1 / 40

CLINICAL BIOCHEMISTRY 1 ___________________________ School of Biomedical Sciences, Curtin University of Technology

CLINICAL BIOCHEMISTRY 1 ___________________________ School of Biomedical Sciences, Curtin University of Technology. By Mrs Chan Gek Lan & Dr Chan Soo Yin 2 Written Assignments 40% Written Examination 60%

lotus
Télécharger la présentation

CLINICAL BIOCHEMISTRY 1 ___________________________ School of Biomedical Sciences, Curtin University of Technology

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. CLINICAL BIOCHEMISTRY 1___________________________School of Biomedical Sciences, Curtin University of Technology By Mrs Chan Gek Lan & Dr Chan Soo Yin 2 Written Assignments 40% Written Examination 60% Ref: Burtis, CA and Ashwood, ER, Tietz Fundamentals of Clinical Chemistry (4th Edition), WB Saunders 1996. Burtis, CA and Ashwood, ER, Tietz Textbook of Clinical Chemistry (3rd Edition), WB Saunders 1999.

  2. INTRODUCTION________________ ANALYTICAL CHEMISTRY Use of analytical methods & good instrumentation Qualitative & quantitative analyses on body fluids CLINICAL CHEMISTRY Understanding of normal biochemical processes Results to diagnose & treat disease

  3. LECTURE 1 • METHOD EVALUATION • SELECTION & INTERPRETATION • RADIOACTIVITY & ITS MEASUREMENT

  4. 1. METHOD EVALUATION___________________________________________________________________________________________________ Method Selection & Evaluation Requirements for Implementation Selecting an Analytical Method Evaluating an Analytical Method Tietz. Fundamentals, Chapters 12

  5. 1A. Method Selection & Evaluation • Key steps in implementing new methods • Select new or revised method with care -> Evaluate analytically • Method involves both methodology & instrument Table 13-1: The Keys to Successful Evaluation of a New Method • Apply a clinical perspective for the whole task. • Set a target – an analytical goal • Conduct the right experiments to collect the necessary data. • Use statistical tools properly so that you estimate the errors correctly. • Make objective conclusions

  6. 1B. Requirements for Implementation • Practical Requirements – specimen, personnel, cost, quality control, safety • Performance Parameters – properties relating to performance of method • Accuracy – closeness of agreement between measured value of an analyte and its ‘true’ value • Analytical Range – range of concentration or other quantity in the sample over which the method is applicable c. Analytical Sensitivity – quantifies change in signal relative to change in quantity, concentration, or property d. Analytical Specificity – ability of an analytical method to determine exclusively the analyte it measures

  7. Cont. Performance Parameters • Blank Measurements – measurements due to reagent & sample constituents f. Detection Limit – the smallest concentration or quantity of an analyte that can be detected • Interferences – the effect of a compound(s) has on the accuracy of measurement • Precision – the ability to produce the same value for replicate measurements i. Recovery – ability of an analytical method to measure an analyte correctly when a known amount of it is added to samples j . Ruggedness – consistently reliable performance when used by different operators & with different batches of reagents

  8. 1C. Selecting an Analytical Method • The principle of the assay • The composition of reagents & reference materials, the quantity provided, & their storage • The stability of regents & reference materials • Possible hazards, appropriate safety precautions • The type, quantity & disposal of waste • Specimen requirements – collection, volume, storage • Anticipated analytical performance – accuracy, precision, range • The reference interval – derivation, values in health & disease • The detailed protocol for performing the test • The availability of technical support, supplies

  9. 1D. Evaluating an Analytical Method • Write an evaluation protocol & a method procedure • Document the validity of the method • Determine the reportable range • Determine the components of precision • Determine the accuracy • Estimate the sensitivity • Estimate the detection limit • Test the specificity • Establish the reference interval

  10. 2. SELECTION & INTERPRETATION • ___________________________________________________________________________ • Sensitivity & Specificity • Receiver operating characteristic curves • Predictive value Tietz. Fundamentals, Chapter 11

  11. SELECTION & INTERPRETATION • ____________________________________________________________________________________________ • Problem -> clinical tests -> different procedures • Qn: Which procedure to use ? • Evaluate test performance -> select Example: Urologist using Assay to detect prostatic carcinoma Table 12-1: Correct Prediction of Prostatic Carcinoma by Two Prostate-specific Antigen Assays *Does not include patients with benign prostatic hypertrophy Qn: Which test is better?

