Clinical and laboratory features associated with myeloperoxidase expression in pediatric B-lymphoblastic leukemia.

BACKGROUND: B-lymphoblastic leukemia (B-ALL) is the most common childhood malignancy, and its diagnosis requires immunophenotypically demonstrating blast B cell lineage differentiation. Expression of myeloperoxidase (MPO) in B-ALL is well-described and it has been recognized that a diagnosis of mixed phenotype acute leukemia should be made cautiously if MPO expression is the sole myeloid feature in these cases. We sought to determine whether MPO expression in pediatric B-ALL was associated with differences in laboratory, immunophenotypic, or clinical features. METHODS: We reviewed clinical, diagnostic bone marrow flow cytometry, and laboratory data for all new B-ALL diagnoses at our pediatric institution in 5 years. Cases were categorized as MPO positive (MPO+) or negative (MPO-) using a threshold of ≥20% blasts expressing MPO at intensity greater than the upper limit of normal lymphocytes on diagnostic bone marrow flow cytometry. RESULTS: A total of 148 cases were reviewed, 32 of which (22%) were MPO+. MPO+ B-ALL was more frequently hyperdiploid and less frequently harbored ETV6-RUNX1; no MPO+ cases had KMT2A rearrangements or BCR-ABL1. Although not significantly so, MPO+ B-ALL was less likely than MPO- B-ALL to have positive end-of-induction minimal residual disease studies (9.4 and 24%, respectively), but relapse rates and stem cell transplantation rates were similar between groups. Aberrant expression of other more typically myeloid markers was similar between these groups. CONCLUSION: In our study cohort, MPO+ B-ALL showed minimal residual disease persistence less often after induction chemotherapy but otherwise had similar clinical outcomes to MPO- B-ALL, with similar rates of additional myeloid antigen aberrancy.

Feasibility of manual white blood cell counts as a predictor of neonatal sepsis in a low-resource setting.

BACKGROUND: Manual white blood cell (WBC) differential counts as a predictor for neonatal sepsis development in a low-resource setting have not been thoroughly evaluated. We hypothesized that manual differentiation (specifically immature:total [I:T] neutrophil ratios) would be feasible and useful as an adjunct to predict early-onset neonatal sepsis (EONS). Secondarily, we hypothesized that vaccination with bacillus Calmette-Guérin (BCG) and oral polio vaccine (OPV) could alter WBC differential counts and thus might reduce its predictive performance. METHODS: We performed a prospective cohort study within a randomized trial, randomizing healthy, high-risk newborns admitted to the nursery at the national hospital in Guinea-Bissau 1:1 to BCG+OPV at admission or at discharge (usual practice). Thin capillary blood films were prepared at 2 d of age in a subset of 268 neonates. WBC counts were assessed by microscopy and neonates were followed up for sepsis development within 2 weeks. RESULTS: Ninety-eight percent (264/268) of smears provided interpretable reads. Of the 264 children, 136 had been randomized to receive BCG+OPV prior to sampling; the remaining 128 were vaccinated at discharge. The I:T ratio (average 0.017) was lower among children who did not develop clinical sepsis but did not predict sepsis (p=0.70). Only three children had an I:T ratio >0.2 (associated with a higher probability of clinical sepsis in previous studies) but did not develop sepsis. Immunization did not alter WBC composition. CONCLUSIONS: Manual WBC differentials are feasible in low-resource settings. WBC differentials are not affected by standard newborn immunization. However, the I:T ratio had no value in predicting subsequent development of sepsis.

Accuracy of Blood Transfusion Records in a Population-based Perinatal Data Registry.

BACKGROUND: Blood transfusion is frequently used as an indicator of severe maternal morbidity during pregnancy. However, few studies have examined its validity in population perinatal databases. METHODS: We linked a perinatal database from British Columbia, Canada, with the province's Central Transfusion Registry for 2004-2015 deliveries. Using the Central Transfusion Registry records for red blood cell transfusion as the gold standard, we calculated the sensitivity, specificity, positive predictive value, and negative predictive value of the perinatal database variable for red blood cell transfusion, overall and by transfusion risk factor status. We used multivariable logistic regression to examine whether outcome misclassification altered the odds ratios for different transfusion risk factors. RESULTS: Among 473,688 deliveries, 4,033 (8.5 per 1,000) had a red blood cell transfusion according to the Central Transfusion Registry. The sensitivity of the perinatal database transfusion variable was 72.3 [95% confidence interval (CI) = 72.2, 72.4]. Sensitivity differed according to the presence of many transfusion risk factors (e.g., 84.9% vs. 72.2% in deliveries with versus without uterine rupture). Odds ratios associated with some transfusion risk factors were exaggerated when the perinatal database transfusion variable was used to define the outcome instead of the Central Transfusion Registry variable, but 95% confidence intervals for these estimates overlapped. CONCLUSION: Blood transfusion was documented with reasonable sensitivity in this large population perinatal database. However, validity varied according to risk factor status. Our findings enable researchers to better account for outcome misclassification in studies of obstetrical transfusion risk factors.

