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. 2021 Sep 9;2(4):716–728. doi: 10.1002/jha2.277

Facilitating EMA binding test performance using fluorescent beads combined with next‐generation sequencing

Andreas Glenthøj 1,✉, Christian Brieghel 1, Amina Nardo‐Marino 1, Richard van Wijk 2, Henrik Birgens 1, Jesper Petersen 1
PMCID: PMC9176113  PMID: 35845192

Abstract

The eosin‐5′‐maleimide (EMA) binding test is widely used as diagnostic test for hereditary spherocytosis (HS), one of the most common haemolytic disorders in Caucasian populations. We recently described the advantages of replacing the use of healthy control blood samples with fluorescent beads in a modified EMA binding assay. In this study we further explore this novel approach. We performed targeted next‐generation sequencing, modified EMA binding test and osmotic gradient ektacytometry on consecutive individuals referred to our laboratory on the suspicion of HS. In total, 33 of 95 carried a (likely) pathogenic variant, and 24 had variants of uncertain significance (VUS). We identified a total 79 different (likely) pathogenic variants and VUS, including 43 novel mutations. Discarding VUS and recessive mutations in STPA1, we used the occurrence of (likely) pathogenic variants to generate a diagnostic threshold for our modified EMA binding test. Twenty‐one of 23 individuals with non‐SPTA1 (likely) pathogenic variants had EMA ≥ 43.6 AU, which was the optimal threshold in receiver operating characteristic (ROC) analysis. Accuracy was excellent at 93.4% and close to that of osmotic gradient ektacytometry (98.7%). In conclusion, we were able to simplify the EMA‐binding test by using rainbow beads as reference and (likely) pathogenic variants to define an accurate cut‐off value.

Keywords: haemolytic anaemia, hereditary anaemias, laboratory haematology, rbc membrane, spherocytosis

1. INTRODUCTION

Hereditary spherocytosis (HS) is a relatively common and well‐characterised hereditary haemolytic disorder. The disease is particularly common in people of northern European descent, with a prevalence of approximately 1:2000 in this population [1, 2]. The genetic background of HS is germline mutations in red blood cell (RBC) cytoskeleton proteins, such as α‐spectrin, β‐spectrin, band 3 and ankyrin [3, 4].

Individuals with HS typically present with Coombs‐negative haemolytic anaemia, high MCHC and splenomegaly. In many cases, however, clinical features and paraclinical findings are equivocal and advanced laboratory tests are necessary to confirm the diagnosis.

The Eosin 5‐Maleimide (EMA) binding test is recommended as the primary screening test for HS, both sensitivity and specificity of this method being over 90% [1]. In this test RBCs are incubated with EMA, which binds extracellular membrane‐associated proteins. EMA fluorescence can be detected by flowcytometry and mainly reflects decreased RBC Band 3, which in HS is reduced compared to healthy controls [2]. Given its simplicity and the wide availability of flow cytometers, this test can be employed in most laboratories at a low cost. Often, results are reported as a ratio of the individual's mean fluorescent intensity (MFI) to that of healthy controls, making the test somewhat comparable across laboratories [5]. This approach does, however, require blood samples from up to six healthy – and ideally age matched ‐ controls, which can be challenging to locate [5, 6, 7]. We recently described a modified version of the EMA binding test, in which we substituted healthy control samples with fluorescent beads. [8]. Although healthy controls were still utilised for calibration, the number of control samples needed was reduced significantly. Performance of this modified EMA binding test was compared to that of the traditional method, using osmotic gradient ektacytometry as validation. We found that accuracy was not compromised, making this approach an attractive and simple alternative [8].

Osmotic gradient ektacytometry is a method for determining RBC deformability and is increasingly used due to the advent of a new generation of ektacytometers [9, 10]. Although this test reliably identifies the RBC characteristics associated with HS, it is incapable of discriminating spherocytes in HS from autoimmune haemolytic anaemia [10].

To facilitate HS diagnosis, targeted next‐generation sequencing (tNGS) is used to detect germline mutations in genes encoding for RBC cytoskeleton proteins [11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21]. tNGS is less time consuming than traditional sequencing techniques, but the technique is associated with high costs and long turnaround. tNGS can be an advantageous diagnostic tool, particularly in transfused individuals where functional testing is affected by donor blood. Nevertheless, the accuracy of tNGS alone has proven somewhat limited for the diagnosis of hereditary anaemias [11, 12, 14, 16–19, 21–32].

Results from the EMA binding test and osmotic gradient ektacytometry are often sufficient to diagnose HS, but both tests have limitations and may produce equivocal results [33, 34, 35]. Many previous studies have evaluated these tests mainly using clinical features of HS as proof of disease, hereby creating an inherent risk of confirmation bias [36].

