We reviewed our leukemia database to reclassify 610 patients previously diagnosed as having acute myeloid leukemia (AML) according to the updated 2016 WHO classification. Nine patients were categorized as having myelodysplastic syndrome and myeloid neoplasms with germline predisposition. AML with recurrent genetic abnormalities accounted for 57.4% (345/601) of the patients under the 2016 WHO classification. AML with mutated NPM1 was the most common form (16.5%), with the majority associated with monocytic differentiation (63.6%). AML with double CEBPA mutations accounted for 8.3% of these cases, and the majority were previously diagnosed as AML with/without maturation (78.0%). These newly classified mutations were mutually exclusive without overlapping with other forms of AML with recurrent genetic abnormalities. AML with mutated NPM1 and AML with myelodysplasia-related changes comprised the oldest patients, whereas AML with RUNX1-RUNX1T1 included the youngest patients. The leukocyte count was highest in AML with mutated NPM1, and the percentage of peripheral blood blasts was the highest in AML with double CEBPA mutations. Our results indicate that implementation of the 2016 WHO classification of AML would not pose major difficulties in clinical practice. Hematopathologists should review and prepare genetic tests for the new classification, according to their clinical laboratory conditions.
Classification ; Humans ; Leukemia ; Leukemia, Myeloid, Acute ; Leukocyte Count ; Myelodysplastic Syndromes

Hereditary spherocytosis (HS) is caused by mutations in the SPTA1, SPTB, ANK1, SLC4A1, and EPB42 genes, all of which encode erythrocyte membrane proteins. Mutations in SLC4A1, which encodes band 3 protein, have rarely been reported as the causative factor among Korean patients with HS. Here, we report two Korean patients with HS carrying mutations in SLC4A1. Patient 1 was a 3-year-old girl with unremarkable past and family histories and was evaluated for anemia that was detected after a complete blood count. She was suspected of having HS considering the spherocytosis of her peripheral blood smear, increased osmotic fragility, hemolytic features in blood chemistry tests, and splenomegaly. Sequence analysis revealed that the patient harbored a single heterozygous missense mutation, c.2278C>T (p.Arg760Trp) in exon 17 of SLC4A1. Patient 2 was a 23-year-old man who had a prior history of intermittent jaundice. Although the patient did not have anemia, a genetic test for HS was performed due to evidence of hemolytic features in the blood chemistry test, splenomegaly, and a family history of HS. The test confirmed a single heterozygous missense mutation, c.2423G>T (p.Arg808Leu) in exon 18 of SLC4A1.
Anemia ; Anion Exchange Protein 1, Erythrocyte ; Blood Cell Count ; Chemistry ; Child, Preschool ; Erythrocyte Membrane ; Exons ; Female ; Humans ; Jaundice ; Mutation, Missense ; Osmotic Fragility ; Sequence Analysis ; Splenomegaly ; Young Adult

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineages. The current study demonstrates that three driver mutations were detected in 82.6% of 407 MPNs with a mutation distribution of JAK2 in 275 (67.6%), CALR in 55 (13.5%) and MPL in 6 (1.5%). The mutations were mutually exclusive in principle except in one patient with both CALR and MPL mutations. The driver mutation directed the pathologic features of MPNs, including lineage hyperplasia, laboratory findings and clinical presentation. JAK2-mutated MPN showed erythroid, granulocytic and/or megakaryocytic hyperplasia whereas CALR- and MPL-mutated MPNs displayed granulocytic and/or megakaryocytic hyperplasia. The lineage hyperplasia was closely associated with a higher mutant allele burden and peripheral cytosis. These findings corroborated that the lineage hyperplasia consisted of clonal proliferation of each hematopoietic lineage acquiring driver mutations. Our study has also demonstrated that bone marrow (BM) fibrosis was associated with disease progression. Patients with overt fibrosis (grade ⩾2) presented an increased mutant allele burden (P<0.001), an increase in chromosomal abnormalities (P<0.001) and a poor prognosis (P<0.001). Moreover, among patients with overt fibrosis, all patients with wild-type JAK2/CALR/MPL (triple-negative) showed genomic alterations by genome-wide microarray study and revealed the poorest overall survival, followed by JAK2-mutated MPNs. The genetic–pathologic characteristics provided the information for understanding disease pathogenesis and the progression of MPNs. The prognostic significance of the driver mutation and BM fibrosis suggests the necessity of a prospective therapeutic strategy to improve the clinical outcome.
Alleles ; Bone Marrow ; Chromosome Aberrations ; Disease Progression ; Fibrosis ; Hematopoietic Stem Cells ; Humans ; Hyperplasia ; Prognosis ; Prospective Studies

