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1.
Ann Clin Lab Sci ; 51(1): 136-139, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33653793

RESUMO

Hereditary spherocytosis (HS) is a congenital disorder of the red blood cell membrane and is characterized by hemolytic anemia, variable jaundice, and splenomegaly. In neonates, the diagnosis of HS can be difficult in the absence of family history. Herein, we describe clinical and molecular genetic findings in a Korean neonate with HS. A one-month-old girl presented with severe anemia and jaundice. Spherocytes were frequently observed on peripheral blood smear, but the erythrocyte osmotic fragility test result was normal. Targeted next-generation sequencing (NGS) revealed the patient was heterozygous for a novel frameshift mutation, c.191_194del (p.Leu64Argfs*7), in exon 3 of ANK1 gene. Family study was performed by direct sequencing, and neither of her parents carried this mutation. The patient also harbored the UGT1A1*6 allele. To the best of our knowledge, this ANK1 mutation identified by targeted NGS has not been reported previously.


Assuntos
Anquirinas/genética , Esferocitose Hereditária/genética , Alelos , Anquirinas/metabolismo , Feminino , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Recém-Nascido , Mutação , República da Coreia , Esferócitos/citologia , Esferocitose Hereditária/diagnóstico , Esferocitose Hereditária/metabolismo
2.
Ann Lab Med ; 41(1): 44-50, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32829578

RESUMO

BACKGROUND: The Advanced RBC Application of the CellaVision DM9600 system (CellaVision AB, Lund, Sweden) automatically characterizes and classifies red blood cells (RBCs) into 21 morphological categories based on their size, color, shape, and inclusions. We evaluated the diagnostic performance of the CellaVision Advanced RBC Application with respect to the classification and grading of RBC morphological abnormalities in accordance with the 2015 International Council for Standardization in Haematology (ICSH) guidelines. METHODS: A total of 223 samples, including 123 with RBC morphological abnormalities and 100 from healthy controls, were included. Seven RBC morphological abnormalities and their grading obtained with CellaVision DM9600 pre- and post-classification were compared with the results obtained using manual microscopic examination. The grading cut-off percentages were determined in accordance with the 2015 ICSH guidelines. The sensitivity and specificity of the CellaVision DM9600 system were evaluated using the manual microscopic examination results as a true positive. RESULTS: In pre-classification, >90% sensitivity was observed for target cells, tear drop cells, and schistocytes, while >90% specificity was observed for acanthocytes, spherocytes, target cells, and tear drop cells. In post-classification, the detection sensitivity and specificity of most RBC morphological abnormalities increased, except for schistocytes (sensitivity) and acanthocytes (specificity). The grade agreement rates ranged from 35.9% (echinocytes) to 89.7% (spherocytes) in pre-classification and from 46.2% (echinocytes) to 90.1% (spherocytes) in post-classification. The agreement rate of samples with within-one grade difference exceeded 90% in most categories, except for schistocytes and echinocytes. CONCLUSIONS: The Advanced RBC Application of CellaVision DM9600 is a valuable screening tool for detecting RBC morphological abnormalities.


Assuntos
Eritrócitos Anormais/citologia , Microscopia/métodos , Acantócitos/classificação , Acantócitos/citologia , Área Sob a Curva , Estudos de Casos e Controles , Eritrócitos Anormais/classificação , Humanos , Microscopia/instrumentação , Curva ROC , Estudos Retrospectivos , Esferócitos/classificação , Esferócitos/citologia
3.
Vet Clin Pathol ; 48(4): 620-623, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31228292

RESUMO

This case report describes a massive honey bee envenomation in a 14-month-old male Belgian Malinois dog from St. Kitts, West Indies. Acute and delayed onsets of hemolytic anemia, echinocytosis, spherocytosis, thrombocytopenia, hemoglobinemia, and hemoglobinuria developed following envenomation. The dog recovered after treatment with glucocorticoids and supportive therapy. Spherocytosis, hemolysis, and thrombocytopenia in patients with massive bee envenomation are likely due to the direct toxic effects of the primary components of bee venom, melittin and phospholipase A2 (PLA2 ). Mellitin causes hemolysis by forming large pores in erythrocytes resulting in leakage of hemoglobin and also causes spectrin stiffening and resultant echinocyte and spherocyte formation. Melittin also stimulates PLA2 , a hydrolase that causes echinocytosis and spherocytosis, in vivo and in vitro, and mitochondrial breakdown in platelets. However, delayed manifestations could be attributed to immune-mediated mechanisms from the generation of antibodies against damaged erythrocytes and platelet membrane proteins.


