Lecture 1 Week 1.pdf

Lecture 1 Performance Standards EQA Analyzer and Assay Configurations Assoc Prof Cyril Mamotte Curtin Medical School Learning Outcomes 1. Understanding and interpretation of allowable limits of performance 2. Knowledge and understanding of external quality assurance (QA) programs and interpretatio...

Lecture 1 Performance Standards EQA Analyzer and Assay Configurations Assoc Prof Cyril Mamotte Curtin Medical School Learning Outcomes 1. Understanding and interpretation of allowable limits of performance 2. Knowledge and understanding of external quality assurance (QA) programs and interpretation of external QA reports 3. Understanding of the basis for large difference in results by different methods 4. Understanding of major differences between general chemistry analyzers and dedicated immunoassay analyzers • Detection and readout systems –signal/noise ratios for colorimetry vs fluorimetry vs chemiluminescence • Distinction between homogenous and heterogenous assays • Assay principles for sensitive immunoassays and their characteristics • Examples of errors in immunoassays Questions • What are “allowable limits of performance”? • The allowable limits of performance for sodium is +/- 3.0 mmol/L. If a lab can measure plasma sodium with a SD of 3.0 mmol/L, is that satisfactory? Explain your answer. • What is a proficiency program, when is it necessary and of what use is it? • Compare the way we use internal and external QC programs. • What type of auto-analyzer would be required to measure a steroid present at a concentration of 12 pmol/L. In addition, what type of assay principle would be required, and what would the calibration curve look like? • Compare the different type of solid phases used by Roche Elecsys and Siemens Immulite assays. • Roche: Streptavidin coated magnetized beads • Siemens Immulite: Beads coated with the capture antibody • How do titrimetric assays differ from competitive assays? • Patient sample is added to a solid phase Ab, following an incubation step, a fixed amount of labelled Ag is added, and binds to vacant capture Ab sites not already bound/filled by the Ag from the patient’s sample. In a competitive assay, patient sample and the fixed amount of labelled Ag are added at the same time and compete for a limited amount of capture Ab. • What is the precision of an assay at the concentration corresponding to its functional sensitivity? • The precision with which we can measure an analyte generally poor at low analyte concentration, and this is reflected in poor CVs. The functional sensitivity is defined at that concentration of an analyte which can be measured with a CV of 20%. • General clinical chemistry analyzers utilize homogenous assays and these account for the bulk of the common laboratory tests we conduct. What does the term homogenous signify, and what advantage do these have compared to heterogenous methods. Examples of tests on general chemistry analyzers: U&Es, glucose, sodium, potassium, LFTs, amylase, lipase, CK, LDH • The assays can be done entirely in the liquid phase, with no requirement to separate any component of the reagents to get a readout. The advantages of such methods are easier to automate hence their • Compare the principle of Siemens Immulite and Roche Elecsys immunoassays. What are the similarities and differences? • The Roche Elecsys system is subjects to two sources of interference that are not seen in most other sensitive immunometric assays. What are these? • What are the two major auto-analyzer types in a clinical biochemistry laboratory and give an example of major differences in the analytical principles of the chemistries they employ. • Consider the following technology? On what type of analyser