In this study, we wish to further investigate the modified EMA binding test using rainbow beads instead of healthy control samples. By defining HS as the presence of diagnostic cytoskeleton protein gene mutations identified using tNGS and validating results using osmotic gradient ektacytometry, we provide a reproducible way of estimating a cut‐off value for the modified EMA‐binding test. Finally, we briefly describe the identified underlying pathogenic mutations.

2. MATERIALS AND METHODS

2.1. Population

We included samples from all individuals referred to our laboratory with suspected HS between 1st May 2017 and 1st July 2018 (Figure 1). As samples were shipped from other institutions, clinical data were not available. Samples have previously been used to test the performance of the EMA binding test using fluorescent beads versus healthy controls [8].

FIGURE 1.

FIGURE 1

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Study flowchart. Samples were subjected to modified EMA‐binding test, osmotic gradient ektacytometry and targeted next‐generation sequencing. Individuals with a positive Coombs test, only SPTA1 mutations, elliptocytosis or variants of uncertain significance (VUS) without a (likely) pathogenic variant were excluded from analysis of the EMA‐binding test threshold value

2.2. Ethics

Data were stored and handled in accordance with permission from the Danish Data Protection Agency (10122009 HEH‐L.HB). All participants or a parent/guardian consented to diagnostic tests for haemolytic anaemia including tests for HS.

2.3. tNGS

Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini QIAcube Kit (Qiagen, Hilden, Germany) according to the manufacturer′s instructions. We used a small panel targeting RBC disorders, including genes covering the cytoskeleton proteins, SPTA1 (α‐spectrin), SPTB (β‐spectrin), ANK1 (ankyrin 1), SCL4A1 (band 3), EPB41 (protein 4.1) and EPB42 (protein 4.2). Targeting, amplification and normalisation was performed according to the manufacturer′s instructions (TruSeq Custom Amplicon v1.5, Illumina, CA, USA). Sequencing was performed on a MiniSEquation (Illumina) via MiniSeq Mid Output Kit (300x paired‐end; Illumina). Sequencing analyses were performed using BaseSpace Variant Interpreter (Illumina) and Integrative Genomics Viewer software [37]. Variants were called with at least 10 variant reads, a minimum read depth of 30x and classified in categories according to recommendations from the American College of Medical Genetics and Genomics [38] using BaseSpace Variant Interpreter (Illumina). Only variants classified as likely pathogenic or pathogenic, referred to as (likely) pathogenic variants, and variants of uncertain significance (VUS) were included in the analyses. Benign mutation and likely benign mutations were excluded. Variants not previously described in ClinVar, dbSNP or COSMIC according to Alamut Visual (Interactive Biosoftware, Rouen, France) were reported as novel mutations.

2.4. EMA binding test

The EMA binding test was performed on EDTA‐stabilised blood within 48 h of sampling. The labeling of RBCs with EMA, usage of mid‐range FL1 Rainbow Fluorescent Particles (BD Biosciences, NJ, USA) and flow cytometry was performed as previously described [8]. A detailed protocol is available online (s). Sample evaluation was performed as a ratio comparison between the MFI of beads and patient. The ratio was calculated as the ΔMFI % (mean fluorescence intensity) using ([MFIRainbow beads ‐ MFIPatient]/MFIRainbow beads) × 100.

EMA=ΔMFI%=1−MFIpatientMFIrainbow×CF×100%

A correction factor was used to adjust the MFI of new rainbow bead MFI lots, compared to the previous lots. To minimise the risk of bead MFI fluctuations, we used a second type of beads as control (FluoroSpheres K0110, Agilent Technologies Denmark ApS, Glostrup, Denmark), assuming the MFI ratio between these two sets of beads would remain constant.

2.5. Osmotic gradient ektacytometry

Osmotic gradient ektacytometry was performed on EDTA stabilised blood within 48 h of sampling, using a LoRRca ektacytometer (RR Mechatronics, Zwaag, Netherlands) as previously described [9]. Two parameters were evaluated on the ektacytometry curve: Omin and EImax. Omin reflects the minimal RBC surface/volume ratio, increasing in conditions with reduced surface/volume ratio such as HS [39]. EImax reflects the maximal deformability of the RBCs. Reduction of EImax typically represents a reduced RBC surface area, as is seen in HS [39]. Ohyper, which reflects hydration status, was not used in this setting as this has been found either high or low in HS [40].

2.6. Statistical analyses

Statistical analyses were performed in ‘R’ version 3.6.3 [41] using packages ggplot2, caret, and pROC.

3. RESULTS

3.1. Population

A total of 99 individuals were included in the study. Fifty‐six (56%) were female and the mean age was 30.7 years (SD 28.3). Three individuals had a positive Coombs test (HS9, HS14 and HS33) and were excluded from further analyses (Figure 1) leaving 96 individuals for further analyses. None of the three Coombs positive individuals had (likely) pathogenic variants.

