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  2. Lecture Outlines • Common terminologies in Chemical Pathology. • Concept of Reference Values and Units of Biochemical Quantities. • Collection, Handling, Registration and Preservation of Specimens in Chemical Pathology Laboratories.
  3. Introduction to clinical laboratories: • Clinical laboratories are important in diseases diagnosis, determination its severity and patient response to specific treatment. • Diagnosis of any disease is first done by physical examination by physician and confirmed by laboratory diagnostic tests. • Laboratory values are very important in determination of disease severity, drug doses and in follow up.
  4. Introduction The sections of clinical laboratory are: • Clinical pathology including Histology and cytology • Haematology including Serology and Blood bank • Clinical biochemistry/Chemical Pathology • Clinical microbiology
  5. Purpose of Clinical Chemistry Tests – It deals with the applications of biochemistry laboratory to find out the cause of a disease. – Measure levels of substances found normally in human blood that have biological functions. Examples: Glucose, Calcium – Detect or measure non-functional metabolites or waste products. Examples: Creatinine, Blood Urea Nitrogen (BUN) – Detect or measure substances that indicate cell damage or disease. Examples: Liver enzymes, such as ALT, Cardiac enzymes, such as CK-MB – Detect or measure drugs or toxic substances. Examples: Dilantin, Drugs of abuse screen
  6. Types of Specimens for Chemical Analysis – Whole blood, serum or plasma. The most common specimen is serum, collected in a tube with no anticoagulant so that the blood will clot. – Urine – often 24 hour collections – Others – Cerebrospinal Spinal Fluid (CSF), joint aspirate fluid, pleural, pericardial or asctific fluid, faeces, and other body fluids or tissues
  7. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • Collection and Handling of Blood Specimens for Chemical Analysis – Blood collection tubes for obtaining serum - Serum Separator Tubes (SST) do not have an anticoagulant but do contain a gel substance which will form an interface between the clot and the serum when the blood specimen is centrifuged. These tubes are sometimes referred to as “Tiger Tops”.
  8. Common terminologies in Chemical Pathology: Phlebotomy refers to blood draw from a vein, artery, or the capillary bed for lab analysis or blood transfusion. Veinpuncture: Normal or Reference Values – range of values for a particular chemistry test from healthy individuals. Controls:
  9. Laboratory request and laboratory report forms Laboratory request form: it fills computerize or paper filled by the doctor then send it to the lab. The lab request contains a list of tests to be performed on specimen of patient. Each lab has its specific request; for example, chemistry request, hematology request… etc. Laboratory report form: it contains the result of patient. Laboratory work flow cycle: The flow cycle includes the entire steps of laboratory test, starting from test ordering by a doctor until reporting the results. Three phases of laboratory testing: Pre-analytical: test ordering, specimen collection, transport and processing Analytical-testing Post-analytical: testing results transmission, interpretation, follow-up, retesting.
  10. Patient preparation for sample collection – time of collection; – effects of eating on chemistry analysis – Some specimens are increased or decreased after eating (ex. Glucose, triglycerides), so it is important to know what the test and collection method call for. Specimens for these tests are usually collected in a fasting state.
