Clinical Biochemistry 3H03 Session 1



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Clinical Biochemistry 3H03 Session 1

  • Dr J. Macri
  • Associate Professor
  • Department of Pathology and Molecular Medicine
  • Clinical Biochemist
  • Hamilton Regional Laboratory Medicine Program

Your Most Important Slide

  • Testing (this applies to my section only)
    • The mid-term and final exam will be based on material from the lectures notes as well as anything covered in class.
  • Mid term
    • Short answer, essay, calculations, multiple choice
  • Final
    • Multiple choice
  • Corresponding reading material
  • Chapters 1-5 in Clinical Chemistry 5th Ed Marshall
  • Note: Not all material covered in the lectures is found in this text. Not all material covered in the text is presented in class.
  • Additional clinical chemistry textbooks are available in the library on reserve.
  • Bottom line for studying is to use the lecture slides and any notes you make while attending class.
  • Office hours: No formal office hours. Feel free to email (macri@hhsc.ca) or call 905 527-4322 ext 46046.
  • I am happy to answer your questions immediately or arrange a time to discuss any issues.
  • Class notes will be able on the website either the day of or following the lecture.

Steps in the Investigation of a Patient

  • Patient History
  • Physical Examination
  • Laboratory Tests
  • Imaging Techniques
  • Diagnosis
  • Therapy
  • Evaluation

Laboratory Medicine

  • A discipline of medicine that functions to provide diagnostic tests which are utilized by physicians to assess the health of an individual.
    • Must more than just a “service”. Dynamic interaction with all hospital departments (Emergency (ER), Intensive Care Unit (ICU), Cardiac Care Unit (CCU) as well as physicians outside of the hospital to maximize health care through:
      • Consultation regarding tests to be requested
      • Education
        • Medical students, residents
        • Medical Technologists
        • Medical Staff
      • Development, Evaluation and Implementation of New Diagnostic Assays
      • Supporting Clinical and Basic Research
      • Interaction with all departments to maintain and/or improve the flow and accuracy of information (i.e test results)
    • Driving force is Patient Care……This must be done effectively and economically
      • Laboratory Medicine Program must operate as a Non-profit business
      • Has a fixed yearly budget to cover staff, equipment and reagents

Hamilton Regional Laboratory Medicine Program (HRLMP)

  • Process started 1998 to combine all Hamilton Hospital laboratories in an effort to maximize resources and improve laboratory service
  • Annual operating budget for the HRLMP is over 50 million dollars
  • Over 600 staff (MDs PhDs, technologists, technicians, clerical)
  • Number of specific procedures (i.e ) performed is over 5000
  • Over a million tests performed per year
  • Operates the Laboratory Reference Centre (LRC) to provide access to specialize testing to other institutes in Central Ontario and rest of Canada
    • Over 125 LRC clients
    • Performed over 246,000 tests and generated a gross income of 4.5 million
  • Clinical Chemistry
  • And Immunology
  • Microbiology
  • Hematology and Transfusion Medicine
  • Genetics
  • Anatomical Pathology

Clinical Chemistry Laboratory Organization

  • Core Lab Facility
  • found at virtually all hospitals
  • operates 24h day 7 days a week to provide the
  • essential most requested tests
  • Highly automated environment
  • Instruments with Multi-analyte capabilities
  • Special Chemistry
    • less frequently ordered tests
    • labour intensive and often manual methods
    • generally non-stat tests (result not required immediately)
  • Point of Care Testing (POCT)
    • Instruments located outside of chemistry laboratory such as CCU, ER, ICU or satellite centre (clinic)

Core Lab

  • High volume tests (many per day). Often require quick turn-around-time (TAT)
  • Many tests where abnormal values are incompatible with life and therefore of critical value to the physician
  • Electrolytes: sodium (Na), potassium (K), Chloride (Cl)
  • Blood gases: pO2, pCO2, pH, HCO3, oxygen saturation
  • Endocrine: Thyroid hormones
        • Prolactin
        • Testosterone
  • Lipids: Total Cholesterol, Low Density Lipoprotein Cholesterol (LDLc), High Density Lipoprotein Cholesterol (HDLc), Triglyceride (Fats)
  • Proteins: Total Protein
  • Glucose
  • Tumour Markers: Prostate Specific Antigen (PSA)
  • Vitamins (Vit B12) and minerals (Calcium, (Ca))
  • Toxicology
    • Ethanol, methanol
    • Drugs of abuse generally conducted as a screen

Core Lab Instrumentation

Core 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).
  • Once resulted are verified, they can be broadcast (sent out)
  • 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
  • Type of analytical techniques found on Multi analyte analyzers
  • Many are immunoassays based with colorimetric detection
  • 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.

