A sensitive and inexpensive high-resolution melting-based testing algorithm for diagnosis of transient abnormal myelopoiesis and myeloid leukemia of Down syndrome.

Patients with Down syndrome (DS) are commonly affected by a pre-leukemic disorder known as transient abnormal myelopoiesis (TAM). This condition usually undergoes spontaneous remission within the first 2 months after birth; however, in children under 5, 20%-30% of cases evolve to myeloid leukemia of Down syndrome (ML-DS). TAM and ML-DS are caused by co-operation between trisomy 21 and acquired mutations in the GATA1 gene. Currently, only next-generation sequencing (NGS)-based methodologies are sufficiently sensitive for diagnosis in samples with small GATA1 mutant clones (≤10% blasts). Alternatively, this study presents research on a new, fast, sensitive, and inexpensive high-resolution melting (HRM)-based diagnostic approach that allows the detection of most cases of GATA1 mutations, including silent TAM. The algorithm first uses flow cytometry for blast count, followed by HRM and Sanger sequencing to search for mutations on exons 2 and 3 of GATA1. We analyzed 138 samples of DS patients: 110 of asymptomatic neonates, 10 suspected of having TAM, and 18 suspected of having ML-DS. Our algorithm enabled the identification of 33 mutant samples, among them five cases of silent TAM (5/110) and seven cases of ML-DS (7/18) with blast count ≤10%, in which GATA1 alterations were easily detected by HRM. Depending on the type of genetic variation and its location, our methodology reached sensitivity similar to that obtained by NGS (0.3%) at a considerably reduced time and cost, thus making it accessible worldwide.

Trastornos mieloproliferativos y leucemia en síndrome de Down. Presentación de dos casos clínicos en el período neonatal / Myeloproliferative disorders and leukemia related to Down syndrome. Report of two clinical cases in neonatal period

ElsíndromedeDownpredisponeatrastornosmieloproliferativos. Se estima que del 5 % al 30 % de los neonatos con esta condición desarrollarán mielopoyesis anormal transitoria. El tratamiento no está estandarizado; la exanguinotransfusión y la citarabina podrían ser efectivos. Se describen dos casos de pacientes con síndrome de Down, quienes durante el período neonatal presentaron leucemia mieloide aguda y mielopoyesis anormal transitoria, los tratamientos utilizados y sus desenlaces. Se considera que la sospecha y el diagnóstico temprano de esta entidad son factores determinantes en el pronóstico.

Down syndrome predisposes to haematological disorders. It is estimated that 5-30% of neonates with this condition will develop transient abnormal myelopoiesis. Treatment is not standardized; exchange transfusion and the use of cytarabine could be effective. We present two clinical cases of patients with Down syndrome, who during the neonatal period showed acute myeloid leukemia and transient abnormal myelopoiesis, the treatments used and their outcomes. Suspicion and early diagnosis of this entity are considered determining factors in prognosis.

[Myeloproliferative disorders and leukemia related to Down syndrome. Report of two clinical cases in neonatal period]. / Trastornos mieloproliferativos y leucemia en síndrome de Down. Presentación de dos casos clínicos en el período neonatal.

Down syndrome predisposes to haematological disorders. It is estimated that 5-30% of neonates with this condition will develop transient abnormal myelopoiesis. Treatment is not standardized; exchange transfusion and the use of cytarabine could be effective. We present two clinical cases of patients with Down syndrome, who during the neonatal period showed acute myeloid leukemia and transient abnormal myelopoiesis, the treatments used and their outcomes. Suspicion and early diagnosis of this entity are considered determining factors in prognosis.

El síndrome de Down predispone a trastornos mieloproliferativos. Se estima que del 5 % al 30 % de los neonatos con esta condición desarrollarán mielopoyesis anormal transitoria. El tratamiento no está estandarizado; la exanguinotransfusión y la citarabina podrían ser efectivos. Se describen dos casos de pacientes con síndrome de Down, quienes durante el período neonatal presentaron leucemia mieloide aguda y mielopoyesis anormal transitoria, los tratamientos utilizados y sus desenlaces. Se considera que la sospecha y el diagnóstico temprano de esta entidad son factores determinantes en el pronóstico.

Complete blood count differences in a cohort of Down syndrome neonates with transient abnormal myelopoiesis screened for GATA1 pathogenic variants.

