http://informahealthcare.com/hem ISSN: 0363-0269 (print), 1532-432X (electronic) Hemoglobin, 2014; 38(3): 165–168 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03630269.2014.888353
ORIGINAL ARTICLE
a-Thalassemia Does Not Seem to Influence Erythrocyte Deformability in Sickle Cell Trait Carriers Amparo Vaya´1, Susana Collado1, Rafael Alis2, Belen Vera3, Marco Romagnoli2,4, and Eva Barraga´n5 1
Haemorheology and Haemostasis Unit, Service of Clinical Pathology, La Fe University Hospital, Valencia, Spain, University Research Institute ‘‘Dr. Vin˜a Giner’’, Molecular and Mitochondrial-Medicine, Catholic University of Valencia ‘‘San Vicente Ma´rtir’’, Valencia, Spain, 3 Haematology Service, La Fe University Hospital, Valencia, Spain, 4 Department of Physical Education and Sports, Catholic University of Valencia ‘‘San Vicente Ma´rtir’’, Valencia, Spain, 5 Molecular Biology Unit, Service of Clinical Pathology, La Fe University Hospital, Valencia, Spain
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2
Abstract
Keywords
Studies dealing with rheological red blood cell (RBC) behavior in sickle cell trait carriers are scarce. Moreover, the association with a-thalassemia (a-thal), which also modifies erythrocyte behavior, has not always been taken into account. We analyzed erythrocyte deformability by means of a shear stress diffractometer, along with hematological and biochemical parameters (glucose and plasma lipids), given their possible influence on erythrocyte deformability, in 14 sickle cell trait carriers and 23 healthy controls. Nine patients were also a-thal carriers and five were not. Among the thalassemia carriers, eight were heterozygous and one was homozygous. When compared with controls, sickle cell trait carriers showed no differences for any of the biochemical parameters analyzed (p40.05), but significantly lower hemoglobin (Hb) (p ¼ 0.003), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) (p50.001) levels, although no differences in erythrocyte deformability were observed at any of the shear stresses tested (p40.05). When comparing sickle cell trait carriers, with and without a-thal, no differences in erythrocyte deformability were observed (p40.05), in spite of the former showing lower MCV and MCH (p50.05) levels. Carriers of a-thal had lower Hb S [b6(A3)Glu ! Val; HBB: c.20A4T] levels (p ¼ 0.013) than non carriers. The existence of a compensating mechanism seems reasonable because, despite presenting lower erythrocyte indices, which could worsen erythrocyte deformability, this rheological property improves when the percentage of Hb S is lower.
a-Thalassemia (a-thal), erythrocyte deformability, hemoglobinopathy, rheology, sickle cell trait
Introduction Sickle cell trait is generally considered to be a clinically benign disorder, although complications such as increased risk of thromboembolism have been described (1). Erythrocyte deformability, when altered, may contribute to vaso-occlusion and to the development of thromboembolic events. Although there are many studies about the rheological behavior of erythrocytes in homozygous sickle cell disease (2–14), the studies performed in sickle cell trait carriers about erythrocyte deformability are scarce (12–15). Moreover the association of sickle cell disease with a- or b-thal is wellknown (15–17), and this association is able to modify rheological blood properties (11,18). The aim of the present study was to analyze erythrocyte deformability along with biochemical and hematological parameters in sickle cell trait carriers. The second aim Address correspondence to Amparo Vaya´, M.D., Ph.D., Hemorheology and Hemostasis Unit, Service of Clinical Pathology, La Fe University Hospital, Avda. de Campanar, 21, 46009, Valencia, Spain. Tel./Fax: +034-963862714. E-mail: [email protected]
History Received 10 October 2013 Revised 24 October 2013 Accepted 25 October 2013 Published online 7 March 2014
was to evaluate whether the presence of thalassemia in sickle cell trait carriers can modify erythrocyte deformability in these subjects.
Material and methods Patients and controls In 14 heterozygous sickle cell trait carriers aged 38 ± 18 years (range 12–58), and in 23 healthy controls aged 39 ± 17 years (range 11–62), we analyzed erythrocyte deformability in a shear stress diffractometer (Rheodyn SSD, Myrenne, Germany) obtaining elongation index (EI) at 12, 30 and 60 Pa (19), along with biochemical and hematological parameters. Sickle cell trait carriers were diagnosed at the Haematology Service, La Fe University Hospital, Valencia, Spain, and controls were obtained from subjects who had minor surgery (strabismus, phimosis, trigger finger, and so on) at our hospital in whom the presence of hemoglobinopathies was ruled out. Patients were not under treatment when sampling took place and they had not received any blood transfusions in the previous 3 months.
