Comparison of white blood cell counts by WNR, WDF, and WPC channels in Sysmex XN hematology analyzer.

INTRODUCTION: The Sysmex XN modular system (Sysmex, Kobe, Japan) uses a novel technology for white blood cell (WBC) count and differential, using separate channels: white cell nucleated (WNR), WBC differential (WDF), and white progenitor cell (WPC) channels. We questioned how concordant WBC counts would be between them. METHODS: In a total of 6327 consecutive specimens, WBC counts were compared between WNR and WDF channels. They were also compared in three groups of WBC counts and two groups of chemotherapy status. In 508 specimens from the 4361 specimens that were run on the XN-20 module, the WPC channel was used for reflex test. Data were compared using Pearson's correlation, absolute difference, and percent difference (%D). RESULTS: WBC counts between WNR and WDF channels showed very high correlations in total specimens (r = 0.9976) and in the groups of WBC counts and chemotherapy. As WBC count increased, absolute difference increased, while %D decreased (P < 0.0001, both). Percent difference was 1.55% in total specimens and showed the highest value in the severe leukopenia group (<1.0 × 10(9)/L, 6.18%). CONCLUSIONS: This is the first large-scale study on novel channel technology for WBC counts in the Sysmex XN. WBC counts by WNR, WDF, and WPC channels are highly correlated, and they are overall interchangeable and reliable.

Evaluation of ABX Pentra DX 120 and Sysmex XE-2100 in umbilical cord blood.

INTRODUCTION: Cord blood (CB) is an important source of hematopoietic stem cells and reflects the hematologic status of neonates. ABX Pentra DX 120 (Horiba Medical, Montpellier, France) and Sysmex XE-2100 (Sysmex, Kobe, Japan) were compared in 200 CB specimens. METHODS: Complete blood count parameters including white blood cell (WBC) differential counts were compared between the two analyzers. Double differential matrix (DDX) by ABX Pentra DX 120 and hematopoietic progenitor cell (HPC) by Sysmex XE-2100 were compared with CD34(+) cells by flow cytometry. RESULTS: Most of the parameters showed acceptable correlation between the two analyzers. Although WBC differential of both analyzers showed acceptable correlation with manual counts, mononuclear cells (MNC) by ABX Pentra DX 120 better correlated with manual count than MNC by Sysmex XE-2100. NRBC by Sysmex XE-2100 better correlated with manual count than NRBC by ABX Pentra DX 120. ABX Pentra DX 120 showed better flagging performances. DDX better correlated with CD34(+) cells than HPC. CONCLUSION: Although the results from both analyzers are mostly interchangeable and reliable in CB specimens, flagging performance of ABX Pentra DX 120 seems to be superior to that of Sysmex XE-2100. DDX by ABX Pentra DX 120 would be valuable to evaluate the quality of CB for further therapeutic utilization.

Establishment of reference interval for immature platelet fraction.

INTRODUCTION: Immature platelet fraction (IPF) is a parameter for reticulated platelets. A high percentage IPF (%-IPF) is indicative of consumptive or recovering thrombocytopenic disorders in contrast to a low %-IPF seen in aplastic states. Absolute IPF (A-IPF) specifically reflects the number of immature platelets in circulation. This study aimed to establish reliable reference intervals for %-IPF and A-IPF. METHODS: Except outliers, platelet counts and IPF were determined in 2152 healthy individuals (1252 men and 900 women) and 133 umbilical cord blood from healthy full-term neonates using XE-2100 hematology analyzer (Sysmex, Kobe, Japan). The reference intervals for %-IPF and A-IPF were defined using nonparametrical percentile methods according to the Clinical and Laboratory Standard Institute (CLSI) guideline. RESULTS: Platelets,%-IPF, and A-IPF all showed nonparametrical distributions. In total individuals, the reference intervals for %-IPF and A-IPF were 0.5-3.3% (0.5-3.1% in men; 0.5-3.4% in women) and 1.25-7.02 × 10(9) /L (1.30-6.80 × 10(9) /L in men; 1.21-7.15 × 10(9) /L in women), respectively. The reference intervals for %-IPF and A-IPF in umbilical cord blood were 0.7-3.8% and 1.93-9.7 × 10(9) /L, respectively. CONCLUSIONS: This study provides the reference interval for IPF, including %-IPF and A-IPF, according to the CLSI guideline. These results could be used as fundamental data for clinical use as well as future researches.

