ARTICLE Red Cell Biology & its Disorders Ferrata Storti Foundation Density, heterogeneity and deformability of red cells as markers of clinical severity in hereditary spherocytosis Rick Huisjes,1 Asya Makhro,2 Esther Llaudet-Planas,3 Laura Hertz,4 Polina Petkova-Kirova,4 Liesbeth P. Verhagen,1 Silvia Pignatelli,1 Minke A.E. Rab,1 Raymond M. Schiffelers,1 Elena Seiler,2 Wouter W. van Solinge,1 Joan-LLuis Vives Corrons,3 Lars Kaestner,4,5 Maria Mañú-Pereira,6 Anna Bogdanova2 and Richard van Wijk1 Haematologica 2020 1 Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands; 2Red Blood Cell Research Group, Institute of Volume 105(2):338-347 Veterinary Physiology, Vetsuisse Faculty and the Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland; 3Red Blood Cell Defects and Hematopoietic Disorders Unit, Josep Carreras Leukemia Research Institute, Badalona, Barcelona, Spain; 4Theoretical Medicine and Biosciences, Medical Faculty, Saarland University, Homburg/Saar, Germany; 5Experimental Physics, Saarland University, Saarbruecken, Germany and 6Rare Anemia Research Unit. Vall d’Hebron Research Institution, University Hospital Vall d’Hebron, Barcelona, Spain ABSTRACT H ereditary spherocytosis (HS) originates from defective anchoring of the cytoskeletal network to the transmembrane protein complexes of the red blood cell (RBC). Red cells in HS are characterized by mem- brane instability and reduced deformability and there is marked heterogene- ity in disease severity among patients. To unravel this variability in disease severity, we analyzed blood samples from 21 HS patients with defects in ankyrin, band 3, α-spectrin or b-spectrin using red cell indices, eosin-5- maleimide binding, microscopy, the osmotic fragility test, Percoll density gra- Correspondence: dients, vesiculation and ektacytometry to assess cell membrane stability, cel- RICHARD VAN WIJK lular density and deformability. Reticulocyte counts, CD71 abundance, band r.vanWijk@umcutrecht.nl 4.1 a:b ratio, and glycated hemoglobin were used as markers of RBC turnover. We observed that patients with moderate/severe spherocytosis have short-living erythrocytes of low density and abnormally high intercellu- Received: April 3, 2018. lar heterogeneity. These cells show a prominent decrease in membrane sta- Accepted: May 28, 2019. bility and deformability and, as a consequence, are quickly removed from the Pre-published: May 30, 2019. circulation by the spleen. In contrast, in mild spherocytosis less pronounced reduction in deformability results in prolonged RBC lifespan and, hence, cells are subject to progressive loss of membrane. RBC from patients with mild doi:10.3324/haematol.2018.188151 spherocytosis thus become denser before they are taken up by the spleen. Based on our findings, we conclude that RBC membrane loss, cellular hetero- Check the online version for the most updated information on this article, online supplements, geneity and density are strong markers of clinical severity in spherocytosis. and information on authorship & disclosures: www.haematologica.org/content/105/2/338 Introduction ©2020 Ferrata Storti Foundation Hereditary spherocytosis (HS) is the most common form of chronic hereditary Material published in Haematologica is covered by copyright. hemolytic anemia in the Caucasian population, with an estimated prevalence of All rights are reserved to the Ferrata Storti Foundation. Use of 1:2000 – 1:5000.1–3 HS usually originates from mutations in ANK1 (ankyrin), published material is allowed under the following terms and conditions: SLC4A1 (band 3), SPTA1 (α-spectrin), SPTB (b-spectrin) or EPB42 (protein 4.2).1 https://creativecommons.org/licenses/by-nc/4.0/legalcode. Anemia in HS may require transfusion(s) and in severe cases splenectomy. A char- Copies of published material are allowed for personal or inter- acteristic feature of HS is red blood cell (RBC) membrane instability, which leads to nal use. Sharing published material for non-commercial pur- membrane loss and formation of dense cells with reduced RBC deformability.4–6 poses is subject to the following conditions: Increased RBC density is an important feature of HS7 and is reflected, for example, https://creativecommons.org/licenses/by-nc/4.0/legalcode, sect. 3. Reproducing and sharing published material for com- by increased mean