Assuntos
Anemia Hemolítica/veterinária , Venenos de Abelha/envenenamento , Abelhas , Doenças do Cão/etiologia , Mordeduras e Picadas de Insetos/veterinária , Esferócitos , Trombocitopenia/veterinária , Anemia Hemolítica/etiologia , Animais , Contagem de Células Sanguíneas/veterinária , Cães , Mordeduras e Picadas de Insetos/complicações , Masculino , Trombocitopenia/etiologia
4.
Orphanet J Rare Dis ; 14(1): 114, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31122244

RESUMO

BACKGROUND: Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS. METHODS: Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform. RESULTS: Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations. CONCLUSIONS: This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.


Assuntos
Fragilidade Osmótica/fisiologia , Esferócitos/metabolismo , Esferocitose Hereditária/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Anquirinas/genética , Anquirinas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Criança , Pré-Escolar , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Lactente , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Mutação/genética , Fragilidade Osmótica/genética , Patologia Molecular , República da Coreia , Espectrina/genética , Espectrina/metabolismo , Esferocitose Hereditária/genética , Adulto Jovem
5.
Sci Rep ; 8(1): 15705, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30356059

RESUMO

Distortions of the normal bi-concave disc shape for red blood cells (RBCs) appear in a number of pathologies resulting from defects in cell membrane skeletal architecture, erythrocyte ageing, and mechanical damage. We present here the potential of acoustic cytometry for developing new approaches to light-scattering based evaluation of red blood cell disorders and of the effects of storage and ageing on changes or damage to RBCs membranes. These approaches could be used to immediately evaluate the quality of erythrocytes prior to blood donation and following transfusion. They could also be applied to studying RBC health in diseases and other pathologies, such as artificial heart valve hemolysis, thermal damage or osmotic fragility. Abnormal distributions of erythrocytes can typically be detected after just 30 to 45 seconds of acquisition time using 1-2 µL starting blood volumes.


Assuntos
Eritrócitos , Citometria de Fluxo/métodos , Som , Anexina A5 , Preservação de Sangue/métodos , Envelhecimento Eritrocítico , Deformação Eritrocítica , Índices de Eritrócitos , Membrana Eritrocítica/ultraestrutura , Transfusão de Eritrócitos , Eritrócitos/ultraestrutura , Citometria de Fluxo/instrumentação , Corantes Fluorescentes , Hemólise , Humanos , Hidrodinâmica , Luz , Lipídeos de Membrana/sangue , Fosfatidilserinas/sangue , Espalhamento de Radiação , Esferócitos/ultraestrutura , Esferocitose Hereditária/sangue
8.
Hematology ; 23(7): 413-416, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29338606

RESUMO

INTRODUCTION: Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia, characterized by anemia, jaundice, and splenomegaly. The diagnosis of HS relies on symptoms of hemolysis, a family history of HS, and a positive laboratory test which is usually the osmotic fragility test (OFT). We conducted a study to assess the utility of mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean sphered cell volume (MSCV), and mean reticulocyte volume (MRV) in the diagnosis of HS and if these are helpful in distinguishing cases of HS from immune hemolytic anemia. METHODS: A total of 102 patients suspected to have HS were enrolled. In addition 10 cases of immune hemolytic anemia (IHA) were included in the study and performance of the above screening tests was evaluated. The diagnosis of HS was based on incubated OFT, eosin 5'-maleimide (EMA) dye binding test, and flowcytometric OFT. RESULTS: A total of 29 patients were diagnosed as having HS. The sensitivity and specificity for diagnosis HS by MCHC > 35 g/dL was 44.82%, and ΔMCV-MSCV > 10 fL has a sensitivity and specificity of 82.75% and 95.9% for diagnosis of HS. Using an algorithm of ΔMCV-MSCV > 10 fL and ΔMRV-MSCV < 25, for the differentiation of HS from IHA had sensitivity of 68.9% and specificity of 98.8%.