are you likely to find this technology? In what clinical setting is this type of technology useful? • What is chemiluminescence, and how does it differ from fluorescence or light colorimetry, and why do immunoassays using chemiluminescence yield a higher sensitivity. • What is a dry chemistry analyzer? • Give a brief outline of how antibodies in some patient samples can interfere in immunoassays. Illustrate your answer. Performance Standards How well should we be able to measure analytes? From RCPA QAP There are two elements to inaccuracy: bias and imprecision. Refresher on the Normal or Gaussian distribution Standard deviation: SD Coefficient of variation: CV = !" x 100 #$%& Data distribution for CV follows similar principles to that for the SD That’s is: ~70% within ± 1SD of the mean ~96% within ± 2SDs etc. -3CV -2CV -1CV +1CV +2CV. +3CV What standard deviation do we need to measure with? -plasma sodium as an exampleWhat proportion of our QC measurements need to fall within these limits. For sodium, the limit is ± 3mmol/L. 95% of the results for a QC in control will fall within ± 2SDs of the mean. Likewise 95% of a patients result needs to lie within ± 2SDs of their true mean The range of ± 3mmol/L needs to fit within ± 2SDs, or within -2SD to +2SD, a range of 4 SDs -> ' (()*/, = 0.75 mmol/L - So SD required = allowable limits/4 -3CV -2CV -1CV +1CV +2CV. 3 mmol needs to fit within these parameters +3CV The SD required varies for different analytes Serum/Plasma Analyte Reference Range RCPA QAP Allowable limits of performance Chloride ± 3 mmol/L Glucose ± 1.0 mmol/L up to 10 mmol/L, ±10% if >10 mmol/L Potassium ± 0.2 mmol/L Sodium ± 3.0 mmol/L Urea ± 1.0 mmol/L up to 10 mmol/L, ±10% if >10 mmol/L Creatinine SD required ~ ¼ of the value shown to the left e.g. ~0.8 mmol/L for Sodium ± 1.0 umol/L up to 100 umol/L, ±10% if >100 umol/L Question The allowable limits of performance for sodium is +/- 3.0 mmol/L If a lab can measure plasma sodium with a SD of 3.0 mmol/L, is that satisfactory? External Quality Assessment = Proficiency testing External Quality Assessment (EQA) • Also called proficiency testing (PT) • Independent assessment of analytical performance • Evaluates a laboratory's testing results by comparing them to those of other laboratories (peers) • How you compare to others using the same methods/reagents • How you compare to those using other methods • • • • • For many assays/measurands/analytes there are performance standards Required for lab accreditation Meeting performance standards is expected A form on long term QC NOT performed on a day to day basis- typically monthly. Can’t be used for real-time monitoring of analytical performance. Sample Distribution, and reporting for RCPA EQA scheme for common analytes • Samples are sent to laboratories in a batch, in duplicate, but all as unknown (labs don’t know which are duplicates) • The lab assays two QC materials, typically once a month, and submit the results. • The labs are the sent a report on their performance: how they performed relative to their peers and stated performance standards • Enables comparison to others using the same instrument or reagents, and against all other labs Notes on assay performance in external QC programs A. How well labs perform is dependent on numerous factors § The inherent characteristics of the method. § How well the method is being applied. § § § § Personnel Training Maintenance Reagents § Labs using the same method can vary in performance B. Finally, some methods have known but acceptable biases, AND in some instances the biases are due to the nature of the QC material being used. Explanation of the report format Note largely Unimodal distribution Method principle Instrument Reagents Result for QC material 72-23 Results for 72-23 vs 72-24 And location with allowable limits Calibrator Result for QC material 72-24 Thyroid hormone example of specific method bias Note bimodal Distribution These types of non Gaussian distributions often seen in immunoassays Example of a) large between lab variation and b) negative proportional bias for this participating lab Questions 1. What is the analytical principle of the assay specified by the lab for measurement of prolactin? 