3.2. Mutations identified

Excluding 26 variants classified as likely benign, we identified a total of 78 variants in 58 of the 96 individuals (Table 1). Of these, 34 were (likely) pathogenic variants and 43 were VUS. Mutations in SPTA1 and SPTB were predominant (Tables I and II). Apart from three intronic mutations (two single nucleotide substitutions and one deletion), all VUS were missense mutations (93%). In contrast, 32 of the 34 (likely) pathogenic variants (94%) were non‐missense mutations. To our knowledge, 42 mutations (26 (likely) pathogenic variants and 16 VUS) had not previously been described and, thus, were regarded as novel mutations (Table 1). One individual ('HS34') carrying a pathogenic EPB41 mutation was excluded from further analyses, as examination of a peripheral blood smear confirmed the diagnosis of hereditary elliptocytosis (Figure 1). Forty‐one patients harbored the common SPTA1 mutation c.6531‐12C > T (α‐spectrinLELY), which is considered benign in itself but may cause overt HS, hereditary elliptocytosis or hereditary pyropoikilocytosis in trans to SPTA1 mutations [42, 43].

TABLE 1.

Specification of mutations in the red blood cell cytoskeleton protein genes (SCL4A1, SPTB, SPTA1, ANK1, EPB41 and EPB42) in 99 patients with suspected hereditary spherocytosis

