The present study was undertaken among 104 subjects admitted to IPD wards and intensive care unit (ICU) of Central India Institute of Medical Sciences (CIIMS) Nagpur, India from November 2012 to January 2014. The Diagnosis of AIS was done in accordance with WHO guidelines for stroke which includes “Rapidly developing signs of focal (or global) disturbance of cerebral function lasting >24 hours (unless interrupted by surgery or death), with no apparent non vascular cause, history, neurological examination and Computerized tomography (CT)”. Baseline data such as age, sex, along with detailed history of risk factors linked to AIS such as hypertension, ischemic heart disease (IHD), smoking, alcohol consumption, any past history of stroke were recorded. Exclusion of subjects mimicking similar outcomes to that of stroke was done based on CT scan analysis. Apart from that, patients in older age groups (85 years or more); haemorrhagic stroke patients; brain malignancies patients; transient ischemic attack; patients who underwent brain operation; patients who presented with severe systemic disease, dementia, psychiatric disease, active infection, refused and patients who took discharge against medical advice were excluded from the study.

Subjects were classified based on time period of admission in Neurology and Emergency wards post events of the stroke. Based on the following admission criteria, the study subjects were classified into two different groups, those admitted within 24 hours (early referrals) after occurrence of stroke event and those admitted after 24 hours post stroke event (late referrals). Details of any thrombolysis treatment given, and decompression surgery done in patients in both groups were also recorded. Severity of stroke on admission was evaluated using National Institute of Health Stroke Scale (NIHSS). The modified Rankin Scale (mRS) was used for evaluation of outcome in AIS patients by a certified investigator at discharge. Based on mRS Score, AIS patients were classified into two outcome groups which includes improved group (n = 87; m-RS score 0-2); and dependent group (n = 17; m-RS score 3-6).

To study impact of admission time on long term functional outcome, follow up study was carried out at 12^th and 18^th months post hospital discharge. Participants were recruited based on earlier records available at hospitals. Telephonic follow up was taken, which consisted of short questionnaire from participants or their kin, about level of improvement/dependency or death post discharge. All follow up cases were further correlated with level of improvement/dependency at time of hospital outcome to study prognostic outcome based on admission time. The detailed recruitment of participants during hospital and follow up including loss of cases in follow up is mentioned in [Table/Fig-1].

[Table/Fig-1]:

Study flow diagram

For evaluation of stroke markers: NSE, S-100 ββ and ITIH4 peptides, blood was collected from both patient groups post admission and at discharge/on death and serum was separated and stored at -20^oC until further use.

The reference sequences of ITIH4 were obtained through NCBI reference sequence databases. The antigenic peptides of ITIH4 were determined on the basis of Kolaskar and Tongaonkar (1990) method by using online software titled “Molecular Immunology Foundation-Bioinformatics software (MIF-Bioinformatics software)”.These antigenic peptide sequences were then subjected to multiple sequence alignment using NCBI BLAST to obtain the sequence similarities with other non redundant protein database sequences. Based on the results of the blast analysis, two antigenic sequences of 9 sequences of ITHIH4 were selected. These peptide sequences were then sent for antibody production in GenicBio lab, Shanghai, China [Table/Fig-2].

An in house ELISA method was employed for detection ITIH4 peptides in AIS patients. Briefly, 96 well microtiter plates were coated with 100μL serum samples and incubated for 45 minutes at 37°C. The plates were washed once with wash buffer, i.e. 0.5% Tween^R 20 in phosphate buffered saline (PBST) and then blocked by addition of 200 μL of blocking buffer (0.5% BSA in PBST). After 90 minutes of incubation, 100μL of primary ITIH4 anti-peptide antibody rose in rabbit) was added and further incubated for 45 minutes. For colour development,100 μL anti-rabbit HOURP conjugated secondary antibody (dilution 1:10,000) was added and incubated at 37°C for 45 minutes. The reaction was stopped with 100μl H₂SO₄ (2.5 N) and intensity of colour developed was measured at 450 nm using an ELISA reader.

NSE serum levels were estimated with Can Ag NSE EIA (Sweden) as per the instructions of kit manufacturer. Test is based on solid phase, non-competitive immunoassay based on two monoclonal antibodies (derived from mice) directed against two separate antigenic determinants of NSE molecule. The monoclonal antibodies (MAb) used bind to γ subunit of enzyme and thereby detect both γ γ and α γ is enzymes of NSE. In brief the method is to transfer the required number of microplate strips to a strip frame. Wash each strip once with the wash solution. Pipette 25 μL of the NSE Calibrators (CAL A, B, C, D, E) and patient specimens (unknowns-Uk) into the strip wells. Incubate the plate for 1 hour (±10 minutes) at room temperature (20-25°C) with constant shaking of the plate using a microplate shaker. After the incubation aspirate and wash each strip 6 times. Add 100 μL of TMB HOURP-Substrate to each well. Incubate for 30 minutes (± 5 minutes) at room temperature with constant shaking. After incubation add 100 μL of stop solution, mix and read the absorbance at 405 nm in a microplate spectrophotometer within 15 minutes after addition of stop solution.