  11. Patient preparation for sample collection • Sometimes serum or plasma appears lipemia (milky) after a patient has eaten a fatty meal. • Lipemia affects most chemistry analyses. The blood must be recollected when the patient is fasting. • A*
  12. Normal or Reference Values • Clinical Chemistry Tests – Normal or Reference Values – range of values for a particular chemistry test from healthy individuals – A reference range is usually defined as the set of values 95 percent of the normal population falls within (that is, 95% prediction interval). – It is determined by collecting data from vast numbers of laboratory tests • Units • Mass concentration (g/dL or g/L) is the most common measurement unit in the United States. Is usually given with dL (decilitres) as the denominator in the United States, and usually with L (litres) in, for example, Sweden. • Molar concentration (mol/L) is used to a higher degree in most of the rest of the world, including the United Kingdom and other parts of Europe and Australia and New Zealand. • International units (IU) are based on measured biological activity or effect, or for some substances, a specified equivalent mass. • Enzyme activity (kat) is commonly used for e.g. liver function tests like AST, ALT, LD and γ-GT in Sweden. • Percentages and time-dependent units (mol/s) are used for calculated derived parameters, e.g. for beta cell function in homeostasis model assessment or thyroid's secretory capacity
  13. Normal or Reference Values • Arterial or venous • If not otherwise specified, a reference range for a blood test is generally the venous range, as the standard process of obtaining a sample is by venipuncture. • An exception is for acid-base and blood gases, which are generally given for arterial blood. • Still, the blood values are approximately equal between the arterial and venous sides for most substances, with the exception of acid-base, blood gases and drugs (used intherapeutic drug monitoring (TDM) assays). • Arterial levels for drugs are generally higher than venous levels because of extraction while passing through tissues
  14. Chemistry Panel grouping • – some tests are “bundled” according to the system or organ targeted. Examples: thyroid panel, liver panel, cardiac panel, kidney panel, basic metabolic panel, etc • Lipid profile • Diabetic profile • Kidney profile • Liver profile • Bone profile • Electrolyte profile
  15. Disposable syringes Vacationer systems Disposable lancets Gauze pads absorbent cotton Tourniquet Alcohol swap Plastic bandage Waste container Phlebotomy or blood collection: The term phlebotomy refers to blood draw from a vein, artery, or the capillary bed for lab analysis or blood transfusion. The phlebotomy equipments: For specimen collection, the following materials will be required: Phlebotomy
  16. Usually vein is used to collect blood by veinpuncture procedure. In adults: most venipuncture procedure use arm vein. On arm, one of three arm veins is used: median cubital vein "located on the middle", cephalic vein or basilic vein "located on both sides". Median cubital vein is the best choice (why?) because it has good blood flow than cephalic and basilica which has slower blood flow. However if veinpuncture procedure is unsuccessful in median capital; cephalic or basilica is used. Artery blood is rarely used in special cases as when blood gases, pH, PCO2, PO2 and bicarbonate is requested. It is usually performed by physicians. Selecting vein site
  17. Preparation of Blood Sample One of three different specimens may be used: • Whole blood • Serum • Plasma First: Whole-blood specimen: It must be analyzed within limited time (why?) – Over time, cells will lyse in whole-blood which will change the concentration of some analytes as potassium, phosphate and lactate dehydrogenase. – Some cellular metabolic processes will continue which will alter analytes concentration like glucose and lactate.
  18. Serum Second Serum: Difference between Serum and plasma: • Serum is the same as plasma except it doesn't contain clotting factors (as fibrin). • Plasma contains all clotting factors. • So, serum and plasma all has the same contents of electrolytes, enzymes proteins, hormones except clotting factors. • Serum is mainly use in chemistry laboratory and serology.
  19. Procedure of Serum preparation • Draw blood from patient. • Select vacutainer with no anticoagulant. • Allow to stand for 20-30 minutes for clot formation. • Centrifuge the sample to speed separation and affect a greater packing of cells. • Clot and cells will separate from clean serum and settle to the bottom of the vessel. • The supernatant is the serum which can be now collected by Dropper or pipette for testing purposes or stored (-20°C to -80°C) for subsequent analysis or use.
  20. Plasma Third Plasma: • The tube will have anti-coagulation • After centrifugation the blood sample got separated into three layers
  21. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry – Blood collection tubes for obtaining plasma
  22. Procedure of plasma preparation • Draw blood from patient. • Select vacutainer with an appropriate anticoagulant. • Mix well with anticoagulant. • Allow to stand for 10 minutes. • Centrifuge the sample to speed separation and affect a greater packing of cells. • The supernatant is the plasma which can be now collected for testing purposes or stored (-20°C to -80°C) for subsequent analysis or use.
