The prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures prior to FVIII inhibitor development.

Preventing factor VIII (FVIII) inhibitors following replacement therapies with FVIII products in patients with hemophilia A remains an unmet medical need. Better understanding of the early events of evolving FVIII inhibitors is essential for risk identification and the design of novel strategies to prevent inhibitor development. The Hemophilia Inhibitor Previously Untreated Patients (PUPs) Study (HIPS; www.clinicaltrials.gov #NCT01652027) is the first prospective cohort study to evaluate comprehensive changes in the immune system during the first 50 exposure days (EDs) to FVIII in patients with severe hemophilia A. HIPS participants were enrolled prior to their first exposure to FVIII or blood products ("true PUPs") and were evaluated for different immunological and clinical parameters at specified time points during their first 50 EDs to a single source of recombinant FVIII. Longitudinal antibody data resulting from this study indicate that there are 4 subgroups of patients expressing distinct signatures of FVIII-binding antibodies. Subgroup 1 did not develop any detectable FVIII-binding immunoglobulin G (IgG) antibodies. Subgroup 2 developed nonneutralizing, FVIII-binding IgG1 antibodies, but other FVIII-binding IgG subclasses were not observed. Subgroup 3 developed transient FVIII inhibitors associated with FVIII-binding IgG1 antibodies, similar to subgroup 2. Subgroup 4 developed persistent FVIII inhibitors associated with an initial development of high-affinity, FVIII-binding IgG1 antibodies, followed by IgG3 and IgG4 antibodies. Appearance of FVIII-binding IgG3 was always associated with persistent FVIII inhibitors and the subsequent development of FVIII-binding IgG4. Some of the antibody signatures identified in HIPS could serve as candidates for early biomarkers of FVIII inhibitor development.

Comparing automated vs manual leukocyte differential counts for quantifying the 'left shift' in the blood of neonates.

OBJECTIVE: The neutrophil 'left shift' can be measured via the immature to total (I/T) neutrophil ratio or the absolute bands per µl using a manual differential count. It can also be measured from an automated differential count by the immature granulocyte percentage (IG%) or the absolute IG per µl. In neonates, it is unknown if the manual or automated differential count is superior. STUDY DESIGN: We directly compared complete blood counts (CBCs) with manual and automated differential counts from infants <90 days old, and documented whether or not each neonate was infected. We developed reference intervals for I/T ratio, bands per µl, IG% and IG per µl using values from non-infected neonates. RESULTS: The database had 10 714 CBCs. The upper reference interval for I/T ratio was 0.29 in the first 48 h and 0.31 thereafter; bands per µl was 3710 µl(-1) in the first 48 h and 1785 µl(-1) thereafter. IG% was 6.2% then 4.2%; IG per µl was 1460 µl(-1) then 613 µl(-1). Statistical performances of the four methods were equivalent for identifying infection. CONCLUSIONS: We developed reference intervals for four methods of quantifying a neonate's 'left shift'. The information from automated differentials is not inferior to that from manual differentials in identifying infections, but automated differentials have the advantages of a larger sample size, being less expensive, and faster performance times.

Reference intervals for reticulocyte parameters of infants during their first 90 days after birth.