corpuscular hemoglobin concentration (MCHC).8,9 mercial purposes is not allowed without permission in writing HS is a very heterogeneous RBC disorder, resulting from a wide range of molecu- from the publisher. lar defects and characterized by a high degree of heterogeneity in RBC properties and disease severity.8–12 In fact, considerable differences in disease severity are reported even between HS patients with identical mutations. It therefore seems rea- 338 haematologica | 2020; 105(2) Phenotypic markers for hereditary spherocytosis sonable to assume that the heterogeneity in disease sever- Separation on a Percoll density gradient and ity is not only a reflection of particular genotypes but is determination of intracellular potassium levels also affected by other factors that control RBC properties. Intact blood samples were layered over a 90% isotonic Percoll Healthy RBC become increasingly dense during their solution containing plasma-like components as described else- lifespan,13,14 but this process is accelerated in HS.1 Shedding where.19 Briefly, Percoll density gradient and RBC separation were of essentially hemoglobin-free vesicles results in an performed during centrifugation at 50,000 g for 15 min. (Sorvall increase in MCHC and a corresponding increase in RBC RC 5C plus, rotor SM-24). Intracellular potassium was measured density and intracellular viscosity.13,15 Electrogenic potassi- using an Instrumentation Laboratory IL943 Flame Photometer, as um leakage also contributes to RBC dehydration in HS described by Jokinen et al.20 patients and is not compensated by an accumulation of Na+.16,17 As a result, the intracellular K+ concentration in the Osmotic gradient ektacytometry, the osmotic fragility RBC of HS patients is approximately 13 mmol/L lower test and eosin-5-maleimide binding than that in the cells of healthy subjects,16 which results in Osmotic gradient ektacytometry measurements of RBC from net ion and water loss. Compensatory activation of Na,K- healthy controls and HS patients were obtained using the ATPase in the RBC of patients is insufficient to prevent the Osmoscan module on a Lorrca MaxSis (Mechatronics, The loss of K+ and dissipation of K+/Na+ gradients. The func- Zwaag, the Netherlands) as described elsewhere.5,21 The osmotic tion of other electroneutral ion transporters (KCC, NKCC, fragility test was carried out as previously described by Parpart et Na/Li exchanger) in RBC of HS patients was reported to al.22 and eosin-5-maleimide (EMA) binding was determined be indistinguishable from that of cells of healthy con- according to previously published protocols.12,23 trols.16,18 In this study, we investigated a unique and genetically Red blood cell production, heterogeneity, vesiculation well-diagnosed group of HS patients in whom we per- and turnover rate markers formed an in-depth analysis of RBC properties, such as RBC were stained with anti-CD71 and isotype controls and membrane instability, cellular density, cellular heterogene- were subsequently measured using a BD FACS Gallios.24 ity, vesiculation, turnover and lifespan. The data obtained Measuring glycated hemoglobin (HbA1c) is an established way to were then correlated to clinical manifestations of HS in acquire information about RBC clearance and RBC age in research both non-splenectomized and splenectomized patients, in on hemolytic anemias.15,25 HbA1c levels were measured using a order to identify markers of disease severity. Our results Menarini/ARKRAY HA-8180V. The band 4.1a:b ratio was detect- indicate that clinical severity in HS cannot be solely attrib- ed in RBC membrane lysates after protein separation by inverse uted to the protein harboring the mutation, but rather to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS- the stability of the whole cytoskeletal network. RBC den- PAGE) (15-7.5%) and visualization of protein bands using sity, heterogeneity and deformability were identified as Coomassie blue staining. RBC projected area and its heterogeneity potential markers of severity. We found that the presence were evaluated by microscopy as described elsewhere.26,27 RBC of dense RBC is strongly associated with milder manifes- vesicles were identified in plasma preserved with citrate-phos- tations of HS. We hypothesize that unstable RBC from phate-dextrose-adenine19 