Detection of Actin Fragments in Serum: A Rapid Screening Test to Aid in the Diagnosis of Chronic Fatigue Syndrome

Chronic Fatigue Syndrome (CFS) is recognized as one of the most common chronic illnesses in the world. CFS is a complex disease syndrome of unknown etiology, afflicting people of all ages. Currently CFS is defined by its symptoms, the hallmark of which is chronic, debilitating fatigue of six months or more in duration.(1) In addition, patients suffer from a number of physical problems including myalgia, arthralgia, cognitive impairment, and sleep disorders.

New Developments Based on RNase L Research

Since the original identification of the abnormal low molecular weight (LMW) RNase L protein by researchers at Temple University in 1995(2), intense research has focused on defining the origins of this protein. Within the 12 months of 2000, researchers at Temple University, Montpellier University, and R.E.D. Laboratories independently determined that the appearance of the LMW RNase L protein was due to proteolysis of native RNase L. Further research performed at R.E.D. Laboratories has identified one of the proteases responsible as calpain (calcium-activated neutral proteinase).

The calpain family of proteins is divided into ubiquitous and tissue-specific subclasses, all of which require calcium ion for activation.(3) Since calpain has been previously demonstrated to degrade a broad array of intra- and extracellular proteins, including cytoskeletal and receptor proteins,(4) researchers hypothesized that other cellular proteins may also be fragmented. One of the first proteins to be examined for possible proteolytic cleavage was actin, since actin is a known substrate of calpain cleavage.(5)

Discovery of Actin Protein Fragments in Cells and in Serum

In its native form, actin exists in a monomeric state with a molecular weight of 42 kDa (referred to as G-actin). To form part of the cytoskeleton, actin polymerizes into long filaments (referred to as F-actin). Any degradation or abnormal proteolysis of actin protein could therefore have dramatic

consequences on the ability of an immune cell to perform its normal function, especially if that function required motility (e.g., phagocytosis). In PBMCs from patients with CFS, fragmentation of actin was detected. The amount of fragmentation correlated significantly with the amount of fragmentation of RNase L protein (p < 0.001, n = 62 specimens).

Researchers then examined the serum for actin protein fragments. Normally, actin is released into the serum as a consequence of cell turnover and/or necrosis and is cleared from circulation by the vitamin D-binding protein (also referred to as the group-specific component, or Gc)(6). Native actin protein binds to the vitamin D-binding protein through actin’s C-terminal amino acid structure. However, the intracellular cleavage of actin results in fragments that do not contain the identical C-terminal structure and thus these fragments are not cleared as rapidly from the serum. Levels of actin protein fragmentation in serum correlates significantly with the amount of both intracellular actin- and RNase L- fragmentation (p < 0.01, n = 175)(7)

The ability to employ a serum-based marker as a screening test for CFS dramatically increases accessibility while significantly reducing the efforts and costs involved in the preparation, storage, shipment, and assay of a PBMC pellet.

The Fragmented Actin Serum Test (FASTest™)

Actin fragments present in serum are measured by Western blot using an antibody specific for native G-actin. Blood is drawn from the patient into a serum separator tube to allow the blood to clot and serum is collected by centrifugation. An aliquot of serum is then mixed with denaturing agents and allowed to migrate through a SDS polyacrylamide gel to separate actin proteins based on molecular weight. After electrophoresis, the proteins are transferred to a solid support which is then allowed to react with antibody and coloration agents. Molecular weight size markers and positive and negative controls are included with each assay.

Results & Interpretation

Results are qualitative. The results of this test should be used in addition to all other relevant clinical data before making a diagnosis and/or a recommendation for treatment. Ranges are subject to change dependent on future data. This test was developed and its performance characteristics determined by R.E.D. Laboratories. It has not been cleared or approved by the U.S. Food and Drug Administration (FDA). The FDA has determined that such clearance or approval is not necessary. This test is used for clinical purposes. It should not be regarded as investigational or for research

Ordering the Test

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Any physician can order this test. To order the test, please use the Request Form provided. In order to use a correct Request Form, please select from the following menu options depending on your location:

USA (zipped):

USA (pdf):



Please send the samples and request forms to:

R.E.D. Laboratories

Pontbeek 61

1731 Zellik


Specimen Requirements, Storage, Shipment & Reporting of Results

To perform the FASTest assay, a minimum of 0.5 mL serum is required. Serum may be stored at 2-8°C (i.e., common refrigerator temperatures) for a period of two weeks before shipping. Sera stored frozen are stable for many months.

Serum specimens may be shipped at room temperature (i.e., ambient), refrigerated temperature, or on dry ice (for instance with other specimens for RNase L testing). Shipping at room temperature is recommended as this is the most convenient method. To ship specimens, the Company recommends the following shippers:

(A pro forma invoice for the shippers is available):

Assay results are available in five working days after the specimens have been received at the laboratory. R.E.D. Laboratories will fax results to the referring party if desired. All results are kept confidential.


1.Holmes, G., et al., “Chronic Fatigue Syndrome: A

Working Case Definition,” Annals of Internal Medicine

108:387-389 (1988).

2.Suhadolnik, R., et al., “Biochemical Evidence for a Novel

Low Molecular Weight 2′-5’A-Dependent RNase L in

Chronic Fatigue Syndrome,” J. Interferon & Cytokine

Research 17:377-385 (1997).

3.Suzuki, K., et al., “A Novel Aspect of Calpain Activation,”

FEBS Letters 433:1-4 (1998).

4.Lee, M-S., et al., “Neurotoxicity Induces Cleavage of p35

to p25 by Calpain,” Nature 405:360-364 (2000).

5.Potter, D., et al., “Calpain Regulates Actin Remodeling

During Cell Spreading,” J. Cell. Biol. 141(3):647-662 (1998).

6.Goldschmidt-Clermont, P., et al., “Gc (Vitamin D-Binding

Protein) Binds the 33.5K Tryptic Fragment of Actin,” Life

Sciences 38:735-742 (1986).

7.Roelens, S., et al., “G-Actin Cleavage Parallels

2-5A-Dependent RNase L Cleavage in Peripheral Blood

Mononuclear Cells. Relevance to a Possible

Serum-Based Screening Test for Dysregulations in the

2-5A Pathway,” submitted to the Journal of Chronic

Fatigue Syndrome.

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