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Serum erythropoietin and outcome after ischaemic stroke: a prospective study

Objectives Erythropoietin (EPO), which is inversely associated with blood haemoglobin (Hb), exerts neuroprotective effects in experimental ischaemic stroke (IS). However, clinical treatment trials have so far been negative. Here, in patients with IS, we analysed whether serum EPO is associated with (1) initial stroke severity, (2) recovery and (3) functional outcome. Design Prospective. Controls available at baseline. Setting A Swedish hospital-initiated study with outpatient follow-up after 3 months. Participants Patients (n=600; 64% males, mean age 56 years, controls n=600) were included from the Sahlgrenska Academy Study on IS (SAHLSIS). Primary and secondary outcome measures In addition to EPO and Hb, initial stroke severity was assessed by the Scandinavian Stroke Scale (SSS) and compared with SSS after 3 months (follow-up) as a measure of recovery. Functional outcome was evaluated using the modified Rankin Scale (mRS) at follow-up. Serum EPO and SSS were divided into quintiles in the multivariate regression analyses. Results Serum EPO was 21% and 31% higher than in controls at the acute phase of IS and follow-up, respectively. In patients, acute serum EPO was 19.5% higher in severe versus mild IS. The highest acute EPO quintile adjusted for sex, age and Hb was associated with worse stroke severity quintile (OR 1.70, 95% CI 1.00 to 2.87), better stroke recovery quintile (OR 1.93, CI 1.09 to 3.41) and unfavourable mRS 3–6 (OR 2.59, CI 1.15 to 5.80). However, the fourth quintile of EPO increase (from acute to follow-up) was associated with favourable mRS 0–2 (OR 3.42, CI 1.46 to 8.03). Only the last association withstood full adjustment. Conclusions The crude associations between EPO and worse stroke severity and outcome lost significance after multivariate modelling. However, in patients in whom EPO increased, the association with favourable outcome remained after adjustment for multiple covariates.


Supplementary information:
Detailed description of study populations and clinical examination (item number) 1. The Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS): The study population comprised Caucasian patients (n=844) who presented with first-ever (n=732) or recurrent (n=112) ischemic stroke (IS) before reaching the age of 70 years consecutively recruited between 1998 and 2008 at four stroke units in Western Sweden and Caucasian population controls (n=668) as previously reported 1,2 . However, only for genetic analysis all these patients were available, whereas fewer subjects were available with serum samples and complete clinical data (n=600 patients and controls, respectively). From these 600 that were selected for analysis, there were some patients had the acute or the three-month sample missing (or vice versa), generating a missing delta value ( Table 2 in the article).
The controls were randomly selected to match cases with regard to age and sex. Controls were from the same geographical region as the patients, and they were recruited through a population-based health survey or from the Swedish Population Register.
2. Clinical definitions and descriptions: Ischemic stroke (IS) was defined as an episode of focal neurological deficits with acute onset and lasting >24 hours or until death, with no apparent non-vascular cause, and no signs of primary hemorrhage on brain imaging. All patients were examined by a physician trained in stroke medicine. Information on the subjects' vascular risk factors was collected as described elsewhere 1 . Hypertension was defined by pharmacological treatment for hypertension, systolic blood pressure ≥ 160 mm Hg, and/or diastolic blood pressure ≥ 90mm Hg. Diabetes mellitus was defined by diet or pharmacological treatment, fasting plasma glucose ≥ 7.0 mmol/L, or fasting blood  Table S1). The few samples below the detection limit (0.7%) were set at 50% of the lower limit of quantification (LLOQ). Although the inter-assay variation was relatively small (9.5%) for EPO, we chose to normalize the obtained values in each plate to the internal quality controls (n=3).

B-Hb was measured on ADVIA 2120i, Siemens Medical Diagnostics AB, or Cell-Dyn
Sapphire, Abbott Diagnostics Division, with a total inter-assay CV of 4%. S-CRP was measured on Cobas 8000, Roche Diagnostics, with a total inter-assay CV of 1.5% (at 8.9 mg/L) and 2.6% (at 66.4 mg/L). 7. All participants provided informed consent prior to enrolment. For participants who were unable to communicate, consent was obtained from their next-of-kin. This study was approved by the Ethics Committee of the University of Gothenburg.  Table 1: Title: EPO variation, detection limits and concentration ranges of EPO.

Text in footnote:
Table 1a: EPO analyzed after various freeze-thaw cycles, after being in fridge and room temperature (RT) for one week (1w). Intra-assay coefficient of variation (CV%) is also presented. Freeze-thaw analysis was made prior to main analysis using double samples of two different subjects.  Supplementary Results text (subsample analysis of ∆EPO with regard to day of sampling): As serum EPO differs somewhat during the first and second week after ischemic stroke (Supplementary Figure 1), the ∆EPO could be skewed and misrepresentative for the associations.
Therefore we made a subsample analysis for the largest group, i.e. for the group with acute blood sampling during days 3-5 with 195 patients with ∆EPO values available. This analysis loses power by having fewer individuals but statistical significance is remained with a somewhat lower P-value but with a somewhat higher OR. For example the 4th quintile of delta EPO is associated with 4.8-fold odds ratio (OR) of having a favorable outcome (p = 0.042, n = 172 with sex, age, Hb and cardiovascular covariates) as compared to the OR = 3.3 for the whole group (p=0.008, n = 366, the same covariates included). These data show that although the found association is remained with a narrower window of blood sampling, the statistical power is lowered by the lower number of participants. Although this gives some support to the suggestion from the Reviewer, the main finding is the same and therefore the Ms presents all the data and not a selection.