ID Gene ID cDNA Protein change Exon Classification Zygosity Translation impact Novel EMA Omin EImax
HS1 SLC4A1 c.118G > A p.(Glu40Lys) Exon 4 Likely benign* Htz missense No 55.2 192 0.506
SPTB c.5290G > T p.(Glu1764*) Exon 25 Likely pathogenic Htz nonsense Yes
SPTA1 c.6549‐4C > G NA Intron 46 VUS Htz intronic No
HS2 SLC4A1 c.1890+1G > T Splice site Intron 15 Likely pathogenic Htz splice Yes 44.9 179 0.553
HS3 SPTB c.1515delT p.(Asn505Lysfs*68) Exon 11 Likely pathogenic Htz frameshift Yes 53.4 170 0.539
HS4 SPTB c.398T > G p.(Met133Arg) Exon 3 VUS Htz missense Yes 42.2 189 0.573
HS5 SLC4A1 c.118G > A p.(Glu40Lys) Exon 4 Likely benign* Htz missense No 49.6 194 0.531
SPTB c.145dupG p.(Ala49Glyfs*3) Exon 1 Likely pathogenic Htz frameshift Yes
HS6 SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No 51.7 179 0.481
ANK1 c.5224C > T p.(Gln1742*) Exon 40 Likely pathogenic Htz nonsense Yes
HS7 SPTA1 c.4605+1G > A Splice site Intron 32 Likely pathogenic Htz splice Yes 42.6 168 0.566
EPB41 c.1700G > A p.(Gly567Asp) Exon 12 VUS Htz missense No
SPTA1 c.6896G > T p.(Cys2299Phe) Exon 50 VUS Htz missense No
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS8 SPTB c.3764+1G > A Splice site Intron 16 Likely pathogenic Htz splice Yes 48.7 183 0.542
SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No
HS10 SLC4A1 c.1030C > T p.(Arg344*) Exon 10 Pathogenic Htz nonsense No 48.9 198 0.538
HS11 SLC4A1 c.1030C > T p.(Arg344*) Exon 10 Likely pathogenic Htz nonsense No 49.0 200 0.524
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS12 SLC4A1 c.2386G > A p.(Gly796Arg) Exon 18 VUS Htz missense Yes 38.4 120 0.598
SPTA1 c.775G > A p.(Ala259Thr) Exon 6 VUS Htz missense No
HS13 SLC4A1 c.118G > A p.(Glu40Lys) Exon 4 Likely benign* Htz missense No 34.6 167 0.613
SPTA1 c.775G > A p.(Ala259Thr) Exon 6 VUS Htz missense No
HS15 SPTB c.3764+1G > A Splice site Intron 16 Likely pathogenic Htz splice Yes 52.1 177 0.553
SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No
HS16 SPTB c.3764+1G > A Splice site Intron 16 Likely pathogenic Htz splice Yes 48.5 185 0.538
HS17 SPTB Large_del NA Exons 2–3 Likely pathogenic Htz frameshift Yes 55.3 18 0.533
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No
HS18 SLC4A1 c.733G > A p.(Val245Met) Exon 9 VUS Htz missense No 49.2 185 0.556
ANK1 c.4915_4921del***CACGAGT p.(His1639Glyfs*90) Exon 39 Likely pathogenic Htz frameshift Yes
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No
HS19 SLC4A1 c.118G > A p.(Glu40Lys) Exon 4 Likely benign* Htz missense No 62.5 199 0.387
SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No
SPTA1 c.2671C > T p.(Arg891*) Exon 19 Likely pathogenic Htz nonsense No
SPTA1 c.4347G > T p.(Lys1449Asn) Exon 31 VUS Htz missense No
SPTA1 c.4339‐99C > T NA Intron 30 Likely benign* Htz intronic No
HS20 SLC4A1 c.2057+1G > A Splice site Intron 16 Likely pathogenic Htz splice Yes 53.6 189 0.578
SLC4A1 c.2057+5G > A NA Intron 16 VUS Htz intronic Yes
HS21 SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No 41.3 168 0.613
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS22 EPB42 c.2054C > T p.(Thr685Met) Exon 13 VUS Htz missense No 40.8 178 0.597
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No
HS23 SPTB Large_del NA Exons 2–3 Likely pathogenic Htz frameshift Yes 48.5 207 0.485
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS25 SPTB c.2431_2450del***GAAGAGTTTCG***GGATTCCCC p.(Glu811Argfs*44) Exon 13 Likely pathogenic Htz frameshift Yes 36.2 166 0.578
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS26 SPTB c.2431_2450del***GAAGAGTTTCG***GGATTCCCC p.(Glu811Argfs*44) Exon 13 Likely pathogenic Htz frameshift Yes 51.8 185 0.491
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS27 SPTB c.2588G > A p.(Trp863*) Exon 13 Likely pathogenic Htz nonsense Yes 56.1 180 0.444
HS28 SPTB c.2588G > A p.(Trp863*) Exon 13 Likely pathogenic Htz nonsense Yes 53.6 176 0.506
HS29 SPTA1 c.2320C > T p.(Arg774*) Exon 17 Likely pathogenic Htz nonsense No 38.3 174 0.597
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No
HS30 SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Pathogenic Hmz missense No 37.0 185 0.581
SPTA1 c.7068A > C p.(Glu2356Asp) Exon 51 VUS Htz missense No
SPTA1 c.3940T > C p.(Ser1314Pro) Exon 28 VUS Htz missense No
SPTA1 c.4339‐99C > T NA Intron 30 Likely benign* Htz intronic No
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS31 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 37.4 152 0.602
HS32 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 32.9 154 0.623
HS33 SLC4A1 c.118G > A p.(Glu40Lys) Exon 4 Likely benign* Htz missense No 31.7 179 0.604
HS34 EPB41 c.820C > T p.(Gln274*) Exon 5 Likely pathogenic Htz nonsense Yes 33.3 171 0.556
HS35 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No 31.7 162 0.608
HS36 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 30.3 185 0.607
HS37 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No 34.4 162 0.607
HS38 ANK1 c.2858+1G > T Splice site Intron 26 Likely pathogenic Htz splice Yes 53.9 185 0.493
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS39 SPTA1 c.1599+1G > T Splice site Intron 12 Likely pathogenic Htz splice Yes 42.0 167 0.576
SPTA1 c.1450G > A p.(Asp484Asn) Exon 11 VUS Htz missense Yes
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No
HS40 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 24.9 170 0.609
HS43 SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No 10.0 165 0.598
HS44 SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No 36.7 165 0.618
