Journal of Perinatology (2014), 1–5 © 2014 Nature America, Inc. All rights reserved 0743-8346/14 www.nature.com/jp
ORIGINAL ARTICLE
Evaluating eosin-5-maleimide binding as a diagnostic test for hereditary spherocytosis in newborn infants RD Christensen1,2,3, AM Agarwal4,5, RH Nussenzveig4, N Heikal4,5, MA Liew4 and HM Yaish3 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. Journal of Perinatology advance online publication, 6 November 2014; doi:10.1038/jp.2014.202
INTRODUCTION Hereditary spherocytosis (HS) is the most common cause of nonimmune hemolytic jaundice identified among neonates who develop kernicterus.1–3 Also, HS is the most common non-immune hemolytic anemia requiring red blood cell (RBC) transfusions during the first month of life.2–4 The adverse outcomes of bilirubin-induced neurological dysfunction and unanticipated emergency hospitalization for transfusion could be minimized or eliminated by early diagnosis of HS and rigorous monitoring of bilirubin and hemoglobin. Diagnosing HS in the first days after birth is challenging. As about 65% of neonates with HS have a parent with HS (autosomal dominant trait), we maintain that all neonates born to an affected parent should have attempts at early diagnosis.3 The 35% of neonates with HS where the family history is negative (because of autosomal recessive inheritance or a de novo mutation) can be particularly challenging to diagnose in the newborn period. The traditional laboratory tests for HS based on erythrocyte fragility are problematic, particularly during the newborn period, because of the physiologically decreased fragility of neonatal erythrocytes. On this basis, osmotic fragility testing is often postponed for several months.4–6 However, in undiagnosed neonates with HS, the first week after birth is the most critical period for
developing kernicterus, and the first month is the most common time for unanticipated anemia requiring emergent transfusion. Therefore, waiting for several months to initiate the HS evaluation is fraught with problems, and reliable means are needed for diagnosing HS during the first days after birth. A relatively new diagnostic test for HS, eosin-5-maleimide (EMA)-flow cytometry, quantifies the erythrocyte band 3 complex using an eosin-5 maleimide dye flow cytometric analysis.7–19 Although this test has been extensively studied among older children and adults with HS, its usefulness to diagnose HS during the neonatal period has not been specifically addressed. Therefore, we conducted the present analysis to assess the performance of EMA flow in detecting HS in newborn infants. METHODS EMA dye binds to the high abundance band 3 and other low abundance transmembrane proteins within the band 3 macro-complex on the surface of RBCs.17,18 We performed EMA flow analysis using blood from three groups of neonates: (i) healthy control term neonates, (ii) neonates for whom the diagnosis of HS was suspected and later confirmed, (iii) neonates for whom the diagnosis of HS was initially suspected but later excluded. This was a convenience sample study. For the healthy control term neonates, otherwise discarded blood was either collected from the
1 The Department of Women and Newborns, Intermountain Healthcare, Salt Lake City, UT, USA; 2Division of Neonatology, University of Utah School of Medicine, Salt Lake City, UT, USA; 3Division of Hematology/Oncology, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT, USA; 4Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA and 5ARUP Laboratories, Salt Lake City, UT, USA. Correspondence: Dr RD Christensen, Intermountain Healthcare, McKay-Dee Hospital Center, 4403 Harrison Blvd, Ogden, 84403 UT, USA. E-mail: [email protected] Received 11 August 2014; revised 29 September 2014; accepted 29 September 2014
EMA flow in neonates with hereditary spherocytosis RD Christensen et al
2 Tested with EMA flow because of suspicion of Hereditary Spherocytosis N=11
Parent with HS YES
N=3
Bili >95th% in first week YES
N=11
Parent with HS NO
N=8
Bili >95th% in first week NO
N=0
Confirmed the diagnosis of HS/HE
Excluded the diagnosis of HS
N=9
N=2
• Spherocytes and anemia persisted, n=9
• Persistent spherocytes on blood films, n=0
• NGS for HS mutations tested, n=8
• Persistent hyperbilirubinemia, n=0
• Genes where mutations were identified :
• Persistent anemia, n=0
Beta spectrin (heterozygous mutation), n=2
• NGS for HS mutations tested, n=0
Band 3 (heterozygous mutation), n=3 Band 3 plus ankyrin, n=1 Alpha spectrin (compound heterozygote), n=3
Figure 1.
Blood from 11 neonates suspected in the first days of life of having HS and tested by EMA flow cytometery.