Special Chemistry Laboratory

  • Generally lower volume tests
  • Not available on auto-analyzer
    • Because the demand is not there
    • The test is not easily automated (extraction steps)
  • Instruments are usually batch analyzers. Either research instruments or diagnostic instruments that perform 1 specific test on multiple samples.
  • More training involved with test protocols and instrumentation than Core Lab instrumentation. Longer time required to reach competency
  • Only larger centres have Special Chemistry Lab because
    • Requires the volume of specimens to justify the test
    • High cost of equipment to relative few specific tests

Special Chemistry Instrumentation and Analytical Methods

  • Just about any analytical technique can be found in a Special Chem Lab
  • Electrophoresis
    • Used to separate serum proteins into 5 distinct bands
    • Used to separate Lipoproteins into 4 distinct bands
    • Often used to separate isoforms of enzymes
  • HPLC
  • Infrared Spectroscopy
    • Used to analyze components of Kidney stones
  • Radioimmunoassay (RIA)
    • 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.
    • Used for quantitative drug measurement

Point of Care Testing (POCT)

  • Tests are of urgent importance, and results will affect the immediate management of the patient
  • Instruments are available that can perform certain tests at remote locations, such as at the bedside on in a clinical care unit
    • 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
  • Many are immunoassay based. Can be qualitative or quantitative

Issues for POC testing

  • Who is going to pay for the instrument and reagents
  • Who is going to conduct the test
  • Who is to perform the necessary quality control, general maintenance and order supplies
  • Does the instrument interface with the hospital LIS.
  • Are the tests performed on the POCT instrument standardized with the same tests performed using the hospital instrumentation

POC Instrumentation

  • Cardiac Markers
  • Urinalysis
  • Drug Screen
  • Visual qualitative tests
  • Results must be recorded
  • and reported manually
  • Can be qualitative, pseudo-quantitative
  • and/or quantitative
  • Hard copy of result generated
  • Possible to interface hospital LIS

Why are laboratory tests ordered

  • Diagnosis
  • Monitor progression of disease
  • Monitor effectiveness of treatment
  • Screening population for diseases
  • To identify complications of treatment
  • For predicting survivability, employability
  • To check the accuracy of an unexpected data
  • To conduct research
  • To prevent malpractice
  • For educating residents
  • To assess nutritional status and health of an health individual
  • Responding to total uncertainty

The Test

  • Measuring an analyte as a Marker to distinguish health and disease
  • Ideal Marker
  • Absolutely specific for a specific disease
  • Easily measurable
  • Quantity reflective of severity of disease
  • Early detection following onset of disease
  • Not affected by other biological disturbances
  • Highly Specific marker:
  • Troponin I. It is a marker of Myocardial infarction (Heart Attack)
  • Found predominately in Cardiac Tissue
  • Released into the blood stream following cell death
  • Non specific marker: low blood pH (acidosis)
  • Very important to know but can be caused by a hosts of events
  • Drugs
  • Respiratory problems
  • Renal problems

Biological Specimens “if you send it to us we will analyze it”

  • Blood
  • Urine
  • Cerebrospinal Fluid
  • Amniotic Fluid
  • Duodenal Aspirate
  • Gastric Juice
  • Gall stone
  • Kidney Stone
  • Stools
  • Saliva
  • Synovial Fluid
  • Tissue Specimen
  • Choice of specimen type depends on
  • Comprise the majority of all
  • specimens analyzed

Blood Composition

  • Plasma
  • Plasma is fluid component of blood.
  • Comprises ~55% of total 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 (WBC)
  • & Platelets
  • Cellular
  • Components
  • Blood Composition
  • Serum
  • Plasma is fluid component of blood.
  • Comprises ~55% of total volume of
  • whole blood. Contains proteins, sugars,
  • vitamins,minerals, lipids, lipoproteins
  • No clotting factors
  • 95% of plasma is water
  • Blood Clot
  • -comprised of clotting factors (Fibrin,platets etc)
  • -RBCs
  • Whole Blood
  • Whole Blood after clotting and centrifugation
  • If blood is collected 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

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

Blood Analysis

  • Testing can be done on whole blood, serum or plasma. Choice depends on a number of factors
  • 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
  • The way the test was developed.
    • Tests are often only validated on either plasma or serum
  • Turn around time
    • Analysis of whole blood is the quickest. No waiting for clot or spinning
    • Plasma requires centrifugation prior to analysis
    • With serum, the blood must clot then you have to centrifuge

Blood Analysis in the Chemistry

  • 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.
  • Important exceptions include
    • Hemoglobin, Red blood cell (RBC) Folate
    • Blood gases
  • Protein electrophoresis was developed based on the analysis of serum. Not done on plasma because of the presence of the protein fibrinogen which distorts the electrophoretic pattern.
  • Many tests can use either serum or plasma

Collection Tubes

  • The most widely used tubes for blood collection are evacuated tubes (Vacutainers)
    • 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 for specific tests (glucose, metals)

Collection Tubes (Vacutainers)