BACKGROUND: Numerous studies tried to find new markers that after hematopoietic stem cell transplantation predict engraftment earlier than the conventional marker, absolute neutrophil count (ANC >500/microL). Early engraftment prediction can be achieved by a marker that reflects the release of neutrophils and monocytes into the leukopenic peripheral blood. METHODS: We analyzed blood cell parameters, including cell population data such as volume, conductivity, and light scatter in 77 patients who underwent HSCT (allogeneic, n=63; autologous, n=11) to detect possible markers. RESULTS: We identified 2 early engraftment markers of neutrophils (NEUTRO) and monocytes (MONO); a pair of mean-volume-neutrophils (MNV) and mean-conductivity-neutrophils (MNC) for NEUTRO; and a pair of mean-volume-monocytes (MMV) and mean-conductivity-monocytes (MMC) for MONO. The new markers showed distinct patterns for early engraftment wherein 1) on the engraftment day, MNV peaked as MNC notched simultaneously for every case, and 2) MMV peaked as MMC notched simultaneously in most cases. Engraftment was predicted 3.8+/-2.7 days earlier than by ANC in 74 successful engraftment cases by using NEUTRO and/or MONO: 1) 72 cases (97.3%), in which NEUTRO and/or MONO predicted earlier engraftment than ANC, 2) 1 case, in which the 3 markers predicted engraftment on the same day, and 3) 1 case, in which NEUTRO predicted engraftment on the same day as ANC and MONO failed to predict engraftment. CONCLUSIONS: By analyzing the data from daily complete blood counts, engraftment can be predicted approximately 4 days earlier than ANC >500/microL using NEUTRO as a base marker and MONO as a supplementary marker.
Adolescent ; Adult ; Child ; Child, Preschool ; Female ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*cytology ; Humans ; Infant ; Leukocyte Count ; Male ; Middle Aged ; Monocytes/*cytology ; Neutrophils/*cytology ; Time Factors ; Transplantation, Autologous ; Transplantation, Homologous ; Young Adult

Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplastic anemia that usually presents early in infancy and is inherited in up to 45% of cases. It is characterized by red cell aplasia, congenital anomalies, and a predisposition to cancer. Corticosteroids and red blood cell transfusions are the mainstays of therapy. We describe a case of 3-month-old infant who presented with severe anemia, elevated levels of HbF and adenosine deaminase and bilateral hydronephrosis, who was later confirmed as DBA by mutation analysis using the direct sequencing method. Direct sequencing analysis of RPS19 gene was performed with both cDNA and genomic DNA extracted from peripheral blood and a c.3G>A point mutation of exon 2 resulting in p.Met1Ile was identified in this patient. The patient showed an inadequate response to steroid therapy and a partial response to RBC transfusion with a follow-up Hb level of 8.3 g/dL on her last visit to the outpatient clinic. DBA is a genetically and phenotypically heterogeneous disease, and we have reviewed the clinical characteristics of 25 Korean patients thus far reported in the literature. To our knowledge, this is the first case of DBA confirmed by mutation analysis in Korea, and mutation identification using molecular method is recommended for confirmation of this genetically and phenotypically heterogeneous disease.
Anemia, Diamond-Blackfan/*diagnosis/genetics/therapy ; Asian Continental Ancestry Group/*genetics ; Bone Marrow/pathology ; Erythrocyte Transfusion ; Exons ; Humans ; Infant ; Point Mutation ; Republic of Korea ; Ribosomal Proteins/*genetics ; Sequence Analysis, DNA

