Transient Unexplained Severe Acute Hyperbilirubinaemia and Cholestasis in a Patient With Hereditary Spherocytosis

The recent discovery of the specific molecular defects in many patients with hereditary spherocytosis and hereditary elliptocytosis/pyropoikilocytosis partially clarifies the molecular pathology of these diseases. HE and HPP are caused by defects in the horizontal interactions that hold the membrane skeleton together, particularly the critical spectrin self-association reaction. Single gene defects cause red cells to elongate as they circulate, by a unknown mechanism, and are clinically harmless. The combination of two defective genes or one severe alpha spectrin defect and a thalassaemia-like defect in the opposite allele (alphaLELY) results in fragile cells that fragment into bizarre shapes in the circulation, with haemolysis and sometimes life-threatening anaemia. A few of the alpha spectrin defects are common, suggesting they provide an advantage against malaria or some other threat. HS, in contrast, is nearly always caused by family-specific private mutations. These involve the five proteins that link the membrane skeleton to the overlying lipid bilayer: alpha and beta spectrin, ankyrin, band 3 and protein 4.2. Somehow, perhaps through loss of the anchorage band 3 provides its lipid neighbours (Peters et al, 1996), microvesiculation of the membrane surface ensues, leading to spherocytosis, splenic sequestration and haemolysis. Future research will need to focus on how each type of defect causes its associated disease, how the spleen aggravates membrane skeleton defects (a process termed 'conditioning'), how defective red, cells are recognized and removed in the spleen, and why patients with similar or even identical defects can have different clinical severity. Emphasis also needs to be given to improving diagnostic tests, particularly for HS, and exploring new options for therapy, like partial splenectomy, which can ameliorate symptoms while better protecting patients from bacterial sepsis and red cell parasites, and perhaps from atherosclerosis (Robinette & Franmeni, 1977) and venous thrombosis (Stewart et al, 1996).

Severe hyperbilirubinemia is the most common cause of neonatal readmission to hospital in Canada even though, in the majority of cases, risk factors can be identified before discharge. Severe neonatal hyperbilirubinemia and kernicterus continue to be reported worldwide in otherwise healthy term infants. We conducted this study to estimate the incidence of severe neonatal hyperbilirubinemia in Canada and to determine underlying causes, improved knowledge of which would be valuable to help identify strategies for risk reduction. Data on term infants 60 days of age and younger with unconjugated hyperbilirubinemia were collected prospectively through the Canadian Paediatric Surveillance Program from 2002 to 2004. Infants were included if they had a peak serum total bilirubin level of more than 425 micromol/L or underwent an exchange transfusion. Infants with rhesus iso-immunization or who were born at less than 36 weeks' gestation were excluded. Of 367 cases reported, 258 were confirmed to be severe neonatal hyperbilirubinemia, for an estimated incidence of 1 in 2480 live births. Causes were identified in 93 cases and included ABO incompatibility (n = 48), glucose-6-phosphate dehydrogenase deficiency (n = 20), other antibody incompatibility (n = 12) and hereditary spherocytosis (n = 7). The mean peak bilirubin level reported was 471 micromol/L (standard deviation [SD] 76 micromol/L, range 156-841 micromol/L). Fifty-seven infants (22.1%) underwent an exchange transfusion. A total of 185 infants (71.7%) were readmitted to hospital, 121 (65.4%) of them within 5 days of age. Severe neonatal hyperbilirubinemia continues to occur frequently in Canada. In the majority of cases, the underlying cause was not identified. The high readmission rate within days after initial discharge indicates a need for a more thorough assessment of newborn infants and consideration of strategies to identify at-risk newborns, such as predischarge measurement of serum bilirubin levels.