Transient abnormal myelopoiesis (TAM) raises the risk for acute myeloid leukemia of Down syndrome (DS) (ML-DS), and both are related to GATA1 pathogenic variants. Here, we analyzed which findings on complete blood count (CBC) are associated with TAM in a cohort of neonates with DS screened for GATA1 pathogenic variants. The CBCs were compared among 70 newborns with DS, including 16 patients (22.9%) with TAM (cases), and 54 patients (77.1%) without TAM (controls). TAM was defined as peripheral circulating blasts (PCBs) ≥ 1%. PCR and direct sequencing were used to screen DNA samples from peripheral blood for GATA1 exon 2 mutations. Multivariate logistic regression analyses determined that the mean count of lymphocytes was significantly higher in DS infants with TAM (p = .035) and that lymphocytosis confers a risk for TAM (adjusted odds ratio = 7.23, 95% confidence intervals: 2.02-25.92). Pathogenic variants of GATA1 were identified in 2 of 70 analyzed DS neonates (2.9%), of which one had ML-DS and another had an asymptomatic TAM. Among those DS infants with TAM, the GATA1 pathogenic variant detection was 12.5%. Our results indicated that lymphocytosis is associated with TAM in neonates with DS. However, since not all infants with an abnormal CBC had TAM, and not all infants with TAM had GATA1 pathogenic variants, we emphasize that only the search for GATA1 pathogenic variants allows the proper identification of the subgroup of DS infants with a real increasing in risk for ML-DS.

Leukemoid reaction: A 21st-century cohort study.

INTRODUCTION: Leukemoid reaction (leukocyte count >50 cells ×109  L) is a rare but extremely relevant finding. Since little has been published on this condition's clinical relevance and prognosis, we investigated leukemoid reaction in patients with a white blood cell count of >50 × 109  L, including etiology and outcomes. METHODS: This retrospective cohort study included all patients at a Brazilian tertiary hospital between January 2016 and July 2018 > 18 years with a total leukocyte count >50 cells×109  L. Demographics, complete blood count, clinical features, and the exams used to diagnose and determine leukemoid reaction etiology were analyzed. A Kaplan-Meyer survival analysis was performed, and a binary logistic regression model identified variables associated with death. RESULTS: Of the 267 cases with white blood cell count of >50 × 109 , 162/267 (60%) were secondary to hematopoietic neoplasm and 105/267 (40%) presenting as a true leukemoid reaction. The primary causes of the true leukemoid reaction cases were infection (59), nonhematopoietic neoplasm (17), or other causes (29). Patient deaths (66) differed significantly between groups (P < .001, log-rank [Mantel-Cox] Test). Lower hemoglobin, older age, and increased segmented neutrophil count were associated with increased risk of death. CONCLUSIONS: This was a modern cohort analysis of leukemoid reactions, inclusive of all etiologies. The most common cause was infection, which involved several microorganisms. Paraneoplastic leukemoid reaction was also common. Both conditions have a poor prognosis with high mortality, being a major medical challenge.

Eritema nudoso secundario a reacción tipo 2 como manifestación de lepra lepromatosa / Erythema nodosum secondary to type 2 reaction as a manifestation of lepromatous leprosy. A case report

Introducción: El eritema nudoso leproso o leprorreacción de tipo 2 supone una reacción de hipersensibilidad nmunológica humoral de tipo III en la lepra lepromatosa y borderline, la cual se presenta clásicamente como nódulos subcutáneos dolorosos en la piel, además de otras manifestaciones sistémicas. En este reporte de caso se mostrará una forma típica de este tipo de leprorreacción. Presentación del caso: Paciente masculino de 28 anos de edad, con diagnóstico de lepra lepromatosa multibacilar en tratamiento, quien fue remitido al Hospital de San José por presentar cuadro clínico de un mes de evolución de fiebre, malestar general, pérdida de peso y aumento de lesiones nodulares en miembros superiores, con hallazgo adicional de bicitopenia y neutropenia severa. Durante la estancia hospitalaria, es valorado por el Servicio de Dermatología quienes consideran el diagnóstico de eritema nudoso leproso e inician tratamiento; además se evidencia mejoría de la neutropenia con posterior aparición de leucocitosis en aumento y en el frotis de sangre periférica, la presencia de blastos, por lo que se sospecha que el paciente curse con una enfermedad hematolinfoide. Por lo anterior, es valorado por el Servicio de Hematología quienes lo descartan, tratándose entonces de una reacción leucemoide probablemente secundaria a su patología de base. Finalmente, el paciente evoluciona favorablemente y es dado de alta sin complicaciones. Conclusión: La leprorreacción de tipo 2, es una reacción inmune mediada por inmunocomplejos que se presenta típicamente como nódulos subcutáneos dolorosos en la piel, además de presentarse con síntomas constitucionales asociados y daño en otros órganos como hígado, riñón y médula ósea. El conocimiento de las leprorreacciones, el diagnóstico y tratamiento oportunos son fundamentales para prevenir la orbimortalidad en estos pacientes.