2. What instrument did the lab use for the analysis? 3. Is the lab measuring prolactin within performance expectations? 4. How does the lab’s results compare to the median results from other labs? What type of bias is apparent? 5. What type of distribution does the data for all labs show? Unimodal, bimodal, trimodal, and why? The Two Major Types of Autoanalysers General Chemistry Analysers Example of Tests U&E Glucose LFT Cardiac enzymes Ca, PO4 Amylase Also capable of doing some immunoassays Homogenous assays- all liquid phase* Paediatric samples: best to use analysers requiring small sample volumes. Immunoassay analysers Examples of tests Thyroid function tests Steroid/pituitary hormones Ferritin bHCG Tumour markers Cardiac Troponin Analytical Method by Analyte Type Small Ions + + - + 2+ Na /K , Cl , Li , Ca , Mg HCO3HPO42- 2+ Methods Ion selective electrodes & colorimetric methods Colorimetric and by calculation from pH + pCO2 Colorimetric Osmometer Blood Gases Methods Low MW organic compounds Methods Proteins Methods pO2, pCO2 Hormones-T4, Steroids, catecholamines Drugs Digoxin, anti-psychotics, sedatives etc Albumin Other major proteins Proteins present at very low concentrations Enzymes Gasometric electrodes Immuno assays, hplc, mass spectrometry Colorimetric (serum/plasma) Low sensitivity immunoassays Higher sensitivity immunoassays Reaction rate measurement + some immunoassays Example of Number of Molecules Per Volume Analyte GH (baseline) fT3 Oestradiol (F) fT4 Oestradiol (M) Aldosterone ACTH Ferritin cTnI (F) Creatinine Albumin Sodium Apo B Transferrin Low Limit nmol/L High Limit nmol/L 0.004 0.008 0.02 0.037 0.08 2 2.1 0.66 Low Limit copies/5uL 0.8 0.0074 1.266 0.05 0.184 0.44 26 12,040,000 24,080,000 60,200,000 111,370,000 240,800,000 6,020,000,000 2,408,000,000 22,274,000 3,810,660,000 150,500,000 553,840,000 1,324,400,000 78,260,000,000 High Limit copies/100 uL 240,800,000 481,600,000 1,204,000,000 2,227,400,000 4,816,000,000 120,400,000,000 48,160,000,000 445,480,000 76,213,200,000f 3,010,000,000 11,076,800,000 26,488,000,000 1,565,200,000,000 126,420,000,000 39,732,000,000 3,160,500,000 60,000 120,000.00 180,600,000,000,000 361,200,000,000,000 451,130 751,880.00 1,357,901,300,000,000 2,263,158,800,000,000 130,000,000 145,000,000.00 391,300,000,000,000,000 436,450,000,000,000,000 3,612,000,000,000,000 27,158,026,000,000,000 7,826,000,000,000,000,000 7,224,000,000,000,000 45,263,176,000,000,000 8,729,000,000,000,000,000 722,400,000,000,000 1,625,400,000,000,000 1,601,320,000,000,000 2,859,500,000,000,000 26,600.00 47,500.00 6,321,000,000 1,986,600,000 Low Limit copies/100 uL 158,025,000 12,000 27,000 0.0525 High Limit copies/5 uL 36,120,000,000,000 81,270,000,000,000 80,066,000,000,000 142,975,000,000,000 ~100,000,000,000 to 400,000,000,000 stars in the milky way ~2,000,000,000,000 galaxies in the universe ~200,000,000,000,000,000,000,000 stars in the universe • https://theconversation.com/how-many-stars-are-there-in-space-165370 Picomolar to nanomolar concentrations Micromolar Concentrations Why Immunoassay analyzers with high sensitivity are required Analyte GH (baseline) fT3 Oestradiol (F) fT4 Oestradiol (M) Aldosterone ACTH Ferritin cTnI (F) Creatinine Albumin Sodium Apo B