BACKGROUND: Manual slide review (MSR) is usually triggered by the results of automated hematolgy analyzers, but each laboaratory has different ciriteria for MSR. This study was carried out to investigate the current status of MSR criteria of automated complete blood cell count (CBC) and white blood cell (WBC) differential results and to propose a basic guideline for MSR. METHODS: Total 111 laboratories were surveyed regarding MSR using questionnaires. The questionnaire asked: kinds of automated hematology analyzers used and the presence of criteria triggering MSR in seven categories: 1) CBC results, 2) 5 differential WBC counts, 3) 3 differential WBC counts, 4) automated reticulocyte counts, 5) delta check, 6) instrument flags (or messages), 7) clinical information (wards or diseases). Based on the survey results, we determined basic and extended criteria for MSR. With these criteria, we consulted nine hematology experts to get a consensus. RESULTS: All 111 laboratories had their own MSR criteria. Among 111 laboratories, 98 (88.3%) used more than three criteria for MSR including CBC results and 5-part WBC differential count results and 95 (85.6%) had criteria of flags triggering MSR. For MSR criteria with numeric values, the 10th, 50th, and 90th percentiles of upper and lower threshold values were obtained. The basic guideline for MSR was made. CONCLUSIONS: We proposed a basic guideline for MSR. This guideline would be helpful to hematology laboratories for their daily operation and providing more rapid and accurate CBC and WBC differential results.
Automation ; Blood Cell Count/instrumentation/*methods/standards ; Humans ; Laboratories, Hospital ; Leukocyte Count/instrumentation/*methods/standards ; Quality Control ; Questionnaires

BACKGROUND: Epstein-Barr virus (EBV) is a well-known causative agent of various diseases including post-transplant lymphoproliferative disorders. Although the level of EBV viral load in donors is expected to have a direct effect on recipients after hematopoietic stem cell transplantation (HSCT), little has been studied providing a clear evidence for that. We performed EBV DNA quantitation in donors and analyzed the effect of donors' EBV viral load on the recipients after HSCT. METHODS: EBV DNA quantitation of peripheral blood in 94 healthy HSCT donors was performed by real-time PCR. We analyzed the distribution of EBV viral load in HSCT donors and EBV positivity in the recipients transplanted from donors who had detectable EBV. RESULTS: Fifteen HSCT donors (16%) showed positive results in EBV real-time quantitative PCR. EBV viral load was below 500 copies/mL in 5 donors and above 500 (680-11,300) copies/mL in 10 donors. Five of the recipients (33.3%) transplanted from these 15 donors showed positivity in EBV PCR after HSCT. All of the EBV PCR positive recipients were transplanted from donors with viral load of >1,000 copies/mL, and 5 (71%) of 7 donors with viral load of >1,000 copies/mL was associated with posttansplant EBV PCR positivity in the recipients. CONCLUSIONS: Higher levels of EBV viral load in donors appear to be associated with EBV transmission to recipients in HSCT. EBV real-time quantitative PCR may be needed for screening EBV DNA level in HSCT donors.
Adolescent ; Adult ; Aged ; Child ; DNA, Viral/*blood ; Female ; *Hematopoietic Stem Cell Transplantation ; Herpesvirus 4, Human/*genetics ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction/*methods ; Tissue Donors ; Transplantation, Homologous ; Viral Load

BACKGROUND: We evaluated the clinical significance of revised 2008 WHO classification needed to diagnose mixed phenotype acute leukemia (MPAL). METHODS: A total of 22 MPAL patients, previously diagnosed by applying the scoring system of the European Group for Immunological Classification of Acute Leukemias (EGIL) were reclassified based on the 2008 WHO classification. RESULTS: In 2008 WHO classification, the number of monoclonal antibodies (mAbs) required for assigning more than one lineage was markedly decreased, from 26 to 11, compared with that of EGIL. Seventeen of the 22 MPAL patients were reclassified as MPAL with following details: 6 MPAL with t(9;22)(q34;q11.2); BCR-ABL1, 1 MPAL with t(v;11q23); MLL rearranged, 7 MPAL, B/Myeloid, not otherwise specified (NOS) and 3 MPAL, T/Myeloid, NOS. Five patients were excluded from MPAL in the revised classification: 4 cytoplasmic myeloperoxidase (cMPO)-negative and 1 CD19-negative. The failure of complete remission achievement and occurrence of relapse were associated with poor prognosis (P=0.0002 and P=0.009, respectively). But the presence of Philadelphia chromsome was not significantly related with patient outcome (P=0.082). One patient with cCD79a, CD20, CD38, cMPO and CD15, whose diagnosis was reclassified from MPAL to AML has survived during the study period. CONCLUSIONS: Because of decreased number of mAbs needed, it is possible that acute leukemia panel is designed to include all mAbs required to diagnose MPAL according to 2008 WHO classification. When diagnosing MPAL, it is critical to figure out positivity in either cMPO or CD19, and AML expressing more than 2 lymphoid antigens are considered as MPAL.
Acute Disease ; Adolescent ; Adult ; Aged ; Antibodies, Monoclonal/immunology ; Child ; Chromosomes, Human ; Female ; Fusion Proteins, bcr-abl/metabolism ; Humans ; Infant ; Leukemia/classification/*diagnosis/mortality ; Male ; Middle Aged ; Phenotype ; Philadelphia Chromosome ; Survival Analysis ; World Health Organization