umbilical cord after birth, or from scavenged blood samples remaining in the hospital laboratory after clinically ordered tests were run. The blood samples were deidentified before submitting them to the research laboratory for EMA-flow cytometry. Blood from neonates in whom HS was suspected were drawn from vascular sticks (0.3 to 0.4 ml) into EDTAcontaining microtubes or into EDTA-containing tubes for qualitative flow cytometry of erythrocyte transmembrane protein band 3, using EMA dye. As the dye binds stoichiometrically to band 3 protein, decreased mean fluorescence intensity is consistent with a red cell cytoskeletal disorder. Briefly, the test was performed as described by King and colleagues.14 One microliter of whole blood was washed in 200 μl of phosphate-buffered saline, the sample was then centrifuged for 5 min at 400 g at room temperature. After centrifugation, the supernatant was removed and 25 μl of phosphate-buffered saline containing 0.5 mg ml − 1 EMA was added. Samples were incubated for 60 min at room temperature in the dark. Following the incubation, the RBCs were washed by adding 200 μl of phosphate-buffered saline + 0.5% (w/v) bovine serum albumin to each sample and centrifuging for 5 min at 400 g at room temperature. This wash step was repeated three times. After the final wash, the RBCs were re-suspended in 200 μl of phosphate-buffered saline + 0.5% (w/v) bovine serum albumin. One hundred thousand events were acquired per sample on a FACS Canto II (BD Biosciences, Sparks, MD) with the acquisition gate set on mainly the singlet RBCs. A snap to polygon is used to gate on the singlet RBCs. Mean florescence intensity and percent co-efficient of variation were evaluated. Analysis also included examination of a side scatter vs EMA fluorescence plot that contains a gate or a ‘footprint’. Essentially, all normal RBCs should be contained within the footprint, whereas abnormal erythrocytes will have a lower EMA fluorescence, with a quantifiable proportion outside the footprint. Three normal controls are run simultaneously with the patient samples to utilize normal Band 3 staining as template for gating patient specimens. The three normal controls must fall within normal limits and should be gated to include at least 99.5% EMA stained RBCs. Specimens from infants up to 12 months of age are tested with at least one washed cord blood as normal control because of some differences in RBC size in infants. Next generation sequencing (NGS) was performed using an original diagnostic panel of 27 genes encoding cytoskeletal proteins, enzymes and UGT1A1 polymorphisms, including mutations in coding regions, splice site Journal of Perinatology (2014), 1 – 5
junctions and deep intronic or regulatory regions.19 Targeted gene capture and library construction for sequencing was performed using HaloPlex as described by the manufacturer (Agilent Technologies, Santa Clara, CA, USA), as we previously described.19 Gestational age of the control neonates and the HS neonates was determined by obstetrical assignment unless this was changed by the neonatologist on the basis of gestational age assessment (based on physical examination and neurological-neurodevelopmental findings). The program used for clinical data collection was a modified subsystem of ‘clinical workstation’. Clinical workstation is a web-based electronic medical record application that stores demographic and clinical information, such as history, physical examination results, laboratory data, problem lists and discharge summaries. 3 M Company (St Paul, MN, USA) approved the structure and definitions of all data points for use within the program. Means and standard deviations were used to express values in groups that were normally distributed, and median and ranges in groups that were not. A Student’s t-test was used to assess continuous variables. Statistical significance was set as Po0.05. The University of Utah Institutional Review Board approved the study as a deidentified data-only investigation.
RESULTS EMA flow cytometric studies were performed on blood samples from 31 newborn infants. Twenty were healthy term control neonates and 11 were neonates for whom HS was initially suspected. As shown in Figure 1, these 11 had severe neonatal hyperbilirubinemia, with a total serum bilirubin level 495th percentile level on an hour-specific nomogram. Also, all 11 had spherocytes reported on the blood film. Three of the 11 had a parent with HS and 8 had no family history of HS. Demographic and clinical information about the 11 initially suspected of having HS is given in Table 1. All 11 received phototherapy in the birth hospital. Nine also received phototherapy at home, four were rehospitalized for intensive phototherapy and one underwent exchange transfusion on © 2014 Nature America, Inc.
No No 34.4 36.0 89.3 96.4 8 0 No No Yes Home Yes Home Rehosp White White Hispanic Hispanic F M 2805 3571 39 1 36 4 10 11
© 2014 Nature America, Inc.
Abbreviations: Exch, exchange; g, grams; Gest, gestational; HE, hereditary elliptocytosis; HS, hereditary spherocytosis; MCHC, mean corpuscular hemoglobin concentration; PK, pyruvate kinase deficiency; RBC, red blood cell; Trans, transfusion; wk, week; wt, weight. The two neonates described in the shaded rows were later found not to have HS. The other nine had the diagnosis of HS confirmed, eight using next generation sequencing and one on the basis of a patent with HS and persistence of spherocytosis and anemia in the neonate. Erythrocyte transfusions given according to Intermountain Healthcare guidelines.20 a Phototherapy: home, home phototherapy; Rehosp, readmitted to hospital for intensive phototherapy.
Beta spectrin and alphaLELY Band 3
Alpha spectrin and alphaLEPRA Alpha spectrin and alphaLELY
HS HS Resolved by 3 months PK and HS HS 95.9 101.6 100.8 0 0 1 No No Yes No No No Yes Home Yes Home Yes White Hispanic White White Hispanic Hispanic M M M 39 4 37 2 33 6 7 8 9
3198 2720 2105
Yes No
Yes Yes Yes 37 3 6
3270
F
Hispanic Pakistani
Yes Home Rehosp
1
95.0
Icteric plasma 33.1 35.9 35.0
No Yes No
Band 3 and alphaLELY
Beta spectrin Band 3 and ANK1
Alpha spectrin and Band 4.1
HE HS HS HS Resolved by 3 months HS No Yes No No No 34.9 36.5 35.1 36.7 35.3 87.2 82.9 104.3 106.3 97.8 1 0 2 0 0 Yes No Yes No No No No No No No Home Rehosp Home Rehosp Home Home (24 days) Yes Yes Yes Yes Yes Hispanic Hispanic White White White White Hispanic White Hispanic Hispanic M M M M M 2844 2630 2375 4443 2113 0 0 3 0 1 37 38 37 39 33 1 2 3 4 5
Exch Trans Photo-therapya Gender Ethnicity Mother Father Birth wt (g) Gest age (wk day) Number
Table 1.
