Differentiation between incomplete Kawasaki disease and secondary hemophagocytic lymphohistiocytosis following Kawasaki disease using N-terminal pro-brain natriuretic peptide.

PURPOSE: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome with many causes, including Kawasaki disease (KD). The purpose of this study was to identify the laboratory tests needed to easily differentiate KD with HLH from incomplete KD alone. METHODS: We performed a retrospective study on patients diagnosed with incomplete KD and incomplete KD with HLH (HLH-KD) between January 2012 and March 2015. We compared 8 secondary HLH patients who were first diagnosed with incomplete KD with all 247 incomplete KD diagnosed patients during the study period. The complete blood count, erythrocyte sedimentation rate, platelet count, and serum total protein, albumin, triglyceride, C-reactive protein, N-terminal pro-brain natriuretic peptide (NT-proBNP), and ferritin levels were compared. Clinical characteristics and echocardiography findings were also compared between the 2 groups. RESULTS: The total duration of fever was longer in the HLH-KD group than in the KD group. White blood cell and platelet counts were higher in the KD group. Alanine aminotransferase, ferritin, and coronary artery diameter were increased in the HLH-KD group compared with those in the KD group. The median of NT-proBNP was significantly higher in the HLH-KD group than in the KD group at 889.0 (interquartile range [IQR], 384.5-1792.0) pg/mL vs. 233.0 (IQR, 107.0-544.0) pg/mL. CONCLUSION: The NT-proBNP level may be helpful in distinguishing incomplete KD from KD with HLH. The NT-proBNP level should be determined in KD patients with prolonged fever, in addition to the white blood cell count, platelet count, and ferritin level, to evaluate secondary HLH.

[Myelodysplastic syndrome mimicking idiopathic thrombocytopenic purpura].

BACKGROUND: In patients with isolated thrombocytopenia, but without significant dysplasia, diagnosis of idiopathic thrombocytopenic purpura (ITP) rather than myelodysplastic syndrome (MDS) may be taken into account. It is important to make an accurate diagnosis because different treatments are used for ITP and MDS. The purpose of this study was to investigate the clinical and hematologic features of patients who were initially diagnosed as ITP but had cytogenetic abnormalities. METHODS: We retrospectively reviewed cytogenetic studies of 100 patients who were diagnosed as ITP from 2004 to 2009 at Mokdong Hospital of Ewha Womans University based on clinical features and hematologic studies. Bone marrow pathology was re-evaluated based on 2008 WHO classification. Cytogenetic analysis was performed by 24-48 hr culture of bone marrow aspirates without using mitogens and 20 metaphases were analyzed. RESULTS: Of the 100 patients diagnosed as ITP initially, three patients (3%) had cytogenetic abnormalities. They had no thrombocytopenia-related symptoms and thrombocytopenia was found accidentally. The numbers of megakaryocytes in bone marrow were increased and dysplasia was not found in megakaryocyte, erythroid, and myeloid cell lineages. The proportion of blasts was within normal limits. Clonal chromosomal abnormalities found were der(1;7)(q10;p10), add(9)(q12), or t(7;11)(p22;q12). Presumptive diagnosis of MDS or diagnosis of idiopathic cytopenia of undetermined significance (ICUS) was made according to 2008 WHO classification. During the follow up, disease progression was not found. CONCLUSIONS: In patients with suspected ITP, cytogenetic analysis should be done. If specific clonal chromosomal abnormality is found, presumptive diagnosis of MDS has to be considered and close follow up is needed.

Immunophenotypic features of granulocytes, monocytes, and blasts in myelodysplastic syndromes.

BACKGROUND: Despite the diagnostic utility of immunophenotyping for myelodysplastic syndromes (MDS), it has not been widely performed, and reports on this are absent in Korea. We aimed to evaluate the immunophenotypic features of non-blastic granulocytes, monocytes, and blasts in patients with MDS and non-clonal disorders using routine flow cytometry (FCM). Moreover, we evaluated the phenotypic abnormalities of mature cells in leukemic patients. METHODS: Marrow aspirates from 60 patients, including 18 with MDS, 18 with leukemia, and 24 with non-clonal disorders (control group), were analyzed using FCM. Blasts, non-blast myeloid cells, and monocytes were gated based on CD45 expression and side scatter (SSC). The phenotypes were then compared among the 3 groups. RESULTS: Compared to non-clonal disorders, the granulocytic lineages of MDS showed decreased SSC (P=0.005), increased CD45 intensity (P=0.020), decreased CD10-positive granulocytes (P= 0.030), and a higher CD56-positive rate (P=0.005). It is noteworthy that similar results were obtained in the leukemia group, and these findings were not related to the phenotypes of the leukemic cells. Using blast and monocytic gating, useful parameters for generating a differential diagnosis were not found. CONCLUSIONS: Gating the granulocytic region is a relatively easy method for MDS immunophenotyping. Among the parameters studied, SSC, CD10, and CD56 were the most useful for differentiating MDS from non-clonal disorders. While immunophenotypic changes in MDS appear to be useful for differentiating MDS from non-clonal disorders, these changes were also noted in the mature cells of leukemic patients.