Severe hemolytic disease of the fetus and newborn due to allo-anti-D in a patient with a partial DEL phenotype arising from the variant allele described as RHD*148+1T (RHD*01EL.31).

BACKGROUND: RhD DEL variants may show complete or partial expression of RhD epitopes. There have been only rare reports of anti-D causing hemolytic disease of the fetus and newborn (HDFN) in this context. We report a case of severe HDFN associated with a recently described DEL variant. CASE REPORT: A multiparous woman presented with an allo-anti-D and showed incongruent phenotyping and genotyping results on initial study. Further investigations identified the RHD mutation, defined as RHD*148+1T and named RHD*01EL.31, which had been previously associated with a DEL phenotype. Extended RhD phenotyping by adsorption-elution showed that there was reactivity with four of nine monoclonal anti-D antibodies, suggesting a partial DEL phenotype. The first child showed no clinical evidence of HDFN, although the cord direct antiglobulin test was positive. The second child developed fetal anemia treated with intrauterine transfusion, and neonatal hyperbilirubinemia requiring exchange transfusion. CONCLUSION: The RHD allele, RHD*148+1T, results in a partial Del phenotype, and the anti-D formed in pregnant women with this phenotype is capable of causing severe HDFN.

Dietary Vitamin K and Association with Hepatic Vitamin K Status in a Yup'ik Study Population from Southwestern Alaska.

SCOPE: The relationship between dietary vitamin K and plasma PIVKA-II concentration, a biomarker of hepatic vitamin K status, in a Yup'ik study population in southwestern Alaska is investigated. METHODS AND RESULTS: A total of 659 male and female, self-reported Yup'ik people, ≥14 years of age, were enrolled. Blood is collected for genotyping and plasma PIVKA-II biomarker analysis. A Yup'ik-specific dietary food frequency questionnaire is used to assess vitamin K intake. Among the participants, 22% report not consuming foods rich in vitamin K during the past year and 36% have a PIVKA-II concentration ≥ 2 ng mL-1 , indicating vitamin K insufficiency. The odds of an elevated PIVKA-II concentration are 33% lower in individuals reporting any versus no consumption of vitamin-K-rich foods. The association is significant after adjusting for CYP4F2*3 genotype. Tundra greens are high in vitamin K1 content, but an exploratory analysis suggests that subsistence meat sources have a greater effect on vitamin K status. CONCLUSIONS: A substantial proportion of the Yup'ik population exhibits vitamin K insufficiency, which is associated with low consumption of vitamin K rich foods and which might affect an individual's response to anticoagulant drugs such as warfarin that target the vitamin K cycle.

Dietary and genetic influences on hemostasis in a Yup'ik Alaska Native population.

Fish and marine animals are important components of the subsistence diet of Alaska Native people, resulting in a high ω3 PUFA intake. The historical record for circumpolar populations highlights a tendency for facile bleeding, possibly related to ω3 PUFA effects on platelet activation and/or vitamin K-dependent clotting factors. To evaluate these two scenarios in Yup'ik people of southwestern Alaska, we examined the association between dietary ω3 PUFA intake and activities of clotting factor II, V, fibrinogen, PT, INR, PTT, and sP-selectin in 733 study participants, using the nitrogen isotope ratio of red blood cells as a biomarker of ω3 PUFA consumption. sP-selectin alone correlated strongly and inversely with ω3 PUFA consumption. Approximately 36% of study participants exhibited PIVKA-II values above the threshold of 2 ng/ml, indicative of low vitamin K status. To assess genetic influences on vitamin K status, study participants were genotyped for common vitamin K cycle polymorphisms in VKORC1, GGCX and CYP4F2. Only CYP4F2*3 associated significantly with vitamin K status, for both acute (plasma vitamin K) and long-term (PIVKA-II) measures. These findings suggest: (i) a primary association of ω3 PUFAs on platelet activation, as opposed to vitamin K-dependent clotting factor activity, (ii) that reduced CYP4F2 enzyme activity associates with vitamin K status. We conclude that high ω3 PUFA intake promotes an anti-platelet effect and speculate that the high frequency of the CYP4F2*3 allele in Yup'ik people (~45%) evolved in response to a need to conserve body stores of vitamin K due to environmental limitations on its availability.