  12. 2A. Sensitivity vs Specificity_________________________________________________ Sensitivity: fraction of those with specific disease that the assay correctly predicts Specificity: fraction of those without the disease that the assay correctly predicts (Table 12-3) Cont. Urologist story: Urologist wants to use assay on BPH patients Benign Prostatic Hyperplasia (noncancerous prostate enlargement) associated with higher PSA If exclude in test -> increase in false-negative If include in test -> increase in false-positive

  13. Table 12-4: Correct Prediction of Prostatic Carcinoma & Benign Prostatic Hyperplasia Fig 12-1: Dot plot of PSA on BPH patients & prostate carcinoma patients • Test A & B are two different cutoffs Lower cutoff -> increase sensitivity -> decrease true-positive

  14. 2B Receiver Operating Characteristic Curves _____________________________________________________________________________________________________________________ • Gives better estimate of sensitivity & specificity • Gives comparison of different tests • Plot sensitivity (y-axis) vs 1-specificity (x-axis) • Y-axis: true-positive rate • X-axis: false positive rate

  15. Cont. ROC Curves Fig. 12-2: ROC curve for PSA • Dotted line: test with no discrimination • Curve above dotted line: performance better than random • Curve from lower left to upper left & then to upper right • Can select cutoff of desired sensitivity or cutoff of desired specificity Fig. 12-3: ROC of Prostattic acid phosphatase (PAP) & PSA • At the same level of sensitivity, PSA assay offers greater specificity

  16. 2C. Predictive Value of a Test • _____________________________________________________________________________________________ Qn: If assay is positive, how likely will patient have disease? Table 12-4Test A: Sensitivity = 78% Specificity = 58% Assume prevalence of 10% of biopsy-positive tumors • Predictive value positive (PV+) = % patients with +ve results who are diseased => No. of TP in 100 screened patients False positive rate = 1 – specificity => No. of FP = FP rate x no. nondiseased patients • PV+ => 17% of men with PSA values greater than 4ug/l in the screening program would have a positive biopsy (vs 10% in total group)

  17. b) Predictive value negative (PV- ) = % patients with –ve test results who are nondiseased TN = specificity x no. disease-free patients FN rate FN = FN rate x no. patients with disease PV- 96% of men with with PSA values of <4ug/l would not have the disease vs 90% in the total group

  18. 3. RADIOACTIVITY & ITS MEASUREMENT _____________________________________________________________________________________________________________________________________________ • Types of Radiation • Units & Specific Activity • Rate of Decay • Interaction of Radiation with Matter • Detection & Measurement of Radioactivity • Radiation Safety • Tietz Fundamentals, Chapter 9 • Tietz Textbook, Chapter 5

  19. Radiation • Cannot feel, taste or smell • Emitted from unstable nucleus -> change in protons & neutrons -> new, more stable nucleus 3A. Types of Radiation • Alpha Particle • Beta Particle • Gamma Rays

  20. a. Alpha particle (Alpha decay) • two protons (charge 2+) & 2 neutrons -> • mass number of 4, atomic number of 2 -> • Identical to helium nucleus He or • Fr unstable heavy elements with atomic numbers >70 Ra  Rn + He • Most -emitters have little clinical applications

  21. b. Beta particle • High-energy electron from unstable nuclei • harge 1- & mass number of 0 -> same as electron e or • Formed when neutrons change into protons n  H + e Neutron new proton electron formed C  N + • Rearrangement of nucleus -> mass number & nucleons unchanged but atomic number +1 • From elements with atomic numbers <60 egs. C, H, I, Fe, S, P • For labeling organic compounds & nucleic acid; protein synthesis studies

  22. Cont. Types of Radiation c. Gamma rays • High energy similar to X-rays • After - or -decay, nucleus may be in excited state -> de-excite by emitting -photon (energy) -> no change in atomic number or mass • Energy only -> no mass or charge • Low ionizing power but high penetration eg. -radiation from Co will penetrate 15cm steel

  23. 3B. Units & Specific Activity Units: • Curie (Ci) expresses nuclear disintegrations 1 Ci = 3.7 X 1010 disintegrations/s • Rad (radiation absorbed dose) measures amount of radiation absorbed per gram of material 1 rad = 10-2 J/kg = 2.4 X 10-3 cal/kg • Rem (radiation equivalent in humans) measures biological damage Rem = rad X RBE • Becquerel measures disintegrations 1Bq = 1 dps SI unit of radioactivity usually in kBq

  24. Cont. Units & Specific Activity Specific Activity • Radioactivity per unit mass • Radioactivity per mass of labeled compound • Radioactivity per unit volume

  25. 3C. Rate of Decay • Characteristic of an individual radionuclide • Is a constant – not affected by temp, pressure, concn, • Activity (A) = strength or rate of decay • Half-life (t) = time reqd for sample activity to decrease to half its initial value An = A0 X 2-t/t where t = elapsed time and An = A0 X 2-n n = number of half-lifes • Equation useful in planning expts & in disposal of radioactive waste What is An after • Seven half-lifes? An = b. Ten half-lifes? An =

  26. 3D. Interaction of Radiation with Matter Alpha & Beta Particles: • Rapidly moving & charged -> force electrons from atoms they pass -> lose energy • Cause ionization & excitation Gamma rays: • If energy is low, photons cause electronic & molecular motions b. Photoelectrical Effect Moderate energetic photon ejects an atomic electron; photon is absorbed c. Compton Effect Highly energetic photon propels an atomic electron in one direction & the photon is scattered in another direction Ejected electron deposits its energy in the surrounding matter by ionization & excitation