IThe instructions, as in the manual (Can Ag S-100 EIA Sweden), was followed for the in vitro assay for the quantitative determination of S-100 ββ in human serum. The assay determines both S-100αβ and S-100ββ without cross reactivity with other forms of S-100 as it is a two-step enzyme immunometric assay based on two monoclonal antibodies derived from mouse specific for two different epitopes of S-100ββ.

In brief, the method is as follows : ELISA wells were washed once with wash Solution. 50 μL of S100ββ calibrators and sample into the strip wells are pipette. 100 μL Biotin Anti-S100ββ ββ was added to each well and the frame was incubated, containing the strips for two hours (± 10 minutes) at room temperature (20–25°C) with constant shaking of the plate using a microplate shaker. After incubation each strip was aspirated and washed 3 times using the wash buffer. 100 μL of tracer working solution was added to each well. The frame was incubated for one hour (±5 minutes) at room temperature with constant shaking. After incubation each strip was aspirated and washed 6 times. 100 μL of TMB/H₂O₂ substrate was added to each well. Incubation was done for 30 minutes (± 5 minutes) at room temperature with constant shaking. 100 μL of stop solution was added. At 405 nm it was mixed and absorbance read, in a microplate spectrophotometer within 15 minutes after addition of Stop solution.

Informed consents were taken from all enrolled participants and their kin for the study. The study was approved by Ethical Committee of Central India Institute of Medical Sciences (CIIMS), Nagpur.

Demographic and baseline characteristics among stroke patients admitted within 24 hours and after 24 hours poststroke occurrence were analysed using chi square test. Adjusted odds ratio for analysis of long term outcome was studied using multivariate analysis by adjusting confounding factors age, sex, diabetes, hypertension, smoking, Alcohol, mRS score, and admission time. Kaplan-Meier analysis was performed to determine the long term survival in early and late referrals post discharge. ITIH4, NSE and S-100 ββ on admission and discharge in serum samples of improved and expired/ dependent AISpatients admitted within 24 hours and after 24 hours were compared using student t-test. P<0.5 was considered statistically significant for all analysis.

Baseline data of 104 participants with stroke in two different groups are given in [Table/Fig-3]. Among 104 participants, majority of cases (n=66) were more than 50 years of age with their ratio slighter greater in late referral cases (68%) than those admitted earlier (58%). Occurrence of stroke cases was more in males (n=73) than females (n=31) among both groups of patients. No significant impact of factors like diabetes, hypertension, IHD, smoking, alcohol was observed between early and late referrals cases admitted with stroke to CIIMS. Around 21% of early admission cases underwent thrombolytic treatment which was significant (p< 0.05) compared to 0% of cases that were admitted after 24 hours. Ratio of stroke severity observed was more in early admission cases (22%) than those of late referral cases (12%). To study prognostic impact of admission time on post stroke occurrence, outcome studies were carried out depending on level of improvement and death/dependency at time of discharge and at 12_th and 18_th month post discharge. [Table/Fig-4a] shows hospital outcome in early and late referral AIS cases admitted to CIIMS. The hospital outcome was available among 104 cases of AIS. Although not significant, dependency rates were comparatively more among late referrals (18%) as compared to early referrals cases (6%). Similarly there were more cases that showed improvement at the time of discharge in early referrals (89%) as compared to that observed in late referrals (77%).

[Table/Fig-4b,c] shows long term outcome in early and late referral at 12_th and 18_th month post hospital discharge. The long term outcomes were available in 60 cases at 12 month and 56 cases at 18_th month out of overall 104 cases. No significant results were obtained in terms of long term outcome at 12_th and 18_th month respectively. Although percentage of cases that remained improved post discharge were more in early referrals (57% & 75%) than late referrals (48% & 67%) at 12_th and 18_th month respectively. On the contrary there were comparatively more cases that became dependent after improvement post hospital discharge in late referrals (32%) compared to early referrals (23%).

Survival analysis was carried out in both early and late referrals using Kaplan-Meier analysis [Table/Fig-5a]. No significant difference in survival time was observed in AIS patients of both the groups. Mean survival time was 17.882 ± 0.73 among late referrals compared to 18.754 ± 0.74 observed in early referral cases over 12_th and 18_th month outcome period [Table/Fig-5b].

