Yukio Hamaguchi

Flow cytometric method for enumeration and classification of reactive immature granulocyte populations.

We developed a flow cytometric method for the enumeration and classification of nonmalignant immature granulocytes (IG). In this study, IG are defined as most immature (IG stage 1: promyelocytes and myelocytes) and as more mature (IG stage 2: metamyelocytes). Blood specimens from 46 patients with documented infectious or inflammatory disease and known presence of IG (by routine manual microscopy) were analyzed. For a reference manual differential count, we used a 400 white blood cell (WBC) differential and separated granulocytes into promyelocytes and myelocytes combined, metamyelocytes, and included band cells in the mature, segmented neutrophil population. The flow cytometric method is based on three-color staining of whole, anticoagulated blood with CD45-PerCP, CD16-FITC, and CD11b-PE-labeled monoclonal antibodies and a three-step gating procedure. The flow cytometric results were confirmed by cell sorting and microscopic evaluation of the sorted cells. A total of 10,000 events, excluding debris, were recorded per specimen and IG stage 1 (CD16-/CD11b-), IG stage 2 (CD16-/CD11b+), and mature neutrophils (CD16+/CD11b+) were categorized. Regression and correlation between flow cytometric IG and the manual differential showed y = 1.34x + 0.95, r(2) = 0.86 for IG stages 1 and 2 combined versus promyelocytes, myelocytes, and metamyelocytes. For IG stage 1 versus microscopic counts of promyelocytes and myelocytes, the results were y = 1.53x + 1.24, r(2) = 0.76; for IG stage 2 versus manual metamyelocyte count, y = 0.77x + 0.21, r(2) = 0.58. Reproducibility of the flow cytometric method showed a coefficient of variation (CV) of 6.8% for all IG combined compared with a CV of 50.2% for manual differential IG count (based on a routine 100 WBC count). Samples were found stable at least 12 h at 25 degrees C and at least 48 h at 4 degrees C for flow cytometry. After staining and lysing, the sample was stable for at least 120 min at room temperature. We analyzed samples from patients with myelodysplastic and myeloproliferative disease separately. We found that CD16- mature neutrophils falsely elevated the flow cytometric IG count. Similar results were obtained in blood from patients treated with granulocyte-colony stimulating factor (G-CSF). Although this restricts the use of the method somewhat, we believe that this flow cytometric method is useful for enumerating reactive IG, as well as for evaluating automated methods for IG identification by hematology analyzers.

New rapid flow cytometric method for the enumeration of nucleated red blood cells

BACKGROUND Nucleated red blood cells (NRBC) in blood specimens compromise the automated white blood cell (WBC) count on most hematology analyzers. This makes it necessary to correct the WBC count by subtracting separately counted NRBC by manual microscopy. In addition, it is clinically important to establish the non-physiological presence of NRBC in blood specimens because of their association with significant hematological and non-hematological disease. Unfortunately, manual microscopic methods lack sensitivity, specificity and reproducibility required for both. METHODS We have developed a new, rapid flow cytometric method for the detection and enumeration of NRBC, based on two-color staining with anti-CD45-fluorescein-isothiocyanate (CD45-FITC) and propidium iodide (PI). EDTA anticoagulated blood samples are incubated for 30 min with CD45-FITC, followed by 30 sec acid-hypotonic lysis, containing PI and subsequent addition of an alkaline-hypertonic solution. The samples are thus ready for flow cytometric analysis. RESULTS The method typically yields up to four populations, (1) red blood cell (RBC) ghosts, debris, lyse-resistant RBC, reticulocytes and platelets, (2) CD45(+) WBC unstained by PI, (3) CD45(+) WBC stained by PI, and (4) CD45(-)/PI(bright) NRBC. Manual microscopic reference NRBC counts of 25 patient specimens showed excellent correlation with flow cytometric NRBC determinations (y = 0.943x+0. 66; r(2) = 0.982). Performance for precision showed a mean coefficient of variation (CV) for the flow cytometric method of =10%, with a mean CV for manual NRBC counts of 40%. CONCLUSIONS We conclude that this method is suitable for NRBC counting in peripheral blood specimens with improved performance in terms of accuracy, reproducibility when compared to manual microscopic methods.

Automated Enumeration of Immature Granulocytes

The performance characteristics of the XE-2100 (Sysmex, Kobe, Japan) automated immature granulocyte (IG) count were studied. The automated IG count was compared with the manual morphology count and with a proposed reference flow cytometric count. The comparison data were analyzed by both least-squares and Passing-Bablok regression analysis. Long-term imprecision using preserved blood quality control specimens at different levels showed a range from 2.59% to 3.57% coefficient of variation (CV) for within-run imprecision and 3.57% to 6.85% CV for total imprecision. The within-run reproducibility performed using fresh blood on 3 different specimens showed a range from 5.55% to 8.24% CV. The counts were stable at both room temperature and after refrigeration for 24 hours.Passing-Bablok regression analysis showed excellent agreement between the proposed reference flow cytometric IG count and the XE-2100 IG count, while there was less agreement with the manual morphology count. Our results indicate that the automated IG count can replace the manual morphology count for IG counting in the clinical laboratory. The results also confirm that the flow cytometric IG count is superior to and can replace the manual morphology count as a reference method for IG counting.