P450-Based Drug-Drug Interactions of Amiodarone and its Metabolites: Diversity of Inhibitory Mechanisms.

In this study, IC50 shift and time-dependent inhibition (TDI) experiments were carried out to measure the ability of amiodarone (AMIO), and its circulating human metabolites, to reversibly and irreversibly inhibit CYP1A2, CYP2C9, CYP2D6, and CYP3A4 activities in human liver microsomes. The [I]u/Ki,u values were calculated and used to predict in vivo AMIO drug-drug interactions (DDIs) for pharmaceuticals metabolized by these four enzymes. Based on these values, the minor metabolite N,N-didesethylamiodarone (DDEA) is predicted to be the major cause of DDIs with xenobiotics primarily metabolized by CYP1A2, CYP2C9, or CYP3A4, while AMIO and its N-monodesethylamiodarone (MDEA) derivative are the most likely cause of interactions involving inhibition of CYP2D6 metabolism. AMIO drug interactions predicted from the reversible inhibition of the four P450 activities were found to be in good agreement with the magnitude of reported clinical DDIs with lidocaine, warfarin, metoprolol, and simvastatin. The TDI experiments showed DDEA to be a potent inactivator of CYP1A2 (KI = 0.46 µM, kinact = 0.030 minute(-1)), while MDEA was a moderate inactivator of both CYP2D6 (KI = 2.7 µM, kinact = 0.018 minute(-1)) and CYP3A4 (KI = 2.6 µM, kinact = 0.016 minute(-1)). For DDEA and MDEA, mechanism-based inactivation appears to occur through formation of a metabolic intermediate complex. Additional metabolic studies strongly suggest that CYP3A4 is the primary microsomal enzyme involved in the metabolism of AMIO to both MDEA and DDEA. In summary, these studies demonstrate both the diversity of inhibitory mechanisms with AMIO and the need to consider metabolites as the culprit in inhibitory P450-based DDIs.

Impact of preoperative laboratory investigation and blood cross-match on clinical management of pediatric neurosurgical patients.

PURPOSE: Studies in the adult literature suggest that preoperative laboratory investigations and cross-match are performed unnecessarily and rarely lead to changes in clinical management. The purposes of this study were the following: (1) to explore whether preoperative laboratory investigations in neurosurgical children alter clinical management and (2) to determine the utilization of cross-matched blood perioperatively in elective pediatric neurosurgical cases. METHODS: We reviewed pediatric patient charts for elective neurosurgery procedures (June 2010-June 2014) at out institution. Variables collected include preoperative complete blood count (CBC), electrolytes, coagulation, group and screen, and cross-match. A goal of the review was to identify instances of altered clinical management, as a consequence of preoperative blood work. The number of cross-matched blood units transfused perioperatively was also determined. RESULTS: Four hundred seventy-seven electively scheduled pediatric neurosurgical patients were reviewed. Preoperative CBC was done on 294, and 39.8 % had at least one laboratory abnormality. Electrolytes (84 patients) and coagulation panels (241 patients) were abnormal in 23.8 and 24.5 %, respectively. The preoperative investigations led to a change in clinical management in three patients, two of which were associated with significant past medical history. Group and screen test was performed in 62.5 % of patients and 57.9 % had their blood cross-matched. Perioperative blood transfusions (71 % of these patients were under 3 years of age) were received by 3.6 % of patients (17/477). The cross-match to transfusion ratio was 16. CONCLUSION: This study suggests that the results of preoperative laboratory exams have limited value, apart from cases with oncology and complex preexisting conditions. Additionally, cross-matching might be excessively conducted in elective pediatric neurosurgical cases.

Microdeletion of exon 3 in the HBA2 gene associated with mild α-thalassemia trait.

A mother and son presented with mild symptoms of thalassemia trait. Polymerase chain reaction (PCR) amplification of their globin genes revealed a previously unreported 203 bp microdeletion in the HBA2 gene (NG_000006.1:g.34305_34507del; HBA2:c301-30_*44del). Both mother and son were heterozygous for the deletion which included DNA coding for all of exon 3. DNA sequence analysis revealed a six nucleotide repeat (5'-CGGGCC-3') flanking the breakpoint, suggesting that the microdeletion may have arisen as a result of reciprocal recombination within the HBA2 alleles.

Two new examples of Hb St. Etienne [beta 92(F8)HisGln] in association with venous thrombosis.