[Table/Fig-5]:

a) Line graph shows Kaplan-Meier estimates of cumulative risk of second stroke in those with an ischemic stroke and history b) shows mean and median survival time observed in short term and at 12th and 18th months post hospital discharge among early and late referrals cases with AIS. b) Kaplan-Meier estimates of cumulative risk of mortality among the early (blue) and late (Green) referrals cases with AIS. b) mean and median survival time observed in short term and at 18th months post hospital discharge among early and late referrals cases with AIS

Multivariate analysis showing adjusted and unadjusted odds ratio of dependent and/expired outcome in early and later referrals AIS cases is depicted in [Table/Fig-6]. After adjusting for confounders, no significant impact was observed in short term outcome (AOR 95% CI 1.811,) and long term outcome at 12 months (AOR 95% CI 1.089) and 18 months (AOR 95% CI 1.419) among early and late admission AIS cases.

To study utility of serum biomarkers for prognosis along with severity of infection in AIS patients, stroke markers like NSE and S-100 ββ along with ITHIH4 peptides were evaluated in both early and late referrals. Both groups of patients were further categorized as improved, dependent/ expired based on hospital outcome from point of admission to discharge. Analysis of 9 ITIH4 peptides revealed significant association of peptides 5 and 7 with level of improvement in early referral cases [Table/Fig-7] Moreover, no such association between peptides was observed in both improved and expired cases of late referrals group. Similarly, estimation of markers NSE and S-100 ββ showed significant association between their serum levels along with level of improvement in early referrals compared to late referrals. Improved cases in early referrals group had significantly low levels of NSE and S-100 ββ compared to expired cases. In contrast, levels of NSE and S-100 ββ were found to be significantly raised in improved late referrals cases on admission, thereby showing impact of early admission on prognostic outcome in AIS stroke. Levels of both NSE and S-100 ββ were found to be significantly raised in expired cases similar to that observed in dependent cases in early referrals group [Table/Fig-8].

[Table/Fig-7]:

Represents Box plots for absorption values of nine ITIH4, on admission and discharge in serum samples of improved and expired/dependent AIS patients admitted within 24 hours (n=45) and after 24 hours (n=54) respectively. Results were expressed as median values with lower and upper quartiles. Whiskers displayed non-outliner maximal and minimal values

[Table/Fig-8]:

Represents Box plots for absorption values of NSE and S-100 ββ post admission and discharge in serum samples of improved and expired/ dependent AIS patients admitted within 24 hours (n=45) and after 24 hours (n=54) respectively. Results were expressed as median values with lower and upper quartiles. Whiskers displayed non-outliner maximal and minimal values

Time interval from onset of stroke to treatment plays a vital role in AIS patients. Admission of patients within window period post occurrence of stroke has been linked with better treatment, outcome and recovery rates [9]. The present study investigated impact of admission time on severity of stroke outcome among patients admitted within 24 hours and after 24 hours after stroke occurrence. 104 patients admitted to neurology department of CIIMS, were categorized into early and late referrals based on arrival time. Biomarkers of stroke: NSE, S-100 ββ and ITIH4 were also investigated in patients of both groups to determine utility of the such markers in prediction of stroke severity with respect to different admission time.

Baseline data of admitted patients showed more number of cases in older age group in both categories of patients, with males predominating than females, which is in accordance with earlier studies [10,11]. Similarly our results are in agreement with other reports which suggest better eligibility for thourombolysis treatment and surgical interventions in patients that arrived earlier as compared to late admission cases. In our study, almost 21% early referrals cases underwent thrombolysis treatment compared to late referrals where none of the cases underwent the same. However, no significant difference among risk factors like alcohol, smoking, and diabetes were observed in either category of patients. Multivariate analysis also revealed no significant impact of above confounders on outcome of AIS in both study groups. Major reason for this may be possibly due to less number of overall cases with smoking, diabetes and alcohol recruited in either group. Studies have shown HTN to be one of the major risk factors linked to AIS outcome [12]. From our observation, although number of patients with hypertension were more or less similar in both groups, hypertension accounted for almost 71% of overall stroke cases in population under study.

Early treatment with recombinant tissue plasminogen activator (rtPA) improves functional outcome by effectively reducing disability and dependency [13]. In our study, we observed similar trend wherein despite higher stroke severity in early referrals cases, we observed marginally better rates of recovery in them in terms of hospital outcome as compared to late referrals who had less severity. The outcome results at long term follow up were compared to that at time of discharge during hospital. Although not significant, there were more number of early referrals that showed improvement at time of discharge, with low conversion rates into dependency during long term outcome. Major reasons for lack of significance can be lesser number of follow up cases in long term outcome. Similarly, it was not possible to trace outcome of all cases in each follow due to unavailability of patients.

