Pre-analytical procedures

1. CSF Tau Protein Phosphorylated at Threonine 231 as a Potential Biomarker of Early Stages of Alzheimer’s diseaseMirjana Babić1, Ivana Kuštek2, Nataša Klepac3, Fran Borovečki3, Patrick R. Hof4, Goran Šimić1* 1 Croatian Institute for Brain Research, University of Zagreb School of Medicine, Croatia 2 Faculty of Science, University of Zagreb, Croatia 3Clinic of Neurology, Clinical Hospital Center Zagreb, Croatia 4Fishberg Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, USA Croatian Science Foundation Abstract Mild cognitive impairment (MCI) is a syndrome characterized by cognitive impairment without dementia, which primarily affects episodic memory. Patients with MCI often have an initial stage of Alzheimer's disease (AD). Tau protein phosphorylated at threonine 231 (pT231) has recently been proposed as a possible biological marker of early AD. In this pilot study we tested whether the measurement of pT231 in the cerebrospinal fluid (CSF) correlates with other pathological features characteristic of AD in patients with MCI. A positive answer to this question would mean that this marker could be used to detect AD in the early stage of the disease. The concentration of pT231 was determined using ELISA kits in the CSF of 34 patients with probable AD, 25 MCI patients, and 7 healthy controls. Levels of pTau231 were significantly higher in the group of AD patients than in MCI patients or healthy control. ROC curve analysis showed that pTau231 could differentiate AD patients from healthy control with a sensitivity of 73.5% and a specificity of 85.7%. The cut-off level for pTau231 (1.904 U/ml) was defined when the sum of specificity and sensitivity was maximized. About 32% of MCI patients had pTau231 levels higher than the cut-off, while the rest of MCI patients (68%) had normal pT231 levels. These data suggest that about one third of MCI patients could be defined as a high-risk group with amnestic MCI. However, a follow-up period of at minimum 5 years is necessary to determine whether these patients will actually convert to AD. In conclusion, pT231 may be a useful biological marker for AD detection in the preclinical stage of the disease. Introduction Methods Results Alzheimer’s disease (AD) is the major primarycause of dementia. Ferri and collaboratorsestimated that 24 million people suffered fromdementia in 2005, with this number reaching81 million by 2040. Clinical diagnosis ofAD, which is still based on symptomatology, isaccurate in only 63 to 90% of dementia cases. A growing number of potential treatmentsfor AD are in different phases of preclinicaland clinical research and thus much effort isdedicated to identify reliable biomarkers toenable an accurate diagnosis of AD. Three main cerebrospinal fluid (CSF)biomarkers of AD, amyloid β1-42 (Aβ1-42), totaltau (tTau), and phosphorylated forms of tau(PTau) reflect two major neuropathologicalhallmarks of AD - neurofibrillary tangles andsenile plaques. These CSF biomarkers arealtered in early stages of AD, even before theoccurrence of the first dementia symptoms,and permit to differentiate patients withprodromal AD (i.e. those with mild cognitiveimpairment, MCI) who often progress to AD,from healthy controls. CSF biomarkers arealso used for differentiation of AD from otherprimary causes of dementia, such as vasculardementia, frontotemporal dementia (FTD), anddementia with Lewy bodies. Reductionof Aβ1-42 in CSF of AD patients is explained byAβ1-42 aggregation into senile plaques, increaseof tTau reflects neuronal degeneration,while elevation of pTau is a consequence ofneurofibrillary degeneration and consequenttangles formation in the brain. Althoughnumerous studies in which diagnostic accuracyof CSF biomarkers was analyzed have beenpublished, an ideal biomarker (with specificityand sensitivity over 85%) could not yet bedefined. Pre-analytical procedures Boxes represent the median, the 25th and the 75thpercentiles, bars indicate the range of data distribution. Circles representoutliers (values more than 1.5 box-length