Transferrin Low Limit nmol/L High Limit nmol/L 0.004 0.008 0.02 0.037 0.08 2 2.1 0.66 0.8 0.0074 1.266 0.05 0.184 0.44 26 Picomolar to nanomolar concentrations Or Integrated “general chemistry/Immunoassay” analyzers homogenous chemistries heterogenous chemistries 0.0525 60,000 120,000.00 451,130 751,880.00 130,000,000 145,000,000.00 12,000 27,000 26,600.00 47,500.00 Require higher sensitivity immunoassays Measured on either dedicated “Immunoassay” analyzers -heterogenous chemistries- Micromolar Concentrations Can be measured on a general chemistry analyzer by immunoturbidimetry Or on a nephelometer Automation Vendors Abbott — https://www.corelaboratory.abbott/int/en/offerings/category/clinical-chemistry Roche — https://diagnostics.roche.com/global/en/products/product-category/clinical-chemistry-andimmunochemistry.html Orthoclinical Diagnostics — https://www.orthoclinical.com/en-au/home Siemens • https://www.siemens-healthineers.com/clinical-chemistry • https://www.siemens-healthineers.com/immunoassay Beckman Coulter Diagnostics • https://www.beckmancoulter.com Abbott Architect https://www.youtube.com/watch?v=flUMWAQlN_s Cobas 8000 https://www.youtube.com/watch?v=lJaTt_S7zW8 Cobas Daily maintenance https://www.youtube.com/watch?v=8FB3LyIxewM Abbott Architect Series Immunoassay analyzers-differences compared to general serum chemistry analyzers Higher cost of reagents Heterogenous assays involving phase separation • Not all reagents in solution phase as for homogenous assays • Separation of analytes/compounds bound to solid phase from that in solution • E.g. capture or solid phase Abs, of free from bound. Often wash steps (to remove residual solution phase reagents • E.g. unbound reporter antibodies). Detection technologies • Luminometry • Fluorimetry Vitros 250 (Dry Chemistry) — Dry Chemistry- using slide technology In very common use for pediatric samples-uses very small sample volumes Vitros 5600 (Dry/Wet Chemistry) The Theranos Mini Lab https://thenextweb.com/news/troubled -medical-startup-theranos-unveils-newblood-testing-device Accessed 19 Juy 2022 Chemistries used on Immunoassays Sandwich Assay (ELISA Example) (Immunometric, Non-competitive, Use an excess of Antibodies) For higher molecular weight antigens-typically polypeptides E E E E E E E P E E E E E E E S P S S E E E E E E E E E E E E E E E P E S E P Sandwich Assay (ELISA Example) (Immunometric, Non-competitive, Ab excess) P E P Calibration Curve S S E E E Signal/Readout E E S P P E Log Concentration S E Types of Solid phases Microplate Tube Beads Competitive ELISA (Ab limited) For large or small molecules Fixed amount of enzyme labelled Ag competes with Ag from Patient sample for binding to a limited amount of Ab -E -E -E -E P -E S -E -E S S -E -E P P -E Signal/Readout -E Calibration Curve Log Concentration Calibration Curve Signal/Readout Titrimetic ELISA -E -E -E Log Concentration P -E -E S P -E S -E Ab not as limited as in a competitive assay And there is an incubation step between adding the sample and the fixed amount of labelled Ag -E -E -E -E -E -E -E -E -E -E -E For traditional ELISAs we measured absorbance to monitor the reaction But this offers very limited sensitivity compared to measuring either a fluorescence signal, or even better measuring chemiluminescence Higher signal/noise ratio Advantage Luminescence Vs Spectrophotometry/Fluorimetry External Light Source External Light Source Measure light absorption Measure fluorescence No external Light Source Measure light emission Advantage Luminescence Vs Spectrophotometry or Fluorimetry Light Light Source Monochromators Light Light Source Illuminating λ ≠ Measured λ @ detector Illuminating λ = Measured λ @ detector Monochromators No light source No background