Confirmatory Testing

Once the clinical suspicion of Pompe disease is raised, confirming the diagnosis requires demonstration of absent or reduced acid alpha-glucosidase (GAA) enzyme activity[1].

Technology allows the measurement of residual GAA enzyme activity in a variety of sample types, including minimally invasive blood-based samples such as dried blood spots, lymphocytes, or fibroblasts[2-5]. These tests are quick and inexpensive, minimizing diagnostic delays for this progressive disease.

Making the Definitive Diagnosis

Historically, muscle tissue obtained from a biopsy was used to evaluate intracellular glycogen content and/or measure GAA activity. However, absence of glycogen accumulation in muscle tissue does not rule out Pompe disease, since glycogen content can vary in muscle tissue[6]. Therefore, muscle biopsy alone cannot be used for confirmatory diagnosis of Pompe disease[6]. Diagnosis should always be confirmed by enzyme assay demonstrating low GAA enzyme activity or by genetic analysis.

Minimally invasive tests can accurately quantify GAA activity in:

  • Dried blood spot (DBS)
  • Lymphocytes
  • Fibroblasts

Diagnosing Pompe Disease & U.S. Lab List (PDF)

A conclusive diagnosis is indicated by absent or markedly reduced GAA enzyme activity. This residual activity can be anywhere from less than 1% (generally in infants) to 40% of normal levels[5], [7].

Pompe disease can also be confirmed by mutation analysis to confirm the presence of two mutated alleles.

Testing Options

Historically, GAA enzyme assay was performed using cultured skin fibroblasts because of its accuracy and reliability[1], [2], [8]. However, sample collection is relatively invasive and it takes approximately six weeks to obtain the results. This long turnaround time can pose serious risks, especially in infants with rapidly progressive disease. The use of blood samples, including dried blood spots, is increasingly becoming standard practice, because it is less invasive, accurate and can usually provide results within a few days.[1], [2]

Although an option, muscle biopsies are generally not preferred for GAA enzyme assay because they are invasive and there is a high risk of false positives due to sample mishandling that can cause low GAA activity. Muscle biopsies are often useful for histological evaluation, but it is important to note that glycogen content can vary among muscles and seemingly normal biopsies do not rule out Pompe disease.[2]

GAA Enzyme Assay Details

Detailed information on the various options for confirmatory GAA enzyme assays is listed below. Note that the turnaround times listed below are average, and will vary depending on laboratory.

Dried Blood Spot (DBS)[1-2], [9-12], [14]

Sample obtained by: heel prick, finger stick, or blood draw

  • Minimally invasive
  • Rapid turnaround time
  • Whole blood or dried blood spot can be shipped. Samples submitted will be tested as a dried blood spot.
  • Useful for screening large number of samples, including newborn screening
  • Diagnosis of Pompe disease should not be based on dried blood spot alone. Historically, a variable of dried blood spot testing has been the condition of the dried-blood spot sample on arrival at the laboratory. A second confirmatory test is recommended for a diagnosis of Pompe disease.

It should be noted that quality assurance programs for blood-based assays are still under development in some regions and therefore secondary confirmatory tests using cultured fibroblasts or genotyping may be necessary for conclusive diagnosis[2].

Lymphocytes[2],[3],[13]

Sample obtained by: blood draw

  • Rapid turnaround time
  • May preclude the need for invasive muscle biopsy sample

Fibroblasts[2],[8],[11]

Sample obtained by: skin biopsy

  • May preclude the need for invasive skin or muscle biopsy sample
  • Requires 4-6 weeks of cell culture
  • If culture is not successful, results will be delayed

Muscle Tissue[2],[8],[13]

Sample obtained by: muscle biopsy

  • Invasive and can require general anesthesia
  • Does not require cell culture
  • Must be frozen in liquid nitrogen and shipped on dry ice

It is important to remember that absence of glycogen accumulation in any particular biopsy sample does not rule out Pompe disease, since glycogen buildup can vary across different muscles and stage of the disease progression.[8] 

Other Tests

Several other tests may be useful to assess the baseline status and monitor disease progression.

Find out more about other useful assessments

Since Pompe disease is inherited, a conclusive diagnosis often prompts for the need to testing other family members, as well as genetic counseling.

References

  1. Zhang H, Kallwass H, Young SP, et al. Comparison of maltose and acarbose as inhibitors of maltase-glucoamylase activity in assaying acid alpha-glucosidase activity in dried blood spots for the diagnosis of infantile Pompe disease. Genet med 2006; 8:302-306.
  2. Winchester B, Bali D, Bodamer OA, et al for The Pompe Disease Diagnostic Working Group. Methods for a prompt and reliable laboratory diagnosis of Pompe disease: report from an international consensus meeting. Mol Genet Metab. 2008;93(3):275-281.
  3. Okumiya T, Keulemans JL, Kroos MA, et al. A new diagnostic assay for glycogen storage disease type II in mixed leukocytes. Mol Genet Metab 2006;88:22-28.
  4. Jack RM, Gordon C, Scott CR, Kishnani PS, Bali D. The use of acarbose inhibition in the measurement of acid alpha-glucosidase activity in blood lymphocytes for the diagnosis of Pompe disease. Genet Med 2006;8:307-312.
  5. Kallwass H, Carr C, Gerrein J, et al. Rapid diagnosis of late-onset Pompe disease by fluorometric assay of alpha-glucosidase activities in dried blood spots. Mol Genet Metab 2007.
  6. Winkel LP, Hagemans ML, van Doorn PA, et al. The natural course of non-classic Pompe’s disease; a review of 225 published cases. J Neurol 2006; 252:875-84.
  7. Kishnani PS, Steiner RD, Bali D, et al. Pompe disease diagnosis and management guideline. Genet Med 2006; 8:267-88.
  8. Hirschhorn, Rochelle and Arnold J. J. Reuser. Glycogen Storage Disease Type II: Acid Alpha-glucosidase (Acid Maltase) Deficiency. In: Scriver C, Beaudet A, Sly W, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. 8th Edition. New York: McGraw-Hill, 2001. 3389-3420.
  9. Li Y, Scott CR, Chamoles NA, et al. Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin Chem 2004; 60:1785-96.
  10. Chamoles NA, Niizawa G, Blanco M, Gaggioli D, Casentini C. Glycogen storage disease type II: enzymatic screening in dried blood spots on filter paper. Clin Chim Acta 2004; 347:97-102.
  11. Umapathysivam K, Hopwood JJ, Meikle PJ. Determination of acid alpha-glucosidase activity in blood spots as a diagnostic test for Pompe disease. Clin Chem 2001; 47:1378-83.
  12. Meikle PJ, Grasby DJ, Dean CJ, et al. Newborn screening for lysosomal storage disorders. Mol Genet Metab 2006; 88:307-14.
  13. Jack RM, Gordon C, Scott CR, Kishnani PS, Bali D. The use of acarbose inhibition in the measurement of acid alpha-glucosidase activity in blood lymphocytes for the diagnosis of Pompe disease. Genet Med 2006;8:307-312.
  14. Goldstein J, Young S, Changela M, Dickerson G, Zhang H, Dai J, et al. Screening for pompe disease using a rapid dried blood spot method: Experience of a clinical diagnostic laboratory. Muscle Nerve 2009;40:32-6.

Find Diagnostic Labs

Get a list of laboratories that can perform enzyme assays and other useful tests to aid in the diagnosis of Pompe disease.

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(Intended for the U.S. only)

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