Fecal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae in Korean community and hospital settings.

PURPOSE: The assessment and early recognition of risk factors for infections due to extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) are important for infection control and proper treatment. The aim of the present study was to investigate the prevalence of fecal carriage of ESBL-E in healthy individuals and hospitalized high-risk patients in Korea and to compare the characteristics of ESBL-E in these two groups. METHODS: A total of 384 samples from 290 healthy individuals and 94 high-risk patients were collected. The screening of ESBL-E was performed using a commercial chromogenic medium. Bacterial identification and antibiotic susceptibility testing were performed using the Vitek 2 system. RESULTS: The prevalence of ESBL-E carriage was 20.3 % in healthy individuals and 42.5 % in high-risk patients. Escherichia coli comprised a large majority (96.6 %) of the isolates from healthy individuals, but Klebsiella pneumoniae was more commonly detected (45.0 %) in high-risk patients than in healthy individuals. K. pneumoniae isolates exhibited significantly higher resistance to ceftazidime, ampicillin, and carbapenem, and E. coli exhibited higher resistance to cefotaxime. E. coli from high-risk patients exhibited significantly higher resistance to levofloxacin and cefepime than that from healthy individuals. CONCLUSIONS: We demonstrated the high prevalence of ESBL-E carriage in Korea and clarified the characteristics of ESBL-E carriage in healthy individuals and high-risk patients. The distribution and antibiotic susceptibility of colonizing ESBL-E were different between the group of healthy individuals and the high-risk patients. Active surveillance of ESBL-E carriage is suggested for infection control, and the use of chromogenic agar appears to be an efficient method.

Isothermal target and probe amplification for Mycobacterium tuberculosis identification from broth cultures.

SETTING: Various methods are used to identify Mycobacterium tuberculosis complex (MTC) from broth cultures. The isothermal target and probe amplification (iTPA) method has recently been introduced as a simple and cost-effective molecular assay. OBJECTIVE: To evaluate the diagnostic performance of the iTPA method. DESIGN: A total of 175 specimens from the Mycobacteria Growth Indicator Tube (MGIT) 960 broth culture system were evaluated. The immunochromatographic test (ICT) and real-time quantitative PCR (RQ-PCR) were compared with the iTPA method. RESULTS: MTC was identified in 71/131 MGIT-positive specimens, including 60 ICT-positive and 11 ICT- negative/PCR-positive specimens. The sensitivity and specificity of the ICT assay were respectively 84.5% (95%CI 74.0-92.0) and 100% (95%CI 94.0-100). These 71 specimens were all MTC-positive with the iTPA method also. Sixty non-tuberculous mycobacteria specimens and 44 MGIT-negative specimens were all MTC-negative with the iTPA method. CONCLUSION: Our data show that the diagnostic performance of the iTPA method is comparable to that of RQ-PCR. The iTPA method could be a reliable and cost-effective option for the identification of MTC from broth culture.

Detection of tuberculosis using artus M. tuberculosis PCR Kit and COBAS AMPLICOR Mycobacterium tuberculosis Test.

