Hobgen test1940-1960

GC-MS (steroïden)1930-today

- Laborious (derivatisation)- Time consuming (long run times)- But still considered reference method

RosalynYalow

RIA

1960-today

Concentration range of analytes in blood

The workhorse of the endocrine laboratory

Immunoassay and its pitfalls

Competitive immunoassay

• Only one epitope• One antibody• Competion for antibody

• Less sensitive• Less specific• Small dynamic range

Sandwich assay

• Two epitopes• Excess capture antibody• Excess detection antibody

• Most sensitive immunoassays• Large dynamic range• High specificity

Immuno-assaypro contra• Relatively sensitive• Easy• Fully automated• Direct measurement

• Lack of accuracy• Lack of sensitivity• Lack of specificity• Limited synamic range• Expensive (kit) reagents• Black box

Serum tubePlastic

Vacuum

Rubber

Separator gel

Surfactants

Clot activator

Plasma tubeLi heparin (green)

fast centrifugation

no clotting

EDTA (purple)

ACTH

renine

aldosteron

Stability and storage

STAT analysis• PTH• Insulin• …

Frozen till analysis

• ACTH• Plasma Renin Activity• Aldosterone• C peptide• Gastrin• Glucagon• …

hemolysisSmall effect on immunoassaysbut:

proteolysisinsulingastringlucagonPTH

cellular releaseNSE

lipemiaSmall effect on immunoassaysBut:

turbidimetricnephelometric

Carry-over

• Most of today’s diagnostic analyzers take every possible precaution to avoid sample carry-over.

• In spite of these efforts, a sample having a VERY high result may affect one or more samples that follow it. e.g. hCG

Cross-reactivity: hCG and LH

Cross-reactivity: steroids

Hormone binding proteins

Albumin, SHBG, TBG, CBG

For total hormone measurement, it is essential to displace all bound hormone from endogenous binding sites

solvent extraction

anilino naphtalene sulfonic acid

For free hormone measurement, displacement can alter the equilibrium

(always happens to a certain degree in free hormone assays + low concentration!)

NEFA in free thyroxine assays

Autoanalyte antibodiesThyroid hormones

Thyroglobulin

Insulin

Prolactin

MacroprolactinBiologically inactive

Cleared slowly

Causes false high prolactin

Heterophile antibodiesHAMA

Bridging of capture anddetector antibodies=> Falsely elevated result

Exclusive binding of capture or detector antibody only=> Falsely lowered result

Anti-Ru antibodies (Roche)

Mainly in areas with textile industry Use of Ru in dying process of clothing

Ru in environment, clothing or food chain

Estimated frequency of interference in first generation Elecsys FT3 assay (Roche Diagnostics): 0.2% (Sapin, Clin Chem Lab Med 2007)

Case report: anti-rhutenium antibodies in 7 y old girl

TSH 0 .66 mIU/L 0.27 - 4.20 Vrij T4 76 pmol/L 12 - 22 Vrij T3 27.4 pmol/L 3.1 - 6.8

Oestradiol (LC-MSMS) < 1 .3 ng/L Oestradiol (Roche) 399 ng/LTestosteron 82 .1 ng/dL 15.0 - 45.0 SHBG 66 nmol/L 41 - 103 Androstenedion 14 .1 ng/dL 30.0 - 200.0

High dose hook effectTumor markers

hCG

Ferritin

Thyroglobulin

High analyte concentration

Poor agreement with reference method

• Poor standardization• Cross reactivity (poor quality of antibody)

e.g. androstenedione, cortisol, oestrone

0 100 200 300 400 5000

50

100

150

200

250

300

350

400

450

Andr RIA

Andr

LC

MS

Lack of sensitivity and bad assay design

Free hormone assaysFree Thyroid Index (FTI)

Total T4Immuno A

Free T4Immuno A

Total T3Immuno A

Free T3Immuno A

LC-MS/MS LC-MS/MS

Mass SpectrometryA reference method for hormones

1) Sample preparation

• Protein precipitation (methanol, acetonitrile, ZnSO4)

• Liquid liquid extraction• Solid phase extraction

2) HPLC

HPLC in practice- Pumps- Fluids

- Tubings- Sample loader- Complex valves

- Column

Massaspectrometer (Q)

CortisolPrednisolon

362,460 Da360,444 Da

++

+MS1

+

Tandem Massaspectrometer (QQQ)

++

++

++

MS1 MS2

+++

Isobaric interference

Mass Spectrometryin practice

- Ion source- Switching valves- Tubings- Vacuum pump- Nitrogen generator

Mass Spectrometryin practice

- Spray probe- Heating probes- Curtain plate- Curtain plate aperture- Orifice

LC-MS/MS in EndocrinologyAnalytical Superiority

Added value LC-MS/MS: Accuracy

van den Ouweland JMW, Kema IP. J Chromatography B 2012

Added LC-MS/MS: Sensitivity

Cortisol Prednisolon

Cross reactivity!

Added value LC-MS/MS: Specificity

???

???

Added value LC-MSMS: steroid profiling

Added value LC-MS/MS: pro and contra

Advantages• Analytical superiority• “Cheap” reagents

• Organic solvents• Deuterated internal standards• Robust colums• Calibrator sets• Pipetting tips and plates

• High troughput possible• Develop new assays

Disadvantages• Expensive instruments• Batch mode• Skilled labor required• LIS interface• Regulartory uncertainty• Ruggedness