HS45 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 37.0 160 0.617
HS46 SPTB c.5266C > T p.(Arg1756*) Exon 25 Pathogenic Htz nonsense No 40.9 185 0.532
HS51 SLC4A1 c.2021T > G p.(Val674Gly) Exon 16 VUS Htz missense Yes 40.5 165 0.608
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No
HS52 ANK1 c.1486G > A p.(Val496Ile) Exon 13 VUS Htz missense No 34.1 142 0.615
HS53 SPTB c.4891C > T p.(Arg1631Cys) Exon 23 VUS Htz missense No 33.4 173 0.599
HS54 SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Hmz missense No 40.0 188 0.554
SPTA1 c.4347G > T p.(Lys1449Asn) Exon 31 VUS Htz missense No
SPTA1 c.4339‐99C > T NA Intron 30 Likely benign* Hmz intronic No
HS55 SPTB c.3479G > A p.(Arg1160His) Exon 15 VUS Htz missense No 35.4 160 0.604
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS56 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 24.5 164 0.609
HS57 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 25.3 172 0.623
HS59 ANK1 c.127‐39554G > A NA Promoter 5`UTR/Intron 1 Likely benign* Htz intronic No 30.2 171 0.618
ANK1 c.127‐39509T > C NA Promoter 5`UTR/Intron 1 Likely benign* Htz intronic No
ANK1 c.5302C > A p.(Gln1768Lys) Exon 40 VUS Htz missense Yes
HS60 SPTA1 c.6896G > T p.(Cys2299Phe) Exon 50 VUS Htz missense No 34.8 166 0.606
EPB41 c.1700G > A p.(Gly567Asp) Exon 12 VUS Htz missense No
HS61 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 21.6 161 0.608
HS62 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 32.6 158 0.612
HS65 SPTB c.3496C > T p.(Gln1166*) Exon 15 Likely pathogenic Htz nonsense Yes 49.5 194 0.544
SPTA1 c.4605+4delA NA Intron 32 VUS Htz intronic No
HS66 SPTB c.5860A > G p.(Thr1954Ala) Exon 27 VUS Htz missense Yes 38.3 147 0.597
SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No
SPTB c.5032G > C p.(Val1678Leu) Exon 24 VUS Htz missense Yes
HS68 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 38.0 160 0.601
HS69 SPTB c.26A > C p.(Asn9Thr) Exon 1 VUS Htz missense No 36.0 154 0.619
HS70 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No 56.3 165 0.524
HS71 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 36.9 167 0.592
HS72 SPTA1 c.1112+1G > T Splice site Intron 8 Likely pathogenic Htz splice Yes 33.4 178 0.571
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No
HS73 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 19.1 196 0.613
HS75 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 38.7 158 0.615
HS78 SPTA1 c.2464+1G > A Splice site Intron 17 Likely pathogenic Htz splice No 37.5 179 0.579
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS80 SPTB c.398T > G p.(Met133Arg) Exon 3 VUS Htz missense Yes 31.0 159 0.594
SPTB c.6856G > A p.(Ala2286Thr) Exon 35 VUS Htz missense No
HS81 SLC4A1 c.2701C > T p.(Arg901Trp) Exon 20 VUS Htz missense No 33.4 160 0.600
SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No
SLC4A1 c.1162C > T p.(Arg388Cys) Exon 11 VUS Htz missense No
SPTA1 c.4339‐99C > T NA Intron 30 Likely benign* Htz intronic No
HS84 EPB42 c.1477G > A p.(Gly493Ser) Exon 10 VUS Htz missense No 24.0 145 0.617
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS85 EPB41 c.1700G > A p.(Gly567Asp) Exon 12 VUS Hmz missense No 51.0 173 0.517
EPB42 c.826C > T p.(Arg276Trp) Exon 6 VUS Htz missense No
HS86 SPTB c.379C > T p.(Arg127Cys) Exon 3 VUS Htz missense No 36.2 180 0.579
ANK1 c.3571C > T p.(Pro1191Ser) Exon 30 VUS Htz missense Yes
HS87 SPTB c.1134_1135delGA p.(Lys379Serfs* 12) Exon 9 Likely pathogenic Htz frameshift Yes 52.3 193 0.508
HS88 ANK1 c.3173G > A p.(Trp1058*) Exon 28 Likely pathogenic Htz nonsense Yes 48.1 190 0.541
ANK1 c.38A > T p.(Asp13Val) Exon 1 VUS Htz missense Yes
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS89 SLC4A1 c.2102G > A p.(Gly701Asp) Exon 17 Pathogenic † Htz missense No 49.0 160 0.561
SPTB c.6626T > C p.(Val2209Ala) Exon 33 VUS Htz missense Yes
SLC4A1 c.92T > C p.(Met31Thr) Exon 3 VUS Htz missense No
HS90 SPTA1 c.4564A > G p.(Thr1522Ala) Exon 32 VUS Htz missense No 33.0 157 0.590
ANK1 c.127‐39554G > A NA Promoter 5`UTR/Intron 1 Likely benign* Htz intronic No
ANK1 c.127‐39509T > C NA Promoter 5`UTR/Intron 1 Likely benign* Htz intronic No
HS91 ANK1 c.127‐39554G > A NA Promoter 5`UTR/Intron 1 Likely benign* Htz intronic No 43.0 194 0.555
ANK1 c.127‐39509T > C NA Promoter 5`UTR/Intron 1 Likely benign* Htz intronic No
ANK1 c.542T > C p.(Leu181Pro) Exon 6 VUS Htz missense Yes
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS92 ANK1 c.491T > C p.(Leu164Pro) Exon 5 VUS Htz missense Yes 44.0 173 0.567
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS93 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 39.5 160 0.604
HS94 SPTA1 c.1112+1G > T Splice site Intron 8 Likely pathogenic Htz splice Yes 34.7 175 0.568
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Hmz intronic No
HS95 SLC4A1 c.443A > G p.(Gln148Arg) Exon 6 VUS Htz missense Yes 46.0 177 0.579
SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No
HS96 SPTA1 c.6531‐12G > A NA Intron 45 Likely benign* Htz intronic No 34.6 163 0.604
HS97 ANK1 c.3508A > T p.(Ser1170Cys) Exon 30 VUS Htz missense Yes 39.8 167 0.600
HS98 SLC4A1 c.1564G > A p.(Glu522Lys) Exon 13 VUS Htz missense Yes 51.1 185 0.560
HS99 SPTA1 c.2909C > A p.(Ala970Asp) Exon 21 Likely benign* Htz missense No 41.1 201 0.511
SPTA1 c.3792_3793dupAA p.(Met1265Lysfs* 4) Exon 27 Likely pathogenic Htz frameshift Yes
SPTA1 c.4339‐99C > T NA Intron 30 Likely benign* Htz intronic No
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*

