2012 lecture 1 intro to lab med NO PICTURES

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Biochemistry 3H03 Clinical Biochemistry Session 1 :

Biochemistry 3H03 Clinical Biochemistry Session 1 Stephen Hill Dept. of Pathology & Molecular Medicine hillstev@hhsc.ca 905-522-1155 x 33098

Instructors:

2 Instructors Dr. S. Hill, 522-1155, Ext. 33098 hillstev@hhsc.ca   Contact me for course administration - missed tests, examinations etc. Material around the content of my section Dr. J. Macri, 527-4322, Ext. 46046 macri@hhsc.ca Contact him for material around the content of his section

Course Mail Box:

3 Course Mail Box In Avenue to Learn Lecture slides will be posted here Question answers copied here for all to see

Lecture Slides:

4 Lecture Slides We will post these on the course site Remember that the slides are outlines of the lecture only They are not intended to be ‘Notes’

Text Book:

5 Text Book Clinical Chemistry 6 th Ed’n, 2008 Marshall WJ Publisher - Mosby  The lectures will follow the text. Please remember that other references will be provided as well and that the lectures may not follow the text exactly. Supplementary material may be provided for some lectures

Test Dates:

6 Test Dates Wednesday February 1 Covers material to date – 25% Monday March 19 Covers material since test #1 – 25% Exam Scheduled by Registrar Covers ENTIRE course 50%

Missed Tests:

7 Missed Tests If you miss a test for medical or compassionate reasons – see your home department, the Biochemistry Department or the Assistant Dean of your Faculty ASAP. Marks for tests missed due to medical or compassionate reasons will be distributed amongst the remaining test and the exam 25, 25, 50% → 35, 65%

Lecture #1 Clinical Biochemistry:

Lecture #1 Clinical Biochemistry Organization of laboratory medicine Specimen collection Steps in laboratory analysis Variability of results 8

What is Laboratory Medicine and Clinical Biochemistry?:

9 What is Laboratory Medicine and Clinical Biochemistry? Branch of Medicine that provides Laboratory results, interpretation and consultation Examines the chemical content of biological fluids. Guide for decision making and management of patients Requires accurate, relevant and timely information

Why do Laboratory Testing?:

10 Why do Laboratory Testing? Diagnosis – establish underlying reason for the signs, symptoms, and complaints. Prognosis – establish risk and potential timeline for future event Monitoring – follow course of condition. Monitor blood concentrations of drugs Screening – look for condition in absence of signs/symptoms Population screening – newborn screening programs Screening based on risk Cholesterol screening over age 40 Screening for breast cancer in women

Steps in obtaining a laboratory test:

11 Steps in obtaining a laboratory test Test is requested by physician and ordered on the computer. Barcode is generated Specimen is collected Specimen and order are transported to the lab The specimen is accessioned in the lab The specimen is prepared for analysis The specimen is analyzed The results are reviewed and verified The results are released to the patient’s record

What Sample should we use to do Clinical Biochemistry? :

12 What Sample should we use to do Clinical Biochemistry? Blood Urine Cerebrospinal Fluid Stool Amniotic Fluid Gastric Juice Gall stone Kidney Stone Saliva Tissue Specimen Choice of sample depends on: Analyte to be measured, biological considerations, ease of collection Comprise the majority of all specimens analyzed

Blood Analysis:

13 Blood Analysis Testing can be done on whole blood, serum or plasma. Choice depends on a number of factors Since most tests in the chemistry lab involve analytes that are dissolved in the fluid portion of blood, serum or plasma are the specimens of choice. Analyte to be measured Most hematology tests requires whole blood Instrumentation used for analysis Most automated instruments are not set up for whole blood analysis Turn around time Analysis of whole blood is the quickest. No waiting for clot or spinning Plasma requires centrifugation prior to analysis Serum – allow blot to clot, then centrifuge

Blood Analysis:

14 Blood Analysis Source Veins Arteries Skin puncture-capillary blood Factors affecting choice of Blood Source and Collection Method Analyte under investigation Patient vascular status ease of collection Collection Method Syringe Evacuated tube Additives Separator gel Intravenous lines

Collection Tubes:

15 Collection Tubes The most widely used tubes for blood collection are evacuated tubes Negative pressure facilitates collection Easy to use, Sterile Universally used colour-coded rubber stoppers to denote tube type. Tubes can contain various anticoagulants for the collection of whole blood or plasma. Tubes can have additives/preservatives for specific tests (glucose, metals) May have a Separator Gel Upon centrifugation forms a barrier between cells and serum or plasma

Blood Composition:

16 Blood Composition Plasma Plasma is fluid component of blood. ~55% of volume of whole blood. Contains proteins, sugars, vitamins, minerals, lipids, lipoproteins and clotting factors . 95% of plasma is water Red Blood cells (RBC) Whole Blood Whole Blood after centrifugation Note: clotting has been prevented White Blood cells & Platelets Cellular Components

PowerPoint Presentation:

17 Blood Composition Serum Plasma is fluid component of blood. Comprises ~55% of total volume of whole blood. Contains proteins, sugars, vitamins, minerals, lipids, lipoproteins 95% of plasma is water No clotting factors Blood Clot -comprised of clotting factors (Fibrin, platelets etc) -RBCs, WBCs Whole Blood Whole Blood after clotting and centrifugation If blood is collected without anticoagulation and allowed to stand it will clot. Formation of an insoluble fibrin clot. If blood is then centrifuged the fluid portion is known as SERUM

Where are Clinical Biochemistry Tests Performed:

18 Where are Clinical Biochemistry Tests Performed In routine, highly automated laboratories – large, high volume mutli-analysers. Operate 24/7 to support ED, ICU etc. Specialty laboratories- lower volume, generally less automation. Point-of-Care testing – simple to operate devices take the laboratory to bedside or clinic.