Introduction: Erythema nodosum leprosum, or type 2 leprosy reaction, is a type III humoral immunological reaction in the lepromatous and borderline categories of the disease. This classically presents as painful subcutaneous nodules on the skin, and other systemic manifestations. In this case report, a typical form of this type of leprosy reaction will be shown. Case presentation: A 28year-old male patient diagnosed and being treated for multibacillary lepromatous leprosy, was referred to the San Jose Hospital one month after onset. He presented with clinical symptoms such as fever, malaise, weight loss, and increased nodular lesions in the upper limbs, with additional finding of bi-cytopenia and severe neutropenia. He was evaluated by the Dermatology Department during his hospital stay, where he was diagnosed with erythema nodosum leprosum and began treatment for it. A further improvement was observed in the neutropenia, with an increase in leucocytosis. The presence of blasts was found in the peripheral blood smears, which led to the suspicion that the patient had a haemato-lymphoid disease. This was evaluated by the haematology department, but was ruled out. Therefore, this condition corresponded to a leukemoid reaction, probably secondary to the underlying disease. Finally, the patient progressed well and was discharged without complications. Conclusions: Type 2 leprosy reaction is an immune reaction mediated by the immune complex that occurs typically as painful subcutaneous nodules on the skin, in addition to the associated constitutional symptoms and damage to other organs such as liver, kidney and bone marrow. Knowledge of leprosy reactions, and their timely diagnosis and treatment are essential in preventing morbidity and mortality in these patients.

Caracterización de mutaciones en el gen GATA-1 en pacientes con Síndrome de Down y diagnóstico de mielopoyesis anormal transitoria o leucemia megacarioblástica aguda / Mutation characterization in the GATA-1 gene in patients with Downs Syndrome diagnosed with transient abnormal myelopoiesis or acute megakaryoblastic leukemia

Los pacientes con síndrome de Down tienen un riesgo más elevado de presentar leucemia megacarioblástica aguda (LMCA). Un 10% de los recién nacidos con ese síndrome presentan un cuadro de mielopoyesis anormal transitoria (MAT), indistinguible de la LMCA, que en general remite espontáneamente. En ambos grupos de pacientes se describió una alta incidencia de mutaciones en el gen GATA-1. Se analizaron 14 muestras de ADN de médula ósea (10 MAT/4 LMCA) correspondientes a 13 pacientes con Síndrome de Down mediante PCR y secuenciación, para describir la frecuencia y las características de las mutaciones en el gen GATA-1 en la población estudiada y sus consecuencias a nivel proteico. Se detectaron mutaciones en 10 de 10 MAT y en 3 de 4 LMCA, que a nivel proteico originarían un codón de terminación prematuro (n= 5), alteraciones en el sitio de corte y empalme (splicing) (n= 6) o cambio de secuencia (n= 3). Se confrmó la alta frecuencia de mutaciones en el gen GATA-1 en recién nacidos con Síndrome de Down y MAT o LMCA.(AU)

Patients with Downs Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Downs Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Downs Syndrome and MAT or AML.(AU)

Caracterización de mutaciones en el gen GATA-1 en pacientes con Síndrome de Down y diagnóstico de mielopoyesis anormal transitoria o leucemia megacarioblástica aguda / Mutation characterization in the GATA-1 gene in patients with Down's Syndrome diagnosed with transient abnormal myelopoiesis or acute megakaryoblastic leukemia

Los pacientes con síndrome de Down tienen un riesgo más elevado de presentar leucemia megacarioblástica aguda (LMCA). Un 10% de los recién nacidos con ese síndrome presentan un cuadro de mielopoyesis anormal transitoria (MAT), indistinguible de la LMCA, que en general remite espontáneamente. En ambos grupos de pacientes se describió una alta incidencia de mutaciones en el gen GATA-1. Se analizaron 14 muestras de ADN de médula ósea (10 MAT/4 LMCA) correspondientes a 13 pacientes con Síndrome de Down mediante PCR y secuenciación, para describir la frecuencia y las características de las mutaciones en el gen GATA-1 en la población estudiada y sus consecuencias a nivel proteico. Se detectaron mutaciones en 10 de 10 MAT y en 3 de 4 LMCA, que a nivel proteico originarían un codón de terminación prematuro (n= 5), alteraciones en el sitio de corte y empalme (splicing) (n= 6) o cambio de secuencia (n= 3). Se confrmó la alta frecuencia de mutaciones en el gen GATA-1 en recién nacidos con Síndrome de Down y MAT o LMCA.

