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Letters to the Editor
We also validate this index in a cohort of patients with non-transfusion dependent HbE/b-thalassemia, in which IE is known to predominate and in a control group with no iron deficiency, no hematological disease or other con- dition which might impact erythropoiesis.
All patients with SCD were recruited from King’s College Hospital, London, UK. Written informed consent was obtained through three approved study protocols (LREC 01-083, 07/H0606/165, and 12/LO/1610). Patient electronic records were reviewed from 2008 to the pres- ent for all measurements of sTfR and ARC taken on the same day in the outpatient setting. Contemporaneous Hemoglobin (Hb) and HbF levels were also recorded and a note made of disease modifying therapy: ongoing regu- lar blood transfusion within 90 days, HU use, or neither. Measurements were excluded if there was evidence of iron deficiency (serum ferritin <30 micrograms/L), or if the patient was younger than 16 years of age at time of sampling.10 Data from a previous study of patients with HbE/b thalassaemia11 were collected and analyzed to validate the index. Data from 22 control samples was also analyzed; these were samples from adult patients taken for clinical reasons. They were without iron defi- ciency or any other condition known to affect erythro- poiesis or red cell survival.
Determination of a thalassemia, G6PD and g(HbF), a composite measure of the major genetic determinants of HbF, in this cohort are described elsewhere.12 sTfR was measured using an ELISA (R&D Systems, Minneapolis, USA) and reticulocytes were measured using automated counting based on RNA staining (Siemens Healthcare, Erlangen, Germany).
Index of ineffective erythropoiesis (IoIE) was calculated by dividing sTfR (nmol/L) by the ARC (x109/L). We defined the limit of the normal range by applying this for- mula to the well-established upper limit of the normal ranges of sTfR (28.1 nmol/L) and ARC (100x109/L), giv- ing a value of 0.28. We validated this with control sam- ples. Where multiple measurements were available, aver- ages were taken. Statistical analyses were performed with GraphPad Prism (version 9). The data were ana- lyzed using the Mann-Whitney unpaired test and the Wilcoxon paired test, as indicated. Correlations were per- formed using the Spearman correlation.
There were 22 controls, 182 patients with HbSS, 87 with HbSC and 12 with HbS/b-thalassemia+ without dis- ease modifying therapy, with a comparable age range and sex ratio (Table 1). As expected, HbSS patients had signif- icantly lower Hb levels and higher ARC, compared to patients with either HbSC or HbS/b+-thalassemia (Figure 1A), and sTfR levels are significantly higher in the HbSS genotype (Figure 1B). There was a strong correlation between ARC and sTfR levels (r=0.3773, P<0.0001, Figure 1D).
The IoIE in the non-treated HbSS group was elevated, at 0.37, compared to the control group (0.209), suggest- ing some degree of IE. This was significantly less than in the validation cohort of 23 non-transfused patients with HbE/b-thalassemia, with a median IoIE of 1.46 (Table 1, Figure 1C). By contrast, in the HbSC group, the IoIE (0.27) was in the normal range though it was slightly higher than in the control group (P=0.0174). The differ- ence between the HbSS and HbSC groups was statistical- ly significant (P<0.0001). Although the numbers were small, the IoIE in the HbS/b+-thalassemia group was higher (0.35) than that of HbSC and significantly higher than the control (P=0.0015), despite similar levels of ane- mia and disease severity compared to HbSC (Figure 1C).
We assessed factors known to influence disease sever-
ity in HbSS with the IoIE. We found a significant negative correlation with Hb (r=-0.32, P=6x10-6), (Figure 1E), and HbF (r=-0.20, P=0.02) (Figure 1F), but no association with G6PD status, (P=0.062), deletional a-thalassemia (P=0.26), or g(HbF) (r=0.02, P=0.82). These findings, together with previous work implicating HbF levels in IE,5 may suggest that increased g globin synthesis in ery- throblasts reduces IE in SCD and contributes to higher Hb levels by improving erythropoiesis, as well as pro- longing red cell survival.
HU and blood transfusion therapy are the two main treatment options in patients with SCD. We evaluated the IoIE in 58 regularly transfused and 50 HU-treated patients. The transfused patients were on long-term reg- ular transfusions, mostly because of cerebrovascular dis- ease to keep the HbS level below 30%. The patients on HU had all been taking it for at least six months and were treated according to clinical response rather than at max- imum tolerated dose. In each of these groups, the meas- urements of Hb, HbF, and ARC differed from baseline in accordance with previously published data (Table 1),13,14 whilst sTfR levels decreased in both groups (Figure 1H). By contrast, the IoIE was significantly lower with trans- fusions (IoIE=0. 26, P=7.2x10-6), but significantly higher with HU treatment (IoIE=0.42, P=0.042) (Figure 1I). We validated these findings in 17 patients for whom both baseline and post-transfusion measurements were avail- able (paired t-test, P=0.019) (Figure 1L) and 20 patients for whom both baseline and HU treated measurements were available (paired t-test, P=0.004) and confirmed that IoIE actively increased with HU (Figure 1J) and decreased with transfusion (Figure 1K). There was no correlation between HbF and IoIE in patients on HU (P=0.072), although numbers were small.
The improvement in IoIE seen in patients on blood transfusions contrasts to that seen with regular blood transfusions to treat HbE/b-thalassemia (n=21), where IoIE remained high at 1.51 (Figure 1K). We suggest this reflects the inherent difference in the causes of IE in tha- lassemia compared to HbSS. In thalassemia there is intrinsic IE due to the imbalance in globin chain synthe- sis, which will not be significantly altered by blood trans- fusion whereas the IE seen in HbSS may be, in part, extrinsically induced by the dysfunctional bone marrow microenvironment, one shown to improve with recur- rent blood transfusion in a recent study of a mouse model.15
The relationship between HbF levels and IE in SCD is complex, as demonstrated by the different effects of nat- urally occurring high HbF levels (reduced IE) and HU (increased IE). This suggests two opposing mechanisms: inherited high HbF levels reduce erythrocyte HbS con- centrations and so reduce intramedullary HbS polymer- ization with improved erythroid survival; conversely, high levels of IE lead to increased selective advantage in favor of cells expressing more HbF,16 resulting in higher circulating HbF levels; this may be one mechanism through which HU mediates its therapeutic increase in HbF, and explains why there is a relative weak correla- tion between HbF and ineffective erythropoiesis.
In summary, we propose the IoIE as a simple, meaning- ful and useful measure of ineffective erythropoiesis. We use it to demonstrate that IE exists in patients with HbSS, but not HbSC, and that the genetic ability to synthesize more HbF is associated with less IE. Furthermore, we make the clinically important observation that HU increases IE whilst blood transfusion reduces IE. Further investigations are required to understand this action of HU in increasing IE in SCD.17 HU has a direct effect on
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haematologica | 2022; 107(1)