by staining them with mouse anti- patients with clinically more severe disease are removed human CD235a-APC and measured using a Beckman Coulter from the circulation before they acquire the features of CytoFLEX flow cytometer (Online Supplementary Figure S1). senescence. Statistical analysis and phenotype correlations One-way analysis of variance with post-hoc correction (Tukey Methods test) was used to compare sample means and the Fisher exact test was applied to determine whether clinical severity was propor- Subjects tionally distributed along the different genotypes. In non-splenec- Patients previously diagnosed with HS were enrolled in the tomized patients, clinical severity was assessed based on: (i) CoMMiTMenT-study (http://www.rare-anaemia.eu/). This study hemoglobin concentration and (ii) reticulocyte count, as previous- was approved by the Medical Ethical Research Board of the ly defined by Bolton-Maggs et al.11 (i.e., mild and University Medical Center Utrecht, the Netherlands, under refer- moderate/severe). To prevent any confounding by splenec- ence code 15/426M and by the Ethical Committee of Clinical tomized patients, phenotype correlations were only carried out Investigations of Hospital Clinic, Spain, (IDIBAPS) under reference for unsplenectomized HS patients. code 2013/8436. Hemocytometry analysis Results Hemocytometry parameters were analyzed on an Abbott Sapphire cell analyzer (Abbott Diagnostics Division, Santa Clara, Baseline characteristics and red cell features CA, USA) and ADVIA 2120 (Hematology System, Siemens of patients with hereditary spherocytosis Healthcare Diagnostics, Forchheim, Germany). Twenty-one patients with HS were included in this study and categorized according to clinical severity (Table Capillary-based measurements of mean corpuscular 1).11 HS was confirmed by targeted next-generation volume and mean corpuscular hemoglobin sequencing of the seven genes most commonly mutated concentration in HS28. Splenectomy or a moderate/severe expression of Triplicate heparinized blood samples were put in capillaries and disease was statistically overrepresented in patients with centrifuged for 5 min at 12,000 rpm (Hematocrit 20, Hettich mutations in ANK1 and SPTB (P<0.05). Therefore, the Zentrifugen). Mean corpuscular volume (MCV) was calculated phenotypic expression of HS due to ANK1 and SPTB using the formula MCV = hematocrit/RBC number. MCHC was mutations appears to be more severe than that of HS due calculated using the formula MCHC = hemoglobin/hematocrit. to SLC4A1 and/or SPTA1 defects. Decreased EMA stain- haematologica | 2020; 105(2) 339 R. Huisjes et al. ing, reflecting band 3 protein loss, was seen in all patients. (50% lysis point) (Figure 1E). Membrane stability was Patients with SPTA1 mutations tended to have greater compromised in all other HS patients. EMA staining (Figure 1A), although the number of As expected, RBC turnover was increased in all patients was too low to draw firm conclusions. The max- patients: reticulocyte counts were high (Table 1) and the imal deformability of RBC, reflected by a decrease in band 4.1a:b ratio, a marker of aging, was lower in all maximal elongation index (EImax) as determined by the patients than in healthy controls (Table 1). Osmoscan, was decreased in all HS patients compared to The heterogeneity of the RBC, reflected by the red that in healthy controls. This decrease was more pro- blood cell distribution width (RDW), was greater in nounced in patients with ANK1 and SPTB mutations than patients with HS than in healthy controls (Figure 1F) as in patients with mutations in SPTA or SCL4A1 (Figure was the MCHC (Figure 1G). Intracellular K+ content was 1B). On the other hand, cells from patients with SLC4A1 reduced in all patients but tended to be higher in HS mutations tended to be more dehydrated as their Ohyper patients with SPTA1 mutations (Figure 1H). values (hypertonic osmolarity at 50% of EImax) were lower An increase in heterogeneity in cell projected areas than those in both control blood samples and patients (mean projected area distribution width, MPA DW) with SPTA1 mutations (Figure 1C). The latter patients (Figure 2A) and a decrease in absolute mean values in pro- also showed the least pronounced loss of surface area-to- jected area (MPA) (Figure 2B) were seen in all