Prevalence of massively diluted bone marrow cell samples aspirated from patients with myelodysplastic syndromes (MDS) or suspected of MDS: A retrospective analysis of nationwide samples in Japan.

Bone marrow (BM) examination is a key element in the diagnosis and prognostic grading of myelodysplastic syndromes (MDSs), and obtaining adequate BM cell samples is critical for accurate test results. Massive haemodilution of aspirated BM samples is a well-known problem; however, its incidence in patients with MDS has not been well studied. We report the first study to examine the incidence of massive haemodilution in nationwide BM samples aspirated from patients diagnosed with or suspected of MDS in Japan. Among 283 cases available for analysis, BM smears from 92 cases (32.5%) were hypospicular (massively haemodiluted) and, particularly, no BM particles were observed in 52 cases (18.4%). Regarding hypospicular cases, we examined how the doctors in charge interpreted the BM smears of their patients. In only 19 of 92 cases (20.7%), doctors realised that the BM smears were haemodiluted. Furthermore, the BM biopsy, which can help diagnose hypospicular cases, was oftentimes not performed when the haemodilution was overlooked by doctors (not performed in 50 of 73 such cases). These real-world data highlight that not only researchers who are working to improve diagnostic tests but also clinicians who perform and use diagnostic tests must realise this common and potentially critical problem.

Myeloblasts transition to megakaryoblastic immunophenotypes over time in some patients with myelodysplastic syndromes.

OBJECTIVES: In myelodysplastic syndromes (MDS), neoplastic myeloblast (CD34+CD13+CD33+ cells) numbers often increase over time, leading to secondary acute myeloid leukemia (AML). In recent studies, blasts in some MDS patients have been found to express a megakaryocyte-lineage molecule, CD41, and such patients show extremely poor prognosis. This is the first study to evaluate whether myeloblasts transition to CD41+ blasts over time and to investigate the detailed immunophenotypic features of CD41+ blasts in MDS. METHODS: We performed a retrospective cohort study, in which time-dependent changes in blast immunophenotypes were analyzed using multidimensional flow cytometry (MDF) in 74 patients with MDS and AML (which progressed from MDS). RESULTS: CD41+ blasts (at least 20% of CD34+ blasts expressing CD41) were detected in 12 patients. In five of these 12 patients, blasts were CD41+ from the first MDF analysis. In the other seven patients, myeloblasts (CD34+CD33+CD41- cells) transitioned to megakaryoblasts (CD34+CD41+ cells) over time, which was often accompanied by disease progression (including leukemic transformation). These CD41+ patients were more frequently observed among patients with monosomal and complex karyotypes. CD41+ blasts were negative for the erythroid antigen, CD235a, and positive for CD33 in all cases, but CD33 expression levels were lower in three cases when compared with CD34+CD41- blasts. Among the five CD41+ patients who underwent extensive immunophenotyping, CD41+ blasts all expressed CD61, but two cases had reduced CD42b expression, three had reduced/absent CD13 expression, and three also expressed CD7. CONCLUSIONS: Myeloblasts become megakaryoblastic over time in some MDS patients, and examining the megakaryocyte lineage (not only as a diagnostic work-up but also as follow-up) is needed to detect CD41+ MDS. The immunophenotypic features revealed in this study may have diagnostic relevance for CD41+ MDS patients.

Clinical, immunophenotypic, and cytogenetic characteristics of high-grade myelodysplastic syndromes with CD41-positive progenitor cells.

BACKGROUND: Patients with myelodysplastic syndromes (MDS) with progenitors expressing CD41 (CD41+ MDS) showed a poor prognosis in a previous study but their detailed characteristics remain unclear. METHODS: One hundred thirty-seven subjects at our institution were diagnosed with excess blasts (EB)-1, EB-2, and acute myeloid leukemia with a low blast count (20%-30%). The immunophenotypes of progenitor cells in their bone marrow (BM) were determined by CD45-gating flow cytometry. A false-positive reaction to CD41 was eliminated by examining the flow cytometry data of lymphocytes and monocytes in addition to progenitors and by examining CD42b in histological sections. The characteristics were compared between CD41+ and CD41- MDS patients. RESULTS: Forty-three patients (31%) were CD41+. Additionally, 91% of the CD41+ MDS patients were very high-risk defined by the Revised International Prognostic Score System, which was higher than in patients with CD41- MDS (p = 0.015). Approximately 60% of the CD41+ MDS patients had a monosomal karyotype and very poor cytogenetics, which was higher than in CD41- MDS patients (p < 0.001). Normal cytogenetics was less common in CD41+ patients (p = 0.0016). Blasts with bleb formation were more abundant in CD41+ MDS patients (p = 0.026). All CD41+ MDS patients were positive for CD13 and were mostly positive for CD33. The frequency of aberrant expression of other antigens on progenitors was similar between CD41+ and CD41- MDS patients. CONCLUSIONS: We determined clinical, immunophenotypic, and cytogenetic characteristics of CD41+ MDS patients. Further studies are needed to improve the survival of these patients.

