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
- 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
- Hematology and Transfusion Medicine
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
- 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 - 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 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
- Whole Blood after centrifugation
- Note: clotting has been prevented
- White Blood cells (WBC)
- & Platelets
- 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 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
- 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)
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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