Using a Next Generation Sequencing Panel to Discover the Obscure Causes of Hereditary Hemolytic Anemias

Severe neonatal unconjugated hyperbilirubinemia is associated with the risk of neurotoxicity and hence warrants prompt diagnostic and therapeutic interventions, including phototherapy and exchange transfusions. Although relatively rare, Hereditary spherocytosis (HS) is one of the common non-alloimmune hemolytic disorders associated with severe neonatal hyperbilirubinemia and hemolytic anemia during childhood. HS results from a mutation in genes encoding 1 or more proteins in the erythrocyte membrane, which leads to a loss of membrane structure and dysfunction of the cell. The clinical spectrum of HS varies widely, leading to under-diagnosis during the neonatal period, and management differs from alloimmune hemolytic disorders. In this case report of a neonate with severe unconjugated hyperbilirubinemia due to HS. Severe unconjugated hyperbilirubinemia due to HS. Phototherapy and exchange transfusion. Subsequent decrease in bilirubin levels and safe discharge of neonate with instructions to follow up with pediatrician, hematology, and genetics with outpatient appointments. In this case report, we emphasize the need for a high index of suspicion among neonatal caregivers and discuss management options. We also discuss the lack of response to intravenous immunoglobulin as a diagnostic clue and the feasibility of performing exchange transfusion using peripheral intravenous access.

BACKGROUNDNeonatal hyperbilirubinemia is a common problem faced by pediatricians. The role of genetic factors in neonatal jaundice has been gradually recognized. This study aims to identify genetic variants that influence the bilirubin level in five patients using next-generation sequencing (NGS).CASE SUMMARYFive neonates with severe hyperbilirubinemia were retrospectively studied. They exhibited bilirubin encephalopathy, hypothyroidism, ABO blood type incompatibility hemolysis, glucose-6-phosphate dehydrogenase (G6PD) deficiency and premature birth, respectively. A customized 22-gene panel was designed, and NGS was carried out for these neonates. Eight variations (G6PD c.G1388A, HBA2 c.C369G, ABCC2 c.C3825G, UGT1A1 c.G211A, SPTB c.A1729G, EPB41 c.G520A, c.1213-4T>G and c.A1474G) were identified in these five neonates. Genetic mutations of these genes are associated with G6PD deficiency, thalassemia, Dubin-Johnson syndrome, Gilbert syndrome, hereditary spherocytosis, and hereditary elliptocytosis. One of the neonates was found to have compound variants of the EPB41 splice site c.1213-4T>G and c.G520A (p.E174K), but no elliptocyte was seen on his blood smear of 4 years old.CONCLUSIONPathological factors of severe neonatal hyperbilirubinemia are complicated. Genetic variants may play an important role in an increased risk of neonatal hyperbilirubinemia, and severe jaundice in neonates may be related to a cumulative effect of genetic variants.

Hereditary spherocytic pyropoikilocytosis: Expanding the clinical and genetic spectrum of red blood cell membrane disorders

Background Conventional diagnosis of hereditary red blood cell (RBC) membrane disorders, in particular hereditary spherocytosis (HS), is labor intensive, time consuming and requires at least 2 ml of blood, which might be impractical in the neonatal period. Participants and methods We evaluated the use of eosin-5-maleimide (EMA) as a rapid screening test for patients with HS. RBCs from 74 healthy controls and 66 anemic children (35 HS and 31 other hemolytic anemias; 10 cases diagnosed as thalassemia, eight cases of autoimmune hemolytic anemia, one case of ovalocytosis and 12 cases of undiagnosed hemolytic anemia) were stained with EMA and analyzed for their mean fluorescence intensity using flow cytometry. Results RBCs from patients with HS showed a greater degree of reduction in mean fluorescence intensity of EMA compared with those from normal controls and patients with other hemolytic diseases. These findings showed that the fluorescence flow cytometric-based method is a simple, sensitive and reliable diagnostic test for RBC membrane disorders using a small volume of blood, and results could be obtained within 2 h. Such a method could serve as a first-line screening for the diagnosis of HS in routine hematology.