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The Institutional Ethics Committee for Clinical Research approved this study. Written informed consent in accordance with the recommendations of the Declaration of Human Rights, the Conference of Helsinki and institutional regulations, was obtained from all patients.
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Laboratory methods Venous blood was drawn from the antecubital vein between 8.0 and 10.0 am after 12 hours of fasting with minimum stasis into vacuum tubes containing EDTA K3 for hematological and rheological tests. Dry tubes were used for the biochemical tests. In accordance with the Committee on Hemorheology Standarization and the new guidelines for hemorheological laboratory techniques (20), all the hemorheological parameters were determined within the first 2 hours of sampling to avoid deterioration of erythrocyte rheological properties. Serum total cholesterol, triglycerides and glucose were determined by enzymatic techniques, and iron was established by colorimetric methods in an Olympus AU 5430 autoanalyzer. Basic hematological parameters, red blood cell count, hemoglobin (Hb) concentration, mean corpuscular volume (MCV), mean corpuscular Hb (MCH) and mean corpuscular Hb concentration (MCHC) were determined by a Sysmex XE-2100 (Roche Diagnostics S.L, Barcelona, Spain). Erythrocyte deformability was established in a shear stress diffractometer (Rheodyn SSD) at 12, 30 and 60 Pa (19). The erythrocyte deformability unit was the elongation index (EI) that rose with increased red blood cell deformability. Hb S [b6(A3)Glu ! Val; HBB: c.20A4T] and Hb F (a2g2) were isolated and quantified by high performance liquid chromatography (HPLC) (Bio-Rad Laboratories, Barcelona, Spain). For the molecular diagnosis of Hb S, genomic DNA was isolated from peripheral blood leukocytes following standard procedures. The identification of the GAG4GTG mutation at codon 6 was based on polymerase chain reaction (PCR) amplification with the following primers: EA74 [50 -GGT TTG AAG TCC AAC TCC T-30 , access number U01317 (61961–61979)] and EA72 [50 -CAC TCA GTG TGG CAA AGG TG-30 access number U01317(62567–63587)], and by direct sequencing using an ABI PRISMÔ 3130 XL DNA Automatic Sequencer (Perkin Elmer, Foster City, CA, USA). These primers also identify the GAG4AAG mutation at codon 6 [Hb C, b6(A3)Glu ! Lys; HBB: c.19G4A], and the most frequent b-thal mutations in eastern Spain. Given the high frequency of a-thal in Hb S carriers, they were also screened for the a3.7 deletion (21). Those who were negative for the a3.7 deletion, were also analyzed by MLPA (multiplex ligation-dependent probe amplification) (22) for other a-globin gene mutations. Statistical analysis The non parametric Mann-Whitney test was used to analyze the differences between the continuous variables in both groups. The 2 test was used to compare differences in the dichotomic variables. A two-tailed p value of less than 0.05 was taken as statistically significant. All the analyses were carried out using the Statistical Package for the Social Sciences, version 15 (SPSS Inc., Chicago, IL, USA).
Hemoglobin, 2014; 38(3): 165–168
Results Among the 14 sickle cell trait carriers, nine patients (65.0%) also carried the a3.7 deletion [eight heterozygotes (a/aa) and one homozygote (a/a)]. No patients were carriers of the b-thal mutations screened. Table 1 shows the biochemical and hematological parameters in the sickle cell trait carriers and controls. Patients showed lower Hb, hematocrit (PCV), MCV and MCH (p50.01). The percentage of Hb S was 32.0% (range 25.0– 39.0%) and Hb F 0.5% (0.5–3.5%). No differences in the EI at any of the shear stresses tested at 12, 30 and 60 Pa were observed when sickle cell trait carriers were compared with controls (p40.05). No differences in plasma lipids or glucose were found when comparing sickle cell trait carriers and controls (Table 1). Table 2 shows the hematological parameters and the EI in patients with (n ¼ 9) and without (n ¼ 5) a-thal. Hb S/a-thal carriers showed lower MCV, MCH and Hb S than those without (p50.01), with no other significant differences found in the other parameters analyzed.