OBJECTIVE: The automated reticulocyte parameters (absolute reticulocyte count, immature reticulocyte fraction (IRF) and reticulocyte hemoglobin content (RET-He)) are of value in managing adults and older children with a variety of hematological disorders. However, the lack of reference intervals for these parameters in neonates and young infants has limited their application to that population. STUDY DESIGN: During a span of 12 months (29 May 2014 to 5 May 2015), a convenience sample of reticulocyte parameters were run from clinically ordered complete blood counts (CBCs) of infants within the first 90 days after birth. Measuring the reticulocyte parameters as a research-only adjunct to the CBC did not require any additional blood or generate a patient charge, and the reticulocyte results were not reported to the provided and did not appear in the clinical records. Values from neonates who had a transfusion or a diagnosis of anemia were subsequently excluded from the reference data set. RESULTS: Nine Intermountain Healthcare clinical laboratories contributed 8438 CBCs to the initial reticulocyte parameter database. From these, 1806 were excluded because of a transfusion or a diagnosis of anemia, leaving 6632 in the reference interval database. The parameters charted over the first 90 days after birth were: (1) blood hemoglobin concentration (g dl(-1)), (2) mean corpuscular volume (fL), (3) reticulocyte count (x10(3) per µl), (4) IRF (%) and (5) RET-He (pg). CONCLUSIONS: The new reference interval charts can help clinicians identify abnormalities in the reticulocyte parameters. This information could be of value in identifying and following neonates with various hematological problems including hemolytic disorders, occult hemorrhage, or iron deficiency or other limitations of erythrocyte production.

Evaluation of von Willebrand factor phenotypes and genotypes in Hemophilia A patients with and without identified F8 mutations.

BACKGROUND: Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in factor VIII (FVIII). von Willebrand disease (VWD) is characterized by a quantitative or qualitative defect in von Willebrand factor (VWF). Patients with VWD with severely low VWF or VWD Type 2N (VWD2N), a VWD subtype distinguished by defective VWF binding to FVIII, may have reduced FVIII levels secondary to their VWD. These patients superficially resemble patients with HA and pose a potential for misdiagnosis. OBJECTIVES: To investigate the unexplained cause of bleeding in HA patients without known FVIII mutations by assessing plasma VWF antigen (VWF:Ag), FVIII binding capacities and VWF genotypes. PATIENTS/METHODS: Thirty-seven of 1027 patients with HA studied as part of the Hemophilia Inhibitor Research Study lacked identifiable F8 mutations. These patients (cases) and 73 patients with identified F8 mutations (controls) were evaluated for VWF:Ag, a patient's VWF capacity to bind FVIII (VWF:FVIIIB) and VWF sequence. RESULTS: Four cases had VWF:Ag < 3 IU dL(-1) and VWF mutations consistent with Type 3 VWD. Six cases and one control were heterozygous for mutations previously reported to cause Type 1 VWD (VWD1) (n = five cases and one control) or predicted to be deleterious by Polyphen2 and SIFT prediction tools (n = 1 case). One control had VWF:Ag < 30 IU dL(-1) and seven patients (four cases and three controls), including two cases who were heterozygous for a known VWD2N mutation, had reduced VWF:FVIIIB. CONCLUSIONS: These data emphasize that some patients diagnosed with HA require VWF assessments in order to achieve a comprehensive diagnosis and an optimal treatment strategy.

End-tidal carbon monoxide as an indicator of the hemolytic rate.

In the first days of life, low grade jaundice is essentially universal. The source of the elevated bilirubin level giving rise to "physiological jaundice of the newborn" is only partly known. We hypothesized that it is, at least in part, the result of active and specific hemolysis involving a physiological mechanism to lower the high fetal hematocrit, appropriate for the relatively low oxygen environment in utero, to a lower level appropriate for the state of oxygen abundance after birth. We tested this by quantifying end tidal carbon monoxide (ETCO) as a marker of the rate of heme metabolism to bilirubin. We found that ETCO values of 20 neonates and children with known hemolytic disorders were higher than 20 age-matched healthy controls (p<0.0001), indicating that this instrumentation recognizes hemolysis in neonates and children. We also found that ETCO reference intervals were indeed higher in healthy neonates during the first three days after birth (5th to 95th percentile reference range, 1.4 to 1.7ppm) than after 1month of age (all ≤1.0ppm, p<0.0001). These results suggest to us that hemolysis is physiological during the first days after birth. The cellular and molecular mechanisms responsible for transient hemolysis after birth are topics of current investigation.

Evaluating eosin-5-maleimide binding as a diagnostic test for hereditary spherocytosis in newborn infants.

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.

Causes of hemolysis in neonates with extreme hyperbilirubinemia.

OBJECTIVE: We instituted a quality improvement process to enhance our capacity to diagnose genetic hemolytic conditions in neonates with extreme hyperbilirubinemia. STUDY DESIGN: During a 1-year period, whenever the total serum bilirubin (TSB) was >25 mg dl(-1) a special evaluation was performed. If we deemed an erythrocyte membrane defect likely, based on red blood cell morphology, EMA-flow cytometry was performed. Otherwise 'next-generation' sequencing was performed using a panel of genes involved in neonatal hyperbilirubinemia. RESULT: Ten neonates had a TSB ⩾ 25 mg dl(-1). Two others were evaluated as part of this process at the request of their attending neonatologists, because each had a TSB >14 mg dl(-1) in the first hours after birth and required phototherapy for ⩾ 1 week. Explanations for the jaundice were found in all 12 neonates. Five had hereditary spherocytosis, three of which also had ABO hemolytic disease. Two had pyruvate kinase deficiency. One had severe G6PD deficiency. The other four had ABO hemolytic disease. CONCLUSION: On the basis of the present small case series, we suggest that among neonates with extreme hyperbilirubinemia, it can be productive to pursue a genetic basis for hemolytic disease.

A neonate with Coombs-negative hemolytic jaundice with spherocytes but normal erythrocyte indices: a rare case of autosomal-recessive hereditary spherocytosis due to alpha-spectrin deficiency.

The diagnosis of hereditary spherocytosis (HS) in a newborn infant is generally made on the basis of a positive family history, spherocytes on blood film and Coombs-negative hemolytic jaundice of variable severity with an elevated mean corpuscular hemoglobin concentration (MCHC) and a low mean corpuscular volume (MCV). In general, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) quantification of erythrocyte membrane proteins is not needed to make the clinical diagnosis of HS. However, we observed that a neonate with no family history of HS, but with abundant spherocytosis on repeated blood films, Coombs-negative hemolytic jaundice and normal MCHC and MCV measurements, where SDS-PAGE revealed alpha-spectrin deficiency, a rare autosomal-recessive variety of HS that generally has a severe clinical phenotype.

A neonate with congenital amegakaryocytic thrombocytopenia associated with a chromosomal microdeletion at 21q22.11 including the gene RUNX1.

We observed a neonate who had severe thrombocytopenia wherein evaluations for neonatal immune-mediated thrombocytopenia and congenital infections were negative, and the marrow findings were consistent with congenital amegakaryocytic thrombocytopenia (CAMT). A genomic microarray identified a microdeletion at 21q22.11 including the gene RUNX1. Two somewhat similar cases were recently reported, but with multiple congenital anomalies that are not present in our case. We propose that a 21q22 deletion resulting in RUNX1 haploinsufficiency can produce a phenotype similar to CAMT with various associated anomalies depending on which adjacent genes are absent or disrupted.

A neonate with the Pelger-Huët anomaly, cleft lip and palate, and agenesis of the corpus callosum, with a chromosomal microdeletion involving 1q41 to 1q42.12.

We observed a neonate with cleft lip and palate, 13 sets of ribs, agenesis of the corpus callosum, slightly small penis, hypoglycemia, and what initially appeared to be a marked leukocyte 'left shift' on complete blood count, but which was actually a Pelger-Huët anomaly. A chromosomal microdeletion was identified at1q41-42.12.

Lead poisoning among Saudi children.

Ten Saudi children with clinical and laboratory findings related to lead intoxication were seen at the King Fahad National Guard Hospital from 1984 to 1988. A presumptive diagnosis of chronic lead poisoning was made retrospectively on 12 other children with hypochromic and microcytic anemia whose other laboratory data were consistent with lead poisoning. The ages of the children ranged from six months to 13 years. After the first child with lead encephalopathy was diagnosed, a high index of suspicion was maintained, thus enabling us to establish an early diagnosis of lead encephalopathy. The findings of this study suggest that excessive lead exposure may still pose a serious public health hazard in Saudi Arabia today which needs to be addressed.

Misdiagnosis of congenital chloride-losing diarrhea.

Congenital chloride-losing diarrhea is a recessively inherited disorder due to the absence of chloride-bicarbonate exchange in the small bowel. Malabsorption of chloride leads to osmotic diarrhea, electrolyte abnormalities, and dehydration. If left untreated, the infants fail to thrive and have a very high mortality. Clinically, affected patients develop secretory diarrhea in utero resulting in distended bowel loops and polyhydramnios. At birth these infants have profuse watery diarrhea that may be confused with urine. Thus, the correct diagnosis is often missed, and they may be subjected to unnecessary interventions. If diagnosed early, the electrolyte abnormalities are easily corrected and the prognosis is good. We report two patients who were initially evaluated for other conditions but later proved to have congenital chloride-losing diarrhea. The cases emphasize the importance of having a high index of suspicion in patients with a history of polyhydramnios, prematurity, and watery stools.

Heterogeneity of gamma-globin chain synthesis in Saudi newborns.

Cord blood samples from 655 unselected neonates born to Saudi mothers at King Fahad National Guard Hospital, Riyadh, Saudi Arabia were analysed to determine the levels of gamma-globin chains in Saudis. The percentage of three types of gamma-chains of human fetal hemoglobin (A gamma T, G gamma and A gamma I) was obtained by high-performance liquid chromatographic (HPLC) method. Although the majority of babies (631/655) had normal G gamma values in the range of 58-74%, there were only 69% with normal G gamma/A gamma ratio. The A gamma T chain or HbF Sardinia was present in 28% of the total neonates with a gene frequency of 0.160. The A gamma T values in this group ranged between 11-42%. Eight babies (1.2%) had G gamma levels 45% or less (mean 41 +/- 3%) and in 16 neonates (2.4%), G gamma values were highly elevated (mean 81.4 +/- 2.8%). The frequency of two G gamma-globin genes was 0.0061 and 0.0122, respectively, which is comparable with other ethnic or racial groups. The differences in G gamma to A gamma ratio in some Saudi babies could be due to an abnormal arrangement of gamma-globin genes of beta-globin gene cluster which is now being investigated.

Increased incidence of hyperbilirubinaemia in 'unchallenged' glucose-6-phosphate dehydrogenase deficiency in term Saudi newborns.

The incidence of severe hyperbilirubinaemia was significantly higher among the G6PD-deficient Saudi infants born at term than in non-deficient babies (34% vs 9%) (p less than 0.005). No apparent offending factors were detected in either the babies or their mothers. All babies who developed hyperbilirubinaemia did so during the 1st week of life. The highest mean bilirubin level for all jaundiced G6PD-deficient babies was recorded on the 4th postnatal day. Although the incidence of severe hyperbilirubinaemia among our neonates was relatively high, only two of them (7%), a boy and a girl, required exchange transfusions. Five of 29 jaundiced babies with G6PD deficiency were readmitted after discharge because of significant jaundice. One required exchange transfusion. Since G6PD deficiency seems to be a relatively common cause of neonatal jaundice in Saudi newborns, early detection of this enzymopathy by cord blood screening is justified to avoid morbidity and deaths.

Idiopathic thrombocytopenic purpura in children.

Idiopathic thrombocytopenic purpura in children 10 years of age or younger was observed to have a more favorable prognosis than in older children. Corticosteroid therapy in children judged to be at increased risk of serious hemorrhage resulted in a significantly greater number of patients with an early increase in platelets than was noted in a control group. All patients with chronic disease who responded to administration of a corticosteroid initially and then relapsed had some response to a subsequent course of therapy, although none had a sustained remission. In such patients, splenectomy was a more effective therapeutic measure than treatment with either a corticosteroid or a cytotoxic agent.