Indicated in Illumina as likely benign but may be pathogenic in homozygous or compound heterozygous state (STPA1:c.6531‐12G > A is only potentially pathogenic in compound heterozygotes).

†

One mutation in HS89 (SCL4A1:c.2102 G > A) was described as pathogenic in Illumina. This pathogenicity is related to distal tubular renal acidosis and was interpreted as VUS.

Abbreviations: EImax, elongation index maximum; Hmz, homozygous; Htz, hetetozygous; NA, not applicable; UTR, untranslated region; VUS, variant of uncertain significance.

Seventeen of the 58 individuals harbored more than one mutation. In total, 34 individuals harbored one or more (likely) pathogenic variants and 24 individuals harbored one or more VUS as the only mutations (Figure 1). Forty‐one individuals had no proven mutations. One SLC4A1:c.2102G > A mutation (HS89) associated with renal tubular acidosis and renal membranopathy was classified as pathogenic. In the context of HS, this mutation was interpreted as a VUS. SPTA1 is associated with autosomal recessive HS [23]. To simplify our efforts to set a diagnostic threshold for the EMA binding test, we considered individuals with only (likely) pathogenic SPTA1 mutation(s) as having 'VUS'.

3.3. EMA binding test and osmotic gradient ektacytometry as predictors of mutation status

We demonstrated significantly higher EMA (ΔMFI%) values in individuals with (likely) pathogenic variants compared to individuals without mutations. Similarly, EMA values were significantly higher in individuals with VUS compared to individuals with no mutations (Figure 2A; p = 0.00044). In ROC curve analysis, we found that a threshold of ≥43.6 was optimal for discriminating between individuals with (likely) pathogenic variants and individuals with no mutations (AUC = 95%) (Figure 2B).

FIGURE 2.

FIGURE 2

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Evaluation of modified EMA binding test and ektacytometry against mutational status. Individual distribution of ΔMFI% values (EMA) and the associated ROC curve (A‐B), Omin values and the associated ROC curve (C‐D), EImax values and the associated ROC curve (E‐F), in 95 individuals with suspected hereditary spherocytosis. Thirty‐eight individuals had no proven mutations in red blood cell cytoskeleton protein genes, 33 had one or more (likely) pathogenic variants (excluding EPB41), and 34 had only SPTA1 mutations or variants of uncertain significance (VUS; not shown)

Omin values were significantly higher in individuals with (likely) pathogenic variants compared to individuals without mutations. This corresponds to an increased osmotic fragility in individuals with (likely) pathogenic variants. A significant difference between Omin values in individuals with VUS and individuals with no underlying mutations was also observed, although to a much lesser extent (Figure 2C; p = 0.0023). In our ROC curve analysis, we found that a threshold of 174 mOsm/kg was optimal for discriminating between individuals with (likely) pathogenic variants and individuals without mutations (Figure 2D; AUC = 95%).

When comparing EImax between individuals with (likely) pathogenic variants and individuals without mutations, values were significantly lower in the subgroup with pathogenic mutations, indicating reduced RBC deformability (Figure 2E). When comparing EImax values from individuals with VUS and individuals without mutations, the difference was still significant (Figure 2E; p < 0.0002). Our ROC curve analysis demonstrated an optimal threshold of <0.579 for discriminating individuals with (likely) pathogenic variants from individuals without mutations (Figure 2F; AUC = 98%).

Applying these thresholds, we subsequently calculated: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for the individual parameters (Table 2). All demonstrated sensitivities, specificities, NPV and PPV above 87%. As a single parameter, EImax, yielded the best results with an accuracy of 95.1%.

TABLE 2.

Sensitivity, Specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of the EMA binding test and osmotic gradient ektacytometry (Omin and EImax) in 95 patients with suspected hereditary spherocytosis

Sensitivity (%) Specificity (%) PPV (%) NPV (%) Accuracy (%)
EMA ≥ 43.6 91.3 94.7 91.3 94.7 93.4
Omin ≥ 174 91.3 92.1 87.5 94.6 91.8
EImax < 0.579 100 92.1 88.5 100 95.1

Omin ≥ 166

EImax < 0.579

 100 97.4 95.8 100 98.7

Omin ≥ 166

EImax < 0.579

EMA ≥ 43.6

 91.3 97.4 95.5 94.9 94.3
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Results are given for each parameter individually and in combination.

Abbreviations: EImax, elongation index maximum; EMA, eosin‐5′‐maleimide; NPV, negative predictive value; PPV, positive predictive value.