Discussion The results of the present study indicate that sickle cell trait carriers do not show an altered erythrocyte deformability at any of the shear stresses tested. This result coincides with the study by Tripette et al. (12). These authors determined erythrocyte deformability by a similar methodology in 33 sickle cell trait carriers and did not observe it to be decreased. It is important to highlight that they excluded the sickle cell trait carriers presenting the a3.7 deletion. Therefore, the erythrocyte indices fell within the normal range. However in the present study, in spite of there being a high percentage of patients with heterozygous Hb S and a-thal (65.0%), as reflected in the lower erythrocyte indices when comparing cases and controls, this was not accompanied by a reduction in erythrocyte deformability. It could be due to the fact that patients in the present study who were carriers of a-thal were all but one (89.0%) heterozygous a-thal carriers, as phenotypically expressed with very mild microcytosis and hypochromia. When the homozygous patient was excluded from the analysis, the results did not change. In addition no differences in erythrocyte deformability were observed when sickle cell trait carriers, with and without a-thal, were compared although the former presented lower MCV and MCH values. It is known that positively charged b chain mutations, such as Hb S in heterozygotes, tend to be relatively low as compared to Hb A, and are lowered further in the presence of a-thal. This is due to the fact that when a-thal is also present, the limiting production of a chains generates a lesser probability of a/bS dimers than a/bA dimers (23,24), leading to lower Hb S levels and in turn, to a less impaired erythrocyte deformability. Among the factors involved in erythrocyte deformability, erythrocyte indices are important as they reveal alterations in the surface/volume ratio of erythrocytes. In the present study, although MCV and MCH levels were lower in patients than in controls, it is important to highlight that MCHC, which is of paramount influence for erythrocyte deformability, was not statistically different when patients and controls were
Erythrocyte Deformability in Sickle Cell Trait
DOI: 10.3109/03630269.2014.888353
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Table 1. Biochemical, Hematological Parameters and Erythrocyte Elongation Index in Sickle Cell Trait Carriers and Controls.
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Parameters Age (years) Gender Glucose (mg/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) Iron (mg/dL) RBC (1012/L) Hb (g/dL) PCV (L/L) MCV (fL) MCH (pg) MCHC (g/dL) Hb S (%) Hb F (%) Erythrocyte EI 12 Pa (%) Erythrocyte EI 30 Pa (%) Erythrocyte EI 60 Pa (%)
Heterozygous Sickle Cell Trait Carriers (n ¼ 14) 38 (12–58) M: 4; F: 10 88.0 (73.0–298.0) 192.0 (124.0–234.0) 72.0 (35.0–315.0) 76.0 (40.0–125.0) 4.8 (4.0–5.4) 12.3 (11.2–14.1) 0.37 (0.34–0.41) 78.0 (69.0–86.0) 26.1 (21.6–29.1) 33.3 (31.1–35.1) 32.0 (25.0–39.0) 0.5 (0.5–3.5) 38.78 (34.92–44.49) 46.96 (43.95–52.23) 51.39 (47.89–56.66)
Controls (n ¼ 23)
p Value
39 (11–62) M: 8; F: 15 89.0 (72.0–103.0) 190.0 (147.0–234.0) 81.0 (53.0–171.0) 81.0 (40.0–144.0) 4.7 (3.8–5.5) 14.0 (12.0–16.7) 0.41 (0.35–0.49) 89.0 (81.0–104.0) 29.9 (26.7–33.7) 33.6 (32.3–35.5)
0.851 0.695 0.388 0.802 0.238 0.381 0.286 0.003 0.002 50.001 50.001 0.397
40.72 (37.68–44.45) 47.43 (43.43–51.66) 50.56 (45.26–55.70)
0.074 0.833 0.314
Hb S: b6(A3)Glu ! Val; HBB: c.20A4T; Hb F: a2g2; erythrocyte EI: erythrocyte elongation index; values as median with range values in parentheses. Table 2. Biochemical, Hematological Parameters and Erythrocyte Elongation Index in Sickle Cell Trait Carriers With and Without a-Thalassemia.
Parameters Age (years) Gender Iron (mg/dL) Hb (g/dL) PCV (L/L) MCV (fL) MCH (pg) MCHC (g/dL) Hb S (%) Hb F (%) Erythrocyte E1 12 Pa (%) Erythrocyte EI 30 Pa (%) Erythrocyte EI 60 Pa (%)
Heterozygous Sickle Cell Trait Carriers With a-Thal (n ¼ 9)
Heterozygous Sickle Cell Trait Carriers Without a-Thal (n ¼ 5)
p Value
45 (12–54) M: 3; F: 6 103.0 (40.0–125.0) 12.4 (11.4–14.1) 0.38 (0.35–0.41) 76.0 (69.0–81.1) 25.5 (21.6–27.1) 33.3 (31.1–34.1) 31.0 (25.0–37.0) 0.5 (0.5–1.4) 38.02 (36.58–44.49) 46.68 (44.99–52.23) 51.29 (49.57–56.66)
37 (13–58) M: 1; F: 4 60.0 (51.0–93.0) 12.3 (11.2–14.1) 0.37 (0.34–0.41) 85.0 (77.0–86.0) 28.4 (27.0–29.1) 33.4 (33.1–35.1) 34.0 (33.0–39.0) 0.5 (0.5–3.5) 39.17 (34.92–43.46) 47.38 (43.95–50.45) 51.52 (47.89–53.19)
0.505 0.597 0.463 0.947 0.606 0.015 0.005 0.256 0.013 0.198) 0.739 0.947 0.947
Hb S: b6(A3)Glu ! Val; HBB: c.20A4T; Hb F: a2g2; erythrocyte EI: erythrocyte elongation index; values as median with range values in parentheses.
