1. Biochemical and genetic evaluation in amyloidosis
Sept 10, 2010
Hee-Jin Kim MD, PhD
Dept of Laboratory Medicine & Genetics
Samsung Medical Center
Sungkyunkwan Univ School of Medicine
Seoul, Korea

2. Introduction Amyloidosis A group of diseases with a common feature of
Extracellular deposition of insoluble fibrils in tissues and organs
The fibrils have a characteristic beta-pleated sheet configuration and produce apple-green birefringens under polarized light on Congo red stain
More than 25 different human proteins have been descri-bed to form amyloid fibrils and cause a variety of amyloid-related diseases

3. Pathology Lab medicine Imaging
Introduction
Diagnostic modalities in amyloidosis
Pathology
Lab medicine
Imaging

4. Amyloidosis The list of amyloid-related diseases in human
Most common cause of amyloidosis
Caused by plasma cell dyscrasia
Biochemical tests are critical for Dx
Most common cause of familial form
of amyloidosis
Particularly relevant to cardiac
amyloidosis
Biochemical and genetic tests are
critical for Dx
Ad Clin Chem 2009:47:1-44

5. Light chain amyloidosis (AL)
The most common type of systemic amyloidosis in human
The nature of amyloid in AL amyloidosis is the immunoglobulin light chain
Produced by a clonal population of plasma cells in the bone marrow (plasma cell dyscrasia)
Most frequently affected organs: kidney and heart
Diagnosis by demonstration of
Amyloid deposit in tissue
Plasma cell dyscrasia in the bone marrow
Monoclonal Ig
- Protein electrophoresis (PEP) & immunofixation EP (IFE)
- Free light chain (FLC) assay

6. Light chain amyloidosis (AL)
PEP and IFE
- Demonstrates the presence of “M-peak”
- Conventional methods

7. Light chain amyloidosis (AL)
“Free” light chain (FLC) assay
- Relatively recently developed diagnostic method
- Involves specific test strategy by targeting epitopes ( ) that are hidden in the intact form of Ig but are exposed when in free form
Nat. Rev. Nephrol. 2009:5;621–627
with modification

8. Light chain amyloidosis (AL)
FLC assay
- Particular relevancy in AL amyloidosis
M-protein may be minimal in AL amyloidosis, and thus the diagnostic utility of PEP/IFE is relatively limited
This is in contrast to other types of plasma cell dyscrasia such as multiple myeloma
- Quantification of κ and λ “free” light chains of Ig
- Immunoturbidometric assay
- FLC “ratio”: kappa/lambda ratio
- A FLC ratio that falls outside reference range: sensitive
and specific for Dx of AL amyloidosis
- Baseline and disease monitoring
- At SMC, Freelite™

9. Light chain amyloidosis (AL)
Diagnostic performance of FLC ratio in different
disease entities of PCD (Data from Mayo and from
other studies)
Clin Chem 2005:51:

10. Light chain amyloidosis (AL)
Based on the accumulation of evidence supporting the diagnostic utility of FLC assay in PCD, the IMWG provided consensus guidelines in 2009

11. Light chain amyloidosis (AL)
IMWG guidelines in 2009
- Summarization of the line of evidence of rates of abnormal FLC ratio in different PCD
Leukemia 2009;23:

12. Light chain amyloidosis (AL)
IMWG guidelines in 2009
- Use of serum Ig FLC assay as baseline values and for disease monitoring
Leukemia 2009;23:

13. Light chain amyloidosis (AL)
IMWG guidelines in 2009
Also included a consensus criteria for treatment response
Leukemia 2009;23:

14. Light chain amyloidosis (AL)
FLC ratio: currently in wide use in Korea
Reference ranges for FLC assays
Adopted from
Clin Chem 2002;48:
Am J Clin Pathol 2003;119:
Item
Reference range
Serum free kappa concentration
mg/L
Serum free lambda concentration
mg/L
Serum free kappa/lambda ratio
Urine free kappa concentration
mg/L
Urine free lambda concentration
mg/L
Urine free kappa/lambda ratio

15. TTR-amyloidosis (ATTR)
The most common type of systemic amyloidosis of familial background in human (autosomal dominant inheritance)
However, the acknowledgement of this disease entity and diagnostic workup is far limited than deserve in Korea
The nature of amyloid is abnormal transthyretin (TTR) due to a point mutation in the TTR gene
Frequently affected organs: heart, kidney, nervous system
Genotype-phenotype correlations have been described
Diagnosis by demonstration of
Amyloid deposit in tissue
Abnormal TTR protein in plasma/tissue
TTR gene mutation

16. TTR-amyloidosis (ATTR)
Mass spectrometry (MS)
Recently gained widespread use for proteomics study
and also for clinical tests
A robust tool to determine the nature of dysprotein
By the observation of the mass difference between wild-type vs mutational protein (e.g., the mean mass of the wild-type TTR: 13,761 Da)
J Chromatography B 2008:870;148–153

17. TTR-amyloidosis (ATTR)
Identification of abnormal TTR protein by using MS
13,761
Clin Chem 2004:50:

18. TTR-amyloidosis (ATTR)
Genotype-phenotype correlations
GeneReviews

19. TTR-amyloidosis (ATTR)
Different types of MS and MS available at SMC
-TTR can be the first target molecule for the application of MS for amyloidosis at SMC
Available at SMC
J Chromatography B 2008:870;148–153

20. TTR-amyloidosis (ATTR)
Molecular genetic testing for TTR mutations
GeneReviews

21. TTR-amyloidosis (ATTR)
A single case report on a Korean patient with hereditary TTR-amyloidosis-only DNA testing

22. TTR-amyloidosis (ATTR)
Integrated protein-genetics laboratory workup for Dx of TTR-amyloidosis
Clin Chem 2004:50:

23. Other familial amyloidosis
- Recent studies have shown that familial amyloidosis may be under-diagnosed
- Role of protein-genetics laboratory workup for DDx
GeneReviews

24. Summary Due to the heterogeneous clinical manifestations,
a high index of suspicion is important for timely Dx and Rx of amyloidosis
When suspected, appropriate laboratory screening workup is needed to detect amyloid early and to type it correctly
FLC assay is an important tool for screening AL amyloidosis, the most common form of amyloidosis
An example of how a specific biomarker can be used for diagnostic and disease monitoring purposes

25. Summary
Screening of abnormal protein by MS along with molecular genetic confirmation can be a robust tool for Dx of TTR-amyloidosis and the resources are currently available at SMC
The strategy can also be applied in other forms of familial/hereditary amyloidosis
Current advances in the drug discovery and trials in amyloid disorders will fuel development and application of new diagnostic modalities for tailored therapy at a clinical basis

26. Thank you