Vrije Lichte Keten assays: nieuwe ontwikkelingen 13 december 2012 - - PowerPoint PPT Presentation

▶
vrije lichte keten assays nieuwe ontwikkelingen
SMART_READER_LITE
LIVE PREVIEW

Vrije Lichte Keten assays: nieuwe ontwikkelingen 13 december 2012 - - PowerPoint PPT Presentation

May 16, 2023 146 likes •421 views

Deelnemersbespreking SKML sectie HIM Vrije Lichte Keten assays: nieuwe ontwikkelingen 13 december 2012 Hans Jacobs UMC St Radboud Afdeling laboratoriumgeneeskunde Laboratorium Medische Immunologie H.Jacobs@labgk.umcn.nl M-proteine

slide-1
SLIDE 1

Deelnemersbespreking SKML – sectie HIM

Vrije Lichte Keten assays: nieuwe ontwikkelingen

13 december 2012 Hans Jacobs UMC St Radboud Afdeling laboratoriumgeneeskunde Laboratorium Medische Immunologie H.Jacobs@labgk.umcn.nl

slide-2
SLIDE 2

Alb α-1 α-2 β Normal γ

Eiwit electroforese #1 #2

ELP G A M

κ

λ

Serum #2 Immunofixatie

IgG-κ

Densitometrie #2 #1

M-proteine diagnostiek

2

slide-3
SLIDE 3

Monoclonal gammopathies

Diagnosed at Mayo Clinic 2002

Multiple Myeloma 18% (273) Amyloidosis (AL) 11% (167) Lymphoma 4% (55) Smouldering myeloma 6% (87) Solitary or extramedullary plasmacytoma 1% (23) Waldenström’s Macroglobulinemia 3% (43) Other 6% (93) MGUS 51% (769) IgA (21%) Biclonal (1%) IgE (0.01%) IgG (59%) IgD (1%) FLC 15% Nonsecretory myeloma (3%)

3

Free Light Chain

λ or κ

slide-4
SLIDE 4

Free Light Chains

Bone marrow and lymphoid organs

Produced 500 mg/day

Kidney

Capacity to absorb and metabolise 10-30 gram/day T1/2 varies from hrs to 2-3 days (renal function)

FLC normal ranges (when measured with Freelite reagents)

Kappa: 3.3 – 19.4 mg/L Lambda: 5.7 – 26.3 mg/L Kappa/Lambda ratio: 0.26 – 1.65

  • Bradwell. sFLC analysis 6th edition

4

slide-5
SLIDE 5

Plasma Cell

Light chain λ of κ Heavy chain IgG / IgA / IgM / IgD / IgE Alb α-1 α-2 β MPR MPR

3.1 g/l

ELP G A M

κ

λ FLC κ FLC λ

‘hidden epitope’

Free Light Chain λ or κ

Free Light Chain diagnostics

5

slide-6
SLIDE 6

Normal γ Alb α-1 α-2 β

SPE

normal

Case: Man, 56 years old. Bone pain & compression # spine Lab: Ca ↑↑, Hb ↓ X-ray: multiple lytic lesions BM biopsy: 58% plasma cells Our Case 410 mg/l 1.9 mg/l 219 REF values Free kappa: 3,3 – 19,4 mg/l Free lambda: 5,7 – 26,3 mg/l Ratio: 0,26 – 1,65 Nephelometry (Freelite)

Bead Bead

Bradwell et al. Clin Chem 1999

Free Light Chain diagnostics

6

slide-7
SLIDE 7

For increased sensitivity: nephelometric FLC analysis (Freelite)

Bradwell et al. Clin Chem 2001 and Blood 2001

Method Nephel.

7

slide-8
SLIDE 8

Freelite assay: FLC conc. correlate to prognosis and disease activitiy

Rajkumar et al. Blood 2005 Dispenzieri et al. Blood 2008

8

Correlation with prognosis for MM: Snozek et al. Leukemia 2008 Correlation with prognosis for Amyloidosis: Palladini et al. JCO 2012 Correlations with other lympoproliferative disorders (review): Charafeddine et al. Am J Clin Pathol 2012 Correlation with disease activity in autoimmune disorder: Gottenberg et al. Ann Rheum Dis 2007

slide-9
SLIDE 9

Advantages:

1) Earlier diagnosis 2) Improved monitoring (international response-criteria) 3) Associated with prognosis (international consensus) 4) High through-put Bradwell et al. 1999 Clin Chem ‘immunoassay for quantification of FLC in serum’ Durie et al. 2006 Leukemia ‘international uniform response criteria for MM’

Freelite assay (The Binding Site): in the clinic

9

slide-10
SLIDE 10

Freelite assay: analytical issues

  • Linearity problems
  • Antigen excess
  • Imprecision
  • Non-accurate

Tate et al. Clin Biochem Rev 2009

10

Plasma cell Ab-production Interference of intact M-proteins Each monoclonal FLC is a unique analyte (hypervariable Fab fragment) Some FLC form dimers Some FLC polymerize

slide-11
SLIDE 11

11

Hoedemakers et al. Clim Chem Lab Med 2011.

