0%) target cell count. The two sickle cell anemia samples showed 0.41% and 3.74% UGC counts and 0.4% and 11.5% target cell counts. A cord blood sample showed 0.01% UGCs and 0% target cells. The three thalassemia samples showed 0.01%, 1.99%, and 7.82% UGC counts and 0%, 1.4%, and 15.5% target cell counts. The samples showing poikilocytosis other than target cells showed normal UGC count (≤0.02%). The positive predictive value of UGCs was 58.2% (124/213) and the negative predictive value was 96.8% (674/696). The UGC counts were well correlated to the manual target cell counts (r=0.944, p=0.000). CONCLUSIONS: This study demonstrates for the first time in the literature that a hematological parameter obtained automatically every time a reticulocyte counting is performed can be used to both screen for the presence of target cells and reliably quantify them.

New proposed guidelines for early identification of successful myeloid and erythroid engraftment in hematopoietic stem cell transplantation.

BACKGROUND: After hematopoietic stem cell transplantation (HSCT), early detection of engraftment would be of critical value for clinicians. The aim of this study was to identify faster parameters for engraftment. METHODS: We evaluated blood cell parameters including complete blood count (CBC), differential counts, and various reticulocyte parameters in 115 patients who received HSCT (allogeneic, n = 93; autologous, n = 22) in the purpose of identifying possible improved laboratory guidelines for engraftment prediction. RESULTS AND CONCLUSION: Days to white blood cell (WBC) count over 100 cells/µl with more than two-fold increase from nadir after transplantation (proposed new WBC guideline) preceded absolute neutrophil count (ANC) >500 cells/µl by 1.7 days. Among erythroid parameters, the earliest marker for erythroid engraftment was high light scattering reticulocytes (HLR) >0.1 (proposed new red blood cell guideline), which preceded reticulocyte counts (RET) >1% and immature reticulocyte fraction >0.5 by 3.9 and 1.6 days, respectively. Among the clinical parameters compared, those with statistically significant influence on myeloid engraftment were donor type (P = 0.009) and conditioning intensity (P = 0.009). As for erythroid recovery, ABO incompatibility was the only significant factor. In conclusion, the new guidelines may ensure engraftment several days earlier than the conventional parameters, which may help clinicians for decision-making on rescue therapy earlier.

Variant of ETV6/ABL1 gene is associated with leukemia phenotype.

The ETV6/ABL1 fusion transcript is thought to be a very rare aberration in hematopoietic malignancies. We describe two new cases of acute leukemia with the ETV6/ABL1 fusion, acute myeloid leukemia with eosinophilia (case 1) and B acute lymphoblastic leukemia (ALL) (case 2), screened by multiplex RT-PCR. The ETV6/ABL1 fusion was also confirmed by fluorescence in situ hybridization using a mixture of BCR/ABL1 and ETV6/RUNX1 probes. A thorough review of all published cases showed that all 7 reported ALL patients possess the type A ETV6/ABL1 fusion transcript, composed of the first 4 exons of ETV6 fused to the second exon of ABL1. The presence of the type A fusion transcript strongly implies ALL manifestation in ETV6/ABL1-positive hematologic malignancies as minor BCR breakpoint in BCR/ABL1-positive ALL.

Analysis of immunoglobulin and T cell receptor gene rearrangement in the bone marrow of lymphoid neoplasia using BIOMED-2 multiplex polymerase chain reaction.

The evaluation of bone marrow (BM) involvement is important for diagnosis and staging in patients with lymphoid neoplasia. We evaluated of immunoglobulin (Ig) and/or T-cell receptor (TCR) gene rearrangements in the BM using the standardized BIOMED-2 multiplex PCR clonality assays and compared the results with microscopic findings such as histology and CD10, CD20, CD79a, CD3 and CD5 immunohistochemistry. A total of 151 samples were enrolled; 119 B cell neoplasia, 29 T cell neoplasia, and 3 Hodgkin's lymphoma. The molecular clonality assay and microscopic diagnosis were concordant in 66.9% (n=101) and discordant in 33.1 % (n=50). Ig/TCR gene clonality assay detected 43 cases of BM involvement which was not presented in the morphology. Two cases among them turned into microscopic BM involvement during a close follow up. Clonal TCR gene rearrangements were detected in 12.6% of B cell neoplasia and Ig gene rearrangement were found in 3.4% of T cell neoplasia. This molecular clonality assay is valuable particularly in diagnosing BM involvement of lymphoid neoplasia if it is morphologically uncertain. But it should be carefully interpreted because molecular clonality may be present in the reactive lymphoproliferation. Therefore, comprehensive analysis with morphologic analysis should be important to reach a final diagnosis.