from the 75th/25thpercentile). The asterisks represent extreme values (value more than three box-lengthfrom the 75th/25thpercentile). All patients with suspected dementia wererecruited from the University Hospital Centre,Zagreb, underwent complete blood tests including electrolytes, albumin, thyroidfunction, levels of vitamin B12, VDRL testfor syphilis, Mini-Mental State Examination(MMSE), and neurological examination. After exclusion of patients with secondarycauses of dementia, selected 59 patients,upon signing the informed consent,underwent lumbar puncture. Out of these, 34 patients fulfilled NINCDS-ADRDA criteriafor probable AD, 25 patients suffered fromMCI. Additionally, seven healthy controlsubjects (HC) with no evidence of dementia,or neurologic and psychiatric symptoms, wereincluded. CSF was taken in the L3/L4 or L4/L5intervertebral spaces, always between 9 a.m.and 11 a.m., and collected in polypropylenetubes. Leukocyte and erythrocyte cell counts, lactate, glucose, total protein concentration,Treponema Pallidum HemagglutionationAssay (TPHA), and IgG index were alsodetermined in native CSF. CSFsamples were centrifuged for 10 minutes at 2,000 g, dispensed into 150 μl aliquots andstored at -80 degrees Celsius. Boxes represent the median, the 25th and the 75thpercentiles, bars indicate the range of data distribution. Circles representoutliers (values more than 1.5 box-length from the 75th/25thpercentile). The asterisks represent extreme values (value more than three box-lengthfrom the 75th/25thpercentile). Analytical procedures CSF PTau231 levels were determined using Invitrogen ELISA kit.ELISA analysis was performed according to theManufacturer’s protocol. Ptau231 concentrations were calculatedon plate reader using curve-fitting softwareand 4-parameter algorithm. Plateswere washed in an automatic washer. Proteinconcentrations were determined using the samealgorithm in GraphPad Prism 5.0 demo versionsoftware (San Diego, CA, USA). Mild cognitive impairment (MCI) is a syndrome characterized by cognitive impairment without dementia, which primarily affects episodic memory. Patients with MCI often have an initial stage of Alzheimer's disease (AD). Tau protein phosphorylated at threonine 231 (pT231) has recently been proposed as a possible biological marker of early AD. In this pilot study we tested whether the measurement of pT231 in the cerebrospinal fluid (CSF) correlates with other pathological features characteristic of AD in patients with MCI.A positive answer to this question would mean that this marker could be used to detect AD in the early stage of the disease. Acknowledgements Statistical analysis cut-off (pTau231) = 1.904 U/ml cut-off (t-Tau) = 213 pg/ml PTau231 levels among AD, MCI, and HC werecompared using a Kruskal-Wallis test(χ2=17.317; df=2; p<0.001), followedby the Mann-Whitney U test for pairwisecomparisons: AD vs MCI - (U=183; Z=-3.712; p<0.001) AD vs HC - (U=81; Z=-0.296; p=0.789) MCI vs HC - (U=38; Z=-2.806; p=0.004) Statistical analyses were performedwith SPSS 19.0.1 (SPSS Inc., Chicago, IL, USA)and MedCalc 12.4.0.0 (Mariakerke, Belgium).P values less than 0.05 were consideredstatistically significant. The authors are grateful to Milica Trbojević-Čepe (University Hospital Center Zagreb) for her help in preparation of this report. All patients signedstandard Patient Consent Form approved by theEthical Committee of the University HospitalCenter Zagreb (approval no. 01-20/53-1/2006signed on 26th June 2006). This research wasalso approved by the central Ethical Committeeof the University of Zagreb Medical School(case no. 380-59/11-500-77/90, class 641-01/11-02 signed on 19th May 2011). The workis funded by the Croatian Science Foundationgrant no. 09/16 “Detection and trackingof biological markers for early therapeuticintervention in sporadic Alzheimer’s disease”to G.Š., and in part by NIH grant P50 AG005138 to P.R.H. The authors declare no conflict of interest. 3 MCI patients (12%) had increased both t-Tau and pTau231, while 8 MCI patients (32%) had increased p-Tau231 only