compared, a fact that may account for not having observed differences in erythrocyte deformability between patients and controls. Given the influence of glucose and lipids on erythrocyte deformability (membrane viscous elasticity properties) (25,26), these biochemical parameters were also determined to rule out their possible influence in sickle cell trait, although no differences in these biochemical parameters were observed (Table 1). In a study on hemoglobinopathies carried out in our country (15), Hb S was associated with a-thal in more than 50.0% of cases. As in the present study, it was observed that when sickle cell trait was associated with a-thal, patients showed statistically lower MCV, MCH, MCHC, and a lower Hb S percentage than those without the association. Other authors (16,17) also found similar results in relation to both Hb S levels and erythrocyte indices, although erythrocyte deformability was not determined in these studies. In a previous study on rheological red blood cell behavior in minor a-thal carriers performed by our group (18),
we observed slightly decreased erythrocyte deformability that could be due to the fact that more than 60.0% of the thalassemic carriers were homozygotes. In fact, the only homozygous a-thal carrier showed lower erythrocyte deformability than the heterozygotes (EI 60 Pa: 47.89%). Given the low sample size of sickle cell trait carriers in the present study, and that they were also classified according to their a-thal status, it would be desirable to perform further studies with a larger number of patients in order to confirm our results. In summary, sickle cell trait carriers do not show decreased erythrocyte deformability. Those associated with a-thal trait do not present a lower EI than those without, in spite of exhibiting mild microcytosis and hypochromia. The sickle cell trait carriers who present with a-thal have lower Hb S levels. So the existence of a compensating mechanism seems reasonable because, despite presenting lower erythrocyte indices that could worsen erythrocyte deformability, this rheological property improves when the percentage of Hb S is
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lower. Since erythrocyte deformability is not impaired in sickle cell trait, it does not seem to be a possible mechanism involved in increased thrombotic risk in these patients.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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References 1. Austin H, Key NS, Benson JM, et al. Sickle cell trait and the risk of venous thromboembolism among blacks. Blood. 2007; 110(3):908–912. 2. Ballas S, Mohandas N. Sickle red cell microrheology and sickle blood rheology. Microcirculation. 2004;11(2):209–225. 3. Chien S, Usami S, Bertles JF. Abnormal rheology of oxygenated blood in sickle cell anemia. J Clin Invest. 1970;49(4):623–634. 4. Clark MR, Mohandas N, Shohet SB. Deformability of oxygenated irreversibly sickled cells. J Clin Invest. 1980;65(1):189–196. 5. Evans E, Mohandas N, Leung A. Static and dynamic rigidities of normal and sickle erythrocytes. Major influence of cell hemoglobin concentration. J Clin Invest. 1984;73(2):477–488. 6. Havell TC, Hillman D, Lessin LS. Deformability characteristics of sickle cells by microelastimetry. Am J Hematol. 1978;4(1):9–16. 7. Lamarre Y, Romana M, Waltz X, et al. Hemorheological risk factors of acute chest syndrome and painful vaso-occlusive crisis in children with sickle cell disease. Haematologica. 2012;97(11): 1641–1647. 8. Martorana MC, Mojoli G, Cianciulli P, et al. Sickle cell anaemia: Haemorheological aspects. Ann Ist Super Sanita. 2007;43(2): 164–170. 9. Nash GB, Johnson CS, Meiselman HJ. Mechanical properties of oxygenated red blood cells in sickle cell (HbSS) disease. Blood. 1984;63(1):73–82. 10. Schmalzer EA, Manning RS, Chien S. Filtration of sickle cells: Recruitment into a rigid fraction as a function of density and oxygen tension. J Lab Clin Med. 1989;113(6):727–734. 11. Serjeant BE, Mason KP, Kenny MW, et al. Effect of a thalassaemia on the rheology of homozygous sickle cell disease. Br J Haematol. 1983;55(3):479–486.