HS patients volume ratio, reflected by a normal Omin (hypotonic compared to those in controls. Patients with SLC4A1 and osmolarity where EI is minimal) (Figure 1D) on the SPTA1 mutations had cellular projected areas more simi- Osmoscan and normal results in the osmotic fragility test lar to those of the healthy control group (Figure 2A, B). Table 1. Hemocytometry, chemistry parameters, cell-age markers and genotypes of patients with mild or moderate/severe hereditary spherocytosis (HS) and splenectomized HS patients included in this study. N. Sex Age Genotype Hb RBC Hct MCV Ret MCHC RDW EMA Omin EImax Ohyper OFT Band CD71 (years) (g/dL) (1012 (%) (fL) (%) (g/L) (%CV) (%) (mOsmo (A.U.) (mOsmo (g/L 4.1/4.2 (%) /L) l/L) l/L) NaCl) ratio (A.U.) Mild HS11 1 ♂ 75 ANK1 c.344T>C p.Leu115Pro 136 4.1 n.a. n.a. 7.2 n.a. 16.2 85 162 0.555 457 7.0 0.70 0.8 2 ♂ 46 SLC4A1 c.1030C>T p.Arg344* 153 4.7 n.a. n.a. 3.2 n.a. 15.2 71 163 0.563 417 6.6 0.87 1.4 3 ♂ 40 SLC4A1 c.1421C>A p.Ala474Asp 140 4.9 n.a. n.a. 4.0 n.a. 12.3 93 162 0.557 427 5.7 0.99 1.2 4 ♀ 55 SLC4A1 c.2057+1G>A (splicing) 136 4.0 36.5 91.1 7.8 372 14.0 73 158 0.554 405 6.4 0.82 2.7 5 ♀ 18 SLC4A1 c.2057+1G>A (splicing) 131 3.7 35.7 96.9 9.4 367 13.1 77 166 0.569 410 6.1 0.84 1.4 6 ♂ 58 SLC4A1 c.2348T>A p.Ile783Asn 132 4.1 36.5 88.4 8.9 363 15.7 68 169 0.548 429 6.8 n.a. 3.5 7 ♂ 40 SPTA1 c.678G>A p.Glu227fs + αLELY 127 4.1 35.9 87.8 5.8 355 15.0 94 175 0.600 453 5.6 0.84 0.9 8 ♂ 54 SPTA1 c.[4339-99C>T; c.4347G>T] 121 3.5 34.7 98.3 3.9 348 15.3 94 160 0.566 458 5.7 n.a. 0.9 p.[(?; Lys1449Asn)]; c.4339-99C>T p.(?) Moderate/severe HS11 9 ♂ 4 ANK 1 c.341C>T p.Pro114Leu 116 3.8 30.0 80.1 9.1 386 14.5 72 162 0.509 398 6.6 0.72 2.2 10 ♀ 3 ANK1 c.1943delC p.Ala648fs 117 4.02 36.3 90.4 11.9 321 21.1 59 164 0.503 424 6.6 n.a. 0.2 11 ♂ 5 ANK1 c.2394_2397delCAGT p.Ser799fs 120 4.1 32.2 78.6 18.4 372 25.4 67 185 0.472 462 7.8 n.a. 7.3 12 ♀ 26 ANK1 c.2559-2A>G (splicing) 102 3.3 29.4 89,5 18.3 346 24.1 84 170 0.537 446 6.4 0.60 5.3 13 ♂ 1 SPTB c.154delC p.Arg52fs 86 3.38 30.2 89.5 11.9 284 24.7 66 168 0.532 456 5.7 n.a. 4.8 14 ♂ 3 SPTB c.2470C>T p.Gln824* 78 2.9 23.7 81.9 10.6 328 23.8 71 180 0.534 475 6.7 n.a. 2.9 15 ♂ 4 SPTB c.5937+1G>A p.(?) 84 3.09 27.7 89.7 16.7 303 23.4 74 173 0.541 459 7.0 n.a. 5.5 16 ♀ 42 SPTA1 c.2755G>T p.Glu919* + αLELY 113 3.5 31.7 91.8 8.2 355 16.0 89 178 0.567 471 5.9 n.a. 2.1 Splenectomized HS11 17 ♂ 31 ANK1 c.341C>T p.Pro114Leu 152 4.9 84.7 41.9 8.4 362 11.8 76 179 0.533 437 7.4 1.08 0.1 18 ♀ 46 ANK1 c.344T>C p.Leu115Pro 143 4.6 n.a. n.a. 2.8 n.a. 12.4 74 158 0.590 423 7.4 1.18 0.2 19 ♀ 84 SLC4A1 c.2057+1G>A (splicing) 160 4.8 121.8 45.1 12.6 354 11.0 67 164 0.565 417 7.1 1.07 0.2 20 ♂ 71 SPTB c.2136_2137delinsTT 164 4.84 100.9 48.8 2.2 337 13.2 77 173 0.509 445 7.3 0.99 0.3 21 ♂ 40 SPTB c.3449G>A p.Trp1150* 163 4.94 92.0 45.5 12.2 359 11.9 73 183 0.512 440 7.8 1.22 0.3 Clinical severity in non-splenectomized HS patients was assigned according to Bolton-Maggs et al.11 on the basis of (i) hemoglobin concentration and (ii) reticulocyte count. Mild HS was defined as hemoglobin levels between 110–150 g/L, moderate HS as hemoglobin levels between 80–120 g/L and severe HS as hemoglobin levels lower than 80 g/L. HS patients with hemoglobin levels between 110 and 120 g/L were categorized as having mild or moderate disease on the basis of their reticulocyte levels (i.e., lower or higher than 6% reticulocytes). Novel mutations are displayed in bold font and the pathogenicity of novel missense variants was predicted with SIFT, PolyPhen-2, and MutationTaster (results not shown). Notation of αLELY represents SPTA1 c.[5572C>G; 6531- 12C>T] p.[(Leu1858Val);(?)]. N: number; Hb: hemoglobin; RBC: red blood cells; Hct: hematocrit; MCV; mean corpuscular volume; Rct: reticulocytes; MCHC: mean corpuscular hemoglobin con- centration; RDW: red cell distribution width; EMA: eosin-5′-maleimide; %CV: percent coefficient of variation; HS: hereditary spherocytosis; n.a.: not available. 