RESUMEN
Clinical flow cytometry plays a vital role in the diagnosis and monitoring of various red blood cell disorders. The high throughput, precision, and automation potential of this technique allows for cost-effective and timely analysis compared to older and more manual test methods. Flow cytometric analysis serves as the gold standard diagnostic method for multiple hematological disorders, especially in clinical scenarios where an assay needs to have high sensitivity, high specificity, and a short turnaround time. In this review, we discuss the role of flow cytometric analysis in paroxysmal nocturnal hemoglobinuria, fetal-maternal hemorrhage, and hereditary spherocytosis.
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Citometría de Flujo , Esferocitosis Hereditaria , Humanos , Citometría de Flujo/métodos , Esferocitosis Hereditaria/diagnóstico , Esferocitosis Hereditaria/sangre , Eritrocitos/citología , Hemoglobinuria Paroxística/diagnóstico , Hemoglobinuria Paroxística/sangre , Enfermedades Hematológicas/diagnóstico , Enfermedades Hematológicas/sangre , Embarazo , Femenino , Transfusión Fetomaterna/diagnóstico , Transfusión Fetomaterna/sangreRESUMEN
Hemoglobin (Hb) disorders are among the most prevalent inherited diseases. Despite a limited number of involved genes, these conditions represent a broad clinical and prognostic spectrum. The menu of laboratory tests is extensive. From widely available modalities, for example, complete blood count to rather sophisticated molecular technologies, the investigation of Hb disorders recapitulates an increasing complexity of laboratory workup in other medical fields. This review highlights a current state of biochemical and molecular investigation of Hb disorders and offers a glimpse on technologies that are yet to be fully embraced in clinical practice.
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Hemoglobinopatías , Talasemia , Humanos , Hemoglobinopatías/diagnóstico , Hemoglobinopatías/genética , Talasemia/diagnóstico , Talasemia/genéticaRESUMEN
Hereditary hemolytic anemia (HHA) is a heterogeneous group of disorders due to genetically caused defects in red blood cell membrane structure, enzymes, heme and globin synthesis, erythroid proliferation, and differentiation. Traditionally, the diagnostic process is complex and includes a plethora of tests from routine to highly specialized ones. The inclusion of molecular testing has significantly improved the diagnostic yield. The value of molecular testing is broader than just rendering the correct diagnosis, as it may also guide therapeutic decisions. As more molecular modalities become available for clinical use, it is imperative to understand their benefits and disadvantages pertaining to the HHA diagnostics. Re-evaluation of the traditional diagnostic workflow may also bring forth additional benefits. This review focuses on the current state of molecular testing for HHA.
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Anemia Hemolítica Congénita , Anemia Hemolítica , Humanos , Anemia Hemolítica Congénita/diagnóstico , Anemia Hemolítica Congénita/genética , Eritrocitos/metabolismo , Membrana Eritrocítica/metabolismo , Técnicas de Diagnóstico Molecular , Anemia Hemolítica/diagnóstico , Anemia Hemolítica/genéticaRESUMEN
ß-thalassemia is a quantitative hemoglobin (Hb) disorder resulting in reduced production of Hb A and increased levels of Hb A2. Diagnosis of ß-thalassemia can be problematic when combined with other structural Hb variants, so that the separation approaches in routine clinical centers are not sufficiently decisive to obtain accurate results. Here, we separate the intact Hb subunits by high-performance liquid chromatography, followed by top-down tandem mass spectrometry of intact subunits to distinguish Hb variants. Proton transfer reaction-parallel ion parking (PTR-PIP), in which a radical anion removes protons from multiply charged precursor ions and produces charge-reduced ions spanning a limited m/z range, was used to increase the signal-to-noise ratio of the subunits of interest. We demonstrate that the δ/ß ratio can act as a biomarker to identify ß-thalassemia in normal electrospray ionization MS1 and PTR-PIP MS1. The application of PTR-PIP significantly increases the sensitivity and specificity of the HPLC-MS method to identify δ/ß ratio as a thalassemia biomarker.