3.4. Combining osmotic gradient ektacytometry and the EMA binding test to predict mutation status

The distribution of all 95 samples, based on the EMA binding test, Omin and EImax values, is illustrated in Figure 3.

FIGURE 3.

FIGURE 3

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Genetic variants and functional testing. (A) Distribution and relationship between the EMA binding test, Omin and EImax in 95 individuals with suspected hereditary spherocytosis. Forty‐one individuals had no proven mutations in red blood cell cytoskeleton protein genes, 33 had one or more (likely) pathogenic variants and 34 had only SPTA1 mutations or variants of uncertain significance (VUS). Full lines correspond to Omin 166 mOsm/kg and EImax 0.579. Dotted line corresponds to Omin 174 mOsm/kg. *Represents two individuals with similar Omin and EImax. (B) Distribution and relationship between the EMA binding test, EImax and mutations in 57 individuals suspected of having hereditary spherocytosis. Thirty‐three individuals had one or more (likely) pathogenic mutations (excluding EPB41), and 34 had only SPTA1 mutations or variants of uncertain significance (VUS)

When combining the calculated thresholds for Omin and EImax (Omin ≥ 174 mOsm/kg and EImax < 0.579), 21 of 23 individuals with (likely) pathogenic variants were double positive (i.e. true positive), and two were false negative (Figure 3A). In addition, one individual without (likely) pathogenic variants remained double positive (Figure 3). Adjusting the Omin ≥ 166 mOsm/kg while keeping EImax < 0.579 improved the sensitivity to 100%, while keeping an excellent specificity of 97.4% (Table 2). Subsequently, we calculated the sensitivity, specificity, PPV, NPV and accuracy using the obtained EMA, Omin, and EImax thresholds in combination. However, this approach resulted in a marked reduction of sensitivity and NPV without improving other measures (Table 2).

Figure 3B shows an excellent relationship between the modified EMA binding test with fluorescent beads and EImax in individuals with (likely) pathogenic variants and VUS, regardless of the type of the mutated gene.

4. DISCUSSION

In this study, we assessed the number of RBC cytoskeleton protein gene mutations in a population of individuals with suspected HS, using pathogenic mutations as the gold standard. This enabled us to set a diagnostic cut‐off value for our newly described modified EMA binding test with fluorescent beads (Figure 2 and Table 2), thereby alleviating it from the otherwise obligatory use of up to six healthy control samples [5]. Using the EMA binding test alone, we obtained a diagnostic accuracy (Table 2) comparable to those previously reported using healthy controls [44, 45, 46, 47]. In many settings, obtaining suitable control samples can be challenging [8]. Furthermore, the inherent variation in control samples complicates interlaboratory comparisons and quality assessment schemes [24]. Our approach has demonstrated a robust performance, comparable to that of the traditional EMA‐binding test with healthy controls [8] and osmotic gradient ektacytometry (the gold standard of membranopathy diagnostics) across a range of causative genes (Figure 4).

As such, this study differs from previous studies in which HS has typically been defined by clinical phenotype or sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) [1, 28, 32, 40, 47, 48]. We are aware that laboratory screening tests must be related to clinical phenotype. Although we did not have access to patient records and clinical data, we must assume that individuals referred to our laboratory on suspicion of HS had clinical symptoms consistent with the disease. All individuals with true HS are expected to have one or more underlying pathogenic germline mutations, although they are not always identified [16]. Using genotype as the gold standard for HS diagnosis when making an ROC analysis for the EMA‐binding test could eliminate confirmation bias. It is likely that some individuals harbor mutations or deletions not detected in the applied tNGS panel and, consequently, remain undiagnosed [16, 28, 32], but this should not have significant impact on the ROC analysis determining the EMA‐binding test cut‐off value. Furthermore, congenital dyserythropoietic anaemia type II often mimics HS on the EMA binding test and osmotic gradient ektacytometry [34, 35], but the causative gene SEC23B was not included in our tNGS panel. As a diagnostic laboratory, we did not have access to data or samples from relatives, which prevented determination of inheritance patterns.

Excluding EPB41, we detected a total of 76 underlying (likely) pathogenic variants and VUS (Table 1), 42 previously undescribed. They were found in SPTB > SPTA1 > SLC4A1 > ANK1 > EPB42 (listed according to mutation frequency). In previous studies, defect or lack of ankyrin has often been reported as the most frequent mutation in HS, particularly in Northern Europe [2, 49, 50]. The order of affected genes in our study differs from those seen in studies in which clinical features and non‐DNA‐based diagnostics define the disease. The high number of SPTA1 mutations found in our population reflects inclusion of the common hypomorph variants such as c.4339‐99C > T (α‐spectrinLEPRA), which in its heterozygous form should not cause overt haemolysis [51].