SETTING: Nucleic acid amplification tests can detect Mycobacterium tuberculosis complex rapidly and reliably. OBJECTIVE: To compare the diagnostic performance of the artus M. tuberculosis PCR Kit and COBAS AMPLICOR Mycobacterium tuberculosis Test. In the artus assay, an appropriate cycle threshold (Ct) value was determined for positivity. DESIGN: A total of 238 clinical respiratory specimens were analysed using both the artus and COBAS AMPLICOR assays. In 221 specimens, these results were further compared with culture results. RESULTS: The overall agreement between artus and COBAS AMPLICOR was 96.2% (229/238). Among the nine (3.8%) discrepant specimens, three (1.3%) were artus-positive and COBAS AMPLICOR-negative, while the other six (2.5%) were artus-negative and COBAS AMPLICOR-positive. Using culture as a standard, the sensitivity and specificity of the artus assay were 97.8% and 85.1%, and those of COBAS AMPLICOR assay were 100% and 86.2%, respectively. The difference was not statistically significant. In the artus assay, the minimum Ct value for the positivity determination was 38. CONCLUSION: The artus and COBAS AMPLICOR assays showed comparable diagnostic performance and can be confidently used for detection of M. tuberculosis complex. In the artus assay, a Ct value of 38 could be suggested as an appropriate cut-off value.

Optimization of laboratory workflow in clinical hematology laboratory with reduced manual slide review: comparison between Sysmex XE-2100 and ABX Pentra DX120.

INTRODUCTION: The validation of automated hematology analyzer results by manual slide review (MSR) is currently an inevitable work process in clinical hematology laboratories. The laboratory workload would be optimized if the requirement for MSR could be reduced without compromising patient care. We investigated whether slide-making rates would be different between two hematology analyzers, which were paired with their own automated slide makers/stainers: Sysmex XE-2100 with SP-1000i (Sysmex, Kobe, Japan) and ABX Pentra DX120 with SPS evolution (ABX-Horiba, Montpellier, France). METHODS: A total of 943 samples were run in parallel on the Sysmex XE-2100 and ABX Pentra DX120. Reflex slides were automatically made in each analyzer according to its own criteria, which reflected the criteria of MSR in our laboratory. The slide-making rates were compared, and the results were further confirmed using the criteria of MSR. RESULTS: The slide-making rates in Sysmex XE-2100, ABX Pentra DX120, and manual review were 22.5% (212/943), 15.91% (150/943), and 11.5% (108/943), respectively. In 774 (82.1%) samples, the three methods showed concordant results, and all made slides in 82 samples. Using the manual method as a standard, the sensitivity and specificity were 86.1% and 85.8% in Sysmex XE-2100 and 89.8% and 93.7% in ABX Pentra DX120. CONCLUSION: Our data show that the slide-making rates are variable in different hematology analyzers. It also implies that although MSR cannot be fully substituted by modern hematology analyzers, it can be effectively reduced to optimize laboratory workload.

High prevalence of autoantibodies in hepatitis A infection: the impact on laboratory profiles.

AIMS: In the absence of IgM antibodies against hepatitis A virus (HAV), HAV infections can be regarded as autoimmune hepatitis when they show positive autoantibodies by indirect immunofluorescence and lack other viral markers. The aim of this study was to evaluate the prevalence, titres and impact of autoantibodies in Korean patients with HAV infection. METHODS: The study involved a retrospective review of the electronic medical records of 73 patients with HAV at Konkuk University Hospital from August 2005 to September 2008. The presence and pattern of anti-nuclear antibody, anti-smooth muscle antibody, anti-mitochondrial antibody and anti-liver/kidney microsomal antibody were assessed by indirect immunofluorescence on Hep-2 cells and mouse/kidney sections. RESULTS: Of the 73 patients with hepatitis A, 65 (89.0%) showed positive indirect immunofluorescence tests. Of note, most of the positive tests (95.5%) showed a cytoplasmic pattern with filamentous staining of cytoplasmic fibres. There was no significant difference between groups in age or sex. In patients positive for autoantibodies, alanine aminotransferase and leucocyte count were significantly higher, while the increase in globulin was not statistically significant. In terms of titres, globulin was significantly higher in patients with > or =1:160 titres than in those with < or =1:80 titres (mean (SD) 3.4 (0.5) versus 2.8 (0.4) g/dl, respectively; p = 0.000). CONCLUSIONS: The study demonstrated a high prevalence of anticytoplasmic autoantibodies in patients with acute hepatitis A. These data would be useful to aid interpretation of indirect immunofluorescence testing in patients with acute hepatitis, especially in areas with a high prevalence of HAV.