Clinical and Oncologic Implications in Epigenetic Down-Regulation of CD26/ Dipeptidyl Peptidase IV in Adult T-Cell Leukemia Cells

CD26/dipeptidyl peptidase IV (DPPIV), a T-cell-activation antigen, is a 110-kD type II surface glycoprotein expressed on various types of normal cells. CD26/DPPIV is considered a multifunction housekeeping protein. Malignant cells often show altered CD26/DPPIV expression or no CD26/DPPIV expression, thus suggesting a useful marker for assessing some T-cell malignancies. In this study, cell surface protein and messenger RNA expression profiles for CD26/DPPIV were examined in 49 patients with adult T-cell leukemia (ATL), 10 carriers of human T-lymphotropic virus I (HTLV-I), and 4 HTLV-I-infected cell lines to assess the utility of CD26/DPPIV expression as a useful molecular marker for ATL pathology. In contrast to normal lymphocytes, ATL cells and HTLV-I-infected cell lines apparently down-regulated or completely lost the CD26/DPPIV antigen. Furthermore, the positive rate and antigen density for CD26/DPPIV in ATL cells gradually declined along with the advancement of ATL stage. Analysis of genomic DNA and the CD26/DPPIV transcript showed that CD26- ATL cells possessed faintly detected transcripts of the gene that were aberrantly methylated at the CpG islands within the promoter region in parallel with the advancement of ATL, a finding supported by a rescue experiment for transcript reexpression using 5-azacytidine as demethylation agent. Moreover, there was no relationship between loss of CD26/DPPIV and HTLV-I tax expression. These results indicate that ATL cells down-regulate CD26 antigens by means of epigenetic machinery and that this antigen abnormality is a useful molecular marker for the pathology of ATL.

Usefulness of Measurement of Reticulated Platelets for Diagnosis of Idiopathic Thrombocytopenic Purpura

Reticulated platelets (RP) and large platelets (LP) were measured by an automated hematology analyzer (modified R-2000) in 287 healthy volunteers and 131 patients with thrombocytopenia or thrombocytosis. RP was significantly higher in patients with idiopathic thrombocytopenic purpura (ITP), especially in active phase, while RP was markedly lower in patients with essential thrombocytosis (ET) or chronic myelocytic leukemia (CML). LP was significantly higher in patients with ITP, especially in active phase, while LP was markedly lower in patients with aplastic anemia (AA), ET, or CML. In ITP, RP and LP were significantly higher in patients positive for anti-glycoprotein (Gp) IIb/IIIa antibody. RP and LP were poorly correlated with platelet-associated IgG (PAIgG). RP and LP were poorly correlated with plasma thrombopoietin levels, and negatively correlated with platelet count. These results show that RP reflects the pathology of thrombocytopenic disorders, and that measurement of RP is useful for the differential diagnosis and analysis of platelet kinetics.

Automated urinalysis. Evaluation of the Sysmex UF-50.

We assessed the Sysmex UF-50 for reproducibility of results and carryover rate by performing between- and within-run precision analyses on 315 urine samples, evaluated the feasibility of using the UF-50 to measure urinary cellular and noncellular components by comparing results from the UF-50 with results of manual urinalysis using the Kova system, and performed side-by-side comparison of the within-run reproducibility from the UF-50, the UF-100, and the Kova system. Results from the UF-50 and UF-100 were highly reproducible, and the carryover rate was 0.5% or less for the urinary components. In between-run precision assays, the coefficients of variation for UF-50 results for all cellular components were less than 10%. The agreement (gamma statistics) between values from the UF-50 and the Kova system was excellent for RBC, WBC, and bacterial counts. The cell counts from the UF-50 for RBCs, WBCs, epithelial cells, and bacteria were 52%, 63%, 54%, and 110%, respectively, of those measured by manual urinalysis. The UF-50 performed quantitative analysis in 72 seconds, compared with 330 seconds for manual methods. The UF-50 is suitable for the first screening to detect hematuria, pyuria, and bacteriuria.

Usefulness of fully automated measurement of reticulated platelets using whole blood.