Demographic and clinical features of 11 neonates suspected of having HS
RBC Trans 1st month
Number Trans in 1st year
1st wk MCV (fL)
1st wk MCHC (g dl − 1)
Parent Eventual with HS diagnosis
Mutation
EMA flow in neonates with hereditary spherocytosis RD Christensen et al
rehospitalitization. Five had a RBC transfusion within the first month. Nine of the 11 were later confirmed as having HS/ hereditary elliptocytosis on the basis of persistence of anemia, reticulocytosis and abnormal erythrocyte morphology, plus finding mutations using NGS that were previously associated with HS (hereditary elliptocytosis in one neonate) (Table 1). One (patient #1) had mutations in alpha spectrin and band 4.1, both of which have been reported in hereditary elliptocytosis. Two (patients #3 and 11) had heterozygous mutations in betaspectrin and band 3, respectively, both previously reported in HS. Two (patients #7 and 8) had mutations in alpha spectrin and also had alphaLELY, previously reported in HS. One (patient #4) had mutations in both band 3 and ankyrin, both previously reported in HS. One (patient #6) had a mutation in band 3 and also had alphaLELY, previously reported in HS. One (patient #10) had a heterozygous mutation in beta-spectrin and also had alphaLELY, previously reported in HS. One (patient #2) was confirmed as having HS on the basis of persistence of anemia and spherocytosis and a parent with HS (thus, NGS was not performed). Two (patients #5 and 9) were initially suspected of having HS but eventually confirmed not to, because on follow-up at 3 months of age, spherocytosis, reticulocytosis and anemia had resolved (thus, they did not have NGS performed). Representative results of normal and abnormal EMA-flow cytometric studies performed during the newborn period are shown in Figure 2. The findings were similar between the 20 healthy controls and the 2 who were initially suspected of having HS but the diagnosis was later excluded, with a normal percent co-efficient of variation and essentially all events falling within the 'footprint’. The flow results of the 9 for whom HS/hereditary elliptocytosis was confirmed were different from the other 22, with a decreased percent co-efficient of variation and events with lower mean fluorescence falling outside the footprint for normal samples. Range of mean florescence intensity for normal newborns (8854 to 11471, 1SD 559.5) did not overlap with the range for the neonates with HS (5889 to 7944, 1 SD157). The course of HS during infancy, among our nine patients, varied much like that reported by Delhommeau et al.21 Transfusions during the first year of life ranged from none to 8 (Table 1). DISCUSSION HS is not a single condition, but is a phenotype of spherical hyperdense erythrocytes with a shortened life span as a result. The condition is caused by a variety of mutations in one or more of the genes encoding erythrocyte cytoskeletal proteins; principally ankyrin, beta-spectrin, band 3, alpha spectrin or protein 4.2.3,22–25 HS is a major contributor to hazardous neonatal hyperbilirubinemia, kernicterus, bilirubin-induced neurological dysfunction (a less severe form of kernicterus) and neonatal anemia.21,26–28 Recognizing HS in the first days of life can prompt neonatal management in a way that could prevent adverse outcomes. Bianchi et al.7 recently reported a comparison of various HS laboratory diagnostic methods. Among 150 patients with a diagnosis of HS, averaging 26 years of age, they evaluated osmotic fragility studies on fresh and incubated blood, the glycerol lysis test, the acidified glycerol lysis test, the Pink test and EMA-flow cytometry.7 They reported the latter to be the best test in terms of disease specificity and sensitivity. EMA dye binds specifically and stoichiometrically to the band 3 transmembrane protein complex on RBCs. The complex to which the dye binds includes the Rh protein, Rh glycoprotein and CD47, which like band 3, are reduced in RBCs of patients with HS. Defects in other erythrocyte cytoskeletal proteins such as band 4.2 and ankyrin also result in decreased EMA binding, possibly because of the interaction of those proteins with band 3. Dye binding is quantified using flow cytometry and is compared with reference normal samples. Journal of Perinatology (2014), 1 – 5
3
EMA flow in neonates with hereditary spherocytosis RD Christensen et al
4
Figure 2. EMA results of a normal neonate and a neonate with HS. FSC vs SSC scatter plots are shown for each neonate (a, d). The gate shaded green in this plot are the singlet RBCs. Data from this gate were then plotted in the EMA histogram plot (b, e). Data from the EMA+ interval gate in the EMA histogram were then plotted in the SSC vs EMA scatter plot (c, f). The gate drawn on the SSC vs EMA scatter plot is the ‘footprint’ where almost all of the normal RBC are. The arrow in panel F points to the RBC that do not fall within the footprint. Footnote: The range of mean florescence intensity for normal newborns (8854 to 11471, 1 SD 559.5) did not overlap with that of neonates with HS (5889 to 7944, 1 SD 157).
Crisp et al.,29 King et al.9,10 and Girodon et al.30 also reported a favorable diagnostic value of EMA-flow cytometry in older children and adult patients with HS. Although presently regarded by many as the best laboratory diagnostic test for HS, the specific application of this test to neonates has not been reported, prompting the present study. In neonates with severe jaundice, HS can be suspected on the basis of a positive family history, spherocytes on blood film, and/or an elevated mean corpuscular hemoglobin concentration.4,5,31 Cases where the family history is negative for HS can be particularly challenging. It has been our observation that an elevated mean corpuscular hemoglobin concentration is not typical in neonates with autosomal recessive HS, such as our patients #7 and 8.32,33 Sequencing relevant genes in the parents of neonates with HS, particularly those neonates with no family history, can clarify the inheritance pattern.32,33 Moreover, the appearance of spherocytes on the blood film of a neonate with jaundice can be a transient finding (as occurred in our patients #5 and 9), and thus finding spherocytes on a blood film in the first days of life is not necessarily diagnostic of HS.6 NGS can confirm HS, but it is more costly and not widely available. Thus, a rapid and highly discriminating test like the EMA-flow is of value when considering the possible diagnosis of HS in a newborn infant. We recognize limitations in our study, particularly the very small sample size of only 11 neonates evaluated for HS. Also, in theory, marked hemolysis could render too few erythrocytes for microanalysis, but this has not appeared as a problem in this or the previously reported EMA-flow cytometric studies. However, we find congruence between our results and previous reports in older children and adults, and on that basis, we speculate that EMA-flow can have value in identifying HS during the neonatal period. Journal of Perinatology (2014), 1 – 5
CONFLICT OF INTEREST Drs. Agarwal, Nussenzveig, Heikal and Liew are employees of ARUP Laboratories, the reference laboratory where the EMA-flow testing is performed. However, none are equity holders in the company and none benefit financially from the test. The other authors (RDC and HMY) have no conflicts of interest to disclose.
REFERENCES 1 Johnson L, Bhutani VK, Karp K, Sivieri EM, Shapiro SM. Clinical report from the pilot USA kernicterus registry (1992–2004). J Perinatol 2009; 29(Suppl 1): S25–S45. 2 Christensen RD, Yaish HM, Nussenzveig RH, Reading NS, Agarwal AM, Eggert LD et al. Acute kernicterus in a neonate with O/B blood group incompatibility and a mutation in SLC4A1. Pediatrics 2013; 132(2): e531–e534. 3 Gallagher PG. Abnormalities of the erythrocyte membrane. Pediatr Clin North Am 2013; 60(6): 1349–1362. 4 Christensen RD, Henry E. Hereditary spherocytosis in neonates with hyperbilirubinemia. Pediatrics 2010; 125(1): 120–125. 5 Sheffield MJ, Christensen RD. Evaluating neonatal hyperbilirubinemia in late preterm Hispanic twins led to the diagnosis of hereditary spherocytosis in them, and in their sibling and in their mother. J Perinatol 2011; 31(9): 625–627. 6 Christensen RD, Yaish HM, Lemons RS. Neonatal hemolytic jaundice: Morphologic features of erythrocytes that will help you diagnose the underlying condition. Neonatology 2014; 105(4): 243–249. 7 Bianchi P, Fermo E, Vercellati C, Marcello AP, Porretti L, Cortelezzi A et al. Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics. Haematologica 2012; 97(4): 516–523. 8 Kedar PS, Colah RB, Kulkarni S, Ghosh K, Mohanty D. Experience with eosin-5'maleimide as a diagnostic tool for red cell membrane cytoskeleton disorders. Clin Lab Haematol 2003; 25(6): 373–376. 9 King MJ, Smythe JS, Mushens R. Eosin-5-maleimide binding to band 3 and Rhrelated proteins forms the basis of a screening test for hereditary spherocytosis. Br J Haematol 2004; 124: 106–113. 10 King MJ, Telfer P, MacKinnon H, Langabeer L, McMahon C, Darbyshire P et al. Using the eosin-5-maleimide binding test in the differential diagnosis of
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hereditary spherocytosis and hereditary pyropoikilocytosis. Cytometry B Clin Cytom 2008; 74(4): 244–250. Kar R, Mishra P, Pati HP. Evaluation of eosin-5-maleimide flow cytometric test in diagnosis of hereditary spherocytosis. Int J Lab Hematol 2010; 32(1 Pt 2): 8–16. Kar R, Rao S, Srinivas UM, Mishra P, Pati HP. Clinico-hematological profile of hereditary spherocytosis: experience from a tertiary care center in North India. Hematology 2009; 14(3): 164–167. Tachavanich K, Tanphaichitr VS, Utto W, Viprakasit V. Rapid flow cytometric test using eosin-5-maleimide for diagnosis of red blood cell membrane disorders. Southeast Asian J Trop Med Public Health 2009; 40(3): 570–575. King MJ, Jepson MA, Guest A, Mushens R. Detection of hereditary pyropoikilocytosis by the eosin-5-maleimide (EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins. Int J Lab Hematol 2011; 33(2): 205–211. D'Alcamo E, Agrigento V, Sclafani S, Vitrano A, Cuccia L, Maggio A et al. Reliability of EMA binding test in the diagnosis of hereditary spherocytosis in Italian patients. Acta Haematol 2011; 125(3): 136–140. Ciepiela O, Kotuła I, Górska E, Stelmaszczyk-Emmel A, Popko K, Szmydki-Baran A et al. Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis. Clin Chem Lab Med 2013; 51(4): 817–823. Golafshan HA, Ranjbaran R, Kalantari T, Moezzi L, Karimi M, Behzad-Behbahani A et al. Evaluation of red cell membrane cytoskeletal disorders using a flow cytometric method in South Iran. Turk J Haematol 2014; 31(1): 25–31. Watanabe T, Ono H, Tajima I, Ishigaki H, Hakamata A, Shirai M et al. Hereditary spherocytosis diagnosed with the eosin-5'-maleimide binding test. Pediatr Int 2014; 56(3): 427–429. Christensen RD, Nussenzveig RH, Yaish HM, Henry E, Eggert LD, Agarwal AM. Causes of hemolysis in neonates with extreme hyperbilirubinemia. J Perinatol 2014; 34(8): 616–619. Henry E, Christensen RD, Sheffield MJ, Eggert LD, Carroll PD, Minton SD et al. Why do four NICUs using identical RBC transfusion guidelines have different gestational age-adjusted RBC transfusion rates?. J Perinatol (epub ahead of print 25 September 2014). Delhommeau F, Cynober T, Schischmanoff PO, Rohrlich P, Delaunay J, Mohandas N et al. Natural history of hereditary spherocytosis during the first year of life. Blood 2000; 95: 393–397.
