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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