METHOD FOR PREDICTING THE RETURN TO FUNCTIONAL AUTONOMY IN A SUBJECT SUFFERING FROM AN ACUTE EVENT

Abstract

A method for predicting the return to functional autonomy in a subject suffering from an acute event, including the steps of i) determining neopterin level in a biological sample obtained from the subject; ii) comparing the level with its predetermined reference neopterin level and iii) predicting that the subject will have a long time to return to functional autonomy when the level of neopterin is higher than its predetermined reference neopterin level or predicting that the subject will have a short time to return to functional autonomy when the level of neopterin is lower than its predetermined reference neopterin level.

Claims

1-15. (canceled)

16. A method for predicting the return to functional autonomy in a subject suffering from an acute event comprising the following steps: i) determining neopterin level in a biological sample obtained from said subject; ii) comparing said level with a predetermined reference neopterin level; and iii) predicting that the subject will have a long time to return to functional autonomy when the level of neopterin is higher than its predetermined reference neopterin level or predicting that the subject will have a short time to return to functional autonomy when the level of neopterin is lower than its predetermined reference neopterin level.

17. The method according to claim 16, wherein the subject is a human subject.

18. The method according to claim 17, wherein the subject is an elderly subject.

19. The method according to claim 18, wherein the subject is older than 65 years old.

20. The method according to claim 17, wherein the subject is older than 75 years old.

21. The method according to claim 16, wherein the acute event is selected from the group comprising hip fracture, pelvic fracture, tibia fracture, fibula fracture, foot fracture, wrist fracture, clavicle fracture and hand fracture.

22. The method according to claim 21, wherein the acute event is hip fracture.

23. The method according to claim 16, wherein the biological sample is selected from the group comprising a blood sample, a serum sample, a plasma sample, an urine sample, a saliva sample, a cerebrospinal fluid sample and a feces sample.

24. The method according to claim 23, wherein the biological sample is a blood sample.

25. The method according to claim 16, wherein the biological sample is fresh, fresh frozen or frozen.

26. The method according to claim 16, wherein the biological sample is obtained at a time chosen from the group comprising: (a) following medical management, (b) between the acute event and a surgery operation aimed to treat the acute event, (c) during the surgery operation, (d) the day following the surgery operation, (e) from one to twelve days following the surgery operation, and (f) from twelve days to twelve months following the surgery operation.

27. The method according to claim 26, wherein the biological sample is obtained at hospital arrival.

28. The method according to claim 26, wherein the biological sample is obtained from seven to ten days following the surgery operation.

29. The method according to claim 26, wherein the biological sample is obtained from six to twelve months following the surgery operation.

30. The method according to claim 16, wherein step i) is performed by ELISA, mass spectrometry, high-performance liquid chromatography or Lateral Flow Immunoassay.

31. The method according to claim 16, wherein the predetermined reference neopterin level is superior to 10 nmol/L.

32. The method according to claim 31, wherein the predetermined reference neopterin level is comprised between 10 and 17 nmol/L.

33. The method according to claim 31, wherein the predetermined reference neopterin level is comprised between 12 and 15 nmol/L.

34. The method according to claim 31, wherein the predetermined reference neopterin level is 15 nmol/L.

35. A kit for predicting the return to functional autonomy in a subject suffering from an acute event.

36. The kit according to claim 35, comprising: a collection mean for the sample, and the reagents necessary to carry out a method for predicting the return to functional autonomy in a subject suffering from an acute event comprising the following steps: i) determining neopterin level in a biological sample obtained from said subject; ii) comparing said level with a predetermined reference neopterin level; and iii) predicting that the subject will have a long time to return to functional autonomy when the level of neopterin is higher than its predetermined reference neopterin level or predicting that the subject will have a short time to return to functional autonomy when the level of neopterin is lower than its predetermined reference neopterin level.

Description

DESCRIPTION OF THE FIGURES

[0099] FIG. 1A-1C are a combination of graphs showing Neopterin levels in elderly at different times. The concentration of neopterin (in nmol/1) was measured at three different times (A) At hospital discharge; (B) at rehab discharge; (C) at six months post-fracture and analyzed for the hip fracture patients according to 2 clinical outcomes: able to walk (black squares, n=84; 28 and 24; respectively) or unable to walk (n=14; 18 and 59; respectively). p are calculated with a non-parametric Mann-Whitney test.