  27. 3E. Detection & Measurement of Radioactivity Most detection techniques by: • Darkening of photographic emulsion • Ionization of gas (portable radiation monitor) • Fluorescent scintillation • Scintillation detectors I. Scintillation Detectors (SD) • Crystal SD & Liquid SD • Principle: Scintillator absorbs radiation -> energy produces a flash of light -> detect/measure • Convert absorbed energy into visible or uv light

  28. A. Crystal Scintillation Detector • Sodium iodide crystal SD • Detect blue-violet (420nm) scintillations • Photons from gamma rays penetrate specimen tube & aluminum to crystal • Beta-radiation cannot penetrate B. Liquid Scintillation Detector • Measures scintillations in transparent vial containing unknown sample & liquid scintillator • Suitable for beta-emitters of low energy & short range eg. tritium & 14C

  29. Cont. LSD • Liquid scintillator/scintillation cocktail • Primary solvent – aromatic hydrocarbon eg. toluene, xylene, pseudocumene • Primary scintillator • PPO (2,5-diphenyloxazole) • Dissolved in primary solvent-> absorbs energy -> converts energy • Secondary solvent – improve solubility of samples; stabilize or emulsify sample • Secondary scintillator (wavelength shifter) – absorb uv photons of PS -> re-emit energy • One or more adjuvants eg. suspension agents, solubilizers & antifreezers

  30. II. Electronics of Scintillation Counting • After each burst of scintillation -> light is collected -> light converted to electrical pulse -> pulses amplified -> sorted by size Photomultiplier • Converts scintillations to electrical pulses • Photons strike cathode -> ejects outer-electrons from it -> electrons accelerated towards dynodes -> increases the number of electrons Preamplifier • To boost the current from the photomultiplier Coincidence Circuit • To reduce the effects of electronic noise & small scintillations in beta counters Pulse-Height Analyzer • To size pulses electronically within an acceptable window

  31. III. Efficiency of Scintillation Counting • Counting Efficiency = Count Rate/Decay Rate • Factors affecting Count Rate: • The fraction of all decays • The fraction of potentially useful radiations that are directed into the scintillator • The fraction of photons entering the scintillator • The scintillation efficiency • The detector threshold

  32. IV. Quenching in LSC A. Quenching: • Factors that reduce scintillation efficiency • Photon emitted in scintillator -> part of the energy is absorbed • Cause batch-to-batch or sample-to-sample variations • Monitor quenching in every assay & where necessary, correct it

  33. B. Types of Quenching: • Chemical Quenching • Caused by molecules in sample of cocktail -> compete with primary scintillator • eg. water, oxygen, nitromethane • Optical Quenching • Absorption of photons by colored compounds in the scintillator eg. heme • Phase Quenching • In multiphase systems • Minute irregularities in physical contact between sample & solvent -> absorb radiation or interferes with energy transfer

  34. C. Quench Correction • Internal standard • Primary reference • Liquid of known reactivity added to • Blank samples (presumed to contain the same quenching factors as the unknown samples • Unknown samples after measuring count rate • Counting Efficiency (internal standard) = Measured count rate/ Known decay rate

  35. Cont. Quench Correction • External standard • Capsule of long-lived, -emitting nuclide eg. Rd, Cs or Ba • -rays produce Compton electrons + some photoelectrons -> scintillation -> quenching affects counting efficiency • Measure shift in the energy spectrum -> determine & correct • Corrected data expressed as decays per min = absolute count rate • Have replaced internal standard as more convenient & adaptable to large automatic counters

  36. Cont. Quench Correction Automatic Quench Correction • Provided on large counters => can print either the measured or corrected count • Require initial calibration using a set of quenching standards each containing • The same, known amount of nuclide but • A different amount of quenching compound • External standards subject to vagaries

  37. 3F. Radiation Safety I. Quantities & Units • Absorbed Dose • SI unit is gray (Gy) • 1 Gy = 1J absorbed per kg • Effect of radiation: • Deposition of large amounts of radiation energy -> disruption of chemical bonds -> cells killed • If less energy absorbed -> damage to cells & tissues apparent in hours • At lower levels -> damage to cells too slight to detect or biological repair is complete -> no discernible damage

  38. Cont. Quantities & Units • Exposure • Amount of charge liberated by ionizing radiation • SI unit is coulomb per kg • Old unit is R • 1R produces ionization equal to 0.258 mC/kg • Exposure of dry air to 1R -> deposition of 0.87 rad • Exposure of soft tissue (denser than air) to 1R -> deposition of 0.93 – 0.97 rad

  39. Cont. Quantities & Units C. Dose Equivalent • Biological effect depends on type of radiation • Dose equivalent = Absorbed dose X Quality Factor • For -particles and -rays, Q=1 • SI unit is Sv • Conventional unit is rem D. Radiation Dose Limits • Total effective dose equivalent = dose from external exposure + dose from internal exposure • Table 5-4 • Radiation dose limit to general public

  40. II. Methods for Limiting Exposure • Limiting External Exposure • Reduce time near source of radiation • Increase distance; use tools • Use shielding B. Limiting Internal Exposure • Limit inhalation • Limit ingestion; wear gloves, gowns, GLP

More Related