Engbers-Buijtenhuijs, P.

Feijen, J.

Haanen, C.

Kamphuis, M.

Poot, A.A.

Sluijs Veer, G. van der

Vermes, I.

Kamphuis, Marloes

van der Sluijs Veer, G.

Poot, Andreas A.

Feijen, Jan

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A novel time resolved fluormetric assay of anoikis using Europium-labelled annexin V in cultured adherent cells

University of Twente Research Information

74 Ned Tijdschr Klin Chem Labgeneesk 2005, vol. 30, no. 218. Effects of processing and storage conditions on CSF Amyloid-ß(1-42) and Tau concentrations: implicationsfor use in clinical practiceN.S.M. SCHOONENBOOM1,2,*, C. MULDER2,*, H. VANDERSTICHELE3, E.J. van ELK2, A. KOK2, G.J. van KAMP2,Ph. SCHELTENS1, M.A. BLANKENSTEIN2*, EQUAL CONTRIBUTIONAlzheimer Center and Department of Neurology1, and Department of Clinical Chemistry2, VU University Medical Center,Amsterdam, The Netherlands; Innogenetics NV3, Ghent, BelgiumIntroduction: Reported concentrations of amyloid-ß(1-42)(Aß42) and tau in cerebrospinal fluid (CSF) differ among re-ports. We investigated the effects of storage temperature, re-peated freeze/thaw cycles, and centrifugation on the concen-trations of Aß42 and tau in CSF.Methods: Stability of samples stored at -80°C was determinedby use of an accelerated stability testing protocol according tothe Arrhenius equation. Aß42 and tau concentrations weremeasured in CSF samples stored at 4, 18, 37, and -80°C. Re-lative CSF concentrations (%) of the biomarkers after onefreeze/thaw cycle were compared with two, three, four, five,and six freeze/thaw cycles. In addition, relative Aß42 and tauconcentrations in samples not centrifuged were compared withsamples centrifuged after 1, 4, 48 and 72 h. Results: Aß42 and tau concentrations were stable in CSF whenstored for a long period at -80°C. CSF Aß42 decreased with20% during the first two days at 4, 18, and 37°C comparedwith -80°C. CSF tau decreased after storage for 12 days at37°C. After three freeze/thaw cycles CSF Aß42 decreased20%. CSF tau was stable during six freeze/thaw cycles. Cen-trifugation did not influence the biomarker concentrations.Conclusion: Repeated freeze/thaw cycles and storage at 4, 18, and37°C influence the quantitative result of the Aß42 test. Preferably,samples should be stored at -80°C immediately after collection. 19. A novel time resolved fluormetric assay of anoikis using Europium-labelled annexin V in cultured adherent cellsP. ENGBERS-BUIJTENHUIJS1,2, M. KAMPHUIS1, G. van der SLUIJS VEER1, C. HAANEN1, A.A. POOT2, J. FEIJEN2, I. VERMES1,2Department of Clinical Chemistry1, Hospital Group Medisch Spectrum Twente, Faculty of Science and Technology,Polymer Chemistry and Biomaterials Group2, University of Twente, Enschede, The NetherlandsIntroduction: Adherent cells are dependent for survival fromcontinuous engagement of cellular integrins to the extra cel-lular matrix. Detachment of adherent cells from the matrixinduces almost immediately apoptosis, a phenomenon desig-nated as ‘anoikis’ or homelessness (1). Anoikis is of pertinentrelevance to tissue homeostasis. We developed a new verysensitive method to analyse anoikis in adherent cell culturesusing the principles of the DELFIA assays (2). Methods: A new and sensitive method to analyse apoptosis andanoikis of adherent cell types using a time resolved fluoromet-ric DELFIA assay with Europium-labelled Annexin V was de-veloped. Anoikis was induced with tumor necrosis factor-alpha/cycloheximide and three cell fractions of the cell cultureswere prepared and analysed. Fraction 1 consisted of adherentcells, analysed while growing on their support (without detach-ment by trypsinisation). Fraction 2 contained detached cellsdue to anoikis (floating cells) and fraction 3 contained apop-totic bodies. Both fractions 2 and 3 were present in the culturemedium and were isolated by differential centrifugation. Results: TNF-alpha treatment of three different types of adher-ent cell cultures induced a significant increase of the amountof floating cells (anoikis) and apoptotic bodies compared tocontrol cell cultures. Also in the adherent cell fractions a smallamount of apoptosis was observed.Conclusion: The novel time resolved assay provides the abilityto analyse the cell death cascade in adherent cell cultures of thesame sample at the same time in a sensitive and reproducibleway. According to our knowledge this is the first direct quanti-tative technique to measure anoikis in adherent cell cultures.Literature: 1. Frisch et al. J Cell Biol 1994, 124: 619. 2. Hemmilä etal. Anal Biochem 1984, 137: 335.20. External Quality Control of Cerebrospinal Fluid Markers for Alzheimer’s DiseaseM.A. BLANKENSTEIN1, A. KOK1, G.J. van KAMP1, N.S. SCHOONENBOOM2, Ph. SCHELTENS2, K. BLENNOW3Departments of Clinical Chemistry1 and Neurology2, VU University Medical Center, Amsterdam, The Netherlands,Department of Clinical Neuroscience3, Sahlgrenska University Hospital, Mölndal, SwedenIntroduction: Amyloidß 1-42 (Aß), Tau (tTau) and phos-porylated Tau-181 (pTau) are gradually being accepted as bio-markers for Alzheimer’s disease (AD). Measurement of theseproteins is typically performed in cerebrospinal fluid (CSF), inthe absence of reliable methods for their detection in moreaccessible body fluids. Different marker levels are reported bydifferent groups, pre-analytical factors have been investigated(1), but QC programmes are currently lacking. This investiga-tion was performed to compare results of different laboratoriesand to establish the feasability of an international QC schemefor AD markers. Methods: CSF specimens with three different levels of theanalytes were distributed to 15 laboratories worldwide in-volved in AD biomarker measurements, which agreed to par-ticipate. Laboratories were asked to report their assay results,information on the assay used and the condition in which thespecimens arrived.Results: Three months after dispatch of the specimens 11 labo-ratories reported their results. Five measured all three markers,six measured one or two analytes. Sometimes a lack of samplewas reported. Coefficients of variation for Aß were 20.3, 52.5and 27.9% at mean levels of 511, 393 and 755 pg/ml respec-tively (n=9). For Ttau the CV’s were 7.9, 28.6 and 15.4% at878, 389 and 219 pg/ml respectively (n=7). For pTau at 107,49 and 35 pg/ml the CV’s were 12.1, 9.6 and 16.0% for 7laboratories. One laboratory reported extremely differing re-sults which were excluded from the present report.Conclusion: Although CSF marker results differed consider-ably between laboratories, the preliminary results of this studyare encouraging. It is concluded that establishment of an inter-national quality assessment schedule for AD markers in CSF isfeasible.Literature: Schoonenboom et al: Clin Chem 2005 In Press 

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A novel time resolved fluormetric assay of anoikis using Europium-labelled annexin V in cultured adherent cells

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