Aβ42 in the cerebrospinal fluid has become widely accepted as an early marker flagging a person’s path toward Alzheimer’s disease. Yet this obstreperous peptide has bedeviled researchers by posting variable measurement results on all levels—between runs, batches, assays, and labs. This stubborn lack of precision is no way to roll out a test for use in multicenter therapy trials, much less for routine clinical diagnosis. Hence a trans-Atlantic effort has sprung up to develop a better way. On October 21, this multiyear effort took a leap forward with the formal acceptance of a mass spectrometry procedure for determining the concentration of Aβ42 in CSF. The recognition came from the Joint Committee for Traceability in Laboratory Medicine (see listing). As part of the European Commission, this regulatory body evaluates reference methods.
“This is a highly important step in the standardization procedures for AD biomarkers,” said Kaj Blennow of the University of Gothenburg, Sweden, who spearheaded the effort with his colleague Henrik Zetterberg.
This step does not mean that labs around the world will now switch to mass spec when measuring CSF Aβ. Rather, the newly anointed reference procedure is part of what the scientists call a calibration hierarchy. This is an established, two-pronged scheme to standardize the measurement of difficult analytes. It calls for the separate development and validation of a method, aka a reference measurement procedure (RMP), and a certified reference material (CRM). The RMP is used to analyze the CRM and to assign it a definitive number value, say, 500 pg/ml. Aliquots are then sent to assay manufacturers, such as Roche, Fujirebio, ADx Neurosciences, and others, which calibrate their commercial assays to these reference aliquots. The idea is that the end user that measures this biomarker—the central hospital lab of a diagnosing physician, a CRO running a clinical trial, a pharma company using its own in-house assay, even an academic lab running many CSF samples for clinical research—will not have to deal with calibration but simply have a more robust, reproducible test. “This calibration scheme will make all assays, regardless from which company, comparable to each other,” Blennow said.
This week’s approval of the RMP marks a halfway point in this process. “The certificate is a clear green light for us to move forward with the value assignment of the reference material. The rest of that work should not be that difficult,” Zetterberg wrote to Alzforum.
Why is this standardization work necessary? Most certified biochemical tests—e.g., blood glucose or cholesterol—are simpler than CSF Aβ or tau in that the analyte can be purified or synthesized and measured with tight reproducibility. Immune assays for protein biomarkers, including established ones such as troponin-t or PSA, vary much more in what they detect in the same sample. Hence they require a more intricate calibration scheme. “We need a gold standard against which every assay can get referenced, like the meter or the kilogram,” Blennow told Alzforum.
Alzforum has previously covered Blennow and Zetterberg’s recruitment of the International Federation of Clinical Chemistry (IFCC), which certifies clinical biochemical tests, and the Institute for Reference Materials and Measurement (IRMM) to help solve this problem. The IRMM is the EU’s version of the U.S. National Institute of Standards and Technology (NIST). It has expertise in developing methods and materials for the most recalcitrant measurement problems and is the leading nonprofit distributor of reference materials. Both bodies collaborate on this project with overlapping working groups. They agreed to step in when the work of the Alzheimer's Association CSF Quality Control program and the Global Biomarker Standardization Consortium (GBSC) began to show that internal feedback mechanisms would be insufficient to reduce variability of the current set of research-grade assays (see Aug 2012 news; May 2014 news; Aug 2015 conference news). Many academic and company researchers who serve on the GBSC now work with IFCC and IRMM to develop the reference procedures and material.
Importantly, the IRMM and NIST have no regulatory authority such as the EMA and FDA. These drug agencies tend to independently repeat each other’s work before issuing approval in their respective jurisdictions. Rather, the IRMM and NIST work with each other when their interests overlap. They are aware of each other’s work but do not repeat it locally. Mark Lowenthal of NIST serves on the IRMM’s CSF standardization group. “We are supportive of the JCTLM’s acceptance of the RMP for Aβ1-42, and subsequent reference material development,” Lowenthal wrote to Alzforum.
