Academy of Diagnostics & Laboratory Medicine - Scientific Short

Is Standardization an Issue for Stroke Biomarkers? The Case of GFAP

Konstantinos Makris and Joan Montaner

Although, no blood biomarker is used for stroke management, some candidates seem promising for specific clinical indications and might be implemented in clinics in the near future. In fact, biomarkers would be useful to improve or speed up stroke diagnosis, identifying specific subtypes of stroke or guiding etiologic diagnosis of stroke and later on predicting the response to reperfusion therapies. Moreover, beyond acute stroke, prediction of stroke outcome or aiding in identification of specific causes of worsening that complicate the course of stroke such as stroke-associated infections would be of extreme help for stroke physicians.(1)

Astrocytes are the most abundant cell types in the brain, providing both structural and functional support for neurons. Astrocytes have a unique structural protein, glial fibrillary acidic protein (GFAP) that was isolated and characterized 50 years ago. (2) Under physiological conditions GFAP is typically not present at detectable levels in the blood however in intracerebral hemorrhage (ICH), shear stress and mechanical forces during hematoma expansion result in astrocyte necrosis and destruction of the blood-brain barrier (BBB), which cause immediate release of GFAP into the systemic circulation. (3) On the other hand in Acute ischemic stroke (AIS), the release of GFAP occurs much more slowly, with peak levels 48-96 hours after onset of AIS. (4) Taking advantage of those differences in the temporal pattern of GFAP release into the bloodstream, several clinical studies have shown the diagnostic promise of GFAP, in stroke subtype differentiation, severity, hematoma volume, and even outcome in ICH, indicating that GFAP may be a useful biomarker in disease characterization and prognosis. However a recent metaanalysis found that its diagnostic accuracy was affected by the type of assay used to measure GFAP. (5)

GFAP is not an easy molecule to measure. Although encoded by a single gene, several isoforms/splice variants along with single nucleotide polymorphisms, posttranslational modifications (glycosylation, citrulination, acetylation) create a variety of molecular species that may enter the circulation after brain injury. Moreover conditions such as traumatic brain injury, stroke, and even neurodegenerative disorders are often associated with neuronal injury or death. In such situations glial damage leads to proteolytic conversion of the intact GFAP (50kDa) into breakdown products of variable molecular weight (38-44 kDa). These breakdown products add to the complexity of molecular species in systemic circulation when the BBB is compromised. (6, 7) Measurement of the breakdown products rather than the intact GFAP molecule could be useful to track the level and kinetics of astrocyte cell damage following brain trauma. (6)

GFAP exemplifies many of the challenges faced during adoption of novel biomarkers. Investigation of molecular species complexity and selection of antibodies are important for the design of robust immunoassays. Today there are several GFAP diagnostic tests (all with indication for research use only) in the market, plus some in-house assays that have been used in research. [reviewed in (5)] In current assays there is limited information if the antibodies only detect intact GFAP or are also able to capture breakdown products (and if so, which). Moreover, since there is no reference method or primary reference material (PRM) in place, the measurement of GFAP is not standardized and samples give incomparable results when assayed with different tests. Finally, all those are in ELISA format but POC tests would be needed if we begin to use those tests in the ambulances to make clinical decisions.

The calibration materials for assays today are either synthetic peptides or purified GFAP. However, no information is available if these peptides are solely intact GFAP or include breakdown products, or if they are representative of the mixture of isoforms and posttranslational modifications encountered in native samples. Value assignment to calibrators is done gravimetrically by the manufacturer due to lack of a reference method or PRM.

A standardization project is necessary to clarify if the development of a reference method and the production of PRM are feasible. More importantly such a project will also define the target analyte for clinical use: the intact molecule or breakdown products. An appropriate PRM can be used by companies to calibrate GFAP methods and provide assays that give comparable results. Last but not least, the development of an external quality assessment scheme (EQAS) is important so laboratories that measure GFAP can measure their bias. Commutability and comparison studies will further enhance such a project.

In addition to these analytical concerns, it is also important to focus on preanalytical variables. All research studies have been performed on stored samples, however studies of short- and long-term stability of GFAP at various temperatures have not been performed. Studies to determine the most suitable sample (serum, EDTA plasma, citrate plasma) are also needed. The biological variation of this analyte is also unknown. This knowledge is crucial to determine if changes in serial GFAP measurements are of clinical importance, not just statistical significance.

All this knowledge will enable us to revise our study selection criteria and perform better metaanalyses. Moreover, with standardization in place we will be able to re-standardize research results with a uniform protocol and provide robust clinical decision limits. That would be the starting point for well-designed prospective multicentric clinical studies. Then we will be able to introduce this assay into clinical practice with confidence. If all the strategy works and GFAP or a panel of biomarkers including GFAP might be used to initiate reperfusion therapies out of the hospitals a revolution in the way we manage stroke nowadays would occur in the field.

REFERENCES

  1. Simats A, Garcia-Berrocoso T, Montaner J. Neuroinflammatory biomarkers: From stroke diagnosis and prognosis to therapy. Biochim Biophys Acta. 2016;1862(3):411-24.
  2. Eng LF, Ghirnikar RS, Lee YL. Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000). Neurochem Res. 2000;25(9-10):1439-51.
  3. Brunkhorst R, Pfeilschifter W, Foerch C. Astroglial proteins as diagnostic markers of acute intracerebral hemorrhage-pathophysiological background and clinical findings. Transl Stroke Res. 2010;1(4):246-51.
  4. Herrmann M, Vos P, Wunderlich MT, de Bruijn CH, Lamers KJ. Release of glial tissue-specific proteins after acute stroke: A comparative analysis of serum concentrations of protein S-100B and glial fibrillary acidic protein. Stroke. 2000;31(11):2670-7.
  5. Perry LA, Lucarelli T, Penny-Dimri JC, McInnes MD, Mondello S, Bustamante A, et al. Glial fibrillary acidic protein for the early diagnosis of intracerebral hemorrhage: Systematic review and meta-analysis of diagnostic test accuracy. Int J Stroke. 2018:1747493018806167.
  6. Yang Z, Wang KK. Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker. Trends Neurosci. 2015;38(6):364-74.
  7. Middeldorp J, Hol EM. GFAP in health and disease. Prog Neurobiol. 2011;93(3):421-43.

Scientific Shorts are brought to you by the

The Academy of Diagnostics & Laboratory Medicine logo

Academy of Diagnostics & Laboratory Medicine Designation

Fellows of the Academy use the designation of FADLM. This designation is equivalent to FACB and FAACC, the previous designations used by fellows of the National Academy of Clinical Biochemistry and AACC Academy. Those groups were rebranded as Academy of Diagnostics & Laboratory Medicine in 2023.