  • Serum Separator Tube (SST)
  • Separator Gel
  • Separator Gel
  • Serum
  • Clot

Collection tubes

  • Red-top tubes contain no anticoagulants or preservatives
  • Red-top tubes are used for collecting serum
    • 10-15 minutes is required to allow blood to clot before centrifuging
    • Used for blood bank specimens, some chemistries

Collection tubes

  • Gold (and “tiger”) top tubes contain a gel that forms a physical barrier between the serum and cells after centrifugation
  • No other additives are present
  • Gel barrier may affect some lab tests

Collection tubes

  • Used for Glucose measurement.
  • After blood collection, glucose concentration decreases significantly because of cellular metabolism
  • Gray-top tubes contain either:
    • Sodium fluoride and potassium oxalate, or
    • Sodium iodoacetate
  • Both preservatives stabilize glucose in plasma by inhibiting enzymes of the glycolytic pathway
    • NaF/oxalate inhibits enolase
    • Iodoacetate inhibits glucose-3-phosphate dehydrogenase

Collection tubes

  • Green-top tubes contain either the Na, K, or lithium (Li) salt of heparin. Most widely used anticoagulant for chemistry tests.
    • Should not be used for Na, K or Li measurement
    • Can effect the size and integrity of cellular blood components and not recommended for hematology studies
  • Heparin accelerates the action of antithrombin III, which inhibits thrombin, so blood does not clot (plasma)
  • The advantage of plasma is that no time is wasted waiting for the specimen to clot

Collection tubes

  • Lavender-top tubes contain the K salt of ethylenediaminetetraacetic acid (EDTA), which chelates calcium (essential for clot formation) and inhibits coagulation
  • Used for hematology, and some chemistries
  • Cannot be used for K or Ca tests

Collection tubes

  • Blue-top tubes contain sodium citrate, which chelates calcium and inhibits coagulation
  • Used for coagulation studies because it is easily reversible.

Collection tubes

  • Brown and Royal Blue top tubes are specially cleaned for trace metal studies
    • Brown-top tubes are used for lead (Pb) analysis
    • Royal blue-top tubes are used for other trace element studies (acid washed)

Test results Variations, Errors, Interferences

  • Variations
  • Clinical variations within an individual and between individuals
  • Analytical variations-no test is perfect. All tests have some degree of variations for repeated measurements of the same sample.
  • The final test result is affected by factors that occur
    • Pre-analytically
    • At the time of the test
    • After the test is completed

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 processed
  • The specimen is analyzed
  • The results are reviewed and verified by an technologists
  • The results are released to the patient’s record

Why Analytical Results Vary

  • Inter-individual Variation
  • Age
  • Sex
  • Race
  • Genetics
  • Long term health status
  • Pre-analytical Variation
  • Transport
  • Exposure to UV light
  • Standing time before separation of cells
  • Centrifugation time
  • Storage conditions
  • Intra-individual Variation
  • Diet
  • Exercise
  • Drugs
  • Sleep pattern
  • Posture
  • Time of venipucture
  • Length of time tourniquet is applied
  • Analytical Variation
  • Random errors
  • Systematic errors
  • Post-analytical
  • Transcriptions errors
  • Results reported to wrong patient

Pre-analytical errors

  • Collection
    • Was the right tube used?
    • Was venipuncture performed correctly?
    • Was the specimen properly stored?
  • Identification
    • Was the blood collected from the correct patient?
    • Was the blood correctly labeled?
      • Patient name, ID, date, time of collection, phlebotomist

Specimen identification

  • One of the common sources of erroneous lab results is misidentified specimens
  • The lab is required to have a clear and rational policy for identifying specimens, and handling misidentified specimens

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 (Fe), cholesterol, bilirubin
  • Decreases potassium

Supine vs. sitting or standing

  • Going from lying (supine) to upright reduces total blood volume by about 700 ml
  • The following may decrease by 5-15% in the supine patient:
    • Total protein
    • Albumin
    • Lipids
    • Iron
    • Calcium
    • Enzymes
  • Specimens requiring special handling
  • Should be placed immediately on ice
    • Lactate
    • Ammonia
    • Acid phosphatase
    • Plasma catecholamines

Significantly affected by hemolysis:

  • Hemolysis-rupture of red blood cell
    • Can be due to improper collection
    • End result is dumping cellular contents into blood. Mild dilution effect in some analytes
  • Significant increase in potassium, magnesium, phosphorous

Interferences

  • Hemolysis
    • 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 colorimetric 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
  • Human Anti Animal Antibodies.
    • Occurs in individual that have been exposed to foreign immunoglobins
    • Can significantly increase or decrease immunoassay based tests since all utilize animal antibodies, particularly mouse. Referred to as Human Anti Mouse Antibodies (HAMA)
    • Tests usually contain reagent to clear HAMA
    • Technicians performs a dilution test to determine if HAMA are present
    • Generally have to send to another lab to test by alternate method or different antibody


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