RESUMEN
INTRODUCTION: Hereditary hemolytic anemias (HHA) comprise a heterogeneous group of disorders resulting from defective red blood cell (RBC) cytoskeleton, RBC enzyme deficiencies, and hemoglobin (Hb) synthesis disorders such as thalassemia or sideroblastic anemia. MATERIALS AND METHODS: Our hemolytic anemia diagnostic next-generation sequencing (NGS) panel includes 28 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The panel covers the complete coding region of these genes, splice junctions, and, wherever appropriate, deep intronic or regulatory regions are also included. Four hundred fifty-six patients with unexplained hemolytic anemia were evaluated using our NGS panel between 2015 and 2019. RESULTS: We identified pathogenic/likely pathogenic variants in 111/456 (24%) patients that were responsible for the disease phenotype (e.g., moderate to severe hemolytic anemia and hyperbilirubinemia). Approximately 40% of the mutations were novel. As expected, 45/456 (10%) patients were homozygous for the promoter polymorphism in the UGT1A1 gene, A(TA)7 TAA (UGT1A1*28). 8/45 homozygous UGT1A1*28 cases were associated with additional pathogenic mutations causing hemolytic anemia, likely exacerbating hyperbilirubinemia. The most common mutated genes were membrane cytoskeleton genes SPTA1, and SPTB, followed by PKLR. Complex interactions between SPTA1 low expression alleles, alpha-LELY and alpha-LEPRA alleles, and intragenic SPTA1 variants were associated with hereditary pyropoikilocytosis and autosomal recessive hereditary spherocytosis in 23/111 patients. CONCLUSIONS: Our results demonstrate that hemolytic anemia is underscored by complex molecular interactions of previously known and novel mutations in RBC cytoskeleton/enzyme genes, and therefore, NGS should be considered in all patients with clinically unexplained hemolytic anemia and in neonates with hyperbilirubinemia. Moreover, low expression alleles alpha-LELY and alpha-LEPRA should be included in all targeted HHA panels.
Asunto(s)
Anemia Hemolítica Congénita , Eliptocitosis Hereditaria , Esferocitosis Hereditaria , Humanos , Anemia Hemolítica Congénita/diagnóstico , Anemia Hemolítica Congénita/genética , Eliptocitosis Hereditaria/diagnóstico , Eliptocitosis Hereditaria/genética , Esferocitosis Hereditaria/diagnóstico , Esferocitosis Hereditaria/genética , Proteínas del Citoesqueleto/genética , Hiperbilirrubinemia , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Azathioprine (AZA) is an immunosuppressant that is widely used to treat many disease states including rheumatoid arthritis. We present a patient who was treated with AZA for rheumatoid arthritis and subsequently hospitalized for severe myelosuppression due to acquired aplastic anemia. Upon genetic testing it was found that the patient was thiopurine methyltransferase (TMPT) deficient, a well-documented risk factor for myelosuppression in patients taking azathioprine. We advocate for TPMT and nudix hydrolase 15 (NUDT15) testing prior to initiation of AZA treatment, or close monitoring with a complete blood count post-AZA initiation to avoid these serious side effects.
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Anemia Aplásica , Artritis Reumatoide , Humanos , Azatioprina/efectos adversos , Anemia Aplásica/inducido químicamente , Anemia Aplásica/genética , Inmunosupresores/efectos adversos , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Pruebas GenéticasRESUMEN
Hemoglobinopathies are one of the most prevalent genetic disorders, affecting millions throughout the world. These are caused by pathogenic variants in genes that control the production of hemoglobin (Hb) subunits. As the number of known Hb variants has increased, it has become more challenging to obtain unambiguous results from routine chromatographic assays employed in the clinical laboratory. Top-down proteomic analysis of Hb by mass spectrometry is a definitive method to directly characterize the sequences of intact subunits. Here, we apply "chimeric ion loading" to characterize Hb ß subunit variants. In this technique, product ions derived from complementary dissociation techniques are accumulated in a multipole storage device before delivery to a 21 T Fourier-transform ion cyclotron resonance mass spectrometer for simultaneous detection. To further improve the efficiency of identification of Hb variants and localization of the mutation site(s), we developed an R programming script, "Variants Identifier", to search top-down data against a database containing accurate intact mass differences and diagnostic ions from investigated Hb variants. A second R script, "PredictDiag", was developed and employed to determine relevant diagnostic ions for additional Hb variants with known sequences. These two R scripts were successfully applied to the identification of a Hb δ-ß fusion protein and other Hb variants. The combination of chimeric ion loading and the above R scripts enables rapid and reliable interpretation of top-down mass spectrometry data, regardless of activation type, for Hb variant identification.
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Hemoglobinas/análisis , Hemoglobinas/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Humanos , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
In North America, jaundiced neonates are not usually tested for G6PD deficiency if the family is of European ancestry. However, we describe such a family where ≥35 males have had severe (Class I) G6PD deficiency. Many of the jaundiced neonates did not have this diagnosis considered, at least three of whom developed bilirubin neurotoxicity. Over seven generations 35 affected males were identified. Three developed signs of kernicterus spectrum disorder; three had exchange transfusions for hyperbilirubinemia; and nine received one or more blood transfusions during childhood.