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22 Gallagher PG. Disorders of red cell volume regulation. Curr Opin Hematol 2013; 20 (3): 201–207. 23 Bogardus HH, Maksimova YD, Forget BG, Gallagher PG. A de novo band 3 mutation in hereditary spherocytosis. Pediatr Blood Cancer 2012; 58(6): 1004. 24 Sangerman J, Maksimova Y, Edelman EJ, Morrow JS, Forget BG, Gallagher PG. Ankyrin-linked hereditary spherocytosis in an African-American kindred. Am J Hematol 2008; 83(10): 789–794. 25 Gallagher PG. Hematologically important mutations: ankyrin variants in hereditary spherocytosis. Blood Cells Mol Dis 2005; 35(3): 345–347. 26 Gundel F, Eber S, Heep A. A new ankyrin mutation (ANK1 EXON E9X) causing severe hereditary spherocytosis in the neonatal period. Ann Hematol 2011; 90(2): 231–232. 27 Berardi A, Lugli L, Ferrari F, Gargano G, D'Apolito M, Marrone A et al. Kernicterus associated with hereditary spherocytosis and UGT1A1 promoter polymorphism. Biol Neonate 2006; 90(4): 243–246. 28 Saada V, Cynober T, Brossard Y, Schischmanoff PO, Sender A, Cohen H et al. Incidence of hereditary spherocytosis in a population of jaundiced neonates. Pediatr Hematol Oncol 2006; 23(5): 387–397. 29 Crisp RL, Solari L, Vota D, Garcia E, Miguez G, Chamorro ME et al. A prospective study to assess the predictive value for hereditary spherocytosis using five laboratory tests (cryohemolysis test, eosin-5’-maleimide flow cytometry, osmotic fragility tests, autohemolysis test, and SDS-PAGE) on 50 hereditary spherocytosis families in Argentina. Ann Hematol 2011; 90(6): 625–634. 30 Girodon F, Garcon L, Bergoin E, Larger M, Delaunay J, Feneant-Thibault M et al. Usefulness of the eosin-5’maleimide cytometric method as a first-line screening test for the diagnosis of hereditary spherocytosis: comparison with ektacytometry and protein electrophoresis. Br J Haematol 2008; 140(4): 468–470. 31 Yaish HM, Christensen RD, Henry E, Baer VL, Bennett S. A simple method of screening newborn infants for hereditary spherocytosis. J Applied Hematol 2013; 4: 27–32. 32 Yaish HM, Christensen RD, Agarwal A. 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. J Perinatol 2013; 33(5): 404–406. 33 Nussenzveig RH, Christensen RD, Prchal JT, Yaish HM, Agrawal AM. Novel alpha spectrin mutation in trans with alpha spectrinLEPRA resulting in severe, nondominant, hereditary spherocytosis. Neonatology 2014; 105(4): 355–357.