340 haematologica | 2020; 105(2) Phenotypic markers for hereditary spherocytosis Patients with HS presented with greater heterogeneity in decrease in hemoglobin concentration and increase in cell density compared with healthy subjects, showing reticulocyte count.11 In our patients hemoglobin concen- more subfractions of the M fraction and a broader distri- tration correlated positively with MCHC (Figure 3A). bution width of the M fraction (Figure 2C-E). These Inverse correlations were observed between hemoglo- changes were more pronounced in patients with muta- bin and RDW (Figure 3B), and between hemoglobin and tions in ANK1 and SPTA1. Furthermore, the M fraction of reticulocyte count (Figure 3C). Furthermore, inverse cor- patients with SLC4A1 mutations was lower than that of relations were observed between hemoglobin and healthy controls or other HS patients (Figure 2F, Online parameters defining RBC hydration status such as intra- Supplementary Figure S2). cellular K+ and Ohyper (Figure 3D, E). In summary, specific changes were observed in param- Decreases in RBC lifespan were assessed by a reduc- eters associated with membrane stability, stiffness and tion in changes in HbA1c in non-splectomized patients deformability, as well as RBC heterogeneity in our cohort with severe HS, whereas in patients with mild disease of HS patients. manifestations or in splenectomized patients HbA1c levels were within the normal range (Figure 4A). Red blood cell markers of severity of hereditary Patients with moderate/severe HS had less deformable spherocytosis RBC, reflected by lower Elmax values, than patients with As described in Table 1, the severity of HS in non- mild HS or splenectomized patients (Figure 4B). The splenectomized patients was determined based on the RBC of patients with mild HS also tended to be more A B C D E F G H Figure 1. Basic characteristics (median ± range) of healthy controls and patients with hereditary spherocytosis. Patients with hereditary spherocytosis (HS) were grouped as a whole and according to their affected genes [ANK1 (ankyrin), SLC4A1 (band 3), SPTB (b-spectrin) and SPTA1 (α-spectrin)]. Blue circles represent healthy controls, black circles represent unsplenectomized HS patients and red circles represent splenectomized HS patients. The gray range indicates the reference range for healthy controls. (A) Eosin-5′-maleimide (EMA)-binding (%), (B) maximum deformability (EImax), (C) hydration state of the red blood cells reflected by Ohyper, (D) osmotic fragility measured by osmotic gradient ektacytometry and reflected by Omin, (E) 50% lysis point in the osmotic fragility test, (F) red blood cell distribution width (RDW) (percent coefficient of variation, %CV), (G) mean corpuscular hemoglobin concentration (MCHC) by capillary measurements (g/L) (%CV), (H) intracellular potassium (mmol/L). Significant differences are depicted with horizontal bars, and significance levels are noted: *P≤0.05, †P≤0.01 or ‡P≤0.001. haematologica | 2020; 105(2) 341 R. Huisjes et al. dehydrated, based on the Ohyper measurements (Figure splenectomy results in an increase in hemoglobin levels 4C). In line with this, patients with mild HS showed and erythrocyte counts (data not shown). In our cohort higher MCHC values than moderately/severely affected splenectomy was also associated with a decrease in RDW patients (Figure 4D) and had a higher density of the M (Figure 5A) and normalization of RBC morphology (Online fraction, based on this latter’s position within the Percoll Supplementary Figures S3 and S4). The survival of RBC gradient (Figure 4E, Online Supplementary Figure S2). from splenectomized HS patients, as assessed by HbA1c Based on reduced MPA, membrane loss was more pro- content or band 4.1a:b ratio, was found to be increased nounced in patients with moderate/severe HS (Figure (Figure 5B, C). 4F). In line with this, the number of RBC vesicles detect- Several parameters remained unaffected by splenecto- ed in the plasma of patients with moderate/severe HS my. Splenectomy did not alter MCHC (Figure D) or intra- was higher than that in patients with mild HS (Figure cellular K+ levels (Figure 5E) and did not correct band 3 loss 4G). However, no difference in EMA staining was (Figure 5F). It also did not affect deformability (no effect observed between patients with mild or on Elmax or Ohyper) (Figure 5G, H). However, the cells did sur- moderate/severe HS (Figure 4C). Intercellular hetero- vive for a longer time in the circulation despite an increase geneity (RDW and MPA DW) was increased in patients in osmotic fragility (Figure 5I). with moderate/severe HS compared to those with mild HS and healthy