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Deficiencia de Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/genética , Polimorfismo de Nucleótido Simple , Femenino , Humanos , Hiperbilirrubinemia/genética , Recién Nacido , Kernicterus/genética , Masculino , América del Norte , Linaje , Población Blanca/genéticaRESUMEN
Anemia is a global health problem in all age groups. According to World Health Organization (WHO), approximately 40% of pregnant women are anemic. Iron deficiency anemia (IDA) due to nutritional deficiency is the most common cause. The incidence of IDA varies worldwide depending on the socioeconomic status, but it remains the leading cause even in developed countries. Physiologic anemia of pregnancy due to relatively higher expansion of blood volume in comparison with elevated red blood cell mass also occurs frequently. Complete blood count (CBC) in the first trimester is recommended for all pregnant women to screen for anemia. The screening of pregnant women for IDA in absence of anemia is still debatable. If IDA is suspected, ferritin level of <30 ng/ml is diagnostic. Iron supplementation is recommended for all pregnant women to compensate the increased demand.
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Anemia/diagnóstico , Servicios de Laboratorio Clínico , Técnicas de Laboratorio Clínico , Complicaciones Hematológicas del Embarazo/diagnóstico , Anemia/etiología , Anemia Ferropénica/diagnóstico , Anemia Ferropénica/etiología , Técnicas de Laboratorio Clínico/métodos , Diagnóstico Diferencial , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Femenino , Humanos , Tamizaje Masivo , Embarazo , Complicaciones Hematológicas del Embarazo/etiologíaAsunto(s)
Eliptocitosis Hereditaria/genética , Mutación del Sistema de Lectura , Mutación Puntual , Espectrina/genética , Globinas beta/genética , Anemia Hipocrómica/genética , Células Cultivadas , Niño , Eliptocitosis Hereditaria/complicaciones , Exones/genética , Femenino , Estudios de Asociación Genética , Heterocigoto , Humanos , Ictericia/etiología , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/complicaciones , Conformación Proteica , Rasgo Drepanocítico/genéticaRESUMEN
X-linked lymphoproliferative disease type 1 (XLP1) is a primary immunodeficiency disorder caused by pathogenic variants in the SH2D1A gene (SH2 domain containing protein 1A). Patients with XLP1 may present acutely with fulminant infectious mononucleosis, hemophagocytic lymphohistiocytosis, and/or B-cell non-Hodgkin lymphoma (B-NHL). We report a boy who developed 2 clonally distinct B-NHL 4 years apart and was found to have previously unrecognized XLP1. The report highlights the importance of clonal analysis and XLP1 testing in males with presumed late recurrences of B-NHL, and the role of allogeneic stem cell transplant (allo-SCT) in XLP1 patients and their affected male relatives.
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Linfohistiocitosis Hemofagocítica/diagnóstico , Linfoma de Células B/patología , Trastornos Linfoproliferativos/diagnóstico , Mutación , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/genética , Adolescente , Niño , Preescolar , Femenino , Humanos , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/terapia , Linfoma de Células B/genética , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/terapia , Masculino , Linaje , PronósticoRESUMEN
Bone marrow necrosis (BMN) is a rare pathologic finding, but when encountered is most often associated with malignancy. In adults, its presence correlates with an inferior prognosis, however in children the prognostic implication is unclear. We performed a retrospective review of 3,760 bone marrow specimens in patients ≤18 years over a 10-year period. BMN was identified in less than 1% of specimens and only in patients with leukemia, lymphoma, or neuroblastoma. BMN contributed to a delay in diagnosis; however, advanced medical imaging may serve as a tool to localize nonnecrotic areas for bone marrow sampling, facilitating an expedited diagnosis.
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Enfermedades de la Médula Ósea/patología , Neoplasias/patología , Adolescente , Enfermedades de la Médula Ósea/etiología , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Masculino , Necrosis , Neoplasias/complicaciones , Pronóstico , Estudios Retrospectivos , Factores de TiempoRESUMEN
High prevalence of hemoglobin (Hb) disorders mandates national programs for screening and genetic counseling in many countries. Increased Hb A2 levels are commonly associated with ß-thalassemias, however, various disorders including alteration of δ chains may result in decreased production of Hb A2, thus hindering the diagnosis of ß-thalassemias. The reported data reflect the experience of a large reference laboratory in the United States. In the current study, we have attempted to assess the prevalence and also tried to characterize the identified mutations in the HBD gene resulting in decreased Hb A2 levels. In our cohort, 1.6% of 6486 patients were found to have Hb A2 values of <1.9%. Bidirectional sequencing of the HBD gene demonstrated mutations in 20 cases (19.0% of the individuals with decreased Hb A2). In addition to the previously reported variants, one novel mutation (Hb A2-Utah or HBD: c.46T>C).