Last year, the reference method was published (Leinenbach et al., 2014) and submitted to the JCTLM. Subsequently, the agency scrutinized the paper, its ample attendant documentation, and tested out the method, but in the end accepted it without further ado. A second, similar method is still undergoing JCTLM review (Korecka et al., 2014). Both methods correlated well in a comparison study, Blennow told Alzforum, noting that ultimately there may be several reference methods but only one certified reference material.
What’s next? Measurement procedure in hand, the material becomes the next priority. Blennow’s center has produced 5 liters of CSF at three Aβ42 concentration levels. IRMM scientists are about halfway through a one-year validation of its homogeneity and stability. Once that is done, in 2016, a round-robin study of participating labs will apply the reference procedure to assign number values and an uncertainty range. Then IRMM can ship the calibration "meter" to manufacturers. If all goes well, routine clinical care centers could see certified assays arriving by early 2017, Blennow said.
Furthermore, the reference method and material will make it possible to set cutoff values for the sets of proposed diagnostic criteria that have started to come into use in therapeutic trials and at some clinical centers (Dubois et al., 2014; Sperling et al., 2011; Albert et al., 2011). Though these criteria call for the use of biomarkers, currently they only require that CSF Aβ be low in preclinical or early AD, but do not state exactly how low. “In the future, it will be defined that Aβ below, say, 500 or 600 is considered indicative of brain amyloidosis, just like there are specific cutoffs for blood glucose and cholesterol,” said Blennow.—Gabrielle Strobel
- Metrology, Certification Heavies Take CSF Tests Under Their Wings
- More Accurate Ways to Measure CSF Aβ Debut
- CSF Aβ Assays Remain Fickle: Robots to the Rescue?
- Leinenbach A, Pannee J, Dülffer T, Huber A, Bittner T, Andreasson U, Gobom J, Zetterberg H, Kobold U, Portelius E, Blennow K, IFCC Scientific Division Working Group on CSF proteins. Mass spectrometry-based candidate reference measurement procedure for quantification of amyloid-β in cerebrospinal fluid. Clin Chem. 2014 Jul;60(7):987-94. Epub 2014 May 19 PubMed.
- Korecka M, Waligorska T, Figurski M, Toledo JB, Arnold SE, Grossman M, Trojanowski JQ, Shaw LM. Qualification of a surrogate matrix-based absolute quantification method for amyloid-β₄₂ in human cerebrospinal fluid using 2D UPLC-tandem mass spectrometry. J Alzheimers Dis. 2014;41(2):441-51. PubMed.
- Dubois B, Feldman HH, Jacova C, Hampel H, Molinuevo JL, Blennow K, DeKosky ST, Gauthier S, Selkoe D, Bateman R, Cappa S, Crutch S, Engelborghs S, Frisoni GB, Fox NC, Galasko D, Habert MO, Jicha GA, Nordberg A, Pasquier F, Rabinovici G, Robert P, Rowe C, Salloway S, Sarazin M, Epelbaum S, de Souza LC, Vellas B, Visser PJ, Schneider L, Stern Y, Scheltens P, Cummings JL. Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria. Lancet Neurol. 2014 Jun;13(6):614-29. PubMed.
- Sperling RA, Aisen PS, Beckett LA, Bennett DA, Craft S, Fagan AM, Iwatsubo T, Jack CR Jr, Kaye J, Montine TJ, Park DC, Reiman EM, Rowe CC, Siemers E, Stern Y, Yaffe K, Carrillo MC, Thies B, Morrison-Bogorad M, Wagster MV, Phelps CH. Toward defining the preclinical stages of Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement. 2011 May;7(3):280-92. Epub 2011 Apr 21 PubMed.
- Albert MS, DeKosky ST, Dickson D, Dubois B, Feldman HH, Fox NC, Gamst A, Holtzman DM, Jagust WJ, Petersen RC, Snyder PJ, Carrillo MC, Thies B, Phelps CH. The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement. 2011 May;7(3):270-9. Epub 2011 Apr 21 PubMed.