Assuntos
Reticulócitos , Esferócitos , Esferocitose Hereditária/sangue , Esferocitose Hereditária/diagnóstico , Adolescente , Adulto , Criança , Pré-Escolar , Índices de Eritrócitos , Feminino , Citometria de Fluxo , Hemólise , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto Jovem
10.
Mol Med Rep ; 17(1): 382-387, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29115431

RESUMO

Patients with combined hereditary spherocytosis (HS) and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) deficiency have been reported sporadically. A discrepancy between the level of elevated serum bilirubin concentration and the degree of anemia may suggest the possibility of a coexistence of these conditions. In the present case report, a 20­year­old female presented with congenital jaundice and anemia, but did not present with the discrepancy between hyperbilirubinemia and anemia in the patient's childhood, and was not previously diagnosed with either HS or UGT1A1 deficiency. During a follow­up of >10 years, the patient's hyperbilirubinemia accumulated progressively, whereas the patient's anemia became relatively mild. Upon further genetic analysis, it was determined that the patient had HS combined with UGT1A1 partial deficiency. Next generation sequencing combined with direct sequencing was used to identify a novel heterozygous mutation (c.G828T; p.Y276X) in the spectrin ß gene, which is causative for HS. Sequence analysis of the patients' UGT1A1 gene revealed a compound heterozygote with c.G211A (p.G71R) and T3279G mutations, which reduced UGT1A1 activity to 30­60% of the normal level. Genetic analysis was crucial for determining the diagnosis and pathogenesis of this unusual case.


Assuntos
Glucuronosiltransferase/genética , Esferocitose Hereditária/diagnóstico , Esferocitose Hereditária/genética , Biomarcadores , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Testes Genéticos , Genótipo , Glucuronosiltransferase/deficiência , Humanos , Mutação , Fenótipo , Espectrina/genética , Esferócitos/patologia , Esferocitose Hereditária/sangue , Adulto Jovem
13.
Chirurgia (Bucur) ; 112(2): 110-116, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28463670

RESUMO

Hereditary spherocytosis (HS) is a disease affecting the red blood cells membrane and belongs to the congenital hemolytic anemias. The clinical spectrum ranges from asymptomatic patients to severe forms requiring transfusions in early childhood. The diagnosis can be based on the physical examination, complete red blood cell count, reticulocytes count, medical history and specific tests, preferentially the EMA test (eosin-5-maleimide binding) test and AGLT (Acidified Glycerol Lysis Time). Splenectomy is considered the standard surgical treatment in moderate and severe forms of hereditary spherocytosis. Total splenectomy exposes the patient to a life - long risk of potentially lethal infections and thus, its usage was reconsidered. Because of this reason, a feasible alternative is the partial splenectomy. The use of partial splenectomy aims to retain splenic immunologic function, while at the same time to decrease the rate of hemolysis. The long - term outcomes of patients with total or subtotal splenectomy for congenital hemolytic anemia, still remain unclear, but the majority of the studies showed a qualitative resolution of anemia and reduction of transfusion rate. Despite the well known advantages of conservative surgery, the optimal choice of treatment and outcomes should be confirmed with the patient.


Assuntos
Contagem de Eritrócitos , Reticulócitos , Esferocitose Hereditária/diagnóstico , Esferocitose Hereditária/cirurgia , Esplenectomia , Contagem de Eritrócitos/métodos , Membrana Eritrocítica , Medicina Baseada em Evidências , Testes Hematológicos , Hemólise , Humanos , Reticulócitos/citologia , Esferócitos/metabolismo , Esplenectomia/métodos , Resultado do Tratamento
14.
Transfusion ; 57(4): 1007-1018, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28150311