Hb St. Etienne [beta92(F8)HisGln] (also known as Hb Istanbul) is a rare unstable beta-globin chain variant that has been described in only three reports involving four patients. We report two individuals in a family of Scottish extraction whose members had been erroneously diagnosed to have hereditary spherocytosis (HS) and have now been shown to be heterozygotes for Hb St. Etienne. They also had venous thrombotic events with minimal provocation. This family illustrates the difficulties in identifying the cause of chronic hemolytic anemia and highlights the possible contribution of chronic hemolysis to increased risk of thrombosis.

Pharmacogenomics of 4-hydroxycoumarin anticoagulants.

Oral anticoagulants of the 4-hydroxycoumarin class, typified by warfarin, are used worldwide to treat thromboembolic disease. These drugs show the beneficial attributes of high efficacy and low cost, but patient management can be complicated by their narrow therapeutic index and wide inter-individual variability in dosing. Our understanding of the latter complication has improved significantly in recent years due to intense investigation of genetic factors influencing drug pharmacokinetics (CYP2C9) and pharmacodynamic response (VKORC1). In particular, the discovery of polymorphisms in the VKORC1 gene that strongly impact oral anticoagulant dose has heightened expectations that genetic testing for a relatively small cadre of warfarin-response genes might substantially enhance patient care in this area, especially during the initiation phase of therapy. However, enthusiasm for genotype-based dosing of oral anticoagulants must be balanced against the ready availability of both a simple phenotypic test (prothrombin time) and an antidote to over-anticoagulation (vitamin K). Wide-spread acceptance of genetically based tests for establishing therapy with warfarin and its congeners will likely require additional evidence that such an approach offers protection against a variety of negative anticoagulation outcomes, especially severe bleeding, as well as offering utility across many racial populations. This article will review recent events in these and other related areas.

Growth factor independence-1 is expressed in primary human neuroendocrine lung carcinomas and mediates the differentiation of murine pulmonary neuroendocrine cells.

Human small cell lung cancers might be derived from pulmonary cells with a neuroendocrine phenotype. They are driven to proliferate by autocrine and paracrine neuropeptide growth factor stimulation. The molecular basis of the neuroendocrine phenotype of lung carcinomas is relatively unknown. The Achaete-Scute Homologue-1 (ASH1) transcription factor is critically required for the formation of pulmonary neuroendocrine cells and is a marker for human small cell lung cancers. The Drosophila orthologues of ASH1 (Achaete and Scute) and the growth factor independence-1 (GFI1) oncoprotein (Senseless) genetically interact to inhibit Notch signaling and specify fly sensory organ development. Here, we show that GFI1, as with ASH1, is expressed in neuroendocrine lung cancer cell lines and that GFI1 in lung cancer cell lines functions as a DNA-binding transcriptional repressor protein. Forced expression of GFI1 potentiates tumor formation of small-cell lung carcinoma cells. In primary human lung cancer specimens, GFI1 expression strongly correlates with expression of ASH1, the neuroendocrine growth factor gastrin-releasing peptide, and neuroendocrine markers synaptophysin and chromogranin A (P < 0.0000001). GFI1 colocalizes with chromogranin A and calcitonin-gene-related peptide in embryonic and adult murine pulmonary neuroendocrine cells. In addition, mice with a mutation in GFI1 display abnormal development of pulmonary neuroendocrine cells, indicating that GFI1 is important for neuroendocrine differentiation.

Detection and genotyping of varicella-zoster virus by TaqMan allelic discrimination real-time PCR.

A proportion of individuals vaccinated with live attenuated Oka varicella-zoster virus (VZV) vaccine subsequently develop attenuated chicken pox and/or herpes zoster. To determine whether postvaccination varicella infections are caused by vaccine or wild-type virus, a simple method for distinguishing the vaccine strain from wild-type virus is required. We have developed a TaqMan real-time PCR assay to detect and differentiate wild-type virus from Oka vaccine strains of VZV. The assay utilized two fluorogenic, minor groove binding probes targeted to a single nucleotide polymorphism in open reading frame 62 that distinguishes the Oka vaccine from wild-type strains. VZV DNA could be genotyped and quantified within minutes of thermocycling completion due to real-time monitoring of PCR product formation and allelic discrimination analysis. The allelic discrimination assay was performed in parallel with two standard PCR-restriction fragment length polymorphism (RFLP) methods on 136 clinical and laboratory VZV strains from Canada, Australia, and Japan. The TaqMan assay exhibited a genotyping accuracy of 100% and, when compared to both PCR-RFLP methods, was 100 times more sensitive. In addition, the method was technically simpler and more rapid. The TaqMan assay also allows for high-throughput genotyping, making it ideal for epidemiologic study of the live attenuated varicella vaccine.