Mean corpuscular volume (MCV) values reflect therapeutic effectiveness in zidovudine-receiving HIV patients.

BACKGROUND: An increase of the mean corpuscular volume (MCV) of erythrocytes and alterations in the lipid profiles have been described in HIV-infected patients under combination of anti-retroviral treatment (cART), particularly zidovudine (AZT). METHODS: In 687 sera from 179 HIV-positive patients, MCV levels were correlated with the clinical outcome or therapeutic effectiveness. The sera were classified into three groups according to AZT treatment; cART with AZT (n = 317), cART without AZT (n = 262), and no anti-retroviral therapy (n = 108). RESULTS: The MCV and lipid profile values were increased after cART. The AZT-based cART group had higher MCV levels (111.6 ± 7.0 vs. 97.8 ± 7.0 fl, P < 0.001) and a higher incidence of macrocytosis (>99 fl; 95.3% vs. 38.2%, P < 0.001) than the non-AZT-based cART group. The receiver operating characteristic curve analysis showed that the area under the curve was 0.835 and the cut-off of MCV (>102 fl) had a sensitivity of 96.1% and specificity of 66.7% for detecting HIV-RNA (-) sera in AZT-based cART group. In the multivariate regression analysis, HIV-viral load and HDL-cholesterol were the only significant correlates to the MCV values in the AZT-base cART group (P < 0.05). CONCLUSION: The MCV values could be used as a surrogate marker to monitor the clinical outcome of HIV-infected patients receiving AZT-based cART.

Phenotypic and genetic characterization of adult T-cell acute lymphoblastic leukemia with del(9)(q34);SET-NUP214 rearrangement.

SET-NUP214 rearrangement is a recently recognized recurrent chromosomal translocation mostly observed in T-ALL. In order to characterize this rare entity, we performed phenotypic and genetic characterization of SET-NUP214 rearrangement through an investigation of a series of 40 consecutive samples of adult T-ALL that was selected among 229 adult ALL cases during 4 years in a single institution. Four cases (10%) of SET-NUP214 translocation were identified in our study. In all cases, diagnosis of T-ALL was established according to the World Health Organization (WHO) classification, and clonal TCR rearrangements were found. The immunophenotypic markers were indicative of the precursor nature of T lymphoblasts, and they expressed one or both of the myeloid-associated antigens (CD13, CD33). Conventional cytogenetic analysis revealed complex chromosomal aberrations in all four SET-NUP214 rearranged cases and del(12)(p13)/ETV6 was frequently involved. Array-CGH demonstrated additional genomic imbalances in addition to deletion 9q34. The genomic breakpoint sequencing identified breakpoints at SET intron 7 and NUP214 intron 17, and random nucleotide addition was found in two cases at the site of rearrangement. Our independently derived data set from a single institution confirms previous findings of SET-NUP214 rearrangement, indicates the relatively high incidence of SET-NUP214 rearrangement in adult T-ALLs, and also demonstrates comprehensive clinical, phenotypic, and genetic characteristics of this entity. Also, our report on genomic breakpoints demonstrates the homogeneity in the localization of the genomic breakpoints at 9q34. Concurrent chromosomal aberrations identified in this study should provide further areas of interest in investigation of SET-NUP214-mediated leukemogenesis.

Novel method to dissociate platelet clumps in EDTA-dependent pseudothrombocytopenia based on the pathophysiological mechanism.

BACKGROUND: EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is an in vitro phenomenon of platelet clumping that leads to spuriously low platelet counts by automatic hematology analyzers. The mechanism is not clearly defined, but is known as an immunologically mediated phenomenon due to the presence of EDTA-dependent antiplatelet auto-antibodies that induce platelet clumping. The purpose of this study was to identify antiplatelet antibodies in EDTA-PTCP samples and to design a method to dissociate platelet clumps based on the pathophysiological mechanism. METHODS: The antiplatelet antibody was investigated using direct and indirect immunofluorescent flow cytometric methods in 23 EDTA-anticoagulated whole blood (WB) samples and 12 serum samples of EDTA-PTCP patients, respectively. A novel mixture containing 9 mmol/L CaCl(2) and 0.1 unit/L sodium heparin, that provides calcium replacement while curbing coagulation, was designed to dissociate platelet clumps. The effect on dissociation was demonstrated in 26 samples of EDTA-PTCP and compared with the established method of kanamycin supplementation. RESULTS: The direct test was positive for IgM and IgG antiplatelet antibody in 60.9% and 4.4% of patients, respectively [mean median fluorescence intensity (MFI) of 223.9 and 128.4, respectively]. The indirect test was positive for IgM antiplatelet antibody in 58.3% of patients (mean MFI of 123.4). The novel method dissociated the platelet clumps with a mean increased platelet count of 242.3% and was equivalent in efficiency to kanamycin supplementation. CONCLUSIONS: The novel method is an easily applicable and efficient measure that allows dissociation of platelet clumps, based on the pathophysiological mechanism of EDTA-PTCP.