controls (Figure 4I, J). In summary, the RBC of patients with more severe Discussion expression of the disease had a reduced lifespan and less stable membrane. Their cells were smaller and more This comprehensive study in a well-characterized heterogeneous in size and density. Strikingly, patients cohort of patients offers insight into the variable pheno- with mild HS had denser RBC with higher MCHC typic manifestations of HS, possible causes of clinical het- (Figure 4D, E). erogeneity and severity, and the impact of splenectomy. We show here that strong markers of moderate/severe Effect of splenectomy on red blood cell markers expression of HS are: (i) lower RBC density, reflected by of disease severity differences in MCHC and fractionation of RBC on the Performed in patients with moderate/severe HS, Percoll density gradient; (ii) reduced RBC deformability A B C D E F Figure 2. Red blood cell heterogeneity in hereditary spherocytosis. Red blood cell (RBC) heterogeneity was measured in healthy controls and patients with hereditary spherocytosis (HS) by microscopy (A, B) and Percoll density gradients (C-F). HS patients were grouped according to their affected genes [ANK1 (ankyrin), SLC4A1 (band 3), SPTB (b-spectrin) and SPTA1 (α-spectrin)]. (A) Mean projected area distribution width (MPA DW). (B) Mean projected area (MPA). (C) An example of a blood sample from a patient with HS and a healthy control. On the samples the young RBC fraction, the main RBC fraction (M fraction) and dense RBC fractions are des- ignated with green, blue and red lines, respectively. The M fraction is subdivided into subfractions (M1, M2, etc.). The position of the M fraction is calculated from the position of the most intense (n arbitrary units, a.u.) subfraction relative to the total length of the Percoll column. The HS patient has seven RBC subfractions, and the position of the most intense subfraction (i.e. subfraction M3) is lower than in the control subject (i.e. subfraction M1). (D) The number of subfractions in the RBC density gradient. (E) Fraction distribution width (%). (F) Position of the M fraction (%). Significant differences are noted: *P≤0.05, †P≤0.01 or ‡P≤0.001. 342 haematologica | 2020; 105(2) Phenotypic markers for hereditary spherocytosis and increased membrane loss, as determined by RBC vesi- noted that patients with SPTA1 mutations had a less cle numbers and a decrease in MPA; and (iii) heterogeneity severe phenotype (based on hemoglobin level, reticulo- in the RBC population reflected by differences in RDW cyte count, RDW, intracellular K+, and EMA staining) and fractionation of RBC using a Percoll gradient. compared to the other patients. This is in contrast with We conclude that patients with moderate/severe HS the more severe disease phenotype of patients with have short-lived RBC of lower density and abnormally SPTA1 mutations reported in other studies.30 Similarly, high intercellular heterogeneity, whereas patients with our patients with SPTB mutations presented with nor- mild HS have a less pronounced reduction in RBC mal MCHC values, whereas in other studies MCHC was deformability resulting in the cells living longer and being shown to be elevated in patients carrying SPTB muta- subject to progressive loss of membrane. RBC from tions.31 Given the relatively small numbers of patients patients with mild HS thus become denser before they are within each group we cannot draw firm conclusions on taken up by the spleen. links between genotype and phenotypic expression. Genotype to phenotype correlations in hereditary Red blood cell density as a marker of clinical severity spherocytosis Decreased hemoglobin, hematocrit and RBC counts While previous studies were limited to protein analysis associated with increased markers of hemolysis and ery- by SDS-PAGE,12 we used next-generation sequencing to thropoietic activity were previously reported as markers establish the cause of HS. This enabled us to define the of HS severity.9 Among the molecular mechanisms defin- primary genetic defect unequivocally, in contrast to other ing severity of HS, a major role was assigned to RBC conventional techniques such as SDS-PAGE, which may membrane instability and loss of membrane proteins28 lead to confounding results as it may be influenced by (MCHC, EMA test, SDS-PAGE), along