Therapeutic use of granulocyte-colony stimulating factor could conceal residual malignant cells in patients with AML1/ETO+ acute myelogenous leukemia.

We have experienced a number of cases of AML1/ETO+ acute myelogenous leukemia that showed remission based on bone marrow (BM) morphological criteria, but that revealed clonal abnormalities in most cells by fluorescence in situ hybridization (FISH). Interestingly, most of these cases had AML with AML1/ETO rearrangement. The malignant cells were differentiated and considered mature cells after granulocyte-colony stimulating factor (G-CSF) treatment. To clarify the possible mechanisms underlying this phenomenon, we investigated the expression levels of G-CSFR in AML cells with AML1/ETO rearrangement by flow cytometry and real-time polymerase chain reaction (PCR). The number of AML1/ETO+ cells expressing G-CSFR at baseline was significantly higher than that of AML1/ETO- AML cells (2673 vs 522). In addition, the G-CSFR gene was more highly expressed in AML1/ETO+ cells than in AML1/ETO- cells by real-time PCR. This study reveals that cases showing remission after treatment with G-CSF mostly had leukemia with AML1/ETO rearrangement. This finding might be explained by the higher expression of G-CSF receptor in AML1/ETO+ cells than in AML1/ETO- cells. We recommend that remission should be confirmed by FISH, because malignant clones can be differentiated and masked in morphological examination or chromosome test, especially for AML with AML1/ETO rearrangement.

Persistence of Escherichia coli O157:H7 in experimentally infected swine.

These experiments determined the ability of Escherichia coli O157:H7 to colonize and persist in pigs simultaneously inoculated with other pathogenic E. coli strains. Three-months-old pigs were inoculated with a mixture of five E. coli strains. The mixture included two Shiga toxigenic E. coli (STEC) O157:H7 strains, two enterotoxigenic E. coli (ETEC) strains and one enteropathogenic E. coli (EPEC) strain. A high dose mixture with all five strains at 10(10)CFU/animal (CFU: colony forming units) and a low dose mixture with the STEC strains at 10(7)CFU and the EPEC and ETEC strains remaining at 10(10)CFU were used. The STEC strains persisted in the alimentary tracts of some pigs at 2 months post-inoculation, following inoculation with both the high and low dose mixtures. When all strains were given at 10(10)CFU (high dose) the STEC strains persisted in greater numbers and in more pigs than did the other E. coli strains. The results demonstrated that persistent colonization (> or =2 months) by E. coli O157:H7 can occur in pigs. These findings were similar to those reported from sheep inoculated with the same mixture of E. coli strains. The results are consistent with reports suggesting that pigs have the potential to be reservoir hosts for STEC O157:H7.

In vitro detection of Shiga toxin using porcine alveolar macrophages.

Porcine alveolar macrophages were found to be highly susceptible to the cytolytic effects of a toxin (Shiga toxin [Stx]) produced by certain strains of Escherichia coli and sometimes associated with clinical disease in pigs and other animals. In comparison with the cells that are most commonly used for Stx detection and titration in vitro (namely, Vero cells), porcine alveolar macrophages appeared to be generally more sensitive and test results could be obtained in less time. Moreover, unlike Vero cells, porcine alveolar macrophages need not be continuously propagated to ensure immediate availability. They can simply be removed from a low-temperature repository, thawed, seeded, and shortly thereafter exposed to the sample in question. These characteristics suggest that porcine alveolar macrophages may be useful in developing a highly sensitive and timely diagnostic test for Stx.

A comparison of bowel care patterns in patients with spinal cord injury: upper motor neuron bowel vs lower motor neuron bowel.