light Absorbance Transmittance Concentration Concentration Luminescence Luminescent labels emit a photon of light Highly sensitive, no light source, hence emission against zero background Luminol — The first chemi-luminescent label — Can be used as a luminogenic substrate Luminol + H2O2 + OH- Peroxidase 3-aminonaphthalate + light (425 nm) Dioxetanes — AMPPD* substrates as an example, can also be used as an ALP luminogenic substrate (AMPPD: adamantyl 1,2-dioxetane aryl phosphate), gives zmol sensitivity — Example Siemens Immulite Acridinium esters, triple ringed organic esters, were the most common type — — Usually used to directly label antibodies (sandwich assays) or Ag for competitive assays Example Abbott Chemiflex system. Ruthenium complexes — Usually used to directly label antibodies (sandwich assays) or Ag for competitive assays — Example: Roche Elecsys Instrumentation for Chemiluminescence Measurements At a fundamental level- Luminometers, but automated Light capture is by a photomultiplier tube There is no light source. Immunoassay analyzers required Analyte GH (baseline) fT3 Oestradiol (F) fT4 Oestradiol (M) Aldosterone ACTH Ferritin cTnI (F) Creatinine Albumin Sodium Apo B Transferrin Low Limit nmol/L High Limit nmol/L 0.004 0.008 0.02 0.037 0.08 2 2.1 0.66 0.8 0.0074 1.266 0.05 0.184 0.44 26 Picomolar to nanomolar concentrations Or Integrated “general chemistry/Immunoassay” analyzers homogenous chemistries heterogenous chemistries 0.0525 60,000 120,000.00 451,130 751,880.00 130,000,000 145,000,000.00 12,000 27,000 26,600.00 47,500.00 Require higher sensitivity immunoassays Measured on either dedicated “Immunoassay” analyzers -heterogenous chemistries- Micromolar Concentrations Can be measured on a general chemistry analyzer by immunoturbidimetry Or on a nephelometer Abbott Architect Sandwich Assay – Generic (Immunometric, Non-competitive, Ab excess) Signal/Readout Calibration Curve Log Concentration Competitive (Ab limited) Signal/Readout Calibration Curve Log Concentration Types of Solid phases Microplate Tube Beads Traditional ELISA Add sample Buffered diluent Solid phase Ab i.e. bound to plastic 96 well plate Empty contents to waste Add wash solution Empty wash Solution to waste Siemens Immulite Analysers Chemiluminescent ELISA, also an immunochemiluminometric (ICMA) assay Add • Sample Spin out contents Add wash • ALP labelled Ab to tube waste solution • Buffered diluent sump • Solid phase Ab • i.e. on bead Spin out wash to Tube waste sump Add ALP or HRP chromogenic substrate Add ALP Chemiluminogenic substrate Siemens Immulite Chemiluminescent ELISA, also an immunochemiluminometric (ICMA) assay Add sample ALP labelled Ab Buffered diluent Solid phase Ab i.e. on bead Spin out contents to tube waste sump Add wash solution Spin out wash to Tube waste sump Add ALP Chemiluminogenic substrate Assay characteristics 1. A sandwich assay = an immunometric assay Excess of reporter and capture or solid phase antibody 2. Also a chemiluminometic assay Since it has a chemiluminescence based reporter system 3. But also an ELISA, it is an Enzyme linked immunoassay 4. It is heterogenous assay, requiring separation unreacted components, i.e. separation of solid phase and liquid phase. 