with decreased secondary protein defects in HS. Regardless of the under- deformability as measured by osmotic gradient ektacy- lying mutation, all patients shared common features such tometry.5 In our cohort, increased disease severity was as increases in reticulocyte counts and MCHC, dehydra- associated with membrane instability and extensive mem- tion and increases of RBC density and heterogeneity and brane and band 3 protein loss over a shorter time (Figure an overall reduction in deformability of the RBC due to 4B, G-I). RBC turnover in patients with severe HS was destabilization of cytoskeletal structures.1,29 We also reflected by a decrease in HbA1c levels, which were high- observed that red cell size, intracellular K+ content and er than those in patients with mild HS (Figure 4A). The reticulocyte counts did not differ between patients with RBC in patients with moderate/severe HS had lower SLC4A1, ANK1, SPTB and SPTA1 mutations (Figures 1H MCHC than those of patients with mild HS (Figures 3A and 2B, Table 1). Within one group of patients with the and 4D). The average M fraction density of RBC from same mutated protein we noted marked differences in patients with moderate/severe HS did not differ from that severity and clinical manifestations of the disease, with of cells of healthy controls, whereas the cells from patients the ANK1 group showing the greatest diversity. We also with mild HS showed an increase in density (Figure 4E) A B C D E Figure 3. Red blood cell parameters and their relationship to clinical severity in unsplenectomized hereditary spherocytosis patients. (A) Mean corpuscular hemo- globin concentration (MCHC) (g/L), (B) red blood cell distribution width (RDW) (percent coefficient of variation, %CV), (C) reticulocytes (%), (D) intracellular potassium (mmol/L), (E) hypertonic osmolarity at 50% of maximal elongation (Ohyper) (mOsmol/L). haematologica | 2020; 105(2) 343 R. Huisjes et al. and lower deformability as they “aged”, spending more ter conservation of RBC deformability (Figure 4B) and time in the circulation getting more “senescent” than the the ability to form dense cells (Figure 4C-E, Online highly unstable cells of patients with severe HS. Thus, Supplementary Figure S2). Direct measurements of mem- RBC lifespan and decrease in Elmax appeared to be reliable brane shedding by monitoring plasma-borne vesicles is markers of disease severity (Figure 4B), regardless of the challenging due to their fast sequestration and clear- genotype. ance.32–34 However, higher levels of circulating RBC vesi- Delayed clearance of RBC in patients with mild HS cles were detected in plasma from patients with moder- allows a more gradual loss of the cell membrane (Figure ate/severe HS (Figure 4G). This finding is in line with an 4G, Online Supplementary Figure S3), which results in bet- increase in other markers of membrane loss, such as A B C D E F G H I J Figure 4. Red blood cell density and stability as markers of severity in hereditary spherocytosis. Patients with hereditary spherocytosis (HS) are grouped according to clinical severity (i.e. mild, moderate/severe and splenectomized) (reference range in gray area). (A) Glycated hemoglobin (HbA1c) (mmol/mol), (B) maxi- mum deformability (EImax), (C) hypertonic osmolarity at 50% of maximal elongation (Ohyper) (mOsmol/L), (D) mean corpuscular hemoglobin concentration (MCHC) (g/L), (E) Position of M-fraction (%), (F) mean cell pro- jected area (MPA) (in arbitrary units, a.u.), (G) red blood cell (RBC) vesicles (particles/1012 RBC/L plasma), (H) eosin-5′-maleimide (EMA)-binding (%), (I) RBC distribution width (RDW) (percent coefficient of variation, %CV), (J) mean cell projected area distribution width (MPA DW). Significant differences are represented by horizontal bars, and significance levels are noted: *P≤0.05, †P≤0.01 or ‡P≤0.001. 