Relationship between ovarian ultrasonographic findings on the seventh post-estrus day and plasma progesterone concentration, nutritional metabolic factors, and pregnancy outcome in dairy cows.

To improve the accuracy of ultrasonographic assessment of luteal function, we investigated the relationship between ovarian ultrasonographic findings on Day 7 (Day 1 = ovulation) and plasma progesterone (P4) concentration, nutritional metabolic factors, and pregnancy outcome. A total of 47 spontaneous estrus events were investigated in 38 lactating Holstein cows (artificial insemination, n = 31; embryo transfer, n = 16). Transrectal ultrasonography was performed on Days 0 and 7 to measure the pre-ovulatory follicle area on Day 0 and the luteal tissue area (LTA), luteal blood flow area (LBF), relative LBF (rLBF) (= LBF/LTA), and dominant follicle area (DFA) on Day 7. Blood samples were collected on Day 7 to measure plasma P4, insulin-like growth factor-I (IGF-I), insulin, and metabolites. Plasma P4 concentration was positively correlated with LTA but was not associated with LBF or rLBF. Plasma P4 concentration was positively correlated with blood glucose and IGF-I and negatively correlated with blood urea nitrogen and free fatty acid, and no significant relationship was found between the ultrasonographic findings of the corpus luteum (CL) and these blood metabolites. Pregnant cows had smaller DFA than non-pregnant cows. In conclusion, LTA measurement can help predict plasma P4 concentration, but it was difficult to detect variations in plasma P4 concentration in relation to changes in energy status by evaluating the CL ultrasonographically. A combined assessment of CL and first-wave dominant follicle may be important in evaluating fertility.

Changes in oxidative stress parameters in healthy and diseased Holstein cows during the transition period in Yamagata Prefecture, Japan.

We investigated changes in oxidative stress markers during the transition period in healthy Holstein cows and those with postpartum diseases. Transition control (TC) Holstein cows (n=9) were evaluated for longitudinal changes during the transition period and postpartum diseased (PD) cows with ketosis (n=10), abomasal displacement (n=9), and acute mastitis (n=10) were evaluated in comparison to control cows (n=10). In the TC group, blood samples were collected at 2 weeks prepartum and at 1, 2, 4, 6, and 8 weeks postpartum. Milk yield and composition were measured at 2 and 4 weeks postpartum. In the PD group, blood samples were collected at the first day of examination during the 60 days postpartum. Peripheral oxidative stress parameters (malondialdehyde, MDA; potential antioxidant capacity, PAO; and glutathione peroxidase) were measured, and biochemical analyses were performed. In the TC group, MDA increased significantly postpartum and was correlated with milk yield, blood glucose (Glu), free fatty acid (FFA), ß-hydroxybutyric acid (BHB), and aspartate aminotransferase. Compared to the control cows, PD cows with ketosis had significantly higher MDA and significantly lower PAO. Moreover, MDA was significantly correlated with Glu, FFA, and BHB. Postpartum increase in MDA might interact with milk yield and Glu, FFA, and BHB in the TC cows, and postpartum diseases, especially ketosis, might signify its increase and interaction with Glu, FFA, and BHB.

Revising flow cytometric mini-panel for diagnosing low-grade myelodysplastic syndromes: Introducing a parameter quantifying CD33 expression on CD34+ cells.