Patients with Down's Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Down's Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Down's Syndrome and MAT or AML.

Mutation characterization in the GATA-1 gene in patients with Down's Syndrome diagnosed with transient abnormal myelopoiesis or acute megakaryoblastic leukemia.

Patients with Down's Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Down's Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Down's Syndrome and MAT or AML.

Mutation characterization in the GATA-1 gene in patients with Downs Syndrome diagnosed with transient abnormal myelopoiesis or acute megakaryoblastic leukemia. / Mutation characterization in the GATA-1 gene in patients with Downs Syndrome diagnosed with transient abnormal myelopoiesis or acute megakaryoblastic leukemia.

Patients with Downs Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Downs Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Downs Syndrome and MAT or AML.

Reacciones leucemoides en el recién nacido / Leukemoid reactions in the newborn

Presentamos el caso de una recién nacida prematura y de bajo peso al nacer que desarrolló cuentas leucocitarias elevadas hasta más de 100,000 x mmc, sin otras anormalidades hematológicas y que resolvió espontáneamente. A propósito del caso se revisó las ca usas de reacciones leucemoides en el período neonatal. En primer lugar la causa más conocida: síndrome míeloprollferatlvo transitorio, descrito en trisomía 21. En pacientes sin anomalías cromosómicas el uso de esteroides para inducir la maduración pulmonar , la corioamnlonitls y la prematurez extrema son exploradas como causas de RL. Luego revisamos el desarrollo de dlsplasia broncopulmonar y el síndrome de respuesta inflamatoria slstémlca en relación a RL...(AU)

Chemiluminescent determination of leukocyte alkaline phosphatase: an advantageous alternative to the cytochemical assay.

The determination of leukocyte alkaline phosphatase (LAP) is used as an aid to diagnose many diseases in the laboratory. For example, it can be used to distinguish chronic myeloid leukemia (CML) from other myeloproliferative disorders (particularly myelofibrosis and polycythemia) and leukemoid reactions (LR). Traditionally, this test is performed with the use of subjective cytochemical assays that assign a score to the level of LAP. Here we present a nonsubjective, quantitative, sensitive, and inexpensive chemiluminescent technique that determines LAP based on the commercial reagent Immulite (AMPPD). To validate this methodology, intact leukocytes obtained from 32 healthy subjects, nine CML patients, and nine LR patients were submitted to the optimized protocol. By measuring the light emission elicited by four concentrations of neutrophils, we were able to estimate the activity of LAP per cell (the slope of the curve obtained by linear regression). A high linear correlation was found between the chemiluminescent result (slope) and the cytochemical score. The slope for healthy individuals ranged between 0.61 and 8.49 (10(-5) mV.s/cell), with a median of 2.04 (10(-5) mV.s/cell). These results were statistically different from those of CML patients (range=0.07-1.75, median=0.79) and LR patients (range= 3.84-47.24, median=9.58; P<0.05).

Malaria grave, pielonefritis, sepsis, reacción leucemoide, abscesos hepáticos. A propósito de un caso clínico / Serious Malaria, pyelonephritis, sepsis, leucemoide reaction, liver abscesses. With regard to a clinical case

Caso problema de una paciente joven procedente de una zona endémica de malaria y una zona emergente de bartonellosis, que inicia un cuadro agudo de fiebre, ictericia y gran compromiso del estado general que la llevo al estado de shock séptico y por lo que fue atendida en este hospital. Durante su ingreso se plantearon patologías y diferentes entidades patológicas y que por prueba terapéutica se llegó al diagnóstico de malaria grave por Plasmodium falciparum. Presentó durante su evolución una serie de complicaciones tales como abscesos hepáticos y anemia severa, quedando por dilucidar si podría corresponder a un caso de bartonellosis.

