S. Jang

Establishment of a reference interval for natural killer cell activity through flow cytometry and its clinical application in the diagnosis of hemophagocytic lymphohistiocytosis

Recently, the Histiocyte Society revised the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) to include low or absent natural killer (NK) cell activity, according to local laboratory reference. The aim of this study was to establish reference interval for functional NK‐cell activity in 63 healthy Korean individuals using a flow‐cytometric assay. We used peripheral blood mononuclear cells (PBMCs) as effector cells and Fluorescein isothiocyanate‐labeled K562 cells as target cells. NK‐cell activity was calculated using the following equation: NK‐cell activity (%) = (test lysis − spontaneous lysis) × 100/(maximum lysis − spontaneous lysis). NK‐cell activity was analyzed in 13 known HLH patients and 16 suspected non‐HLH patients using a flow‐cytometric assay. The mean (±SD) cytotoxicity of PBMCs from healthy individuals was 20.9 ± 5.3% and the reference interval was 11.8–31.9%. The mean NK‐cell activity of HLH patients (8.3 ± 8.9%) was significantly lower (P = 0.001) than that of non‐HLH patients (20.1 ± 7.8%). The sequential changes in NK‐cell activity in the HLH group corresponded to clinical and laboratory findings following treatment. We successfully developed a functional NK‐cell activity test for use in the clinical laboratory and obtained a reference interval of NK‐cell activity from healthy donors. This assay, and associated reference interval, was used to analyze 30 clinically relevant specimens and the results were shown to be well correlated.

Sepsis affects most routine and cell population data (CPD) obtained using the Sysmex XN-2000 blood cell analyzer: neutrophil-related CPD NE-SFL and NE-WY provide useful information for detecting sepsis.

The Sysmex XN‐2000 analyzer can assess 36 routine and 57 cell population data (CPD) items. In this study, we evaluated these items as sepsis biomarkers.

Body fluid cellular analysis using the Sysmex XN-2000 automatic hematology analyzer: focusing on malignant samples

The majority of previous studies on body fluid (BF) mode of automatic hematology analyzer used nonmalignant BF samples. Here, we evaluated the BF mode on the recently launched Sysmex XN for counting blood cells, especially for malignant samples.

Automated digital cell morphology identification system (CellaVision DM96) is very useful for leukocyte differentials in specimens with qualitative or quantitative abnormalities.

The CellaVision DM96 system (CellaVision AB, Lund, Sweden) was developed as one of the automated digital cell morphology analyzer for determining leukocyte differential counts in peripheral blood smears (PBS) and we evaluated this system.

Application of an immune-magnetic cell sorting method for CD138-positive plasma cells in FISH analysis of multiple myeloma

Introduction:  Interphase fluorescence in situ hybridization (FISH) analysis of multiple myeloma (MM) may indiscriminately count signals of nonplasma cells, thus decreasing specificity and sensitivity. We aimed to evaluate the usefulness of an immune‐magnetic sorting method for plasma cells in FISH analysis of MM and define optimal sample preparation conditions.

Morphologic detection of blast cells in the cerebrospinal fluid at diagnosis of adult acute lymphoblastic leukemia appears to be associated with adverse prognosis

Appropriate treatment of central nervous system (CNS) involvement in adult acute lymphoblastic leukemia (ALL) is important for patient prognosis, but the diagnostic criteria of CNS involvement has not been established.

A rare case of Lennert's type peripheral T-cell lymphoma with t(14;19)(q11.2;q13.3).

Although most patients with peripheral T‐cell lymphoma (PTCL) show clonal rearrangement of T‐cell receptor genes, few PTCLs show recurrent chromosomal abnormalities. We describe here a rare chromosomal rearrangement, t(14;19)(q11.2;q13.3), in a Lennert’s lymphoma, a variant of PTCL, not otherwise specified. Sequential fluorescence in situ hybridization assays showed that the breakpoint in 19q13.3 was located distal to the BCL3 and PVRL2 genes, both of which may be candidate proto‐oncogenes. These findings suggest that another gene is involved in the pathogenic characteristics observed in this patient with Lennert’s lymphoma.

FISH analysis of MLL gene rearrangements: detection of the concurrent loss or gain of the 3' signal and its prognostic significance.

The rearrangement of the mixed‐lineage leukemia (MLL) gene occurs through translocations and insertions involving a variety of partner chromosome genes. However, there are few studies on aberrant MLL signal patterns such as concurrent 3ʹ MLL deletion.

A case of de novo aleukemic mast cell leukemia without c-KIT mutations in exons 8 and 17.