Red dotted lines illustrate that differences in FLC concentrations can be observed between the two commercial immunoassays (up to 10 times !)

Recently Siemens has introduced a second commercial immunoassay to measure FLC (N Latex assays)

Te Velthuis et al. Clim Chem Lab Med (2011)

slide-12
SLIDE 12

12

First (personal and international) experiences in clinical labs with the N latex assays

Compared to Freelite assay:

  • Improved linearity but also STRONG non-linearity in some samples, especially when also

intact M-protein is present (Jacobs et al. Clin Chim Acta 2012)

slide-13
SLIDE 13

FLC linearity

Jacobs et. al. Clin. Chim. Acta. 2012

FLC linearity in presence of intact monoclonal Ig

Freelite !

slide-14
SLIDE 14

14

First (personal and international) experiences in clinical labs with the N latex assays

Compared to Freelite assay:

  • Improved linearity but also STRONG non-linearity in some samples, especially when also

intact M-protein is present (Jacobs et al. Clin Chim Acta 2012)

  • Build in antigen excess protection
slide-15
SLIDE 15

Antigen excess in FLC measurements

N latex Freelite Kappa 2 Lambda

See also Murata et. al. 2010. Clin. Chem.

N = 93

N latex Freelite Kappa 24 g/L 32 g/L Lambda 13 g/L 78 g/L

Highest measured concentration

11.2 g/L 11.5 g/L

Antigen excess

slide-16
SLIDE 16

16

First (personal and international) experiences in clinical labs with the N latex assays

Compared to Freelite assay:

  • Improved linearity but also STRONG non-linearity in some samples, especially when also

intact M-protein is present (Jacobs et al. Clin Chim Acta 2012)

  • Build in antigen excess protection
  • Higher batch-to-batch precision (Pretorius et al. Ann Clin Biochem 2012)
  • mAb impose risk of missing FLCclone (Hutchison et al. BMC Clin Pathol 2012)
  • Reference values are similar but not identical
slide-17
SLIDE 17

17

Adapted from dr. Jillian Tate, presentation during webinar sept 2012

Reference values sFLC

slide-18
SLIDE 18

18

Reference values sFLC

Adapted from dr. Jillian Tate, presentation during webinar sept 2012

slide-19
SLIDE 19

19

Adapted from dr. Jillian Tate, presentation during webinar sept 2012

0.26–1.65

Summary FLC assays

slide-20
SLIDE 20

20

Is harmonization possible?

Strong concentration differences observed when compared to Freelite assay

a) Both assays report results in mg/L b) Which result is correct?! International standard is lacking… c) Reference values are similar but not identical d) The above provides a big problem

  • For translation of data from literature
  • For patients switching from hospital

Standardization is urgently needed (but will be difficult).

slide-21
SLIDE 21

Can immunoglobulins and FLC be quantified using mass spectrometry?

MRM technique in a triple-quadrupole instrument with stable isotope

  • standards. Proven extremely powerful to accurately quantitate proteins.

Meng et al. J. of Proteomics 2011

Mass-select peptide Fragment peptide Detect fragment peptide

MRM = multiple reaction monitoring (= SRM = selected reaction monitoring)

21

Tripple quad MS

slide-22
SLIDE 22

Can MS-suitable peptides from Ig and FLC be selected? Kappa Lambda

MS-suitable trypsin digestive peptides available in all intact Ig’s BUT ALSO in Light Chains!!!

22

slide-23
SLIDE 23

Quantification of FLC-ratio in pt samples using MRM MS

  • 1. Healthy control
  • 2. Pt. on dialysis
  • 3. MM-pt. FLC Kappa
  • 4. MM-pt. FLC Lambda
  • 5. MM-pt. IgG-K (with few FLC K)
  • 6. MM-pt. IgG-L (with abundant FLC L)

23

slide-24
SLIDE 24

Sigma FLC lambda 1.0 g/L Sigma FLC kappa 1.0 g/L

Method comparison MRM MS versus immunoassay

1.0 1.2 5.6 8.7 1.0 0.8 16.2 1.7 1.2 0.7

24

slide-25
SLIDE 25

Scope

25

Aim: reference method for FLC (and Ig) measurements…

slide-26
SLIDE 26

Radboud University Nijmegen Medical Centre

Department of Laboratory Medicine

Corrie de Kat Angelino Renate van der Molen Ron Wevers Irma Joosten

Acknowledgements

Department of Hematology

Sandra Croockewit

Erasmus MC Rotterdam

Department of Neurology

Martijn van Duijn Theo Luider

MS project starting grants

NVKC Noyons Stipendium Relares Grant