5. Like all “sandwich” assays, it can’t be used to assay low molecular weight compounds-e.g. steroid hormones. Note: Heterogenous assays typically also have wash steps Abbott Architect Immunoassay (Chemiflex system) Competitive or titrimetric chemiluminescent assay Diluent buffer + Sample cortisol (added first, incubated) Acridinium ester labelled cortisol (added after incubation) Wash beads to remove Unreacted or unbound fraction Add pre-trigger (H2O2) and trigger (NaOH) solution -> light emission Abbott Architect Immunoassay (Chemiflex system) Competitive or titrimetric chemiluminescent assay Flash of light Momentary 1. A titrimetric assay –similar characteristics to a competitive assay Sample antigen added first, labelled antigen binds remaining sites Lower capture Ab than in immunometric assays *In strictly competitive assays, the sample and labelled antigen are added together 2. A chemiluminescent assay, but not a chemiluminometric assay Luminescence Assay characteristics 3. Not an ELISA, no enzyme labelled antigen or antibodies used 4. It is heterogenous assay, requiring separation unreacted components, i.e. separation of solid phase and liquid phase. 5. Like all competitive or titrimetic assays, can be used to assay for large or low MW compounds Note: Heterogenous assays typically also have wash steps Log Concentration a) Luminescence b) Time a) Abbott Chemiflex –labelled is an acridinium ester; chemiluminescence triggered by H2O2 and NaOH b) Siemens Immulite-labe is ALP and using a chemiluminescent ALP substrate Roche Immunochemiluminometric assay (ELECSYS system) Add • Sample • Ru2+ labelled reporter Ab (Ru2+-<) • Biotin labelled Ab (>-B) • Buffered diluent Add streptavidin coated magnetized beads Immobilized Magnetized Beads drawn to Electrode/magnet Wash away Unreacted Sample/reagents Add TPA and voltage To stimulate chemiluminescence Ru2+-<Ag>-B >-B Ru2+-<Ag>-B Ru2+-< >-B Ru2+-<Ag>-B Ru2+-< >-B >-B Ru2+-<Ag>-B Ru2+-< >-B Ru2+-<Ag>-B Roche Immunochemiluminometric assay (ELECSYS system) Add • Sample • Ru2+ labelled reporter Ab (Ru2+-<) • Biotin labelled Ab (>-B) • Buffered diluent Add streptavidin coated magnetized beads Immobilized Magnetized Beads drawn to Electrode/magnet 2. Also a chemiluminometic assay Since it has a chemiluminescence based reporter system 3. But it’s NOT an ELISA, not an enzyme labelled immunosorbent assay 4. It is heterogenous assay, requiring separation unreacted components, i.e. separation of solid phase and liquid phase. Add TPA and voltage To stimulate chemiluminescence Ru2+-<Ag>-B >-B 1. A sandwich assay = an immunometric assay Excess of reporter and capture or solid phase antibody Ru2+-<Ag>-B Ru2+-< >-B Ru2+-<Ag>-B Ru2+-< >-B >-B Ru2+-<Ag>-B Ru2+-< >-B Ru2+-<Ag>-B Assay characteristics Wash away Unreacted Sample/reagents Electrochemiluminesence Ruthenium complexes, e.g Ru (bpy)3 2+ • (Ru2+ ) -> (Ru3+ ) -> (Ru2+)* -> Ru2+ + light Roche Competitive or Titrimetric chemiluminescent system (ELECSYS detection system) Add • Sample • Ru2+ labelled Ag • Biotin labelled Ab (>-B) • Buffered diluent Add streptavidin Coated magnetized beads S S S Add TPA and voltage To stimulate chemiluminescence Ru2+-<Ag>-B S S Ru2+-<Ag>-B S >-B S Wash away Unreacted Sample/reagents Ru2+-< >-B S Ag >-B Ru2+-Ag>-B Ru2+-Ag>-B Ag Ag Ru2+-<Ag>-B S Immobilized Magnetized Beads drawn to Electrode/magnet S Assay characteristics If competitive, would use more limited or lower amount of Ab, and sample and labelled Ag would be added at the same time 2. Also a chemiluminescent assay Since it has a chemiluminescence based reporter system Luminescence 1. A competitive or titrimetric assay 3. NOT an ELISA, since no enzyme labels are used 4. It is heterogenous assay, requiring separation unreacted components, i.e. separation of solid phase and liquid phase. Log Concentration Errors in Immunoassays -The hook effect- Two step assays, adding the sample, followed by a wash prior to adding the second Ab are not prone to the hook effect From: Hormone Immunoassay Interference: A 2021 Update. Ghazal K, Brabant S, Prie D, Piketty ML. Ann Lab Med. 2022 Jan 1;42(1):3-23. doi: 10.3343/alm.2022.42.1.3. Review. Errors in Immunoassays -Interfering Antibodies from patient samplesFrom: Hormone Immunoassay Interference: A 2021 Update. Ghazal K, Brabant S, Prie D, Piketty ML. Ann Lab Med. 2022 Jan 1;42(1):3-23. doi: 10.3343/alm.2022.42.1 .3. Review. Errors in Immunoassays -Interference in Roche Elecsys assaysDecreased signal in the following cases a) Patients taking large doses of biotin (vit B7) b) Anti-ruthenium antibodies in patient samples Lower signal output means -> overestimation for competitive assays -> underestimation for sandwich or immunometric assays Questions • What are “allowable limits of performance”? • The allowable limits of performance for sodium is +/- 3.0 mmol/L. If a lab can measure plasma sodium with a SD of 3.0 mmol/L, is that satisfactory? Explain your answer. • What is a proficiency program, when is it necessary and of what use is it? • Compare the way we use internal and external QC programs. • What type of auto-analyzer would be required to measure a steroid present at a concentration of 12 pmol/L. In addition, what type of assay principle would be required, and what would the calibration curve look like? • Compare the different type of solid phases used by Roche Elecsys and Siemens Immulite assays. • Roche: Streptavidin coated magnetized beads • Siemens Immulite: Beads coated with the capture antibody • How do titrimetric assays differ from competitive assays? • Patient sample is added to a solid phase Ab, following an incubation step, a fixed amount of labelled Ag is added, and binds to vacant capture Ab sites not already bound/filled by the Ag from the patient’s sample. In a competitive assay, patient sample and the fixed amount of labelled Ag are added at the same time and compete for a limited amount of capture Ab. • What is the precision of an assay at the concentration corresponding to its functional sensitivity? • The precision with which we can measure an analyte generally poor at low analyte concentration, and this is reflected in poor CVs. The functional sensitivity is defined at that concentration of an analyte which can be measured with a CV of 20%. • General clinical chemistry analyzers utilize homogenous assays and these account for the bulk of the common laboratory tests we conduct. What does the term homogenous signify, and what advantage do these have compared to heterogenous methods. Examples of tests on general chemistry analyzers: U&Es, glucose, sodium, potassium, LFTs, amylase, lipase, CK, LDH • The assays can be done entirely in the liquid phase, with no requirement to separate any component of the reagents to get a readout. The advantages of such methods are easier to automate hence their • Compare the principle of Siemens Immulite and Roche Elecsys immunoassays. What are the similarities and differences? • The Roche Elecsys system is subjects to two sources of interference that are not seen in most other sensitive immunometric assays. What are these? • What are the two major auto-analyzer types in a clinical biochemistry laboratory and give an example of major differences in the analytical principles of the chemistries they employ. • Consider the following technology? On what type of analyser are you likely to find this technology? In what clinical setting is this type of technology useful? • What is chemiluminescence, and how does it differ from fluorescence or light colorimetry, and why do immunoassays using chemiluminescence yield a higher sensitivity. • What is a dry chemistry analyzer? • Give a brief outline of how antibodies in some patient samples can interfere in immunoassays. Illustrate your answer. Maths for the scale of what we need to measure The Math 1 mol = 6.02 × 1023 molecules thus 1 mol/L = 6.02 x 1023 molecules 1 𝜇mol/L = (10-6 x 6.02 × 1023 )/L = (6.02 x 1017)/L = (6.02 x 1011)/uL = 6.02 x 