STUDY DESIGN: A face-to-face interview survey. OBJECTIVE: To compare bowel care patterns in spinal cord injury (SCI) patients based on type of neurogenic bowel. SETTING: Department of Physical Medicine and Rehabilitation of a tertiary university hospital in Suwon, Korea. METHODS: Among chronic SCI patients, 22 patients with upper motor neuron bowel (UMNB) and 20 patients with lower motor neuron bowel (LMNB) participated in an interview survey for the evaluation of bowel care patterns. RESULTS: The patients with LMNB demonstrated increased frequency of defecation, increased frequency of fecal incontinence, increased use of oral medications for bowel care, increased required time for defecation and more diet modification than those with UMNB (P < 0.05). However, there was no significant difference in the subjective difficulty of bowel care. Among several available bowel care methods, suppositories were used most frequently by the UMNB group, whereas the Valsalva maneuver was the most frequently used method by the LMNB group. CONCLUSIONS: Patients with LMNB tend to suffer more difficulties in management of their neurogenic bowel than those with UMNB. Therefore, more intensive and aggressive bowel care programs should be provided for SCI patients with LMNB.

Intervention with Shiga toxin (Stx) antibody after infection by Stx-producing Escherichia coli.

Shiga toxins (Stxs) produced by Escherichia coli (STEC) cause systemic vascular damage, manifested as hemolytic uremic syndrome in humans and as edema disease in pigs. Edema disease, a naturally occurring disease of pigs, was used to determine whether Stx antibodies, administered after infection and after the onset of Stx production, could prevent the systemic vascular damage and clinical disease caused by Stxs. A total of 119 STEC-infected pigs were treated with low, medium, or high doses of Stx antibody or with placebo. After inoculation with STEC, antibodies or placebo was injected intraperitoneally at 2 days postinoculation (DPI; low dose) or 4 DPI (medium and high doses). Edema disease was prevented with the low- and high-dose Stx antibody treatments administered at 2 and 4 DPI, respectively. High-dose antibody treatment also reduced the incidence and extent of vascular lesions. The degree of protection depended on the dose of antibody and the time of administration.

Persistent colonization of sheep by Escherichia coli O157:H7 and other E. coli pathotypes.

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10(7) or 10(10) CFU/strain/animal. The other strains were given only at 10(10) CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10(7) or 10(10) CFU. One of the ETEC strains also persisted when inoculated at 10(10) CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10(7) CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.

Cattle lack vascular receptors for Escherichia coli O157:H7 Shiga toxins.

Escherichia coli O157:H7 causes Shiga toxin (Stx)-mediated vascular damage, resulting in hemorrhagic colitis and the hemolytic uremic syndrome in humans. These infections are often foodborne, and healthy carrier cattle are a major reservoir of E. coli O157:H7. We were interested in knowing why cattle are tolerant to infection with E. coli O157:H7. Cattle tissues were examined for the Stx receptor globotriaosylceramide (Gb(3)), for receptivity to Stx binding in vitro, and for susceptibility to the enterotoxic effects of Stx in vivo. TLC was used to detect Gb(3) in tissues from a newborn calf. Gb(3) was detected by TLC in kidney and brain, but not in the gastrointestinal tract. Immunohistochemistry was used to define binding of Stx1 and Stx2 overlaid onto sections from cattle tissues. Stx1 and Stx2 bound to selected tubules in the cortex of the kidney of both newborn calves (n = 3) and adult cattle (n = 3). Stx did not bind to blood vessels in any of the six gastrointestinal and five extraintestinal organs examined. The lack of Gb(3) and of Stx receptivity in the gastrointestinal tract raised questions about the toxicity of Stx in bovine intestine. We found that neither viable E. coli O157:H7 nor Stx-containing bacterial extracts were enterotoxic (caused fluid accumulation) in ligated ileal loops in newborn calves. The lack of vascular receptors for Stx provides insight into why cattle are tolerant reservoir hosts for E. coli O157:H7.

Vascular ultrastructure and DNA fragmentation in swine infected with Shiga toxin-producing Escherichia coli.