RESUMO

BACKGROUND: Storage lesion may explain the rapid clearance of up to 25% of transfused red blood cells (RBCs) in recipients. Several alterations affect stored RBC but a quantitative, whole cell-based predictor of transfusion yield is lacking. Because RBCs with reduced surface area are retained by the spleen, we quantified changes in RBC dimensions during storage. STUDY DESIGN AND METHODS: Using imaging flow cytometry we observed the dimension and morphology of RBCs upon storage, along with that of conventional biochemical and mechanical markers of storage lesion. We then validated these findings using differential interference contrast (DIC) microscopy and quantified the accumulation of microparticles (MPs). RESULTS: Mean projected surface area of the whole RBC population decreased from 72.4 to 68.4 µm2 , a change resulting from the appearance of a well-demarcated subpopulation of RBCs with reduced mean projected surface (58 µm2 , 15.2%-19.9% reduction). These "small RBCs" accounted for 4.9 and 23.6% of all RBCs on Days 3 and 42 of storage, respectively. DIC microscopy confirmed that small RBCs had shifted upon storage from discocytes to echinocytes III, spheroechinocytes, and spherocytes. Glycophorin A-positive MPs and small RBCs appeared after similar kinetics. CONCLUSION: The reduction in surface area of small RBCs is expected to induce their retention by the spleen. We propose that small RBCs generated by MP-induced membrane loss are preferentially cleared from the circulation shortly after transfusion of long-stored blood. Their operator-independent quantification using imaging flow cytometry may provide a marker of storage lesion potentially predictive of transfusion yield.


Assuntos
Preservação de Sangue , Micropartículas Derivadas de Células , Citometria de Fluxo/métodos , Esferócitos/citologia , Biomarcadores/sangue , Feminino , Humanos , Cinética , Masculino , Esferócitos/metabolismo , Fatores de Tempo
16.
J Biomech Eng ; 138(5): 051002, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26926169

RESUMO

In this paper, an attempt has been made to study sedimentation of a red blood cell (RBC) in a plasma-filled tube numerically. Such behaviors are studied for a healthy and a defective cell which might be created due to human diseases, such as diabetes, sickle-cell anemia, and hereditary spherocytosis. Flow-induced deformation of RBC is obtained using finite-element method (FEM), while flow and fluid-membrane interaction are handled using lattice Boltzmann (LB) and immersed boundary methods (IBMs), respectively. The effects of RBC properties as well as its geometry and orientation on its sedimentation rate are investigated and discussed. The results show that decreasing frontal area of an RBC and/or increasing tube diameter results in a faster settling. Comparison of healthy and diabetic cells reveals that less cell deformability leads to slower settling. The simulation results show that the sicklelike and spherelike RBCs have lower settling velocity as compared with a biconcave discoid cell.


Assuntos
Eritrócitos/citologia , Eritrócitos/patologia , Análise de Elementos Finitos , Fenômenos Mecânicos , Modelos Biológicos , Plasma/citologia , Anemia Falciforme/sangue , Fenômenos Biomecânicos , Sedimentação Sanguínea , Diabetes Mellitus/sangue , Deformação Eritrocítica , Membrana Eritrocítica/metabolismo , Eritrócitos/fisiologia , Humanos , Esferócitos/citologia , Esferócitos/patologia
17.
Ann Hematol ; 94(12): 1959-64, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26336967

RESUMO

Exact diagnosis of hereditary spherocytosis (HS) is widely considered unreliable around birth. However, early postnatal diagnosis at the beginning of congenital hemolysis may be essential for managing neonatal anemia and hemolytic icterus, identifying those at high risk for severe hyperbilirubinemia, irreversible kernicterus, or sudden need for red cell transfusion. We analyzed 37 blood samples from neonates or infants up to six weeks of life that had been collected in-house or shipped to our laboratory due to suspected red cell membrane disorder. By combining assessment of red cell morphology, acidified glycerol lysis test (AGLT), and eosin-5'-maleimide (EMA) binding assay, we were able to clearly exclude HS in 22 and confirm HS in 10 patients, of which one had undergone red cell transfusion prior to blood sampling. Assessment of red cell morphology and normal test results allowed diagnosis of infantile pyknocytosis or Heinz body anemia in three neonates. Re-evaluation of five patients with inconsistent results of AGLT and EMA binding led to confirmation of HS in two cases. Automated analysis of hematologic parameters revealed elevated proportion of hyperdense cells to be a highly significant indicator for HS in neonatal infants. We showed that assessment of red cell morphology in combination with AGLT and EMA binding assay is a reliable basis for confirming or rejecting suspected diagnosis of HS even in neonates. Our data underline the necessity for blood sampling and laboratory exploration in suspected red cell membrane or enzyme defects at the earliest occasion.


