Page 105 - 2020_02-Haematologica-web

Phenotypic markers for hereditary spherocytosis
sonable to assume that the heterogeneity in disease sever- ity is not only a reflection of particular genotypes but is also affected by other factors that control RBC properties.
Healthy RBC become increasingly dense during their lifespan,13,14 but this process is accelerated in HS.1 Shedding of essentially hemoglobin-free vesicles results in an increase in MCHC and a corresponding increase in RBC density and intracellular viscosity.13,15 Electrogenic potassi- um leakage also contributes to RBC dehydration in HS patients and is not compensated by an accumulation of Na+.16,17 As a result, the intracellular K+ concentration in the RBC of HS patients is approximately 13 mmol/L lower than that in the cells of healthy subjects,16 which results in net ion and water loss. Compensatory activation of Na,K- ATPase in the RBC of patients is insufficient to prevent the loss of K+ and dissipation of K+/Na+ gradients. The func- tion of other electroneutral ion transporters (KCC, NKCC, Na/Li exchanger) in RBC of HS patients was reported to be indistinguishable from that of cells of healthy con- trols.16,18
In this study, we investigated a unique and genetically well-diagnosed group of HS patients in whom we per- formed an in-depth analysis of RBC properties, such as membrane instability, cellular density, cellular heterogene- ity, vesiculation, turnover and lifespan. The data obtained were then correlated to clinical manifestations of HS in both non-splenectomized and splenectomized patients, in order to identify markers of disease severity. Our results indicate that clinical severity in HS cannot be solely attrib- uted to the protein harboring the mutation, but rather to the stability of the whole cytoskeletal network. RBC den- sity, heterogeneity and deformability were identified as potential markers of severity. We found that the presence of dense RBC is strongly associated with milder manifes- tations of HS. We hypothesize that unstable RBC from patients with clinically more severe disease are removed from the circulation before they acquire the features of senescence.
Methods
Subjects
Patients previously diagnosed with HS were enrolled in the CoMMiTMenT-study (http://www.rare-anaemia.eu/). This study was approved by the Medical Ethical Research Board of the University Medical Center Utrecht, the Netherlands, under refer- ence code 15/426M and by the Ethical Committee of Clinical Investigations of Hospital Clinic, Spain, (IDIBAPS) under reference code 2013/8436.
Hemocytometry analysis
Hemocytometry parameters were analyzed on an Abbott Sapphire cell analyzer (Abbott Diagnostics Division, Santa Clara, CA, USA) and ADVIA 2120 (Hematology System, Siemens Healthcare Diagnostics, Forchheim, Germany).
Capillary-based measurements of mean corpuscular volume and mean corpuscular hemoglobin concentration
Triplicate heparinized blood samples were put in capillaries and centrifuged for 5 min at 12,000 rpm (Hematocrit 20, Hettich Zentrifugen). Mean corpuscular volume (MCV) was calculated using the formula MCV = hematocrit/RBC number. MCHC was calculated using the formula MCHC = hemoglobin/hematocrit.
Separation on a Percoll density gradient and determination of intracellular potassium levels
Intact blood samples were layered over a 90% isotonic Percoll solution containing plasma-like components as described else- where.19 Briefly, Percoll density gradient and RBC separation were performed during centrifugation at 50,000 g for 15 min. (Sorvall RC 5C plus, rotor SM-24). Intracellular potassium was measured using an Instrumentation Laboratory IL943 Flame Photometer, as described by Jokinen et al.20
Osmotic gradient ektacytometry, the osmotic fragility test and eosin-5-maleimide binding
Osmotic gradient ektacytometry measurements of RBC from healthy controls and HS patients were obtained using the Osmoscan module on a Lorrca MaxSis (Mechatronics, The Zwaag, the Netherlands) as described elsewhere.5,21 The osmotic fragility test was carried out as previously described by Parpart et al.22 and eosin-5-maleimide (EMA) binding was determined according to previously published protocols.12,23
Red blood cell production, heterogeneity, vesiculation and turnover rate markers
RBC were stained with anti-CD71 and isotype controls and were subsequently measured using a BD FACS Gallios.24 Measuring glycated hemoglobin (HbA1c) is an established way to acquire information about RBC clearance and RBC age in research on hemolytic anemias.15,25 HbA1c levels were measured using a Menarini/ARKRAY HA-8180V. The band 4.1a:b ratio was detect- ed in RBC membrane lysates after protein separation by inverse sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS- PAGE) (15-7.5%) and visualization of protein bands using Coomassie blue staining. RBC projected area and its heterogeneity were evaluated by microscopy as described elsewhere.26,27 RBC vesicles were identified in plasma preserved with citrate-phos- phate-dextrose-adenine19 by staining them with mouse anti- human CD235a-APC and measured using a Beckman Coulter CytoFLEX flow cytometer (Online Supplementary Figure S1).
Statistical analysis and phenotype correlations
One-way analysis of variance with post-hoc correction (Tukey test) was used to compare sample means and the Fisher exact test was applied to determine whether clinical severity was propor- tionally distributed along the different genotypes. In non-splenec- tomized patients, clinical severity was assessed based on: (i) hemoglobin concentration and (ii) reticulocyte count, as previous- ly defined by Bolton-Maggs et al.11 (i.e., mild and moderate/severe). To prevent any confounding by splenec- tomized patients, phenotype correlations were only carried out for unsplenectomized HS patients.
Results
Baseline characteristics and red cell features of patients with hereditary spherocytosis
Twenty-one patients with HS were included in this study and categorized according to clinical severity (Table 1).11 HS was confirmed by targeted next-generation sequencing of the seven genes most commonly mutated in HS28. Splenectomy or a moderate/severe expression of disease was statistically overrepresented in patients with mutations in ANK1 and SPTB (P<0.05). Therefore, the phenotypic expression of HS due to ANK1 and SPTB mutations appears to be more severe than that of HS due to SLC4A1 and/or SPTA1 defects. Decreased EMA stain-
haematologica | 2020; 105(2)
339