In our study, 32 of 34 individuals with detected (likely) pathogenic variants had heterozygous non‐missense mutations, and two individuals had homozygous SPTA1 missense mutations: c.2909 C > A (α‐spectrinBug Hill; HS30 and HS54 in Table 1). c.2909 C > A was originally classified as pathogenic (autosomal recessive) [52], but this is likely due to frequent co‐occurrence of c.4339‐99C > T in cis [51]. In contrast, all VUS were missense mutations, except three intron mutations (two in the SPTA1 gene and one in the SLC4A1 gene).

As α‐spectrin is synthesised in excess [53], heterozygous SPTA1 pathogenic mutations are considered clinically benign but may be pathogenic in homozygous and compound heterozygous state. Accordingly, individuals heterozygous for (likely) pathogenic STPA1 mutations were not used in ROC analysis but several had borderline ΔMFI% changes (Table 1). This is in line with some degree of RBC surface area loss and even mild clinical haemolysis as previously described [28]. Four individuals in our study only harbored a heterozygous SPTA1 mutation (HS29, HS72, HS78, HS94 in Table 1), and one was homozygous for two common missense mutation in SPTA1: α‐spectrinLEPRA and α‐spectrinBug Hill combined with two VUS (HS30). None of these exceeded the EMA cut‐off value. The remaining five individuals with SPTA1 mutations all had EMA values ≥ 40. Five of these had multiple SPTA1 mutations: 3 α‐spectrinLEPRA combined with truncating mutations (HS19, HS99) or with a homozygous SPTA1 missense mutation (HS54), one individual with a pathogenic splice‐site mutation combined with 2 VUS interpreted as compound heterozygous (HS7), and one individual with a splice site mutation as the only alteration (SPTA1:c.1599+1G > T)(HS39). The prevalence of α‐spectrinLELY was notably high (43%) compared to studies of the background population [54].

Although all 10 individuals with pathogenic SPTA1 mutations were double positive on ektacytometry (Omin ≥ 166 mOsm/kg and EImax < 0.579), it is worth noting that five of these were not detected by the EMA‐binding test applied. Positive ektacytometry in these 10 individuals likely suggests that pathogenic mutations functionally decrease the stability of the RBC cytoskeleton.

As single parameters, results were comparable for Omin and the EMA binding test, whereas EImax was superior compared to both, when separating individuals with (likely) pathogenic variants from those without mutations (Table 2). Combining Omin and EImax provided an excellent accuracy of 98.7% and adding EMA on top provided no benefit (Table 2).

Our accuracy measures may not be entirely representative, as we discarded individuals with homozygous SPTA1 mutations and VUS. Likely, several of these have HS as evaluated by their EMA binding test and ektacytometry (Table 1). In contrast, some causative mutations and deletions could have been missed by our tNGS approach. Furthermore, our study was not powered to calibrate the EMA threshold for the individual mutated genes. In this study, however, we aimed to set a diagnostic threshold for the EMA binding test without using healthy controls – and as such not to determine its precise accuracy. For this purpose, these limitations are accep table in our opinion.

In conclusion, our data demonstrate the reliability of the modified EMA binding test with rainbow beads when defining a cut‐off for HS by mutational status. When established, this approach makes the test more manageable and less time‐consuming. Ensuring consistency of data over time requires careful evaluation of new batches of rainbow beads and EMA dye. Interlaboratory work is ongoing to test whether this novel approach can be applied in a multicenter setting.

FUNDING INFORMATION

This study was supported by funding from the Department of Hematology, Rigshospitalet, Copenhagen, Denmark.

CONFLICT OF INTEREST

Andreas Glenthøj: Agios, bluebird bio, Bristol Myers Squibb, Novartis: consultancy. Research grant: Alexion, Saniona. Honoraria: Novo Nordisk. The authors declare no conflict of interest relevant to the manuscript.

AUTHOR CONTRIBUTIONS

Henrik Birgens, Jesper Petersen and Andreas Glenthøj planned this study. Jesper Petersen performed all tNGS and ektacytometry analyses. Jesper Petersen, Andreas Glenthøj and Christian Brieghel performed the statistical analysis. Henrik Birgens, Jesper Petersen, Christian Brieghel, Andreas Glenthøj, Amina Nardo‐Marino and Richard van Wijk analysed data and wrote the manuscript. Andreas Glenthøj prepared all figures. All authors contributed to the final approved version of this report.

ACKNOWLEDGEMENTS

The authors would like to thank Precision Medicine, Kings College Hospital and Red Cell Biology, King's College London for their assistance with the interpretation of tNGS data.

Glenthøj A, Brieghel C, Nardo‐Marino A, van Wijk R, Birgens H, Petersen J. Facilitating EMA binding test performance using fluorescent beads combined with next‐generation sequencing. eJHaem. 2021;2:716–728. 10.1002/jha2.277

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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