BACKGROUND Hereditary spherocytosis (HS) is an autosomal dominant inherited disorder that causes severe hyperbilirubinemia in neonates. There is no factual data about the prevalence in Indonesia. It is common that neonates with suspected hereditary spherocytosis are not diagnosed or treated adequately in developing countries such as Indonesia. CASE REPORT A 6-day-old baby was referred from a secondary public hospital to our tertiary hospital in Malang, East Java with severe hyperbilirubinemia unresponsive to the 2 days of conventional phototherapy. Initial laboratory examination showed total serum bilirubin level 28.83 mg/dL and indirect bilirubin level 25 mg/dL. Complete blood count showed hemoglobin level of 10.3 g/dL with high MCHC 36.9 g/dL and increased RDW 18.7%. The HS ratio (MCHC per MCV) was 0.41. The blood smear showed spherocytes with positive family history from the mother and grandmother. There were no specific tests such as EMA binding, cryohemolysis, or analysis of erythrocyte membrane protein available in our hospital. The patient was then treated with 2 sessions of intensive phototherapy with phototherapy unit bilisphere 360 LED. The total serum bilirubin level dropped to 12.19 mg/dL. In this case, we decided to perform intensive phototherapy first, not only because of facility-based constraints to do timely exchange transfusion, but also due to the low socio-economic and educational background of the parents. CONCLUSIONS There are some challenges in diagnosing and treating HS adequately in Indonesia. Limitations of specific tests, inadequacy of conventional phototherapy, lack of awareness of and adherence to guidelines, and facility-based inability to perform timely exchange transfusion all can contribute to severe hyperbilirubinemia and its sequelae.

Hereditary spherocytosis is a common hemolytic anemia with an estimated incidence of 1/2500 births. It is caused by a molecular defect in one or more of the proteins of the red blood cell cytoskeleton. Mutations in the ABCB11 gene, encoding the bile salt export pump, can entail progressive familial intrahepatic cholestasis and benign recurred intrahepatic cholestasis. A18 year old Turkish patient with hereditary spherocytosis was admitted to hospital with pruritus and severe jaundice. Ultrasound examination presented stones in gallbladder and bile duct. After endoscopic retrograde cholangiography with extraction of small bile duct stones abdominal pain resolved and liver enzymes normalized within a few days, but bilirubin and bile acids remained highly elevated. Liver biopsy revealed a severe canalicular cholestasis. Genetic analysis showed the compound heterozygous variants ABCB11 A444V and 3084A>G. Treatment with ursodesoxycholic acid and intermittent therapy with prednisone reduced pruritus and jaundice with concomitant improvement of blood test. Here we report the first case of a patient with combined hereditary spherocytosis and compound heterozygous ABCB11 gene variants predisposing to intrahepatic cholestasis. Therefore, patients with hemolytic disorders should be investigated for bile acid transporter diseases in case of hyperbilirubinemia and severe cholestasis.

Dubin-Johnson syndrome is a chronic, benign, intermittent jaundice, mostly of conjugated hyperbilirubinemia. The level of bilirubin is not expected to be more than 20 mg/dl in this syndrome. In this article, we report a patient who was evaluated for hyperbilirubinemia and liver function test abnormalities and diagnosed with Dubin-Johnson syndrome coexisting with hereditary spherocytosis. We suggest that other diseases should be investigated if patients with Dubin-Johnson syndrome present with severe hyperbilirubinemia. Dubin-Johnson syndrome accompanied by hemolytic diseases might also have high coproporphyrin levels (as in Rotor's syndrome) than expected in pure Dubin-Johnson syndrome.