[0100] FIG. 2A-2C are a combination of graphs showing that Neopterin levels in elderly are linked to a time to return to functional autonomy. The concentration of neopterin of the patients was measured at arrival to hospital (in nmol/1), and compared to three clinical parameters at different time (A) Maximal recovery at hospital discharge (B) Walking Delay at 30 days after arrival at hospital (C) Short Physical Performance Battery (SPPB) at 30 days after arrival at hospital. p are calculated with a non-parametric Mann-Whitney test.

[0101] FIG. 3A-3C are a combination of graphs showing that Neopterin levels in elderly are linked to a time to return to functional autonomy, with others tests and indexes with reference to FIGS. 2A-2C. The concentration of neopterin of the patients was measured at arrival to hospital (in nmol/1), and compared to three clinical parameters at different time (A) Activities of Daily Life (B) Instrumentalized Activities of Daily Life (C) Short Physical Performance Battery (SPPB) at 30 days after surgery. p are calculated with a non-parametric Mann-Whitney test.

[0102] FIG. 4 is a Receiver Operating Characteristics (ROC) curve depicting sensitivity and specificity of prediction model for Functional Autonomy-related to ability to walk at D30 based on neopterin concentration measured at arrival to hospital. AUC=0.91; Specificity=100%; Sensitivity=75%; threshold: =15 nM.

DETAILED DESCRIPTION—EXAMPLE

[0103] More than two hundred elderly individuals >75 years old were screened to participate in our study. One third of the cohort was constituted of healthy individuals whereas the other ones were admitted in emergency department for hip fracture. Of note, patients suffering from osteoporosis were not included; therefore only accidental hip fracture patients were included in our cohort (n=120). Longitudinal follow-up post fracture was designed with blood samples taken at their arrival to hospital (DO; PRE), after surgery (POST), at their discharge from the hospital (D7-D10; EXIT) and at long-term post fracture (M6-M12; FOLLOW UP).

[0104] Plasma as well as PBMCs have been cryopreserved until use. Experiments always included samples from healthy controls compared to hip-fracture patients (all timepoints being performed simultaneously).

[0105] We choose to focus on the level of neopterin reached by the patients at their arrival to hospital.

[0106] We analyzed neopterin as a predictive marker of functional recovery post hip fracture, according to the following protocol.

[0107] Materials and Methods

[0108] Experiments were performed using a commercially available kit from IBL-international by following manufacturer's instructions (described below).

[0109] Principle of the Test

[0110] Neopterin ELISA kit is based on competitive binding of human Neopterin from serum samples and enzyme-labeled Neopterin to Neopterin specific antibodies immobilized on microtiter plates. After a washing step, chromogenic substrate is added and color developed. The enzymatic reaction (blue color) is inversely proportional to the amount of Neopterin present in the sample. The reaction is terminated by adding stopping solution (converts blue to yellow). Absorbance is then measured on an ELISA reader at 450 nm and the concentration of Neopterin in samples and control is read off the standard curve.

[0111] Sensitivity

[0112] The lower detection limit is calculated from the standard curve by determining the resulting concentration of the mean OD of Calibrator A (based on 10 replicate analyses) minus 2 SD. The sensitivity of the Neopterin ELISA kit is 0.7 nmol/L.

[0113] Specificity (Cross Reactivity)

[0114] The following compounds were tested for cross-reactivity with the Direct Neopterin ELISA kit. No significant interference was detected at the following concentration. Hemoglobin 35 mg/dL, Bilirubin 2.25 mg/d£, Triglyceride 125 mg/dL.

[0115] Specimen Collection and Handling

[0116] Blood was collected by venipuncture, allowed to clot, and serum was separated by centrifugation at room temperature. The serum was not heat inactivate. If sera cannot be immediately assayed, these could be stored at −20° C. for up to six months. While the sample can be frozen and thawed, repeated freezing and thawing of samples should be avoided. Do not use specimens containing NaN3. Samples appearing turbid should be centrifuged before testing to remove any particulate material.

[0117] Our experiments were performed on frozen human plasma. The plasma samples have been aliquoted, protected from light and cryopreserved at −80° C. for one year before usage. Thawing has been done by putting samples in a fridge allowing slow thawing. Thereafter, thawed plasma have been centrifuged at 1000 rpm during 10 min in the dark.

[0118] Reagents Preparation

[0119] Stock Wash buffer was diluted (1:20) with water and stored at 4° C. for 1 month.

[0120] All reagents were at room temperature prior to their use.

[0121] For the following experiment, human plasma aliquots were used undiluted and manipulated in the dark. If necessary, samples to be used would have been diluted with assay buffer (1:10).