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12. Tripette J, Alexy T, Hardy-Dessources MD, et al. Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle-hemoglobin C disease. Haematologica. 2009;94(8): 1060–1065. 13. Usami S, Chien S, Bertles JF. Deformability of sickle cells as studied by microsieving. J Lab Clin Med. 1975;86(2):274–279. 14. Vaya A, Collado S, Dasi MA, et al. Erythrocyte deformability and aggregation in homozygous sickle cell disease. Clin Hemorheol Microcirc. 2013 Apr 19. [Epub ahead of print]. 15. Ataulfo Gonza´lez F, Bla´zquez C, Ropero P, et al. Asociacio´n de hemoglobinopatı´a S y alfatalasemia. Ana´lisis de 45 casos. Med Clin (Barc). 2005;124(19):726–729. 16. el-Hazmi MA, Warsy AS. On the molecular interactions between a-thalassaemia and sickle cell gene. J Trop Pediatr. 1993; 39(4):209–213. 17. Mouele R, Pambou O, Feingold J, Galacteros F. a-Thalassemia in Bantu population from Congo-Brazzaville: Its interaction with sickle cell anemia. Hum Hered. 2000;50(2):118–125. 18. Vaya A, Suescun M, Hernandez JL, et al. Rheological red blood cell behavior in minor a-thalassaemia carriers. Clin Hemorheol Microcirc. 2011;48(4):241–246. 19. Ruef P, Poschl JMB, Linderkamp O. The rheodyn SSD for measuring erythrocyte deformability. Biorheology. 1995;32(2):357–358. 20. Baskurt OK, Boynard M, Cokelet GC, et al. New guidelines for hemorheological laboratory techniques. Clin Hemorheol Microcirc. 2009;42(2):75–97. 21. Dode C, Krishnamoorthy R, Lamb J, Rochette J. Rapid analysis of a3.7 thalassaemia and aaaanti3.7 triplication by enzymatic amplification analysis. Br J Haematol. 1993;83(1):105–111. 22. Schouten JP, McElgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002;30(12):e57. 23. Bunn HF, McDonald MJ. Electrostatic interactions in the assembly of haemoglobin. Nature. 1983;306(5942):498–500. 24. Huisman THJ. Percentages of abnormal hemoglobins in adults with a heterozygosity for an a-chain and/or a b-chain variant. Am J Hematol. 1983;14(4):393–404. 25. Mohandas N. Molecular basis for red cell membrane viscoelastic properties. Biochem Soc Trans. 1992;20(4):776–782. 26. Forsyth AM, Braunmuller S, Wan J, et al. The effects of membrane cholesterol and simvastatin on red blood cell deformability and ATP release. Microvasc Res. 2012;83(3):347–351.
ORIGINAL ARTICLE
a-Thalassemia Does Not Seem to Influence Erythrocyte Deformability in Sickle Cell Trait Carriers Amparo Vaya´1, Susana Collado1, Rafael Alis2, Belen Vera3, Marco Romagnoli2,4, and Eva Barraga´n5 1
Haemorheology and Haemostasis Unit, Service of Clinical Pathology, La Fe University Hospital, Valencia, Spain, University Research Institute ‘‘Dr. Vin˜a Giner’’, Molecular and Mitochondrial-Medicine, Catholic University of Valencia ‘‘San Vicente Ma´rtir’’, Valencia, Spain, 3 Haematology Service, La Fe University Hospital, Valencia, Spain, 4 Department of Physical Education and Sports, Catholic University of Valencia ‘‘San Vicente Ma´rtir’’, Valencia, Spain, 5 Molecular Biology Unit, Service of Clinical Pathology, La Fe University Hospital, Valencia, Spain
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2
Abstract
Keywords
Studies dealing with rheological red blood cell (RBC) behavior in sickle cell trait carriers are scarce. Moreover, the association with a-thalassemia (a-thal), which also modifies erythrocyte behavior, has not always been taken into account. We analyzed erythrocyte deformability by means of a shear stress diffractometer, along with hematological and biochemical parameters (glucose and plasma lipids), given their possible influence on erythrocyte deformability, in 14 sickle cell trait carriers and 23 healthy controls. Nine patients were also a-thal carriers and five were not. Among the thalassemia carriers, eight were heterozygous and one was homozygous. When compared with controls, sickle cell trait carriers showed no differences for any of the biochemical parameters analyzed (p40.05), but significantly lower hemoglobin (Hb) (p ¼ 0.003), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) (p50.001) levels, although no differences in erythrocyte deformability were observed at any of the shear stresses tested (p40.05). When comparing sickle cell trait carriers, with and without a-thal, no differences in erythrocyte deformability were observed (p40.05), in spite of the former showing lower MCV and MCH (p50.05) levels. Carriers of a-thal had lower Hb S [b6(A3)Glu ! Val; HBB: c.20A4T] levels (p ¼ 0.013) than non carriers. The existence of a compensating mechanism seems reasonable because, despite presenting lower erythrocyte indices, which could worsen erythrocyte deformability, this rheological property improves when the percentage of Hb S is lower.
a-Thalassemia (a-thal), erythrocyte deformability, hemoglobinopathy, rheology, sickle cell trait
Introduction Sickle cell trait is generally considered to be a clinically benign disorder, although complications such as increased risk of thromboembolism have been described (1). Erythrocyte deformability, when altered, may contribute to vaso-occlusion and to the development of thromboembolic events. Although there are many studies about the rheological behavior of erythrocytes in homozygous sickle cell disease (2–14), the studies performed in sickle cell trait carriers about erythrocyte deformability are scarce (12–15). Moreover the association of sickle cell disease with a- or b-thal is wellknown (15–17), and this association is able to modify rheological blood properties (11,18). The aim of the present study was to analyze erythrocyte deformability along with biochemical and hematological parameters in sickle cell trait carriers. The second aim Address correspondence to Amparo Vaya´, M.D., Ph.D., Hemorheology and Hemostasis Unit, Service of Clinical Pathology, La Fe University Hospital, Avda. de Campanar, 21, 46009, Valencia, Spain. Tel./Fax: +034-963862714. E-mail: [email protected]
History Received 10 October 2013 Revised 24 October 2013 Accepted 25 October 2013 Published online 7 March 2014
was to evaluate whether the presence of thalassemia in sickle cell trait carriers can modify erythrocyte deformability in these subjects.
Material and methods Patients and controls In 14 heterozygous sickle cell trait carriers aged 38 ± 18 years (range 12–58), and in 23 healthy controls aged 39 ± 17 years (range 11–62), we analyzed erythrocyte deformability in a shear stress diffractometer (Rheodyn SSD, Myrenne, Germany) obtaining elongation index (EI) at 12, 30 and 60 Pa (19), along with biochemical and hematological parameters. Sickle cell trait carriers were diagnosed at the Haematology Service, La Fe University Hospital, Valencia, Spain, and controls were obtained from subjects who had minor surgery (strabismus, phimosis, trigger finger, and so on) at our hospital in whom the presence of hemoglobinopathies was ruled out. Patients were not under treatment when sampling took place and they had not received any blood transfusions in the previous 3 months.
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A. Vaya´ et al.
The Institutional Ethics Committee for Clinical Research approved this study. Written informed consent in accordance with the recommendations of the Declaration of Human Rights, the Conference of Helsinki and institutional regulations, was obtained from all patients.
Hemoglobin Downloaded from informahealthcare.com by Thammasat University on 10/04/14 For personal use only.
Laboratory methods Venous blood was drawn from the antecubital vein between 8.0 and 10.0 am after 12 hours of fasting with minimum stasis into vacuum tubes containing EDTA K3 for hematological and rheological tests. Dry tubes were used for the biochemical tests. In accordance with the Committee on Hemorheology Standarization and the new guidelines for hemorheological laboratory techniques (20), all the hemorheological parameters were determined within the first 2 hours of sampling to avoid deterioration of erythrocyte rheological properties. Serum total cholesterol, triglycerides and glucose were determined by enzymatic techniques, and iron was established by colorimetric methods in an Olympus AU 5430 autoanalyzer. Basic hematological parameters, red blood cell count, hemoglobin (Hb) concentration, mean corpuscular volume (MCV), mean corpuscular Hb (MCH) and mean corpuscular Hb concentration (MCHC) were determined by a Sysmex XE-2100 (Roche Diagnostics S.L, Barcelona, Spain). Erythrocyte deformability was established in a shear stress diffractometer (Rheodyn SSD) at 12, 30 and 60 Pa (19). The erythrocyte deformability unit was the elongation index (EI) that rose with increased red blood cell deformability. Hb S [b6(A3)Glu ! Val; HBB: c.20A4T] and Hb F (a2g2) were isolated and quantified by high performance liquid chromatography (HPLC) (Bio-Rad Laboratories, Barcelona, Spain). For the molecular diagnosis of Hb S, genomic DNA was isolated from peripheral blood leukocytes following standard procedures. The identification of the GAG4GTG mutation at codon 6 was based on polymerase chain reaction (PCR) amplification with the following primers: EA74 [50 -GGT TTG AAG TCC AAC TCC T-30 , access number U01317 (61961–61979)] and EA72 [50 -CAC TCA GTG TGG CAA AGG TG-30 access number U01317(62567–63587)], and by direct sequencing using an ABI PRISMÔ 3130 XL DNA Automatic Sequencer (Perkin Elmer, Foster City, CA, USA). These primers also identify the GAG4AAG mutation at codon 6 [Hb C, b6(A3)Glu ! Lys; HBB: c.19G4A], and the most frequent b-thal mutations in eastern Spain. Given the high frequency of a-thal in Hb S carriers, they were also screened for the a3.7 deletion (21). Those who were negative for the a3.7 deletion, were also analyzed by MLPA (multiplex ligation-dependent probe amplification) (22) for other a-globin gene mutations. Statistical analysis The non parametric Mann-Whitney test was used to analyze the differences between the continuous variables in both groups. The 2 test was used to compare differences in the dichotomic variables. A two-tailed p value of less than 0.05 was taken as statistically significant. All the analyses were carried out using the Statistical Package for the Social Sciences, version 15 (SPSS Inc., Chicago, IL, USA).
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Results Among the 14 sickle cell trait carriers, nine patients (65.0%) also carried the a3.7 deletion [eight heterozygotes (a/aa) and one homozygote (a/a)]. No patients were carriers of the b-thal mutations screened. Table 1 shows the biochemical and hematological parameters in the sickle cell trait carriers and controls. Patients showed lower Hb, hematocrit (PCV), MCV and MCH (p50.01). The percentage of Hb S was 32.0% (range 25.0– 39.0%) and Hb F 0.5% (0.5–3.5%). No differences in the EI at any of the shear stresses tested at 12, 30 and 60 Pa were observed when sickle cell trait carriers were compared with controls (p40.05). No differences in plasma lipids or glucose were found when comparing sickle cell trait carriers and controls (Table 1). Table 2 shows the hematological parameters and the EI in patients with (n ¼ 9) and without (n ¼ 5) a-thal. Hb S/a-thal carriers showed lower MCV, MCH and Hb S than those without (p50.01), with no other significant differences found in the other parameters analyzed.
Discussion The results of the present study indicate that sickle cell trait carriers do not show an altered erythrocyte deformability at any of the shear stresses tested. This result coincides with the study by Tripette et al. (12). These authors determined erythrocyte deformability by a similar methodology in 33 sickle cell trait carriers and did not observe it to be decreased. It is important to highlight that they excluded the sickle cell trait carriers presenting the a3.7 deletion. Therefore, the erythrocyte indices fell within the normal range. However in the present study, in spite of there being a high percentage of patients with heterozygous Hb S and a-thal (65.0%), as reflected in the lower erythrocyte indices when comparing cases and controls, this was not accompanied by a reduction in erythrocyte deformability. It could be due to the fact that patients in the present study who were carriers of a-thal were all but one (89.0%) heterozygous a-thal carriers, as phenotypically expressed with very mild microcytosis and hypochromia. When the homozygous patient was excluded from the analysis, the results did not change. In addition no differences in erythrocyte deformability were observed when sickle cell trait carriers, with and without a-thal, were compared although the former presented lower MCV and MCH values. It is known that positively charged b chain mutations, such as Hb S in heterozygotes, tend to be relatively low as compared to Hb A, and are lowered further in the presence of a-thal. This is due to the fact that when a-thal is also present, the limiting production of a chains generates a lesser probability of a/bS dimers than a/bA dimers (23,24), leading to lower Hb S levels and in turn, to a less impaired erythrocyte deformability. Among the factors involved in erythrocyte deformability, erythrocyte indices are important as they reveal alterations in the surface/volume ratio of erythrocytes. In the present study, although MCV and MCH levels were lower in patients than in controls, it is important to highlight that MCHC, which is of paramount influence for erythrocyte deformability, was not statistically different when patients and controls were
Erythrocyte Deformability in Sickle Cell Trait
DOI: 10.3109/03630269.2014.888353
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Table 1. Biochemical, Hematological Parameters and Erythrocyte Elongation Index in Sickle Cell Trait Carriers and Controls.
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Parameters Age (years) Gender Glucose (mg/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) Iron (mg/dL) RBC (1012/L) Hb (g/dL) PCV (L/L) MCV (fL) MCH (pg) MCHC (g/dL) Hb S (%) Hb F (%) Erythrocyte EI 12 Pa (%) Erythrocyte EI 30 Pa (%) Erythrocyte EI 60 Pa (%)
Heterozygous Sickle Cell Trait Carriers (n ¼ 14) 38 (12–58) M: 4; F: 10 88.0 (73.0–298.0) 192.0 (124.0–234.0) 72.0 (35.0–315.0) 76.0 (40.0–125.0) 4.8 (4.0–5.4) 12.3 (11.2–14.1) 0.37 (0.34–0.41) 78.0 (69.0–86.0) 26.1 (21.6–29.1) 33.3 (31.1–35.1) 32.0 (25.0–39.0) 0.5 (0.5–3.5) 38.78 (34.92–44.49) 46.96 (43.95–52.23) 51.39 (47.89–56.66)
Controls (n ¼ 23)
p Value
39 (11–62) M: 8; F: 15 89.0 (72.0–103.0) 190.0 (147.0–234.0) 81.0 (53.0–171.0) 81.0 (40.0–144.0) 4.7 (3.8–5.5) 14.0 (12.0–16.7) 0.41 (0.35–0.49) 89.0 (81.0–104.0) 29.9 (26.7–33.7) 33.6 (32.3–35.5)
0.851 0.695 0.388 0.802 0.238 0.381 0.286 0.003 0.002 50.001 50.001 0.397
40.72 (37.68–44.45) 47.43 (43.43–51.66) 50.56 (45.26–55.70)
0.074 0.833 0.314
Hb S: b6(A3)Glu ! Val; HBB: c.20A4T; Hb F: a2g2; erythrocyte EI: erythrocyte elongation index; values as median with range values in parentheses. Table 2. Biochemical, Hematological Parameters and Erythrocyte Elongation Index in Sickle Cell Trait Carriers With and Without a-Thalassemia.
Parameters Age (years) Gender Iron (mg/dL) Hb (g/dL) PCV (L/L) MCV (fL) MCH (pg) MCHC (g/dL) Hb S (%) Hb F (%) Erythrocyte E1 12 Pa (%) Erythrocyte EI 30 Pa (%) Erythrocyte EI 60 Pa (%)
Heterozygous Sickle Cell Trait Carriers With a-Thal (n ¼ 9)
Heterozygous Sickle Cell Trait Carriers Without a-Thal (n ¼ 5)
p Value
45 (12–54) M: 3; F: 6 103.0 (40.0–125.0) 12.4 (11.4–14.1) 0.38 (0.35–0.41) 76.0 (69.0–81.1) 25.5 (21.6–27.1) 33.3 (31.1–34.1) 31.0 (25.0–37.0) 0.5 (0.5–1.4) 38.02 (36.58–44.49) 46.68 (44.99–52.23) 51.29 (49.57–56.66)
37 (13–58) M: 1; F: 4 60.0 (51.0–93.0) 12.3 (11.2–14.1) 0.37 (0.34–0.41) 85.0 (77.0–86.0) 28.4 (27.0–29.1) 33.4 (33.1–35.1) 34.0 (33.0–39.0) 0.5 (0.5–3.5) 39.17 (34.92–43.46) 47.38 (43.95–50.45) 51.52 (47.89–53.19)
0.505 0.597 0.463 0.947 0.606 0.015 0.005 0.256 0.013 0.198) 0.739 0.947 0.947
Hb S: b6(A3)Glu ! Val; HBB: c.20A4T; Hb F: a2g2; erythrocyte EI: erythrocyte elongation index; values as median with range values in parentheses.
compared, a fact that may account for not having observed differences in erythrocyte deformability between patients and controls. Given the influence of glucose and lipids on erythrocyte deformability (membrane viscous elasticity properties) (25,26), these biochemical parameters were also determined to rule out their possible influence in sickle cell trait, although no differences in these biochemical parameters were observed (Table 1). In a study on hemoglobinopathies carried out in our country (15), Hb S was associated with a-thal in more than 50.0% of cases. As in the present study, it was observed that when sickle cell trait was associated with a-thal, patients showed statistically lower MCV, MCH, MCHC, and a lower Hb S percentage than those without the association. Other authors (16,17) also found similar results in relation to both Hb S levels and erythrocyte indices, although erythrocyte deformability was not determined in these studies. In a previous study on rheological red blood cell behavior in minor a-thal carriers performed by our group (18),
we observed slightly decreased erythrocyte deformability that could be due to the fact that more than 60.0% of the thalassemic carriers were homozygotes. In fact, the only homozygous a-thal carrier showed lower erythrocyte deformability than the heterozygotes (EI 60 Pa: 47.89%). Given the low sample size of sickle cell trait carriers in the present study, and that they were also classified according to their a-thal status, it would be desirable to perform further studies with a larger number of patients in order to confirm our results. In summary, sickle cell trait carriers do not show decreased erythrocyte deformability. Those associated with a-thal trait do not present a lower EI than those without, in spite of exhibiting mild microcytosis and hypochromia. The sickle cell trait carriers who present with a-thal have lower Hb S levels. So the existence of a compensating mechanism seems reasonable because, despite presenting lower erythrocyte indices that could worsen erythrocyte deformability, this rheological property improves when the percentage of Hb S is
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lower. Since erythrocyte deformability is not impaired in sickle cell trait, it does not seem to be a possible mechanism involved in increased thrombotic risk in these patients.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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