Assuntos
Amarelo de Eosina-(YS)/análogos & derivados , Membrana Eritrocítica , Glicerol/química , Esferócitos , Esferocitose Hereditária/diagnóstico , Amarelo de Eosina-(YS)/química , Membrana Eritrocítica/química , Membrana Eritrocítica/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Esferócitos/química , Esferócitos/metabolismo
18.
Indian J Pathol Microbiol ; 58(3): 307-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26275251

RESUMO

CONTEXT: Mean sphered cell volume (MSCV) and mean reticulocyte volume (MRV) are additional reticulocyte parameters generated while processing the blood samples on Beckman coulter LH 755 in the reticulocyte mode using the volume, conductivity and scatter technology. It has been observed that the difference between mean corpuscular volume (MCV) and MSCV is higher in the cases of hereditary spherocytosis (HS) and this difference is increasingly being utilized as a screening tool for spherocytes. In addition now there have been new observations that reticulocyte volume in cases of HS is less as compared to normal reticulocyte. AIMS: Our aim was to test the usefulness of reticulocyte parameters like MSCV and MRV in distinguishing cases of HS and autoimmune hemolytic anemia (AIHA). MATERIALS AND METHODS: This is a retrospective and partly prospective study where peripheral blood ethylenediaminetetraacetic acid samples from cases of HS (n = 57) and AIHA (n = 29) were processed on LH 755 in both the differential and the reticulocyte mode. The data generated were analyzed and compared with data from normal healthy donors (n = 46). RESULTS: Using an algorithm of MCV - MSCV >10 and MRV - MSCV <25, a sensitivity of 84.2% and specificity of 94.7% was observed in cases of HS. CONCLUSIONS: With the reticulocyte analysis, we may now have a simple and cheap additional tool for screening of HS.


Assuntos
Anemia Hemolítica Autoimune/diagnóstico , Anemia Hemolítica Autoimune/patologia , Reticulócitos/fisiologia , Esferócitos/patologia , Esferocitose Hereditária/diagnóstico , Esferocitose Hereditária/patologia , Adolescente , Adulto , Tamanho Celular , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
19.
J Perinatol ; 35(5): 357-61, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25357094

RESUMO

OBJECTIVE: Neonates with undiagnosed hereditary spherocytosis (HS) are at risk for developing hazardous hyperbilirubinemia and anemia. Making an early diagnosis of HS in a neonate can prompt anticipatory guidance to prevent these adverse outcomes. A recent comparison study showed that a relatively new diagnostic test for HS, eosin-5-maleimide (EMA)-flow cytometry, performs better than other available tests in confirming HS. However, reports have not specifically examined the performance of this test among neonates. STUDY DESIGN: We compared EMA-flow cytometry from blood samples of healthy control neonates vs samples from neonates suspected of having HS on the basis of severe Coombs-negative jaundice and spherocytes on blood film. The diagnosis of HS was later either confirmed or excluded based on clinical findings and next generation sequencing (NGS) after which we correlated the EMA-flow results with the diagnosis. RESULT: EMA-flow was performed on the blood of 31 neonates; 20 healthy term newborns and 11 who were suspected of having HS. Eight of the 11 were later confirmed positive for HS and one was confirmed positive for hereditary elliptocytosis (HE). All nine had persistently abnormal erythroid morphology, reticulocytosis and anemia, and eight of the nine had relevant mutations discovered using NGS. The other was confirmed positive for HS on the basis that a parent had HS, and the neonate's spherocytosis, reticulocytosis and anemia persisted. The 20 healthy controls and the 2 in whom HS was initially suspected but later excluded all had EMA-flow results in the range reported in healthy children and adults. In contrast, all nine in whom HS or HE was confirmed had abnormal EMA-flow results consistent with previous reports in older children and adults with HS. CONCLUSION: Although our sample size is small, our findings are consistent with the literature in older children and adults suggesting that EMA-flow cytometric testing performs well in supporting the diagnosis of HS/HE during the early neonatal period.


Assuntos
Amarelo de Eosina-(YS)/análogos & derivados , Triagem Neonatal , Esferocitose Hereditária/diagnóstico , Esferocitose Hereditária/genética , Estudos de Casos e Controles , Amarelo de Eosina-(YS)/análise , Citometria de Fluxo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Esferócitos
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