Automated Lab Instrumentation :

19 Automated Lab Instrumentation Bar-coded test tubes are loaded onto to the instrument. Menu Driven Test selection All pipetting, mixing and measurements are automatic Random Access (can perform specific tests on a specific sample) Analyzer is interfaced with Laboratory Information System (LIS). All reagents for specific tests such as control calibrators, buffers come as kits that a loaded directly onto the instruments. Instruments constantly monitors amount of consumables on-board and volume of liquid and solid waste generated

Analytical techniques found on Multi analyte analyzers:

20 Analytical techniques found on Multi analyte analyzers Colourometric analysis – chemical reaction generates colour proportional to the concentration of the analyte of interest – spectrophotometric detection. Immunoassays - antibodies used to capture and detect the analyte. Ion-selective electrodes. Designed with a membrane that is specific for a particular analyte (Na, K, Cl). Change in potential is measured when electrode is placed in sample.

Specialty Laboratories:

21 Specialty Laboratories Generally lower volume tests Not available on automated analyzers Because the demand is not there The test is not easily automated (extraction steps) Requires the volume of specimens to justify the test High cost of equipment to relative few specific tests

Analytical Techniques in Specialty Laboratories :

22 Analytical Techniques in Specialty Laboratories Colourometric analysis Electrophoresis Used to separate serum proteins into 5 distinct bands Often used to separate isoforms of enzymes HPLC - Used to measure vitamins Infrared Spectroscopy - Used to analyze components of kidney stones Immunoassays including Radioimmunoassay (using radioactive detection) Used less and less but still employed for those analytes present in minute amounts (pmol) in the blood (ie. testosterone) GC-MS (Gas chromatography-mass spectroscopy) and/or LC-MS (liquid chromatography- mass spectroscopy. Molecular Biology

Point of Care Testing (POCT) :

23 Point of Care Testing (POCT) It is sometimes more convenient an efficient to perform the test at the bedside, in the clinic, in the physician’s office, or at home Some examples: Blood glucose Urinalysis Blood gases Electrolytes Cardiac markers (Troponin I & T) Drug screens POC are nearly always more expensive, than the same tests performed in the central laboratory Anywhere from 2x- 10x the cost for the same test . Test quality is usually, but not always, lower.

Issues for POC testing:

24 Issues for POC testing Cost System management In Ontario, lab testing in a hospital is responsibility of the laboratory, regardless of who performs it – in lab or in clinic Connectivity to hospital computer system Standardization to laboratory instruments POC for blood is usually done on whole blood as opposed to serum/plasma

Test results Variability, Errors, Interferences :

25 Test results Variability, Errors, Interferences Results of biochemical analysis are ESTIMATES of concentration only. Biological variability within an individual and between individuals. Analytical variability All tests have some degree of variation upon repeated measurements of the same sample. –PRECISION. Ability of the analytical method to reflect the true concentration ACCURACY or BIAS. The final test result is affected by factors that occur Before analysis At the time of the analysis After the test is completed

Biological Variability:

26 Biological Variability Inter-individual Age Sex Race Genetics Intra-individual Age Diet Exercise Drugs Sleep pattern Posture Monthly Cycles Seasonal Cycles

Supine vs. sitting or standing:

27 Supine vs. sitting or standing Going from lying (supine) to upright reduces total blood volume by about 700 ml The following may increase by 5-15% by changing form lying to standing Total protein Albumin Lipids Iron Calcium Enzymes

Analytical Variability:

Analytical Variability 28 Pre-analytical Correct patient Transport Exposure to UV light Standing time before separation of cells Centrifugation time Storage conditions Analytical Random errors Systematic errors Post-analytical Transcriptions errors Results reported to wrong patient

Pre-analytical errors:

29 Pre-analytical errors Identification (common source of error) Was the blood collected from the correct patient? Was the blood correctly labeled? Patient name, ID, date, time of collection Collection Was the right tube used? Was venipuncture performed correctly? Was the specimen prope rly stored?

Prolonged venous stasis:

30 Prolonged venous stasis Blocking the flow of blood with the tourniquet with eventually lead to a sieving effect. Small molecules, water and ions are forced out blood vessels and larger molecules are concentrated Increases Total Protein, proteins, iron cholesterol, bilirubin Decreases potassium

Analytical Performance :

Analytical Performance Precision – ability to achieve the same result upon repeated measurement Accuracy or Bias – ability of the method to produce a result that is close to “True”. Analytical Sensitivity – ability to measure low concentrations and/or small differences in concentration Analytical Specificity – ability to measure only the component of interest, and freedom from interference by other substances 31

Analytical Uncertainty:

32 Analytical Uncertainty Errors in the determination of the concentration of the analyte of interest. Due to Errors or variability in the analytical process Inappropriate process control Interferences Biological or induced in the sample collection or preparation.

PowerPoint Presentation:

33

Common Biological Interferences:

34 Common Biological Interferences Hemolysis – rupture of red bloods cells releases cellular contents into plasma or serum. Improper collection or sample handling Increases in K , Mg, PO 4 The release of hemoglobin into blood can effect the reactions comprising specific tests Causes serum or plasma to be red and can effect tests that are colorimetric Lipemia (lots of fats) and proteinemia (lots of protein) Causes serum or plasma to be become turbid. This can effect colourimetric and turbidometric based tests Also can cause a dilution effect. Fats and proteins are large and displace water in plasma. Can give falsely low results especially for Na Bilirubin – a highly yellow coloured constituent of blood. High concentrations will interfere with colourometric reactions

PowerPoint Presentation:

35 Specimens requiring special handling Should be placed immediately on ice Lactate Ammonia Acid phosphatase Plasma catecholamines

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