Erythropoietic Stimuli and Response in Hereditary Spherocytosis

Background: Hereditary spherocytosis in the Hispanic population does not often present with severe hyperbilirubinemia. Spectrin and band 3 mutations are most frequent in this population. Case report: We present a Hispanic full-term female newborn with early onset significant hyperbilirubinemia without a history of familial hemolytic disorders. She was diagnosed with hereditary spherocytosis based on laboratory findings, including presence of spherocytes on a peripheral smear, and was later found by next-generation sequencing to have Tokyo-1 mutation, an ANK1 gene mutation, that was previously only reported in Japanese population. Conclusion: Our report adds to the currently limited literature of the genetic spectrum and characteristics of hereditary spherocytosis in the Hispanic population. The absence of a positive family history does not preclude hereditary spherocytosis as a differential for pathologic neonatal hyperbilirubinemia.

Objective. Comparative studying of levels of some biochemical indices in experimental acute biliary pancreatitis and obstructive cholestasis. Materials and methods. An acute biliary pancreatitis was simulated on 32 rats and obstructive cholestasis – on 23. On the first, third and 7th days after the blood indices of general bilirubin, albumin and the urea, as well as activity of alaninaminotranspherase, aspartataminotranspherase, alkaline phosphatase and amylase were studied. Results. In the acute biliary pancreatitis on the second day of the experiment 10 (31.3%) rats have deceased, and in obstructive cholestasis - 1 (4.3%) rat (p < 0.01). In both groups of laboratory animals the signs of cholestasis were determined, and in the acute biliary pancreatitis – signs of pancreatitis. On the first day in acute biliary pancreatitis in comparison with acute cholestasis there were noted the enhanced values of the amylase activity (p < 0.001) and the urea content (p < 0.05); on the third day – the lower values of the general bilirubin content (p < 0.001), higher values of the alaninaminotranspherase activity (p < 0.05), the urea content (p < 0.01) and activity of amylase (p < 0.05); on the 7-th day – lower values of general bilirubin (p < 0.05), higher values of activity of alkaline phosphatase and aspartataminotranspherase (p < 0.05). Conclusion. More severe hyperbilirubinemia is observed in obstructive cholestasis. In the acute biliary pancreatitis the trustworthy enhanced values of the alaninaminotranspherase (on the third day) and aspartataminotranspherase (on the 7-th day) activities, the urea content (on the third day), as well as activity of alkaline phosphatase (on the 7th day) are noted.

Toward the Creation of Diagnostic Algorithms for Congenital Hemolytic Anemia Using Advanced Parameters from Modern Hematology Analyzers

A flow cytometry-based test using eosin-5-maleimide (EMA) dye was used for diagnosis of hereditary spherocytosis (HS). The mean fluorescence intensiy (MFI) of EMA tagged erythrocytes is lower in HS than that in other hemolytic and nonhemolytic anemias. We enrolled 114 subjects comprising 20 confirmed HS, 20 suspected HS/hemolytic anemia (HA), 20 normal controls, 20 other hemolytic anemias [13 autoimmune hemolytic anemia, three congenital dyserythropoietic anemia (CDA), one pyruvate kinase deficiency, two microangiopathic hemolytic anemia], 18 microcytic anemia and 16 macrocytic anemia cases. All samples were subjected to flow cytometry as per standard protocol. The mean MFI of normal control subjects was 11 861.5 (SD 883.5) and of confirmed HS was 7949.3 (SD 1304.1). Using this test, of 20 patients suspected to be HS/HA but with no confirmatory diagnosis, eight patients were diagnosed as HS. Using logistic regression analysis, the optimum cut-off MFI value between HS and normal controls was 10126. The area under the ROC curve was 0.99. The statistical significance of MFI values was obtained by t-test or Wilcoxon rank sum test as applicable. Compared with normal controls, the MFI values in HS were lower and in megaloblastic anemia were higher which was statistically highly significant (P<0.01), and the MFI values in CDA were lower which was statistically significant (P<0.05). False-positive values were obtained in three cases of AIHA and two cases of CDA. The sensitivity and specificity was 96.4% and 94.2% respectively. The EMA-based flow cytometry test is a highly sensitive and specific method for the diagnosis of HS.