Sir, Mast cell leukemia (MCL) is a very rare form of aggressive systemic mastocytosis (ASM), occurring in <0.5% of patients with mastocytosis [1, 2]. The diagnosis of MCL is based on the presence of ≥20% atypical MC in the bone marrow (BM) and/or ≥10% in the peripheral blood (PB). The diagnosis of ‘aleukemic’ variant of MCL is made when the number of circulating MC is <10% [1, 2]. Given that SM is known to be frequently associated with c-KIT mutations, most commonly D816V mutations [1, 2], the absence of c-KIT D816V mutations in MCL cases is worth reporting. We report here a case of de novo aleukemic MCL without c-KIT mutations in exons 8 and 17. A 72-year-old woman was admitted to the authors’ institution due to persistent cytopenia (WBC 3.3 9 10/L, hemoglobin 6.3 g/dL, platelets 2.6 9 10/L), weight loss (12 kg), and gastric discomfort for 6 months. Her PB smears showed the presence of immature MC exhibiting signs of marked atypia, characterized as hypogranular cytoplasm and irregularly shaped monocytoid and bilobated nuclei (defined as promastocytes)[1, 2], with the frequency of 3% (figure 1a). Her BM aspiration and touch print possessed immature MC that showed identical morphology to that found in PB, with the frequency of 87.4%, as well as a few typically round mononuclear mature MC with metachromatic granules in the cytoplasm (figure 1b–d). The immunophenotyping of MC found in the BM aspiration showed strong expression of CD13(57.4%) and CD33 (35.5%), reduced expression of CD117 (24.3%), and no expression of CD34 (0.9%), HLA-DR (1.8%), and other B and T cell markers including CD2 (5.4%). The BM biopsy was hypercellular (approximately 100%) and comprised almost exclusively of CD117 and mast cell tryptase immunohistochemical (IHC) stain strongly positive atypical MC present in a dense aggregates (figure 1e–g). The MC found in BM biopsy showed weak positivity in CD25 IHC stain, but were negative in CD2 IHC stain (figure 1h,i). Esophagogastroduodenoscopy (EGD) revealed diffuse atrophic mucosal change on the antrum of stomach and multiple mucosal erosions in duodenum, suggesting atrophic gastritis and duodenitis. The HemaVision screening test (DNA technology, Aarhus, Denmark), qualitative multiplex reverse transcriptase polymerase chain reaction diagnostic test designed to screen for 28 different translocations or chromosomal rearrangements, was performed, and the result was negative for all detectable fusion transcripts (figure 1j). Her karyotype was 46,XX,inv(9)(p12q13),inv(11)(p11.2q23) [4]/46,XX,inv(9)(p12q13)[16]. This result indicates the presence of abnormal karyotype with inv(11)(p11.2q23) in 20% of total metaphases, as well as constitutional variant with inv(9)(p12q13) present in all metaphases. The c-KIT mutation analysis in exons 8 and 17 based on direct sequencing was performed by previously described methods [3], and the result was wild type for both exons (figure 1k). Based on these results, she was diagnosed as de novo aleukemic MCL without c-KIT mutations in exons 8 and 17. According to recently published article that reviewed MCL, only 30 and 11 cases with de novo and secondary MCL have been reported up to now, respectively [2]. They hypothesized that patients with de novo MCL have more aggressive symptom of pathologic MC activation such as gastroduodenal ulcer (due to release of large amount of histamine) than those with secondary MCL [2]. This hypothesis can be supported by our case because she exhibited severe symptoms and sign of duodenal ulcer at admission, and her EGD finding did support the diagnosis. The most recent 2008 WHO classifications define abnormal MC as the expression of CD2 and/or CD25 [1]. Although most MC found in MCL express both CD25 and CD2, recent studies reported that the expression of CD25 and CD2 was not found in 25% and 48% of MCL cases [2], and even one-third of MCL cases reported in the literatures were double negative for both antigens [4, 5]. The clonality of the MC found in our case could be demonstrated from the CD25 IHC positivity in BM biopsy, which satisfies the WHO definition for MCL.

Direct international normalized ratio determination using multicalibrators is more responsive than the conventional method for measuring prothrombin time.

Direct international normalized ratio (INR) determination using certified INR plasmas was shown to improve precision and accuracy. We evaluated the utility of a multicalibrator in determining INR. INR values were measured in 493 blood samples from patients subjected to anticoagulation therapy (320) and control subjects (173). Study was performed using CA‐7000 coagulation analyzer (Sysmex, Japan) with Thromborel S (Dade Behring, Germany). Direct INR values were obtained using PT‐Multi Calibrator (Dade Behring) composed of five lyophilized calibrant plasmas. Conventional INR values were calculated from mean normal prothrombin time and instrument/reagent‐specific international sensitivity index (ISI). We compared the difference between the INR results obtained with the two methods. The mean INR value of direct INR method was significantly higher than that of conventional method. The differences in values (direct INR – conventional INR) generated using the two methods increased in proportion to the INR values. Elevation of INR was observed in data obtained with the direct INR method, compared with conventional INR values. Accordingly, we conclude that direct INR method is more responsive than conventional method in determining INR.