Detection of anti-aquaporin-4 antibodies in neuromyelitis optica: comparison of tissue-based and cell-based indirect immunofluorescence assays and ELISA.

NMO-IgG against aquaporin-4 (AQP4) is a specific marker for neuromyelitis optica (NMO). We evaluated the performance of different NMO-IgG detecting methods. In 124 sera (from 54 with NMO spectrum disorders including nine with NMO, ten with multiple sclerosis including two with OSMS, and 60 with other neurological diseases), NMO-IgG was measured with tissue-based indirect immunofluorescence (IIF-tissue) using mouse cerebellum, cell-based IIF (IIF-AQP4) using transfected HEK293 cells which express human AQP4, and AQP4 autoantibody detecting enzyme linked immunosorbent assay (ELISA-AQP4). The sensitivities and specificities of three assays were 44.4-55.6% and 87.0-92.2% for detecting NMO, and 11.1-20.4% and 95.7-97.1% for detecting NMO spectrum disorders. Although there was no significant difference, the patients with NMO or NMO spectrum disorders showed higher rates of seropositivity in the ELISA-AQP4 vs. IIF assays. Out of the 19 sera with NMO-IgG, in at least one test, only six (31.6%) were found to be positive by all three assays. Among the three methods, the ranges of co-negativities, co-positivities, and agreement were 77.4-97.4%, 42.9-75.0%, and 91.1-95.2% (kappa 0.475-0.641), respectively. In patients who had positive ELISA-AQP4 results, IIF-AQP4 positivity was associated with NMO (P = 0.01). In summary, we observed an increased prevalence of NMO-IgG in patients with NMO and NMO spectrum disorders. ELISA-AQP4 may be more sensitive and specific when confirmed by IIF-AQP4.

Leukocyte cell population analysis from the coulter automatic blood cell analyzer DxH800 to monitor the effect of G-CSF.

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) induces the formation of toxic granulation neutrophils (TGNs), which are found in many inflammatory responses. Cell population data (CPD) may be able to clarify the effect of G-CSF, and potentially help doctors in discriminating the effect of G-CSF from other inflammatory situations. METHODS: To achieve this, we performed analyses of leukocyte CPD from normal controls and healthy donors that had received G-CSF for peripheral blood stem cells (PBSCs) mobilization (G-CSF group). RESULTS: Two hundred and seventy-one subjects were enrolled as normal controls, and 21 subjects were enrolled in the G-CSF group. Mean volume (MN-V)-neutrophils (NE), mean axial light loss (MN-AL2)-NE, and all standard deviation (SD) parameters increased significantly, whereas all light scattering parameters, mean median angle light scatter (MN-MALS)-NE, mean upper median angle light scatter (MN-UMALS)-NE, mean lower median angle light scatter (MN-LMALS)-NE, and mean low angle light scatter (MN-LALS)-NE reduced significantly in the G-CSF group. MN-V-lymphocytes (LY) from the G-CSF group showed no significant difference (P = 0.143), whereas MN-V-monocytes (MO) were significantly decreased (P < 0.001). Receiver operating characteristic (ROC) curves for the discrimination of the G-CSF group from normal controls showed excellent sensitivity in SD-LALS-NE (at 30.85, sensitivity 95.2%, specificity 76.0%), MN-AL2-NE (at 134.5, sensitivity 90.5%, specificity 83.0%), and SD-AL2-NE (at 16.4, sensitivity 95.2%, specificity 95.2). Several CPD parameters of lymphocytes and monocytes, as well as neutrophils can be used as markers for determining the effect of G-CSF. CONCLUSION: Our data show that many CPD of leukocytes can be considered to be useful parameters of the effect of G-CSF.