Shiga toxins (Stx) produced by Escherichia coli cause systemic vascular damage that manifests as edema disease in swine and hemolytic uremic syndrome in humans. In vitro, Stx inhibit protein synthesis and, depending on circumstances, induce necrosis, apoptosis, or both. The mechanism of in vivo Stx-mediated vascular damage is not known. The ability of Stx to cause apoptosis of vasculature in vivo was studied in pigs with edema disease that was produced by oral inoculation with Stx-producing E. coli. Arterioles of ileum and brain were evaluated by terminal dUTP nick-end labeling (TUNEL) assay for DNA fragmentation in myocytes (10 infected pigs, 5 control pigs) and by transmission electron microscopy for ultrastructural changes characteristic of apoptosis (17 infected pigs, 8 control pigs). In comparison with controls, increased numbers of TUNEL-positive arterioles were detected in 6/10 (60%) subclinically affected pigs 14-15 days after inoculation. Ultrastructurally, lesions in myocytes consisted of lysis (necrosis), with cytoplasmic debris and nuclear fragments contained between intact basement membranes. Endothelial cell changes ranged from acute swelling to necrosis and detachment from basement membrane. Subclinically affected pigs (n = 14) tended to have changes predominantly in myocytes, whereas pigs with clinical illness (n = 3) more commonly had changes in endothelial cells. The arteriolar lesions and clinical signs of edema disease are attributed to the effects of Stx on vasculature. Therefore, our findings suggest that the Stx-induced arteriolar lesions seen in this study were primarily necrotic, not apoptotic. We suspect that necrosis was the principal cause of the DNA fragmentation detected.

Escherichia coli O157:H7 causes more-severe systemic disease in suckling piglets than in colostrum-deprived neonatal piglets.

Our objective was to determine if suckling neonatal piglets are susceptible to enterohemorrhagic Escherichia coli (EHEC) O157:H7 disease. Surprisingly, EHEC O157:H7 caused more-rapid and more-severe neurological disease in suckling neonates than in those fed an artificial diet. Shiga toxin-negative O157:H7 did not cause neurological disease but colonized and caused attaching-and-effacing intestinal lesions.

Shiga toxin-producing Escherichia coli infection: temporal and quantitative relationships among colonization, toxin production, and systemic disease.

Edema disease, a naturally occurring disease of swine caused by Shiga toxin-producing Escherichia coli (STEC), was used as a model for the sequence of events that occur in the pathogenesis of STEC infection. The mean time from production of levels of Shiga toxin 2e (Stx2e) detectable in the feces (day 1) to the onset of clinical disease (neurologic disturbances or death) was 5 days (range, 3-9). Bacterial colonization and titers of Stx2e in the ileum peaked at 4 days after inoculation in pigs without signs of clinical disease and at 6 days after inoculation in clinically affected pigs. Animals with the greatest risk of progressing to clinical disease tended to have the highest fecal toxin titers (>/=1:4096). Stx2e was detected in the red cell fraction from blood of some pigs showing clinical signs of edema disease but was not detected in the serum or cerebrospinal fluid.

Ultrastructure and DNA fragmentation analysis of arterioles in swine infected with Shiga toxin-producing Escherichia coli.

Shiga toxins (Stx) produced by E. coli are potent cytotoxins that affect the vascular system. In humans, systemic toxemia causes renal glomerular damage manifested as hemolytic uremic syndrome. In swine, Stx-producing E. coli (STEC) cause edema disease that is characterized microscopically by segmental arteriolar smooth muscle cell (SMC) lesions. Our objectives were to characterize ultrastructurally and by TUNEL the type of death (apoptosis or necrosis) that occurs in SMCs during edema disease. Increased DNA fragmentation consistent with apoptosis was detected by TUNEL in arterioles of challenged pigs 14-15 days post inoculation. Ultrastructurally 3 grades of SMC lesions were distinguished: 1) Partial loss of SMCs, intercellular space filled with granular cellular debris admixed with membrane bound vacuoles; 2) Complete loss of SMCs; only granular cellular debris and clear vacuoles remained within basement membrane; 3) Inflammation of media; SMCs replaced by a rim of cellular debris located in the periphery of vessel wall. The most common lesion detected was grade 1 (9 ilea and 4 brains). We did not find apoptotic nuclear changes in SMCs or apoptotic inclusion bodies within resident cells. Our study indicates, that (1) Stx produced during edema disease does not cause SMC apoptosis 14-15 dpi; (2) SMCs undergo an array of changes from degeneration to necrosis.