Journal of Perinatology (2014), 1 – 5
ORIGINAL ARTICLE
Evaluating eosin-5-maleimide binding as a diagnostic test for hereditary spherocytosis in newborn infants RD Christensen1,2,3, AM Agarwal4,5, RH Nussenzveig4, N Heikal4,5, MA Liew4 and HM Yaish3 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. Journal of Perinatology advance online publication, 6 November 2014; doi:10.1038/jp.2014.202
INTRODUCTION Hereditary spherocytosis (HS) is the most common cause of nonimmune hemolytic jaundice identified among neonates who develop kernicterus.1–3 Also, HS is the most common non-immune hemolytic anemia requiring red blood cell (RBC) transfusions during the first month of life.2–4 The adverse outcomes of bilirubin-induced neurological dysfunction and unanticipated emergency hospitalization for transfusion could be minimized or eliminated by early diagnosis of HS and rigorous monitoring of bilirubin and hemoglobin. Diagnosing HS in the first days after birth is challenging. As about 65% of neonates with HS have a parent with HS (autosomal dominant trait), we maintain that all neonates born to an affected parent should have attempts at early diagnosis.3 The 35% of neonates with HS where the family history is negative (because of autosomal recessive inheritance or a de novo mutation) can be particularly challenging to diagnose in the newborn period. The traditional laboratory tests for HS based on erythrocyte fragility are problematic, particularly during the newborn period, because of the physiologically decreased fragility of neonatal erythrocytes. On this basis, osmotic fragility testing is often postponed for several months.4–6 However, in undiagnosed neonates with HS, the first week after birth is the most critical period for
developing kernicterus, and the first month is the most common time for unanticipated anemia requiring emergent transfusion. Therefore, waiting for several months to initiate the HS evaluation is fraught with problems, and reliable means are needed for diagnosing HS during the first days after birth. A relatively new diagnostic test for HS, eosin-5-maleimide (EMA)-flow cytometry, quantifies the erythrocyte band 3 complex using an eosin-5 maleimide dye flow cytometric analysis.7–19 Although this test has been extensively studied among older children and adults with HS, its usefulness to diagnose HS during the neonatal period has not been specifically addressed. Therefore, we conducted the present analysis to assess the performance of EMA flow in detecting HS in newborn infants. METHODS EMA dye binds to the high abundance band 3 and other low abundance transmembrane proteins within the band 3 macro-complex on the surface of RBCs.17,18 We performed EMA flow analysis using blood from three groups of neonates: (i) healthy control term neonates, (ii) neonates for whom the diagnosis of HS was suspected and later confirmed, (iii) neonates for whom the diagnosis of HS was initially suspected but later excluded. This was a convenience sample study. For the healthy control term neonates, otherwise discarded blood was either collected from the
1 The Department of Women and Newborns, Intermountain Healthcare, Salt Lake City, UT, USA; 2Division of Neonatology, University of Utah School of Medicine, Salt Lake City, UT, USA; 3Division of Hematology/Oncology, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT, USA; 4Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA and 5ARUP Laboratories, Salt Lake City, UT, USA. Correspondence: Dr RD Christensen, Intermountain Healthcare, McKay-Dee Hospital Center, 4403 Harrison Blvd, Ogden, 84403 UT, USA. E-mail: [email protected] Received 11 August 2014; revised 29 September 2014; accepted 29 September 2014
EMA flow in neonates with hereditary spherocytosis RD Christensen et al
2 Tested with EMA flow because of suspicion of Hereditary Spherocytosis N=11
Parent with HS YES
N=3
Bili >95th% in first week YES
N=11
Parent with HS NO
N=8
Bili >95th% in first week NO
N=0
Confirmed the diagnosis of HS/HE
Excluded the diagnosis of HS
N=9
N=2
• Spherocytes and anemia persisted, n=9
• Persistent spherocytes on blood films, n=0
• NGS for HS mutations tested, n=8
• Persistent hyperbilirubinemia, n=0
• Genes where mutations were identified :
• Persistent anemia, n=0
Beta spectrin (heterozygous mutation), n=2
• NGS for HS mutations tested, n=0
Band 3 (heterozygous mutation), n=3 Band 3 plus ankyrin, n=1 Alpha spectrin (compound heterozygote), n=3
Figure 1.
Blood from 11 neonates suspected in the first days of life of having HS and tested by EMA flow cytometery.
umbilical cord after birth, or from scavenged blood samples remaining in the hospital laboratory after clinically ordered tests were run. The blood samples were deidentified before submitting them to the research laboratory for EMA-flow cytometry. Blood from neonates in whom HS was suspected were drawn from vascular sticks (0.3 to 0.4 ml) into EDTAcontaining microtubes or into EDTA-containing tubes for qualitative flow cytometry of erythrocyte transmembrane protein band 3, using EMA dye. As the dye binds stoichiometrically to band 3 protein, decreased mean fluorescence intensity is consistent with a red cell cytoskeletal disorder. Briefly, the test was performed as described by King and colleagues.14 One microliter of whole blood was washed in 200 μl of phosphate-buffered saline, the sample was then centrifuged for 5 min at 400 g at room temperature. After centrifugation, the supernatant was removed and 25 μl of phosphate-buffered saline containing 0.5 mg ml − 1 EMA was added. Samples were incubated for 60 min at room temperature in the dark. Following the incubation, the RBCs were washed by adding 200 μl of phosphate-buffered saline + 0.5% (w/v) bovine serum albumin to each sample and centrifuging for 5 min at 400 g at room temperature. This wash step was repeated three times. After the final wash, the RBCs were re-suspended in 200 μl of phosphate-buffered saline + 0.5% (w/v) bovine serum albumin. One hundred thousand events were acquired per sample on a FACS Canto II (BD Biosciences, Sparks, MD) with the acquisition gate set on mainly the singlet RBCs. A snap to polygon is used to gate on the singlet RBCs. Mean florescence intensity and percent co-efficient of variation were evaluated. Analysis also included examination of a side scatter vs EMA fluorescence plot that contains a gate or a ‘footprint’. Essentially, all normal RBCs should be contained within the footprint, whereas abnormal erythrocytes will have a lower EMA fluorescence, with a quantifiable proportion outside the footprint. Three normal controls are run simultaneously with the patient samples to utilize normal Band 3 staining as template for gating patient specimens. The three normal controls must fall within normal limits and should be gated to include at least 99.5% EMA stained RBCs. Specimens from infants up to 12 months of age are tested with at least one washed cord blood as normal control because of some differences in RBC size in infants. Next generation sequencing (NGS) was performed using an original diagnostic panel of 27 genes encoding cytoskeletal proteins, enzymes and UGT1A1 polymorphisms, including mutations in coding regions, splice site Journal of Perinatology (2014), 1 – 5
junctions and deep intronic or regulatory regions.19 Targeted gene capture and library construction for sequencing was performed using HaloPlex as described by the manufacturer (Agilent Technologies, Santa Clara, CA, USA), as we previously described.19 Gestational age of the control neonates and the HS neonates was determined by obstetrical assignment unless this was changed by the neonatologist on the basis of gestational age assessment (based on physical examination and neurological-neurodevelopmental findings). The program used for clinical data collection was a modified subsystem of ‘clinical workstation’. Clinical workstation is a web-based electronic medical record application that stores demographic and clinical information, such as history, physical examination results, laboratory data, problem lists and discharge summaries. 3 M Company (St Paul, MN, USA) approved the structure and definitions of all data points for use within the program. Means and standard deviations were used to express values in groups that were normally distributed, and median and ranges in groups that were not. A Student’s t-test was used to assess continuous variables. Statistical significance was set as Po0.05. The University of Utah Institutional Review Board approved the study as a deidentified data-only investigation.