344 haematologica | 2020; 105(2) Phenotypic markers for hereditary spherocytosis changes in gross morphology toward a spherocytic form (Figure 3B). However, since absolute RDW values are (Online Supplementary Figure S3), as well as a higher num- known to vary between laboratories and depend on the ber of microcytes, as determined by quantitative digital age and physical activity of the subjects,35 we also used microscopy (Online Supplementary Figure S4). microscopy, confirming that patients with severe HS did indeed have a broader range of RBC shapes and MPA DW Red blood cell heterogeneity and deformability (Figure 4J, Online Supplementary Figure S3). as a marker of clinical severity Another parameter we found to reflect disease severity Word of caution regarding automated detection of in HS is RBC heterogeneity. Increases in RDW, hematocrit and mean corpuscular hemoglobin RDW/hemoglobin and MCHC/RDW ratios were suggest- concentration in patients with hereditary spherocytosis ed to be markers of the clinical severity of HS.35 We also MCHC was previously suggested to be prognostic for found in our study that RDW correlates with HS severity the severity of HS in non-splenectomized patients.9,11 We A B C D E F G H I Figure 5. Role of splenectomy on red blood cell parameters in hereditary spherocytosis. Blue circles represent healthy controls, black circles represent (unsplenec- tomized) hereditary spherocytosis (HS) patients and red circles represent splenectomized HS patients. HS patients are grouped according to clinical severity (i.e. mild, moderate/severe and splenectomized). (A) Red cell distribution width (RDW (percent coefficient of variation, %CV), (B) glycated hemoglobin (HbA1c) (mmol/mol), (C) band 4.1a/4.1b ratio, (D) mean corpuscular hemoglobin concentration (MCHC) (g/L), (E) intracellular potassium (mmol/L), (F) eosin-5′-maleimide (EMA)-binding hemoglobin (%), (G) maximum deformability (EImax), (H) hydration state of the red blood cells reflected by hypertonic osmolarity at 50% of maximal elongation (Ohyper) (mOsmol/L), (I) 50% lysis point in the osmotic fragility test (g/L NaCl). Significant differences are noted: *P≤0.05, †P≤0.01 or ‡P≤0.001. Table 2. Summary of parameters that characterize disease severity in hereditary spherocytosis. Disease Severity MCHC Density RDW Reticulocytes Deformability Membrane loss (Percoll, Ohyper) (Elmax) (vesiculation) ↑↑ ↑↑ ↑ ↓ ↑ ↑ ↑ ↑↑ ↑↑ ↓↓ ↑↑ Mild = ↑↑ ↑ Moderate/severe Splenectomized = = = = The ↑ symbol indicates increased compared to healthy controls, ↓ indicates decreased compared to healthy controls and = indicates the same as in healthy controls. MCHC: mean corpuscular hemoglobin concentration; RDW: red cell distribution width; EImax: maximal elongation index, haematologica | 2020; 105(2) 345 R. Huisjes et al. used a capillary-based method with determination of the ciated with prolonged survival of RBC in the circulation, hematocrit to measure MCHC. In line with previous allowing greater loss of membrane, which results in small- observations,36–38 we found that automated measurements er and denser RBC. Shorter-lived, unstable RBC from of MCHC of pathological RBC are imprecise for HS patients with severe HS phenotype are more heteroge- patients (see Online Supplementary Figure S5). Inaccuracy of neous and less dense, as reflected by lower MCHC. automated MCHC detection of dehydrated RBC has been Parameters that specifically mark clinical severity in HS discussed for over 30 years.36 It results from a substantial are summarized in Table 2 and are RBC density (MCHC, overestimation of MCV, both values being reliant on Percoll, Ohyper), RBC deformability (EImax), and RBC hetero- hematocrit detection or calculation methods.39,40 As men- geneity (RDW). These parameters may be used to monitor tioned previously, others have shown that increased the success of supportive therapy and assist in the develop- hemoglobin concentrations correlate with milder disease ment of new personalized treatment regimens. severity scores in HS.9,11 We show that MCHC, calculated based on the capillary hematocrit, correlated with blood Funding hemoglobin content (Figures 1G, 3A, and 4D). Funding for the research leading to these results was received from the European Seventh Framework Program under grant Conclusions agreement number 602121 (CoMMiTMenT) and from the This study reveals the factors defining RBC longevity European Union’s Horizon 2020 Research and Innovation and erythropoietic activity in patients with HS. These fac- Programme under grant agreement number 675115 — RELE- tors include membrane stability, which in turn depends on VANCE — H2020-MSCA-ITN-2015/H2020-MSCA-ITN- the localization of mutations affecting vertical or horizon- 2015. 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