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Hemoglobina A2/metabolismo , Talasemia beta/sangre , Talasemia beta/genética , Globinas delta/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Niño , Preescolar , Índices de Eritrocitos , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Hemoglobina A2/genética , Hemoglobinopatías/sangre , Hemoglobinopatías/diagnóstico , Hemoglobinopatías/epidemiología , Hemoglobinopatías/genética , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mutación , Estados Unidos/epidemiología , Adulto Joven , Globinas alfa , Talasemia beta/diagnóstico , Talasemia beta/epidemiología , Globinas delta/metabolismoRESUMEN
We report a novel glucose-6-phosphate dehydrogenase (G6PD) variant (c.1375C>G) discovered in a 3-day-old Hispanic male child from Salt Lake City, UT, USA. This newborn presented with severe hyperbilirubinemia (29.8 mg/dL or 510 µmol/L) and marked hemolysis evidenced by elevated end-tidal carbon monoxide concentration (5.9 ppm, normal <1.7 ppm). Despite a very low prevalence of G6PD deficiency in Hispanic populations, we pursued testing for this condition and found he had low erythrocyte G6PD enzyme activity (2.8 U/g Hb, normal 9.9-16.6 U/g Hb) and a novel G6PD variant. His mother was heterozygous for this same variant, and she had a moderate decrease in G6PD enzyme activity (7.1 U/g Hb). On the basis of these findings, we propose this variant as a novel pathogenic mutation.
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Deficiencia de Glucosafosfato Deshidrogenasa , Glucosafosfato Deshidrogenasa , Hiperbilirrubinemia , Femenino , Glucosafosfato Deshidrogenasa/genética , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Heterocigoto , Hispánicos o Latinos/genética , Humanos , Hiperbilirrubinemia/genética , Recién Nacido , MasculinoRESUMEN
Dizygotic twin males, born at 34 weeks gestation, had prolonged jaundice, microcytic, hypochromic anemia, FABarts hemoglobin, elevated end-tidal CO, and blood films consistent with hereditary pyropoikilocytosis. DNA sequencing revealed both had a heterozygous alpha spectrin (SPTA1) mutation (c.460_462dup) inherited from their asymptomatic mother, plus a 3-base pair duplication in alpha globin (HBA2) (c.364_366dupGTG) inherited from their asymptomatic father.
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Anemia Hemolítica/complicaciones , Anemia Hipocrómica/complicaciones , Eliptocitosis Hereditaria/complicaciones , Ictericia/complicaciones , Anemia Hemolítica/sangre , Anemia Hemolítica/genética , Anemia Hipocrómica/sangre , Anemia Hipocrómica/genética , Eliptocitosis Hereditaria/sangre , Eliptocitosis Hereditaria/genética , Humanos , Recién Nacido , Ictericia/sangre , Ictericia/genética , Masculino , Mutación Puntual , Espectrina/genética , Gemelos Dicigóticos/genéticaRESUMEN
PURPOSE: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC. EXPERIMENTAL DESIGN: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC. RESULTS: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. CONCLUSIONS: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/mortalidad , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/mortalidad , Macrófagos Asociados a Tumores/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/análisis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores de Tumor/análisis , Carcinoma de Células Renales/diagnóstico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/terapia , Línea Celular Tumoral , Quimioterapia Adyuvante , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/análisis , Neoplasias Renales/diagnóstico , Neoplasias Renales/genética , Neoplasias Renales/terapia , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Nefrectomía , Pronóstico , RNA-Seq , Estudios Retrospectivos , Análisis de la Célula Individual , Análisis de Supervivencia , Macrófagos Asociados a Tumores/inmunologíaRESUMEN
Anemia is a major health burden worldwide and affects approximately one-third of world's population. It is not a diagnosis; it is a manifestation of an underlying pathophysiology leading to either decreased hemoglobin (Hb), hematocrit (Hct), or red blood cells (RBCs). Iron deficiency anemia is still the most common cause of anemia worldwide. The symptoms are usually due to the underlying compensatory responses to decrease in oxygen delivery to the tissues. Laboratory investigation should start with complete blood count (CBC), reticulocyte count (RC), and peripheral smear evaluation. Further testing depends on these indices, that is, iron parameters and hemoglobinopathies/thalassemia evaluation in microcytic hypochromic anemia, vitamin B12, and folic acid level in macrocytic anemia. Increased RC denotes adequate bone marrow response and points toward hemolytic process and vice versa. Anemia diagnosis can be complex and confusing for the practicing physician. This review tries to give a practical simplistic approach to the diagnosis, focusing mainly on the basic parameters, that is, CBC, RC, and peripheral smear etc. Moreover, we have also tried to provide an update on the pyruvate kinase deficiency, as there has been recent exciting development in the management of these patients.