RESULTS EMA flow cytometric studies were performed on blood samples from 31 newborn infants. Twenty were healthy term control neonates and 11 were neonates for whom HS was initially suspected. As shown in Figure 1, these 11 had severe neonatal hyperbilirubinemia, with a total serum bilirubin level 495th percentile level on an hour-specific nomogram. Also, all 11 had spherocytes reported on the blood film. Three of the 11 had a parent with HS and 8 had no family history of HS. Demographic and clinical information about the 11 initially suspected of having HS is given in Table 1. All 11 received phototherapy in the birth hospital. Nine also received phototherapy at home, four were rehospitalized for intensive phototherapy and one underwent exchange transfusion on © 2014 Nature America, Inc.
No No 34.4 36.0 89.3 96.4 8 0 No No Yes Home Yes Home Rehosp White White Hispanic Hispanic F M 2805 3571 39 1 36 4 10 11
© 2014 Nature America, Inc.
Abbreviations: Exch, exchange; g, grams; Gest, gestational; HE, hereditary elliptocytosis; HS, hereditary spherocytosis; MCHC, mean corpuscular hemoglobin concentration; PK, pyruvate kinase deficiency; RBC, red blood cell; Trans, transfusion; wk, week; wt, weight. The two neonates described in the shaded rows were later found not to have HS. The other nine had the diagnosis of HS confirmed, eight using next generation sequencing and one on the basis of a patent with HS and persistence of spherocytosis and anemia in the neonate. Erythrocyte transfusions given according to Intermountain Healthcare guidelines.20 a Phototherapy: home, home phototherapy; Rehosp, readmitted to hospital for intensive phototherapy.
Beta spectrin and alphaLELY Band 3
Alpha spectrin and alphaLEPRA Alpha spectrin and alphaLELY
HS HS Resolved by 3 months PK and HS HS 95.9 101.6 100.8 0 0 1 No No Yes No No No Yes Home Yes Home Yes White Hispanic White White Hispanic Hispanic M M M 39 4 37 2 33 6 7 8 9
3198 2720 2105
Yes No
Yes Yes Yes 37 3 6
3270
F
Hispanic Pakistani
Yes Home Rehosp
1
95.0
Icteric plasma 33.1 35.9 35.0
No Yes No
Band 3 and alphaLELY
Beta spectrin Band 3 and ANK1
Alpha spectrin and Band 4.1
HE HS HS HS Resolved by 3 months HS No Yes No No No 34.9 36.5 35.1 36.7 35.3 87.2 82.9 104.3 106.3 97.8 1 0 2 0 0 Yes No Yes No No No No No No No Home Rehosp Home Rehosp Home Home (24 days) Yes Yes Yes Yes Yes Hispanic Hispanic White White White White Hispanic White Hispanic Hispanic M M M M M 2844 2630 2375 4443 2113 0 0 3 0 1 37 38 37 39 33 1 2 3 4 5
Exch Trans Photo-therapya Gender Ethnicity Mother Father Birth wt (g) Gest age (wk day) Number
Table 1.