Long term outcomes carried out at 12_th and 18_th post discharge indicated no significant difference in death /dependency. Kaplan-Meier estimates of cumulative risk of second stroke in patients after discharge indicated similar survival rates in both early and late referral cases. These results suggest that although admission time may play a vital role in terms of therapeutic interventions in short term outcome, it may have little impact on death/dependency in terms of long term stroke outcome.

Limited evidences are available on impact of admission time on functional outcome in AIS patients. Other studies in these regard suggest variable impact of admission time in AIS cases. Study done by Haeusler et al., on impact of admission time on non working hours after thrombolysis for stroke showed limited impact of admission time of short term outcome in stroke [14]. Another study by Salter et al., suggests better functional gains and shorter stay in patients admitted earlier after 30 days post stroke occurrence [5]. Despite debatable evidences on admission time on stroke outcome, early admission to stroke care unit is generally encouraged. In the current study, our results suggests limited impact of admission time on stroke outcome and recovery in both cases however as discussed earlier, patients have better chances of recovery despite high stroke severity if admitted earlier due to greater possibility of undergoing tPA therapy. However, further studies in this regard needs to be carried out to further justify the impact on admission time on short and long term outcome in AIS patients.

Another aspect of the study was to evaluate biomarkers of neuronal damage in AIS patients to study impact of time of admission on level of prognosis in both patient groups. Both early and late referrals were further categorized as improved and dependent/ expired based on mRS scale at the time of discharge. Stroke markers, NSE, S-100 ββ and ITIH4 peptides were evaluated at time of admission as well as discharge in improved and cases that were dependent or expired. Analysis showed significant association of ITIH4 peptides with level of improvement in early referrals cases. Similarly improved cases in early referrals were associated with low NSE and S-100 ββ levels thereby showing better prognosis. On the contrary, improved cases in late referrals were associated with similar ITIH4 levels at admission and discharge. Although improved late referrals were associated with low NSE and S-100 ββ, they showed comparatively higher levels of both markers on admission, compared to improved cases in early referral group, suggesting that late admission is associated with more neuronal damage and poor prognosis. The results also suggest that although baseline data showed limited impact of time of admission on post stroke outcome, analysis of stroke markers provide significant information on prognostic outcome and stroke severity associated with time of admission in AIS patients.

NSE is a dimeric isoenzyme of the glycolytic enzyme enolase and is found mainly in the neurons and cells of the neuroendocrine system [15]. Thus high levels of NSE in serum/CSF often indicate neuronal damage in case of neurological disorders such as stroke [16]. Some studies have shown a positive correlation between NSE levels and infarct volume in patients of AIS [17,18]. S100 ββ is a calcium-binding protein expressed mainly in human astroglial cells. Because astroglia are as sensitive as neurons to hypoxia, serum S100 ββ levels have the potential to be a surrogate marker for neuronal damage and damage to the blood-brain barrier [19]. Our results are in agreement with above evidences, wherein improved cases in late referrals had higher NSE and S-100 ββ levels on admission compared to early referrals group, suggesting that late admission of AIS cases are associated with more neuronal damage than cases which are admitted earlier.

Another marker that we analysed was ITIH4, which is 120kDa anti- inflammatory marker earlier reported by our group. Studies by Kashyap P et al., have shown ITIH4 to be specifically expressed in serum of healthy patients and down regulated in AIS cases [8]. In the present study, peptides of ITIH4 were designed and evaluated in patient groups. Results suggests a significant association of ITIH4 levels with level of improvement especially in cases admitted earlier which is in agreement with our earlier published observation. Moreover, results suggests that peptides based assay of stroke markers may play an important role in stroke diagnosis by offering dual advantage of being cost effective and an alternate to antigen based assay for stroke prognosis during hospital outcome.

The present study had certain limitations. Major limitation includes the low sample size. Another limitation is the lack of follow up in all the patients in prediction of long term outcome. The reason for this is the change of address and contact numbers of majority of patients’ admitted in our institute, since most of them were from outside the state which limited their recruitment for follow up study. Lack of estimation of stroke markers in follow up samples including at hospital discharge was also one of the limitations associated with the study. However, lack of such studies carried out in Central India in this regard warrants an edge over other studies despite the number of limitations associated with it.

To conclude, early admission of AIS patients are associated with better hospital outcome and low levels of NSE, S-100 ββ and ITIH4 however it has no major impact on long term outcome in patients. Further studies focusing on prognostic aspect with large sample size with adequate follow up is therefore needed to justify the results.