[0122] Storage and Stability

[0123] The microtiter well plate and all other reagents are stable at 2-8° C. until the expiration date printed on the label. The whole kit stability is usually 6 months from the date of shipping under appropriate storage conditions. The unused portions of the standards should be stored at 2-8° C. or stored frozen in small aliquots.

[0124] Test Procedure

[0125] All reagents were allowed to reach room temperature before use. The required number of coated strips were removed and arranged on the microtiter well plate.

[0126] The microtiter well strips to be used on the plate were labeled and the wash buffer was diluted with water (1:20).

[0127] 20 μl of standards, controls, and samples were pipeted into appropriate wells in duplicate.

[0128] 100 μl of ready-to-use enzyme conjugate were added into each well, followed by 50 μl of ready-to-use Neopterin antiserum into each well, before gently mixing for 5-10 seconds. The plate was covered and incubated for 90 minutes at 18-25° C. on orbital shaker (500 rpm) in the dark.

[0129] The well contents were aspirated and the plate blotted on absorbent paper before immediately washing the wells 4 times with 300 μl of 1× wash buffer.

[0130] 150 μl of TMB substrate solution was added before gently mixing for 5-10 seconds. The plate was covered and incubated for 10 mins in the dark at 18-25° C.

[0131] The reaction was stopped by adding 150 μl of stop solution to all wells at the same timed intervals as in step 5. Gentle mixing was performed for 5-10 seconds to have uniform color distribution (blue color turned yellow).

[0132] Absorbance was measured at 450 nm using an ELISA reader within 15 min.

[0133] Calculation of Results

[0134] The absorbance of all duplicates were averaged before subtracting the averaged non-specific binding (NSB) absorbance from the average obtained above. This yields the net absorbance. Net absorbance was divided by the net zero standard absorbance (Bo) to obtain the percent bound (% B/Bo).

[0135] Formula:

[0136] Abs. (sample)−Abs. (NSB)

[0137] % B/Bo=×100 Abs. (zero standard)−Abs. (NSB)

[0138] Abs.=average absorbance of duplicate wells

[0139] NSB=non-specific binding (also known as the blank) Sample=particular serum or standard being calculated Zero Standard=0 nmol/L standard or 100% binding wells.

[0140] Construct a plot of the percent bound (Y-axis) versus the concentration of the neopterin standards ((X-axis) starting with the 0.5 nmol/L point. Either logit-log or semi-log graph paper may be used. This yields the standard curve.

[0141] Using the standard curve, the neopterin concentration of each sample was determined.

[0142] Expected Reference Neopterin Level

[0143] Usually, healthy subjects show the following values: healthy subjects neopterin level (Normal): <10 nmol/L (0.3-3.0 ng/mL).

[0144] Conversion: Neopterin (nmol/L)×0.253=ng/mL

[0145] Results & Discussion

[0146] As shown in FIG. 1, the level of neopterin was statistically different between the hip fracture patients who were able to walk compared to those who were unable to perform the tests. This observation was valid for their ability to walk at hospital discharge (FIG. 1A.; p=0.002), after their stay in rehab unit (FIG. 1B.; p<0.0001), and at Month 6 post fracture (FIG. 1C.; p=0.004).

[0147] A cutoff value between a population of functionally autonomous subjects and a population of dependent subjects was determined at 15 nmol/L (75% sensitivity; 100% specificity; AUC=0.91) based on Receiver Operating Characteristic analysis related to ability to walk at D30 post surgery (FIG. 4).

[0148] We also found correlations between the level of neopterin measured at arrival to hospital and: [0149] the maximal recuperation score (FIG. 2A, p=0.006; r=−0.47) [0150] the time necessary to walk 5 m at day 30 post-surgery (FIG. 2B, p=0.002; r=0.46) [0151] the score obtained for the Short Physical Performance Battery (SPPB) measured at D30 post-surgery (FIG. 2C, p=0.002; r=−0.5)

[0152] Additional comparisons between the level of neopterin measured at arrival to hospital and functional autonomy of the subjects were performed, emphasizing the correlation between neopterin and return to functional autonomy: [0153] the score obtained for the Activities of Daily Life (ADL) measured at D30 post-surgery (FIG. 3A, p=0.0008) [0154] the score obtained for the Instrumentalized Activities of Daily Life (IADL) measured at D30 post-surgery (FIG. 3B, p=0.009) [0155] the score obtained for the Short Physical Performance Battery (SPPB) measured at D30 post-surgery (FIG. 3C, p=0.01)