Severe congenital neutropenia is a rare hematological disease characterized by a selective decrease in circulating neutrophils, maturation arrest of granulocytic precursors at the promyelocyte stage, and recurrence of infections. A 2-month-old male infant (patient A) and a 14-month-old female child (patient B) were referred to our hospital due to severe neutropenia. Sequencing analysis of ELA2 and HAX1 genes was performed. Two single nucleotide polymorphisms of HAX1 gene were found. They were 5,104T-->G point mutation of exon 1 and 5,474A-->G point mutation of intron 1 in HAX1 gene. The mutation of ELA2 gene was not found. The patient A showed a good response to granulocyte colony-stimulating factor (G-CSF) treatment and the absolute neutrophil count recovered to 1,195/microliter. But the patient B showed a partial response to G-CSF treatment and experienced several episodes of herpetic gingivostomatitis, oral ulcer, acute pharyngotonsillitis and otitis media during follow-up.
Adaptor Proteins, Signal Transducing/genetics ; Bone Marrow/pathology ; Female ; Granulocyte Colony Stimulating Factor, Recombinant/adverse effects/therapeutic use ; Humans ; Infant ; Male ; Neutropenia/congenital/drug therapy/*genetics ; Neutrophils/cytology/pathology ; Oral Ulcer/etiology ; Otitis Media/etiology ; Polymorphism, Single Nucleotide ; Serine Endopeptidases/genetics ; Stomatitis, Herpetic/etiology

BACKGROUND: Real-time PCR for quantification of JAK2 V617F has recently been introduced and used to evaluate the importance of mutant allele burden in both diagnosis and disease progression in myeloproliferative diseases (MPDs). We evaluated the usefulness of JAK2 MutaScreen(TM) kit that uses a real-time semiquantitative PCR method and has been designed to screen JAK2 V617F mutant allele burden. METHODS: Forty MPD patients were included in this study. We screened JAK2 V617F and determined the mutant allele burden using JAK2 MutaScreen(TM) kit. The mutant allele burden was estimated by six-scaled standards of JAK2 V617F mutant allele (2%, 5%, 12.5%, 31%, 50%, and 78%). For evaluation of test performance, an allele-specific PCR (AS-PCR) was carried out in all samples by using Seeplex JAK2 Genotyping kit. We assessed the clinical differences in distinct disease entities of MPDs according to JAK2 V617F mutant allele burden. RESULTS: JAK2 V617F mutation was detected in 30 cases, including 10 of 11 cases (91%) of polycythemia vera (PV), 13 of 20 cases (65%) of essential thrombocythemia (ET), and 2 of 3 cases (67%) of chronic idiopathic myelofibrosis (CIMF). The concordance rate between the two tests was 95% (38/40). JAK2 V617F mutant allele burden was greater than 50% in 17 cases, and 10 of them (59%) were PV. In contrast, mutant allele burden was less than 50% in 13 cases and 11 of them (85%) were ET. CONCLUSIONS: JAK2 MutaScreen(TM) kit that utilizes a real-time semi-quantitative PCR method is a useful tool for diagnosing MPDs precisely. It can be used to assess the grade of mutant allele burden as well as to screen JAK2 V617F simultaneously.
Adult ; Aged ; *Alleles ; Amino Acid Substitution ; DNA Mutational Analysis ; Disease Progression ; Female ; Humans ; Janus Kinase 2/*genetics ; Male ; Middle Aged ; Mutation ; Myeloproliferative Disorders/*diagnosis/genetics ; Polymerase Chain Reaction/*methods ; Reagent Kits, Diagnostic