  23. In the laboratory Specimen rejection criteria: • Specimen improperly labeled or unlabeled. • Specimen improperly collected or preserved. • Specimen submitted without properly completed request form. • Haemolyzed sample (show tubes). • Lipeamic sample
  24. Haemolysis Haemolysis : • It means liberation of haemoglobin due to rupture of red blood cells (RBCs). • Due to haemolysis plasma or serum appears pink to red color. • It causes elevation in: K+, Ca2+, phosphate, SGOT, SLDH and acid phosphatase. • Haemolysis is occurred due to sampling, transporting and storage (too hot or too cold). • According to the degree of haemolysis it is classified as H+, H++ and H+++. H+ may be accepted for some tests that are not affected by RBCs contents as glucose and lactate, H++ and H+++ not acceptable for any test. Changes in the serum color indicate one of the following: • Haemolyzed: serum appears pink to red due to rupture of RBCs • Icteric: serum appears yellow due to high bilirubin. • Lipemic: serum appears milky or turbid due to high lipid.
  25. Blood collection tubes:
  26. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry
  27. Blood collection tubes: Two major types of blood collecting tubes: • Serum separating tubes (SST) • Plasma separating tubes (PST)
  28. Top Color Additives Principle Uses Lavender EDTA -The strongest anti-coagulant - Ca+2 chelating agent - To preserve blood cells components - Hematology - Blood bank (ABO) - HbA1C (Glycosylated Hb) Light Blue Sodium Citrate Ca+2 chelating agent - PT: Prothrombin Time - PTT: Partial Thromboplastin Time ( in case of unexplained bleeding and liver disease) Green Sodium Heparin or Lithium Heparin Heparin binds to Thrombin and inhibits the second step in the coagulation cascade (Prothrombin Thrombin) Fibrinogen Fibrin Enzymes Hormones Electrolytes (Na+, K+, Mg+, Cl- Heparin Plasma Separating Tubes (PST)
  29. Top Color Additives Principle Uses Black Sodium Citrate Ca+2 chelating agent ESR ( Erythrocyte Sedimentation Rate) to test how much inflammation in the patient, unexplained fever, Arthritis, Autoimmune Disorder Gray -Sodium Fluoride -Potassium Oxalate Glycolysis inhibitor Anti-Coagulant Glucose tests Royal Blue Heparin Na-EDTA Anti-Coagulant Tube should not be contaminated with metals Toxicology Trace Elements and metals Yellow ACD ( Acid-Citrate Dextrose) Anti-Coagulant DNA Studies Paternity Test HLA Tissue Typing (Human Leukocyte Antigen) The body used this protein to differentiate the self-cells from non-self cells
  30. Top Tubes Additives Principle Uses Red ------ Sometimes it has gel or silicon at the bottom of tube to reduce hemolysis Enhancing the formation of blood clot Serology -Antibodies -Hormones -Drugs Virology Chemistry Blood cross matching before blood transfusion Gold ------- It has gel at the bottom of the tube to separate serum from the blood Serum separating from the blood through the gel in the tube Serology Chemistry Serum Separating Tubes (SST)
  31. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • Commonly Performed Chemistry Tests or Analytes – Proteins – essential components of cells and body fluids. Some made by body, others acquired from diet. Provides information about state of hydration, nutrition and liver function, since most serum proteins are made in the liver.