Demographic and clinical features of 11 neonates suspected of having HS
RBC Trans 1st month
Number Trans in 1st year
1st wk MCV (fL)
1st wk MCHC (g dl − 1)
Parent Eventual with HS diagnosis
Mutation
EMA flow in neonates with hereditary spherocytosis RD Christensen et al
rehospitalitization. Five had a RBC transfusion within the first month. Nine of the 11 were later confirmed as having HS/ hereditary elliptocytosis on the basis of persistence of anemia, reticulocytosis and abnormal erythrocyte morphology, plus finding mutations using NGS that were previously associated with HS (hereditary elliptocytosis in one neonate) (Table 1). One (patient #1) had mutations in alpha spectrin and band 4.1, both of which have been reported in hereditary elliptocytosis. Two (patients #3 and 11) had heterozygous mutations in betaspectrin and band 3, respectively, both previously reported in HS. Two (patients #7 and 8) had mutations in alpha spectrin and also had alphaLELY, previously reported in HS. One (patient #4) had mutations in both band 3 and ankyrin, both previously reported in HS. One (patient #6) had a mutation in band 3 and also had alphaLELY, previously reported in HS. One (patient #10) had a heterozygous mutation in beta-spectrin and also had alphaLELY, previously reported in HS. One (patient #2) was confirmed as having HS on the basis of persistence of anemia and spherocytosis and a parent with HS (thus, NGS was not performed). Two (patients #5 and 9) were initially suspected of having HS but eventually confirmed not to, because on follow-up at 3 months of age, spherocytosis, reticulocytosis and anemia had resolved (thus, they did not have NGS performed). Representative results of normal and abnormal EMA-flow cytometric studies performed during the newborn period are shown in Figure 2. The findings were similar between the 20 healthy controls and the 2 who were initially suspected of having HS but the diagnosis was later excluded, with a normal percent co-efficient of variation and essentially all events falling within the 'footprint’. The flow results of the 9 for whom HS/hereditary elliptocytosis was confirmed were different from the other 22, with a decreased percent co-efficient of variation and events with lower mean fluorescence falling outside the footprint for normal samples. Range of mean florescence intensity for normal newborns (8854 to 11471, 1SD 559.5) did not overlap with the range for the neonates with HS (5889 to 7944, 1 SD157). The course of HS during infancy, among our nine patients, varied much like that reported by Delhommeau et al.21 Transfusions during the first year of life ranged from none to 8 (Table 1). DISCUSSION HS is not a single condition, but is a phenotype of spherical hyperdense erythrocytes with a shortened life span as a result. The condition is caused by a variety of mutations in one or more of the genes encoding erythrocyte cytoskeletal proteins; principally ankyrin, beta-spectrin, band 3, alpha spectrin or protein 4.2.3,22–25 HS is a major contributor to hazardous neonatal hyperbilirubinemia, kernicterus, bilirubin-induced neurological dysfunction (a less severe form of kernicterus) and neonatal anemia.21,26–28 Recognizing HS in the first days of life can prompt neonatal management in a way that could prevent adverse outcomes. Bianchi et al.7 recently reported a comparison of various HS laboratory diagnostic methods. Among 150 patients with a diagnosis of HS, averaging 26 years of age, they evaluated osmotic fragility studies on fresh and incubated blood, the glycerol lysis test, the acidified glycerol lysis test, the Pink test and EMA-flow cytometry.7 They reported the latter to be the best test in terms of disease specificity and sensitivity. EMA dye binds specifically and stoichiometrically to the band 3 transmembrane protein complex on RBCs. The complex to which the dye binds includes the Rh protein, Rh glycoprotein and CD47, which like band 3, are reduced in RBCs of patients with HS. Defects in other erythrocyte cytoskeletal proteins such as band 4.2 and ankyrin also result in decreased EMA binding, possibly because of the interaction of those proteins with band 3. Dye binding is quantified using flow cytometry and is compared with reference normal samples. Journal of Perinatology (2014), 1 – 5
3
EMA flow in neonates with hereditary spherocytosis RD Christensen et al
4
Figure 2. EMA results of a normal neonate and a neonate with HS. FSC vs SSC scatter plots are shown for each neonate (a, d). The gate shaded green in this plot are the singlet RBCs. Data from this gate were then plotted in the EMA histogram plot (b, e). Data from the EMA+ interval gate in the EMA histogram were then plotted in the SSC vs EMA scatter plot (c, f). The gate drawn on the SSC vs EMA scatter plot is the ‘footprint’ where almost all of the normal RBC are. The arrow in panel F points to the RBC that do not fall within the footprint. Footnote: The range of mean florescence intensity for normal newborns (8854 to 11471, 1 SD 559.5) did not overlap with that of neonates with HS (5889 to 7944, 1 SD 157).
Crisp et al.,29 King et al.9,10 and Girodon et al.30 also reported a favorable diagnostic value of EMA-flow cytometry in older children and adult patients with HS. Although presently regarded by many as the best laboratory diagnostic test for HS, the specific application of this test to neonates has not been reported, prompting the present study. In neonates with severe jaundice, HS can be suspected on the basis of a positive family history, spherocytes on blood film, and/or an elevated mean corpuscular hemoglobin concentration.4,5,31 Cases where the family history is negative for HS can be particularly challenging. It has been our observation that an elevated mean corpuscular hemoglobin concentration is not typical in neonates with autosomal recessive HS, such as our patients #7 and 8.32,33 Sequencing relevant genes in the parents of neonates with HS, particularly those neonates with no family history, can clarify the inheritance pattern.32,33 Moreover, the appearance of spherocytes on the blood film of a neonate with jaundice can be a transient finding (as occurred in our patients #5 and 9), and thus finding spherocytes on a blood film in the first days of life is not necessarily diagnostic of HS.6 NGS can confirm HS, but it is more costly and not widely available. Thus, a rapid and highly discriminating test like the EMA-flow is of value when considering the possible diagnosis of HS in a newborn infant. We recognize limitations in our study, particularly the very small sample size of only 11 neonates evaluated for HS. Also, in theory, marked hemolysis could render too few erythrocytes for microanalysis, but this has not appeared as a problem in this or the previously reported EMA-flow cytometric studies. However, we find congruence between our results and previous reports in older children and adults, and on that basis, we speculate that EMA-flow can have value in identifying HS during the neonatal period. Journal of Perinatology (2014), 1 – 5
CONFLICT OF INTEREST Drs. Agarwal, Nussenzveig, Heikal and Liew are employees of ARUP Laboratories, the reference laboratory where the EMA-flow testing is performed. However, none are equity holders in the company and none benefit financially from the test. The other authors (RDC and HMY) have no conflicts of interest to disclose.
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