1013/100 uL 1 𝜂mol/L = (10-9 x 6.02 × 1023 )/L = (6.02 x 1014)/L = (6.02 x 108)/uL = 6.02 x 1010/100 uL 1 𝜌mol/L = (10-12 x 6.02 × 1023 )/L = (6.02 x 1011)/L = (6.02 x 105)/uL = 6.02 x 107/100 uL Free hormones range in concentration from 𝜌mol/L to 𝜂mol/L concentrations Assays typically use 5 𝜇L to 100 𝜇L volumes At the lower end there are > between 10 million to 60 million molecules or copies of a hormone in a reaction Maths for the scale of what we need to measure The Math 1 mol = 6.02 × 1023 molecules thus 1 mol/L = 6.02 x 1023 molecules 1 𝜇mol/L = (10-6 x 6.02 × 1023 )/L = (6.02 x 1017)/L = (6.02 x 1011)/uL = 6.02 x 1013/100 uL 1 𝜂mol/L = (10-9 x 6.02 × 1023 )/L = (6.02 x 1014)/L = (6.02 x 108)/uL = 6.02 x 1010/100 uL 1 𝜌mol/L = (10-12 x 6.02 × 1023 )/L = (6.02 x 1011)/L = (6.02 x 105)/uL = 6.02 x 107/100 uL Free hormone concentrations range in concentration from 𝜌mol/L to 𝜂mol/L concentrations Assays typically use 5 𝜇L to 100 𝜇L volumes At the lower end > between 10 million to 60 million molecules or copies of a hormone in a reaction Example of Number of Molecules Per Volume Analyte GH (baseline) fT3 Oestradiol (F) fT4 Oestradiol (M) Aldosterone ACTH Ferritin cTnI (F) Creatinine Albumin Sodium Apo B Transferrin Low Limit nmol/L High Limit nmol/L 0.004 0.008 0.02 0.037 0.08 2 2.1 0.66 Low Limit copies/5uL 0.8 0.0074 1.266 0.05 0.184 0.44 26 12,040,000 24,080,000 60,200,000 111,370,000 240,800,000 6,020,000,000 2,408,000,000 22,274,000 3,810,660,000 150,500,000 553,840,000 1,324,400,000 78,260,000,000 High Limit copies/100 uL 240,800,000 481,600,000 1,204,000,000 2,227,400,000 4,816,000,000 120,400,000,000 48,160,000,000 445,480,000 76,213,200,000f 3,010,000,000 11,076,800,000 26,488,000,000 1,565,200,000,000 126,420,000,000 39,732,000,000 3,160,500,000 60,000 120,000.00 180,600,000,000,000 361,200,000,000,000 451,130 751,880.00 1,357,901,300,000,000 2,263,158,800,000,000 130,000,000 145,000,000.00 391,300,000,000,000,000 436,450,000,000,000,000 3,612,000,000,000,000 27,158,026,000,000,000 7,826,000,000,000,000,000 7,224,000,000,000,000 45,263,176,000,000,000 8,729,000,000,000,000,000 722,400,000,000,000 1,625,400,000,000,000 1,601,320,000,000,000 2,859,500,000,000,000 26,600.00 47,500.00 6,321,000,000 1,986,600,000 Low Limit copies/100 uL 158,025,000 12,000 27,000 0.0525 High Limit copies/5 uL 36,120,000,000,000 81,270,000,000,000 80,066,000,000,000 142,975,000,000,000 ~100,000,000,000 to 400,000,000,000 stars in the milky way ~2,000,000,000,000 galaxies in the universe ~200,000,000,000,000,000,000,000 stars in the universe • https://theconversation.com/how-many-stars-are-there-in-space-165370 Picomolar to nanomolar concentrations Micromolar Concentrations Maths for the scale of what we need to measure The Math 1 mol = 6.02 × 1023 molecules thus 1 mol/L = 6.02 x 1023 molecules 1 𝜇mol/L = (10-6 x 6.02 × 1023 )/L = (6.02 x 1017)/L = (6.02 x 1011)/uL = 6.02 x 1013/100 uL 1 𝜂mol/L = (10-9 x 6.02 × 1023 )/L = (6.02 x 1014)/L = (6.02 x 108)/uL = 6.02 x 1010/100 uL 1 𝜌mol/L = (10-12 x 6.02 × 1023 )/L = (6.02 x 1011)/L = (6.02 x 105)/uL = 6.02 x 107/100 uL Free hormone concentrations range in concentration from 𝜌mol/L to 𝜂mol/L concentrations Assays typically use 5 𝜇L to 100 𝜇L volumes At the lower end > between 10 million to 60 million molecules or copies of a hormone in a reaction Acridinium Ester Light Emission Acridinium ester An unstable N-methylacridon Returns to ground state and releases light Chemiluminogenic akaline phosphatase substrate Chemiluminogenic1 • 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) From: . Bronstein, I., et al. (1989). "1,2-dioxetanes: novel chemiluminescent enzyme substrates. Applications to immunoassays." J Biolumin Chemilumin 4(1): 99-111.