  32. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry – Electrolytes – sometimes called “lytes” • Includes sodium (Na), potassium (K), chloride (Cl) and bicarbonate (HCO3-) • Collectively these have a great effect on hydration, acid-base balance and osmotic pressure as well as pH and heart and muscle contraction • Levels differ depending on if inside vs. outside cells • Important in transport of substances into and out of cells
  33. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry – Minerals • Calcium – Used in coagulation and muscle contraction – 99% is in skeleton and is not metabolically active – Influenced by vitamin D, parathyroid hormone, estrogen and calcitonin – Hypercalcemia – occurs in parathyroidism, bone malignancies, hormone disorders, excessive vitamin D, and acidosis; may cause kidney stones – Hypocalcemia – can cause tetany; occurs in hypoparathyroidism, vitamin D deficiency, poor dietary absorption and kidney disease
  34. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • Phosphorus – 80% in bone and rest in energy compounds such as ATP – Influenced by calcium and certain hormones • Iron – Essential for hemoglobin – Deficiency results in anemia; may be caused by lack of iron in diet, poor absorption, poor release of stored iron or loss due to bleeding – Increased in hemolytic anemia, increased iron intake or blocked synthesis of iron-containing compounds, such as in lead poisoning
  35. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry – Kidney Function Tests • Serum Creatinine – Best test for overall kidney function; not affected by diet or hormone levels – Waste product of muscle metabolism – Serum creatinine rises when kidney function is impaired
  36. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • BUN (Blood Urea Nitrogen) – BUN is surplus amino acids that are converted to urea and excreted by kidneys as a waste product – BUN influenced by diet and hormones, so it is NOT as good an indicator of renal function as serum creatinine levels – BUN increased in kidney disease, high protein diet, and after administration of steroids – BUN decreased in starvation, pregnancy and in persons on a low protein diet
  37. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • Uric Acid – Formed from breakdown of nucleic acids and excreted as a waste product by kidneys – Increased in kidney disease, but most often used to diagnosis gout (pain in joints, mainly big toe, due to precipitated uric acid crystals) – Also increased in increased cell destruction, such as after massive radiation or chemotherapy
  38. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry – Liver Function Tests • Liver functions: – Synthesizes glycogen from glucose – Makes plasma proteins (albumin, lipoproteins, coagulation proteins) – Forms cholesterol and degrades it into bile acids, which emulsifies fats for absorption – Stores iron, glycogen, vitamins and other substances – Destroys old blood cells and recycles components of hemoglobin
  39. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry
  40. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • Total Bilirubin – Waste production of hemoglobin breakdown – Increased in excessive RBC breakdown, such as hemolytic anemia, or impaired liver function or some sort of obstruction, such as a tumor or gall stone
  41. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • Liver Enzymes – levels increase following damage to liver tissues – Alkaline Phosphatase (ALP or AP) - Greatly increased in liver tumors and lesions; moderately increased in diseases such as hepatitis – Alanine Aminotransferase (ALT; formerly called SGPT) - Increases up to 10x in cirrhosis, infections or tumors and up to 100x in viral or toxic hepatitis
  42. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry – Asparate Aminotransferase (AST; formerly called SGOT) - Increased in liver disease, but also in heart attacks – Gamma Glutamyl Transferase (GGT) - Often used to monitor patients recovering from hepatitis and cirrhosis – Lactate Dehydrogenase (LD) - Increased in liver disease and following heart attacks
  43. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • Cardiac Function Tests – Creatine Kinase (CK) - Widely used to diagnosis and monitor heart attacks – Troponins » Only present in heart muscle, making it a more accurate indicator of heart attack than CK » Cardiac Troponin T (cTnT) » Cardiac Troponin I (cTnI)
  44. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • Lipid Metabolism Tests – Cholesterol » Present in all tissues » Serves as the skeleton for many hormones » Recommended to be less than 200 mg/dL in adults) » LDL = “bad” cholesterol; HDL = “good” cholesterol
  45. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry – Triglycerides » Main storage form of lipids, comprising 95% of fat tissue » Hyperlipidemia – having high blood levels of triglycerides – may increase risk of heart attack • Carbohydrate Metabolism Tests – Glucose - Largely regulated by insulin
  46. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry
  47. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • Thyroid Function Tests – Thyroid Stimulating Hormone (TSH) - Inverse relationship to thyroid function (the higher the TSH, the lower the thyroid function and vice versa) – Other less common thyroid tests include T3 and T4 – Hypothyroidism – underactive thyroid gland – Hyperthyroidism – overactive thyroid gland
  48. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry • For more information on most clinical laboratory tests, visit: Click on any lab test or condition or disease and find all sorts of information!
  49. Unit #6D – Clinical Laboratory Testing - Basic Clinical Chemistry Photos of some clinical chemistry laboratories
  50. Thanks for your attention Any questions??
  51. ASSIGNMENTS • 1) Write the reference values for all variables/analyte that can be measured in the Chemical Pathology Laboratory. 2) Tabulate the effects of environmental factors, genetic factors and cultural behaviours on the analysis of analytes in the Chemical Pathology laboratory emphasizing whether they are increased, decreased or unchanged.