Diagnosis of myelodysplastic syndromes (MDS) is not straightforward when objective data, such as blast excess and abnormal cytogenetics, are lacking. Expert laboratories use flow cytometry (FCM) to help diagnose MDS. However, most of FCM protocols for MDS are complex, requiring a high level of expertise and high cost. We have reported a FCM mini-panel consisting of four FCM parameters (so-called Ogata score), which is simple to conduct and inexpensive. In this paper, to refine this mini-panel, we have introduced a new FCM parameter, which quantifies CD33 expression on CD34+ cells (called Granulocyte/CD34 cell CD33 ratio). Bone marrow cells from MDS without blast excess (low-grade MDS) and controls were stained with CD34, CD45, and CD33 and analyzed for five parameters ("Granulocyte/CD34 cell CD33 ratio" plus four parameters in the Ogata score). By a multivariate logistic regression model, only three parameters, including "Granulocyte/CD34 cell CD33 ratio" had statistically significant power for diagnosing low-grade MDS. Based on the results, we constructed a new scoring system, which showed approximately 50% sensitivity and more than 95% specificity in diagnosing low-grade MDS. Our revised mini-panel is suitable for screening samples suspected for MDS and provides a basis for further improvement in diagnostic FCM protocols for MDS.

Herd-level risk factors associated with Leptospira Hardjo infection in dairy herds in the southern Tohoku, Japan.

A cross-sectional study was designed to generate information on the herd level prevalence and the risk factors for Leptospira serovar Hardjo (L. Hardjo) in Yamagata, the southern Tohoku, Japan. Bulk tank milk samples from 109 dairy herds were used to test the herd level sero-prevalence of L. Hardjo using a commercial ELISA kit, which detects both L. interrogans serovar Hardjo and L. borgpetersenii serovar Hardjo. A questionnaire survey was conducted at the sampled farms, and univariable and multivariable analyses were performed. Spatial clustering of L. Hardjo at the herd level was examined using spatial scan statistics. Seventy-one herds were found to be positive for L. Hardjo, and the apparent herd prevalence was 65.1% (95% CI: 56.2-74.1%). The risk factors for sero-positivity were larger herd size (p=0.004) and cows with a history of staying in Hokkaido (p <0.001). The spatial scan statistic detected a most likely cluster (relative risk=1.87, log likelihood ratio=9.93, radius=13.70km, p<0.01) in the southern part of the study area where there are large herd sizes and farm density is high. This study revealed that L. Hardjo is prevalent throughout Yamagata, and large scale herd owners introducing cows from Hokkaido in particular should be aware of the risk of infection.

Water intoxication in adult cattle.

Water intoxication is a common disorder in calves and is usually characterized by transient hemoglobinuria. In contrast, the condition is very rare in adult cattle, with few reports on naturally occurring cases. In the present report, four female Japanese Black cattle, aged 16-25 months, showed neurological signs when they drank water following a water outage. Hemoglobinuria was not grossly observed, while severe hyponatremia was revealed by laboratory tests. Autopsy indicated cerebral edema with accumulation of serous fluid in expanded Virchow-Robin spaces. These results indicate the possibility of water intoxication associated with cerebral edema due to severe dilutional hyponatremia in adult cattle.

Evaluation of ABCG2 and p63 expression in canine cornea and cultivated corneal epithelial cells.

OBJECTIVE: To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration. PROCEDURES: Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR. RESULTS: The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages. CONCLUSIONS: ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively.

A comparison of neurosphere differentiation potential of canine bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells.

Stem cell transplantation is one of the most promising yet enigmatic treatments for spinal cord injury (SCI), a common problem in dogs. As pre-differentiated mesenchymal stem cells (MSCs) can be expanded and differentiated into neurospheres in vitro, before being transplanted back, they may prove to be more beneficial for treating SCI. Therefore, we compared the endogenous differentiation potential, including the neuronal cell differentiation, of neurospheres from canine bone marrow MSCs (cBMMSCs) with that of the adipose tissue-derived MSCs (cADMSCs). Nestin-positive neurospheres were generated from MSCs derived from the bone marrow and adipose tissue. Neuronal cells were differentiated from the neurospheres derived from both these tissues. Gene expression analysis revealed that Nestin, ßIII-tubulin, NCAM, OCT4 and SOX2 were expressed in MSCs and the corresponding neurospheres. Notably, cBMMSC-derived neuronal cells expressed higher levels of ßIII-tubulin. The mRNA expressions of NANOG, Nestin, OCT4 and SOX2 were upregulated in neurospheres derived from both. Immunofluorescence analysis detected the expression of neuronal markers, namely, ßIII-tubulin, GFAP, S100, NF200 and MAP2, in differentiated neuron-like cells. Our findings highlight that both cBMMSCs and cADMSCs could be differentiated into neurospheres and neuron-like cells, and therefore, these cells are suitable candidates for cell transplantation. Further, cADMSCs form a more suitable cell source, as larger number of cells could be harvested from cADMSC-derived neurospheres. Future studies employing in vivo transplantation models to investigate the effectiveness of MSCs for treating SCI are warranted.