Síndrome hipereosinofílica idiopática associada à doença eosinofílica disseminada em um cão / Hypereosinophilic idiopathic syndrome associated with disseminated eosinophilic disease in a dog

Relata-se um caso de síndrome hipereosinofílica idiopática associada à doença eosinofílica disseminada em um cão, macho, mestiço Pastor Alemão, com cinco anos de idade. Os sinais clínicos incluíam apatia, anorexia, intolerância ao exercício, caquexia, dispnéia e taquicardia. Laboratorialmente, havia reação leucemóide eosinofílica e na citologia da medula óssea observou-se acentuada hiperplasia eosinofílica. Radiologicamente, detectou-se uma área radiopaca intratorácica bilateral cranial ao coração. A punção aspirativa intratorácica demonstrou grande quantidade de eosinófilos, o que permitiu um diagnóstico clínico de infiltração pulmonar com eosinofilia. O diagnóstico foi confirmado histologicamente.

Reacción leucemoide secundaria al uso prenatal de betametasona en un prematuro / The leukemoid reaction in premature infant secondary to prenatal betamethasone

Introducción. En 1972 se hizo el primer estudio sobre la utilidad de los esteroides prenatales, en la prevención de la enfermedad de membrana hialina. Al incrementarse su uso a finales de los años 70, se publicó por primera vez en un prematuro efectos secundarios sobre la cuenta leucocitaria (reacción leucemoide). El objetivo de este artículo es presentar el caso de un prematuro con reacción leucemoide transitoria, atribuible al uso de betametasona antenatal. Caso Clínico. Recién nacido pretérmino, masculino con 30 semanas de edad gestacional y peso de 1700 gramos, que nació en el Hospital de Ginecología y Obstetricia de Monterrey. Antecedente prenatal de ruptura prematura de membranas de una semana de evolución, así como la administración de betametasona intramuscular 72 horas antes del nacimiento. Presentó dificultad respiratoria al nacer, que requirió apoyo ventilatorio por una semana, a las 48 horas de vida presentó una leucocitosis de 75,000/mm3, que encendió a 117 mil al quinto día, para descender gradualmente a los 19 días de vida. Se reportaron cultivos negativos para sepsis, la evolución clínica fue favorable, dado de alta a los 36 días de vída con un peso de 2036 gramos. Conclusión. Consideramos de interés la presentación de este caso, ya que con el empleo más frecuente de los esteroides prenatales, es posible esperar más casos de ®reacción leucemoide¼ transitoria en el neonato prematuro

Incidence, significance, and kinetic mechanism responsible for leukemoid reactions in patients in the neonatal intensive care unit: a prospective evaluation.

OBJECTIVE: To prospectively investigate the incidence, significance, and kinetic mechanism responsible for leukemoid reactions in patients in the neonatal intensive care unit (NICU). DESIGN: We prospectively studied all infants admitted to the NICU at the University of Florida who, during a period of 12 consecutive months, had a leukemoid reaction. All those identified had a standardized evaluation consisting of (1) karyotype analysis, (2) bacterial cultures, (3) evaluations for toxoplasmosis, other (congenital syphilis and viruses), rubella, cytomegalovirus, and herpes simplex virus) (TORCH), (4) determination of blood viscosity, (5) use of marrow aspirates for morphology, clonogenic progenitor cell assays, and cell-cycle analysis of progenitors, (6) determination of serum concentrations of granulocyte and granulocyte-macrophage colony-stimulating factors, and (7) serial complete blood cell counts until the leukemoid reaction remitted. RESULTS: During 12 months, 707 patients were admitted to the NICU and 4262 complete blood cell counts were performed on samples from these patients. A leukemoid reaction was identified in nine patients, all of whom were preterm (born at 24 to 38 weeks' gestation). Peak blood leukocyte concentrations were 51.7 +/- 15.6 x 10(3)/microl (mean +/- SD). The leukemoid reactions were detected during the first 4 days of life in seven patients, on day 9 in one, and on day 25 in one. An abnormal karyotype (47, XY, +21) was present in one infant. Mothers of four infants had received betamethasone antenatally. None had elevated whole blood viscosity or positive findings on bacterial or TORCH evaluations. None of the bone marrow findings were consistent with steroid-induced leukocytosis; all studies indicated accelerated neutrophil production. Serum concentrations of granulocyte-macrophage colony-stimulating factor were either negligible or nondetectable. Serum granulocyte colony-stimulating factor was elevated in three patients, low in two, and nondetectable in four. The leukemoid reactions persisted for 5 to 32 days, the longest being in the patient with trisomy 21. CONCLUSIONS: Leukemoid reactions were not particularly rare in our NICU (1.3% of patients). The reactions were not associated with hyperviscosity and, except in one patient with a karyotype abnormality, were transient. The responsible kinetic mechanism was increased neutrophil production, not steroid-induced leukocytosis.