Malaria detection with the Sysmex XE-2100 hematology analyzer using pseudoeosinophilia and abnormal WBC scattergram.

Recent investigation using the Sysmex XE-2100 hematology analyzer (Sysmex Corporation, Japan) has demonstrated erroneously high eosinophil counts and abnormal white blood cell (WBC) scattergrams in malaria cases. This study was conducted to assess the diagnostic efficiency of the Sysmex XE-2100 analyzer for malaria. One hundred forty-four patients initially diagnosed with Plasmodium vivax infection, 319 patients with febrile illness, and 24 patients who underwent malaria treatment were analyzed. Atypical features on Sysmex XE-2100 analyzer were categorized as pseudoeosinophilia (a gap of more than 5% in eosinophil counts between the Sysmex XE-2100 analyzer and microscopic examination) and abnormal WBC scattergram. Pseudoeosinophilia or abnormal WBC scattergram were detected in 100 of 144 malaria-positive samples (sensitivity 69.4%, specificity 100%). The samples with pseudoeosinophilia or abnormal WBC scattergrams showed significantly higher parasite counts than the samples without pseudoeosinophilia or an abnormal WBC scattergram (P<0.05). All 24 samples from patients for whom the malaria smear was repeated after malaria treatment was initiated showed a normalized eosinophil count and a normal WBC histogram. In conclusion, attention to differential count and WBC scattergrams provided by the Sysmex XE-2100 would be a valuable tool in malaria detection.

hTERT mRNA levels by real-time RT-PCR in acute myelogenous leukemia.

The purpose of this study was to investigate whether levels of hTERT mRNA, as determined by real-time RT-PCR, are associated with prognosis and clinical course in AML patients. Fifty-four bone marrow specimens from 21 patients diagnosed with de-novo AML were included. The level of hTERT mRNA was measured with the Telo TAGGG hTERT Quantification Kit (Roche Diagnostics, Mannheim, Germany), using a LightCycler Instrument (Roche Diagnostics). The level of hTERT mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of hTERT mRNA by the level of the porphobilinogen deaminase (PBGD) housekeeping gene in the same samples [1,000x(hTERT/PBGD)]. The expression rates of hTERT mRNA were significantly higher at diagnosis (73%) and during relapse (80%) than during remission (27%) (P<0.05). The median RR for diagnosis or relapse was significantly higher than that for patients in remission (P<0.05). hTERT mRNA expression was not correlated with CD34 expression, blast counts, white blood cell counts, or chromosomal abnormality (P>0.05). Two patients who showed hTERT mRNA expression during remission (RR 3.14 and 7.15, respectively) relapsed after 1 month. Among seven patients with high hTERT mRNA levels (RR>9.51), 4 failed to achieve complete remission (CR), whereas 4 of 5 patients without hTERT mRNA expression at diagnosis or during relapse achieved CR (P>0.05). Patients showing a trend of increasing hTERT mRNA levels failed to reach a second CR after relapse, while those with a trend toward decreasing hTERT mRNA did achieve CR. Among eight samples showing hTERT mRNA expression in remission (RR>0), 5 were obtained from patients who had received GCSF within 14 days. The expression rate and level of hTERT mRNA during remission were significantly higher in patients who had previously received GSCF (56%, RR=0.15) than in other patients (15%, RR=0) (P<0.05). Serial and quantitative analysis of hTERT mRNA may be a useful marker for prediction of prognosis and monitoring in AML patients.

Adherent cells generated during long-term culture of human umbilical cord blood CD34+ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion.

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study, UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin, flt3 ligand, and granulocyte-colony stimulating factor. By week 4-5, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor, human vascular cell adhesion molecule-1, human intracellular adhesion molecule-1, human CD31, E-selectin, and human macrophage. Furthermore, when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer, better expansion of total nucleated cells, CD34(+) cells, and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells, which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells, we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method, one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors, establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering.