SEARCH

SEARCH BY CITATION

Keywords:

  • sickle cell disease;
  • CD163;
  • haemolysis;
  • vasculopathy

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Sickle cell disease (SCD) is characterized by vasculopathy, which has been causally linked to intravascular haemolysis and high levels of free plasma haemoglobin. Soluble CD163 (sCD163) is implicated in the clearance of free plasma haemoglobin and high plasma concentrations have been linked to arterial disease. We therefore investigated the value of sCD163 as a biomarker in children with SCD, and also measured haptoglobin levels in this population. We measured sCD163 in 25 control children with no haemoglobinopathy, 41 with sickle cell anaemia (HbSS) in the steady state, 27 with HbSS taking hydroxycarbamide, and 7 with HbSC disease. There was no significant difference between sCD163 levels in steady-state HbSS (1·78 mg/l) and controls (1·81 mg/l) (P = 0·86). However, sCD163 levels were significantly lower in those HbSS children taking hydroxycarbamide (1·35 mg/l) compared to both steady state HbSS (P = 0·004) and controls (P = 0·036). In children on hydroxycarbamide, sCD163 correlated negatively and highly significantly with percentage HbF (R = −0·76, P < 0·001), and this relationship was absent in those not taking hydroxycarbamide (R = 0·07, P = 0·65). sCD163 is a potentially useful biomarker in children with SCD, and may have a role in monitoring responses to hydroxycarbamide.

CD163 is a member of the scavenger-receptor cysteine-rich family of proteins. It is expressed on cells of the monocyte-macrophage lineage and is the main haemoglobin-haptoglobin (HbHp) receptor (Kristiansen et al, 2001). In addition to clearing haemoglobin from the plasma, it mediates the interaction between macrophages and erythroblasts (Fabriek et al, 2007), is a TWEAK [TNF (tumour necrosis factor)-like weak inducer of apoptosis] receptor (Bover et al, 2007), acts as a direct receptor for bacteria (Fabriek et al, 2009) and is an immunomodulator (Van Gorp et al, 2010). CD163 is actively shed from the surface of monocytes/macrophages and soluble CD163 (sCD163) occurs at high levels of up to 2 mg/l in normal human plasma (Moller et al, 2002). The functions of sCD163 are unknown, although it has been suggested that it might bind HbHp complexes and reduce cellular iron uptake (Weaver et al, 2006) and has also been shown to inhibit T-lymphocyte activation and proliferation (Hogger & Sorg, 2001). Plasma levels of sCD163 have been investigated as potential markers of inflammation and macrophage activation, and show marked increases in some conditions, such as Gaucher disease (Moller et al, 2004), sepsis (Moller et al, 2006), liver disease (Moller et al, 2007) and peripheral arterial disease (Moreno et al, 2010).

sCD163 is highly relevant when considering the pathophysiology and complications of SCD. There is a significant chronic inflammatory component in SCD due to continuous ischaemic tissue damage, with more inflammation occurring during acute complications (Belcher et al, 2003); sCD163 may therefore be higher in children with more severe disease and predictive of poor outcome. Haemolysis is an important component of the pathophysiology of SCD, causing the release of free haemoglobin in to the plasma, where it binds strongly to and inactivates nitric oxide (NO) (Reiter et al, 2002); this functional NO deficiency causes endothelial dysfunction, which may contribute to progressive vascular damage. There is evidence that the rate of haemolysis, as measured by plasma lactate dehydrogenase (LDH) levels, correlates with some complications in SCD, particularly pulmonary hypertension (Gladwin et al, 2004), leg ulcers, priapism (Kato et al, 2006) and cerebral vasculopathy (O’Driscoll et al, 2008). However, there is very little evidence concerning differences in the rate of clearance of free haemoglobin from the plasma, which is also likely to be a determinant of any damage caused by haemolysis. sCD163 levels may indicate variability in inflammation and the clearance of free plasma haemoglobin, and explain some of the clinical variability which characterizes SCD.

We have therefore measured the sCD163 level in children with SCD and controls to investigate whether it might be a useful marker of disease activity, the effects of treatment with hydroxycarbamide (HC) and its relationship to any complications.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients

The study was approved by King’s College Hospital Research Ethics Committee (06/Q0703/33). The children involved were all seen in the Department of Child Health at King’s College Hospital, a teaching hospital in south London. Following informed consent, blood was collected from several groups of children: steady-state (no symptoms of SCD for at least one month), taking HC, acute pain and on regular transfusions. Control samples were collected from children having blood tests for indications not associated with inflammation, haemolysis or anaemia, including those having preoperative assessment and elective allergy testing. Clinical information was collected and transcranial Doppler measurements of carotid and intracerebral blood flow recorded.

Laboratory analysis

Routine blood tests were performed, including full blood count, reticulocyte count, renal and hepatic function. HbF percentages were measured using high-performance liquid chromatography. Following completion of these tests, plasma was separated from the EDTA-anticoagulated blood within five hours of venesection, and stored at −70°C. Samples were then shipped on dry-ice to Aarhus where sCD163 and haptoglobin levels were measured.

Measurement of sCD163

sCD163 was measured essentially as described previously (Moller et al, 2002). Rabbit anti-CD163 was coated onto micro-titre wells and plates transferred to a BEP-2000 enzyme-linked immunosorbent assay (ELISA)-analyser (Dade Behring, Eschborn, Germany). Samples (diluted 1:101) were added in duplicates and incubated for 1·5 h at 37°C. Monoclonal anti-CD163 (GHI/61, 3 μg/ml) was incubated for 1 h at 37°C, and peroxidase-labelled antibody (goat anti-mouse immunoglobulins, DAKO P447, 0·125 μg/ml; Dako, Glostrup, Denmark) was incubated for 1 h at 37°C. Tetramethylbenzidine (TMB) substrate solution (Kem-En-Tec, Taastrup, Denmark. Cat. no. 4380) was used. The assay was calibrated using serum traceable to purified human CD163. The interassay variation over the current 4 runs was 2·6–4·5%.

Measurement of haptoglobin

The concentration of Hp was determined by a sensitive ELISA using biotinylated anti-human Hp (DAKOCytomation A0030). Briefly anti-Hp (1:3000) was coated onto micro-titre wells overnight. Samples (diluted 1:500 – 1:25,000) were added in duplicates and incubated for 1 h at room temperature (RT). Biotinylated anti-Hp (1:3000) was incubated for 1 h at RT, and detected by avidin-peroxidase and TMB substrate solution. The assay was calibrated using Roche c.f.a.s. (calibrator for automated systems) protein calibrator (2·14 g/l; Roche, Hvidovre, Denmark. Cat. no 11355279). The interassay variation over the current 6 runs was 13%.

Statistical analysis

The links between sCD163, haptoglobin, and routine laboratory and clinical parameters were assessed using the statistical programme spss 17·0 (SPSS Inc., Chicago, IL, USA). The plasma haptoglobin levels were skewed (Kolmogorov-Smirnov test), but normalized by logarithmic transformation; therefore haptoglobin concentrations were transformed using natural logarithms before analysis (ln haptoglobin). Results with P < 0·05 were considered statistically significant. Analyses included Pearson correlation coefficients, linear regression and independent samples T-test.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Samples were collected from 41 children with sickle cell anaemia (HbSS) in the steady-state, 27 with HbSS taking HC, 25 controls with HbA only, 4 with HbSS and acute pain, 4 with HbSS on regular blood transfusions and 7 with HbSC in the steady-state (Table I). Most analyses were only performed on the first three groups, because of the small numbers of others.

Table I.   Mean sCD163, haptoglobin and other parameters in children with SCD, with standard deviation in brackets.
 ControlsHbSS steady-stateHbSS HCHbSC steady stateHbSS acute painHbSS transfused
  1. HC, hydroxycarbamide; LDH, lactate dehydrogenase; WBC, white blood cell count; MCV, mean cell volume; ALT, alanine transaminase; TAMMV, time averaged mean of the maximum velocity.

  2. *Laboratory normal ranges, not derived from these controls.

  3. Not measured in this patient group. Transfused patients were receiving regular blood transfusions and the sample taken immediately pre-transfusion.

N254127744
Age (years)8·0 (5·8)9·7 (4·6)10·2 (3·3)6·9 (5·4)12·2 (1·3)9·2 (4·6)
Soluble CD163 (mg/l)1·81 (0·97)1·78 (0·71)1·35 (0·48)1·44 (0·35)2·44 (0·59)0·90 (0·10)
Hb (g/l)126 (13)78 (12)86 (14)102 (11)74 (5·0)95 (11)
WBC (× 109/l)8·0 (2·9)12·2 (6·8)9·1 (3·4)6·4 (1·6)13·0 (2·0)12·1 (4·2)
Neutrophils (× 109/l)3·3 (1·8)5·6 (3·0)4·3 (2·4)2·2 (0·7)6·9 (1·3)6·9 (3·2)
Monocytes (× 109/l)0·49 (0·23)0·99 (0·45)0·79 (0·45)0·52 (0·23)0·98 (0·40)0·75 (0·50)
Reticulocytes (× 109/l)89 (24)381 (145)258 (112)119 (26)586 (93)396 (105)
MCV (fl)80 (6·3)76 (9·6)90 (11)64 (3·3)87 (7·0)83 (2·7)
LDH (iu/l)<240*600 (167)580 (147)354 (104)
Bilirubin (μmol/l)3–20*55 (38)52 (37)19 (11·9)43 (19)53 (29)
ALT (iu/l)5–55*26 (12)26 (17)18 (6)
HbF (%)<1%*8·4 (5·4)11·6 (6·5)3·7 (3·5)4·4 (4·6)
Total HbF (g/l)<1·0*6·8 (4·8)10·5 (7·1)3·5 (2·9)4·4 (5·2)
Haptoglobin (mg/l)369 (630)2·0 (1·0)3·1 (6·2)359 (925)1·8 (0·4)1·9 (1·3)
TAMMV (cm/s)129 (28)130 (35)99 (25)193 (19)

Soluble CD163 levels

There was no statistical difference between sCD163 concentrations in steady-state HbSS children and the control population (1·78 mg/l vs. 1·81 mg/l, P = 0·86). However, those on HC had significantly lower levels of sCD163 than both steady-state HbSS (1·35 mg/l vs. 1·78 mg/l, P = 0·004) and controls (P = 0·036). Although numbers were too small to allow statistical analysis, acute pain was associated with increased sCD163 levels, and regular blood transfusion lower levels (Table I). Age and sex showed no relationship to sCD163 in either population. Five children with HbSS in the steady state had palpably enlarged spleens, and their mean sCD163 was 1·70 mg/l (range 1·08–2·70), not significantly different to those with nonpalpable spleens. Children with HbSC had lower mean sCD163 (1·44 mg/l) levels than HbSS, although this was not statistically significant (Table I).

In steady-state HbSS children, sCD163 levels correlated significantly with white cell count (R = 0·33, P = 0·034), reticulocyte count (R = 0·41, P = 0·009), alanine transaminase (R = 0·38, P = 0·023) and ln haptoglobin (R = 0·38, P = 0·016). There was no correlation with HbF percentage (R = 0·074, P = 0·65) or total HbF (R = 0·014, P = 0·93) (Table II). None of these factors remained significant on multiple linear regression against sCD163. Similar analysis for the children taking HC showed significant correlation between sCD163 and haemoglobin (R = −0·49, P = 0·009), white cell count (R = 0·50, P = 0·009), neutrophils (R = 0·46, P = 0·015), monocytes (R = 0·46, P = 0·016), and percentage HbF (R = −0·76, P < 0·001) and total HbF (R = −0·77, P < 0·001). There was no correlation with mean cell volume (MCV; R = −0·26, P = 0·18). (Table II) On multiple regression analysis, HbF remained strongly significant (P = 0·005) whilst the other factors lost their significance (Fig 1).

Table II.   Pearson correlation coefficients of soluble CD163 levels with various parameters in control samples, patients with HbSS in the steady state, and those with HbSS on hydroxycarbamide.
 ControlsHbSSHbSS on HC
NRPNRPNRP
  1. HC, hydroxycarbamide; WBC, white blood cell count; MCV, mean cell volume; LDH, lactate dehydrogenase; ALT, alanine transaminase; TCD, transcranial Doppler scan; TAMMV, time-averaged mean of the maximum velocity.

  2. P is the 2-tailed significance, and is shown in bold if <0·05.

Red cell
 Haemoglobin24−0·0510·81141−0·2700·08727−0·4910·009
 Reticulocytes   410·4080·009270·3100·124
 MCV24−0·2340·272410·5670·09227−0·2640·183
 HbF %   390·6540·07427−0·763<0·001
 Total HbF   390·0140·93327−0·769<0·001
White cells
 Total WBC240·3000·154410·3310·034270·4950·009
 Neutrophils230·0720·744410·1950·223270·4620·015
 Monocytes230·3400·113410·1620·311270·4610·016
Haemolysis and hepatic
 LDH   400·2010·213260·2090·305
 ALT   360·3770·023250·1990·341
 Ln haptoglobin250·3870·056410·3760·016270·0840·677
 TCD
 TAMMV   330·2560·15114−0·0390·895
image

Figure 1.  Scatter plot of sCD163 levels against percentage HbF in children with HbSS in the steady state (A) and those with HbSS taking hydroxycarbamide (B). The lines are fitted by linear regression computed using the least squares method and a constant.

Download figure to PowerPoint

Haptoglobin concentrations

Haptoglobin levels were significantly higher in controls and those with HbSC than in children with HbSS in steady-state and on HC (Fig 2). In steady-state HbSS, ln haptoglobin correlated significantly with sCD163 concentrations (R = 0·38, P = 0·016), although no similar relationship was present in the other groups. There was no statistically significant relationship between haptoglobin and LDH, a marker of haemolysis. There was no difference in haptoglobin levels between those taking and not taking HC (ln haptoglobin 0·72 vs. 0·61, P = 0·37).

image

Figure 2.  Box and whisker plot of ln haptoglobin levels in different categories of sickle cell disease patient. Mean control values were significantly higher than steady state HbSS (P < 0·001) and HbSS on hydroxycarbamide (hydroxycarbamide (HC); P < 0·001). There was no statistical difference between haptoglobin levels in steady state HbSS and HbSS on HC (P = 0·37).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Haemolysis and free plasma haemoglobin are increasingly implicated in the vasculopathy of SCD (Kato et al, 2006), although whether their action is mediated through NO is controversial (Bunn et al, 2010). It is therefore important to understand how the clearance of free plasma haemoglobin occurs in SCD, and whether variability in this is responsible for some of the phenotypic differences. Although the clearance of plasma haemoglobin is partly understood in health, there may be significant differences in SCD due to the increased rate of haemolysis, chronic inflammation and hyposplenism. CD163 is central to this process, being the main HbHp receptor. The measurement of sCD163 in the plasma of children with SCD has not previously been reported and has the potential to shed light on haemolysis-related endothelial dysfunction; for example, high sCD163 levels have been found to be a marker for coronary atherosclerosis (Aristoteli et al, 2006).

We did not find any relationship between the rate of haemolysis, haemolytic complications and sCD163 concentrations. For example, there was no significant difference between levels found in steady-state HbSS and healthy controls. Similarly, there was no correlation between sCD163 levels and markers of haemolysis, such as LDH or vasculopathy, as indicated by transcranial Doppler scan velocities. This lack of association suggests that sCD163 does not play an important functional role in binding to HbHp complexes in chronic haemolysis, as has also been suggested by in vitro studies showing that sCD163 binds HbHp complexes weakly compared to the membrane-bound form (Moller et al, 2010). However, it is unclear from our study whether the monocyte-macrophage expression of CD163 might vary in SCD and contribute to phenotypic differences; this possibility is indirectly supported by evidence that corticosteroids increase the expression of CD163 on monocytes (Schaer et al, 2002) and are also of therapeutic benefit in acute pain (Griffin et al, 1994) and acute chest syndrome (Bernini et al, 1998). It is also possible that although sCD163 levels were normal in SCD, this is a balance between both increased production and increased removal by HbHp complexes.

Surprisingly our study showed that HC significantly reduced concentrations of sCD163 in children with SCD, below that found in the steady-state and also below concentrations in paediatric controls. This decrease showed a strong correlation with increasing HbF levels, but no correlation with MCV. It is possible that the fall in sCD163 is related to the myelosuppressive effects of HC, being directly linked to the reduced numbers of monocytes and macrophages. There was no significant difference in monocyte numbers between those taking and not taking HC (0·79 × 109/l vs. 0·99, P = 0·09), although there was a trend towards lower monocytes in those on HC, and an effect on resident macrophages that are supposed to be the main source of sCD163 cannot be excluded. It is possible that the higher HbF levels result in less chronic inflammation and vascular damage, and that the lower sCD163 levels are indicative of this. An alternative explanation is that HC has a direct effect on sCD163 concentration that is proportionate to its effects on HbF levels; this could be caused either by direct suppression of sCD163 production or reduced rates of release from monocyte/macrophage membranes. Another possibility is that HC directly nitrosylates the cysteine-rich sCD163 molecule, causing its degradation. sCD163 has the potential to be a useful biomarker to monitor HC therapy, and may also shed further light on mechanisms of action of HC in SCD, including its role in promoting HbF. Future studies could address its prognostic significance in comparison with HbF.

Using conventional assays, haptoglobin is undetectable in 75% of patients with SCD (Podmore et al, 2007). However, using this highly sensitive assay, haptoglobin was measurable in all our patients, but did not correlate with any known markers of haemolysis, such as LDH, or other clinical events. As expected, haptoglobin levels were lower than normal in HbSS, and to a lesser extent in HbSC. There was no significant difference in haptoglobin levels between those taking and not taking HC. However, haptoglobin levels were detectable using this assay, suggesting that the HbHp system continues to offer some protection in SCD against the damaging effects of free plasma haemoglobin. It is, however, possible that the low Hp levels determined in patients with SCD partly reflect haptoglobin-related protein due to cross reactivity of the antibody used in the ELISA. We did not examine the role of haptoglobin polymorphisms, haptoglobin-related-protein or haemopexin in this study.

Our study suggests that sCD163 is a potentially useful biomarker in SCD, and may be particularly relevant in monitoring the effects of HC. The significant correlation between HbF percentages and sCD163 concentrations in children on HC may lead to further insights into the mechanisms of action of HC. Further studies of sCD163 and haemoglobin scavenging in SCD are likely to be informative.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We would like to thank the patients who took part in this study, the clinical staff involved in their care, and Professor Swee Lay Thein for support in completing this study.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Aristoteli, L.P., Moller, H.J., Bailey, B., Moestrup, S.K. & Kritharides, L. (2006) The monocytic lineage specific soluble CD163 is a plasma marker of coronary atherosclerosis. Atherosclerosis, 184, 342347.
  • Belcher, J.D., Bryant, C.J., Nguyen, J., Bowlin, P.R., Kielbik, M.C., Bischof, J.C., Hebbel, R.P. & Vercellotti, G.M. (2003) Transgenic sickle mice have vascular inflammation. Blood, 101, 39533959.
  • Bernini, J.C., Rogers, Z.R., Sandler, E.S., Reisch, J.S., Quinn, C.T. & Buchanan, G.R. (1998) Beneficial effect of intravenous dexamethasone in children with mild to moderately severe acute chest syndrome complicating sickle cell disease. Blood, 92, 30823089.
  • Bover, L.C., Cardó-Vila, M., Kuniyasu, A., Sun, J., Rangel, R., Takeya, M., Aggarwal, B.B., Arap, W. & Pasqualini, R. (2007) A previously unrecognized protein-protein interaction between TWEAK and CD163: potential biological implications. Journal of Immunology, 178, 81838194.
  • Bunn, H.F., Nathan, D.G., Dover, G.J., Hebbel, R.P., Platt, O.S., Rosse, W.F. & Ware, R.E. (2010) Pulmonary hypertension and nitric oxide depletion in sickle cell disease. Blood, 116, 687692.
  • Fabriek, B.O., Polfliet, M.M., Vloet, R.P., van der Schors, R.C., Ligtenberg, A.J., Weaver, L.K., Geest, C., Matsuno, K., Moestrup, S.K., Dijkstra, C.D. & van den Berg, T.K. (2007) The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor. Blood, 109, 52235229.
  • Fabriek, B.O., van Bruggen, R., Deng, D.M., Ligtenberg, A.J., Nazmi, K., Schornagel, K., Vloet, R.P., Dijkstra, C.D. & van den Berg, T.K. (2009) The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria. Blood, 113, 887892.
  • Gladwin, M.T., Sachdev, V., Jison, M.L., Shizukuda, Y., Plehn, J.F., Minter, K., Brown, B., Coles, W.A., Nichols, J.S., Ernst, I., Hunter, L.A., Blackwelder, W.C., Schechter, A.N., Rodgers, G.P., Castro, O. & Ognibene, F.P. (2004) Pulmonary hypertension as a risk factor for death in patients with sickle cell disease. New England Journal of Medicine, 350, 886895.
  • Griffin, T.C., McIntire, D. & Buchanan, G.R. (1994) High-dose intravenous methylprednisolone therapy for pain in children and adolescents with sickle cell disease. New England Journal of Medicine, 330, 733737.
  • Hogger, P. & Sorg, C. (2001) Soluble CD163 inhibits phorbol ester-induced lymphocyte proliferation. Biochemical and Biophysical Research Communications, 288, 841843.
  • Kato, G.J., McGowan, V., Machado, R.F., Little, J.A., Taylor, J., Morris, C.R., Nichols, J.S., Wang, X., Poljakovic, M., Morris, S.M. & Gladwin, M.T. (2006) Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease. Blood, 107, 22792285.
  • Kristiansen, M., Graversen, J.H., Jacobsen, C., Sonne, O., Hoffman, H.J., Law, S.K. & Moestrup, SK. (2001) Identification of the haemoglobin scavenger receptor. Nature, 409, 198201.
  • Moller, H.J., Peterslund, N.A., Graversen, J.H. & Moestrup, S.K. (2002) Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma. Blood, 99, 378380.
  • Moller, H.J., de Fost, M., Aerts, H., Hollak, C. & Moestrup, S.K. (2004) Plasma level of the macrophage-derived soluble CD163 is increased and positively correlates with severity in Gaucher’s disease. European Journal of Haematology, 72, 135139.
  • Moller, H.J., Moestrup, S.K., Weis, N., Wejse, C., Nielsen, H., Pedersen, S.S., Attermann, J., Nexo, E. & Kronborg, G. (2006) Macrophage serum markers in pneumococcal bacteremia: Prediction of survival by soluble CD163. Critical Care Medicine, 34, 25612566.
  • Moller, H.J., Gronbaek, H., Schiodt, F.V., Holland-Fischer, P., Schilsky, M., Munoz, S., Hassanein, T. & Lee, W.M.; U.S. Acute Liver Failure Study Group. (2007) Soluble CD163 from activated macrophages predicts mortality in acute liver failure. Journal of Hepatology, 47, 671676.
  • Moller, H.J., Nielsen, M.J., Maniecki, M.B., Madsen, M. & Moestrup, S.K. (2010) Soluble macrophage-derived CD163: a homogenous ectodomain protein with a dissociable haptoglobin-hemoglobin binding. Immunobiology, 215, 416412.
  • Moreno, J.A., Dejouvencel, T., Labreuche, J., Smadja, D.M., Dussiot, M., Martin-Ventura, J.L., Egido, J., Gaussem, P., Emmerich, J., Michel, J.B., Blanco-Colio, L.M. & Meilhac, O. (2010) Peripheral artery disease is associated with a high CD163/TWEAK plasma ratio. Arteriosclerosis, Thrombosis and Vascular Biology, 30, 12531262.
  • O’Driscoll, S., Height, S.E., Dick, M.C. & Rees, D.C. (2008) Serum lactate dehydrogenase deficiency as a biomarker in children with sickle cell disease. British Journal of Haematology, 140, 206209.
  • Podmore, A.H.B., Dalton, S., Henthorn, J.S. & Davies, S.C. (2007) Levels of haptoglobin and haemopexin in patients with sickle and other haemolytic diseases, and haptoglobin genotype frequency. British Journal of Haematology, 137, 71.
  • Reiter, C.D., Wang, X., Tanus-Santos, J.E., Hogg, N., Cannon, R.O. III, Schechter, A.N. & Gladwin, M.T. (2002) Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nature Medicine, 8, 13831389.
  • Schaer, D.J., Boretti, F.S., Schoedon, G. & Schaffner, A. (2002) Induction of the CD163-dependent haemoglobin uptake by macrophages as a novel anti-inflammatory action of glucocorticoids. British Journal of Haematology, 119, 239243.
  • Van Gorp, H., Delputte, P.L. & Nauwynck, H.J. (2010) Scavenger receptor CD163, a jack-of-all-trades and potential target for cell-directed therapy. Molecular Immunology, 47, 16501660.
  • Weaver, L.K., Hintz-Goldstein, K.A., Pioli, P.A., Wardwell, K., Qureshi, N., Vogel, S.N. & Guyre, P.M. (2006) Pivotal advance: activation of cell surface Toll-like receptors causes shedding of the hemoglobin scavenger receptor CD163. Journal of Leukocyte Biology, 80, 2635.