Reticulated platelets (RP) were measured with an automated hematology analyzer (modified R-2000) in 287 healthy volunteers and in 212 patients with thrombocytopenia. In healthy volunteers, the RP was 0.48 ± 0.26% in men and 0.48 ± 0.32% in women. No significant difference in the RP values due to gender or age (21-60 years) was observed. Furthermore, the reverse correlation was observed between platelet counts and RP. The RP was high in patients with idiopathic thrombocytopenic purpura (ITP), those with high fibrinogen and fibrin degradation products (FDP), and those with high C-reactive protein (CRP), but low in patients after chemotherapy. The RP was highest in active phase of ITP, and relatively high in the partial remission phase of aplastic anemia. In patients after chemotherapy, the patients had a minimum phase of RP and then a maximum phase of RP before platelet counts increased. RP was significantly high in the maximum phase and significantly low in the minimum phase. The relationships between platelet count and RP were negatively correlated in patients with ITP, high FDP, or high CRP, but were not correlated in patients with aplastic anemia, liver disease, or after chemotherapy. These results show that RP reflects the pathology of thrombocytopenic disorders and the measurement of RP is useful for the differential diagnoses and analysis of platelet kinetics.

Enumeration of Bacterial Cell Numbers and Detection of Significant Bacteriuria by Use of a New Flow Cytometry-Based Device

ABSTRACT A new, automated flow cytometry-based urine bacterium analyzer (UBA) was developed. We assessed the UBA for linearity of measurement, reproducibility of results, carryover rate, and correlation of measured results with those determined by urine culture. We also evaluated its ability to screen urine samples for significant bacteriuria. The UBA showed excellent linearity and a minor carryover rate. Results from the UBA were highly reproducible, and in between-run precision assays, the coefficients of variation for the UBA results were smaller than those for the urine culture results. Two hundred seventy-three urine specimens from patients attending the outpatient clinics of two university-based hospitals were examined. The results for the UBA were compared with those for urine culture. The UBA detected significant bacteriuria with a sensitivity of 96.6%, a specificity of 79.9%, a positive predictive value of 57.0%, a negative predictive value of 98.8%, a false-positive rate of 15.8%, a false-negative rate of 0.7%, and an accuracy of 83.5%. These results were comparable to or better than those obtained with previously reported screening procedures. The UBA can perform accurate enumeration of bacterial cells automatically in 90 seconds and can be used for the screening of significant bacteriuria.

Automated Sperm Concentration Analysis With a New Flow Cytometry-Based Device, S-FCM

The S-FCM uses flow cytometry technology to measure sperm concentrations. Semen samples from 104 men attending a male infertility clinic were used to evaluate the reproducibility of results and the carryover rate with the S-FCM by performing between- and within-run imprecision analyses. In addition, sperm concentrations measured with the S-FCM were compared with those obtained by manual analyses with the Makler chamber and the improved Neubauer hemacytometer. The results showed that automated analyses with the S-FCM were highly reproducible and the carryover rate was 0.17% or less. In within-run imprecision assays, the coefficients of variation for the S-FCM were less than 5% at all sperm concentrations, while those for the Makler chamber were between 17.7% and 28.7% at lower sperm concentrations. The overall correlation between values measured with the S-FCM and those measured with the Makler chamber and improved Neubauer hemacytometer was excellent, but at lower sperm concentrations the correlation was lower. The S-FCM performed sperm concentration analyses in 110 seconds compared with 5 minutes for the Makler chamber and 10 minutes for the improved Neubauer hemacytometer. The S-FCM is suitable for quantitative measurement of lower sperm concentrations.

Highly Sensitive Detection of Hepatitis B Virus Surface Antigen by Use of a Semiautomated Immune Complex Transfer Chemiluminescence Enzyme Immunoassay

ABSTRACT The performance of hepatitis B surface antigen (HBsAg) screening assays is continuously improved to reduce the risk of transfusion-associated hepatitis B. In this study, a semiautomated immune complex transfer chemiluminescence enzyme immunoassay (ICT-CLEIA) for the detection of HBsAg, which is as sensitive as hepatitis B virus (HBV) DNA PCR, was developed; the ICT-CLEIA assay performance was compared with the performance of the Architect HBsAg QT assay and HBV DNA PCR. The specificities in the initial assay and after retesting were 99.50% (1,988/1,998 samples) and 99.95% (1,997/1,998 samples), respectively. The analytical detection limit was determined to be 0.2 mIU/ml using the 2nd International WHO HBsAg standard, and the cutoff value (0.5 mIU/ml) of the ICT-CLEIA assay was 8.0 standard deviations (SD) above the mean of the HBsAg-negative specimens. The ICT-CLEIA assay could detect HBsAg even in the presence of anti-HBs antibodies and demonstrated a 23.6-day-shorter window period using commercially available HBsAg seroconversion panels than the Architect HBsAg QT assay. Furthermore, the monitoring of the viral kinetics by the ICT-CLEIA assay and the HBV DNA PCR produced very similarly shaped curves during both the HBsAg seroconversion and reverse seroconversion periods. Therefore, the ICT-CLEIA assay may be useful not only for an earlier detection of HBV reactivation but also for the monitoring of hepatitis B patients.