Clinical Chemistry Basics & QC PDF

CLINICAL CHEMISTRY Julius P. Mario, RMT, Ph.D. The purpose & function of laboratorians through clinical pathology & laboratory medicine are to assist clinicians in: 1. Confirming or rejecting a diagnosis 2. Providing guidelines in patient management 3. Establishing a prognosis...

CLINICAL CHEMISTRY Julius P. Mario, RMT, Ph.D. The purpose & function of laboratorians through clinical pathology & laboratory medicine are to assist clinicians in: 1. Confirming or rejecting a diagnosis 2. Providing guidelines in patient management 3. Establishing a prognosis 4. Detecting disease through case finding or screening 5. Monitoring follow-up therapy  Clinical Chemistry The systematic study of biochemical processes associated with health & disease & the measurement of constituents in body fluids or tissues to facilitate diagnosis of disease.  SCOPE OF CLINICAL CHEMISTRY Biochemistry Computers Instrumentation CLINICAL CHEMISTRY Pharmacology Endocrinology Immunology Toxicology Analytical Chemistry It is much easier to detect a MALFUNCTION than to elucidate or document the cause. Malfunction of a cell maybe caused by a variety of factors 1. Destruction by trauma or by invasive agents 2. Genetic deficiency of a vital enzyme 3. Insufficient supply of one or more essential nutrients 4. Insufficient blood supply 5. Insufficient oxygen supply 6. Malignancy 7. Accumulation of waste products 8. Failure of a control system 9. Defect in the cellular recognition of certain signals Point-of-care Testing (POCT) Also known as near-patient testing, alternate-site testing or patient-focused testing Used in emergency dept., operating suites, clinics, health maintenance organization (HMO), physicians, offices & nursing homes Addresses acute patient needs Instrumentation includes portable chemistry analyzers, glucometers, BG Analyzers, hemoglobin meters & coagulation testing Specimens or samples are analyzed while substances in them are measured or quantitated. Substances measured in serum fall generally into the following categories: 1. Substances normally present with a function in the circulation  glucose  electrolytes  TP  TAG, cholesterol  albumin  hormones  individual proteins  vitamins 2. Metabolites- nonfunctioning waste products in the process of being cleared  urea  ammonia  creatinine  bilirubin  uric acid 3. Substances released from cells as a result of cell damage & abnormal permeability or abnormal cellular proliferation enzymes such as LD, ALT, AST, CK, AMS, GGT, ALP & ACP ferritin 4. Drugs & toxic substances  antibodies  therapeutic drugs  substances of abuse  poisons Quick Quiz Tissue injury is greatly associated with elevated levels of blood a. electrolytes b. hormones c. enzymes d. albumin Electrochemical methods are ideal for the measurement of serum/plasma a. glucose b. enzymes c. proteins & amino acids d. metal ions  Laboratory Tests Chemical tests Direct or Indirect Colorimetric Redox Condensation Degradative tests (Enzymatic) One-step or Multi-step NADH Consumption Measurement of Activity (not concentration) Multi-method Titrimetric Electrochemical Quick Quiz Identify the analyte measured using the following tests and the type of each test: 1. Jaffe reaction 2. Hantzsch test 3. GOD-PAP 4. Clark-Collip 5. Schales and Schales 6. Evelyn-Malloy  LAB REQUESTS “STAT” = immediate; urgent requests; emergency = usually less than 1 hour TAT PRIORITY = important for clinical decisions = less than 2 hour TAT ROUTINE = non urgent; standard or special tests = generally 4-6 hours Glasswares Plasticwares Breakable Highly shock proof; shatterproof Reusable Disposable Ideal for acidic solutions Ideal for alkaline solutions High thermal & corrosion Relatively inert resistance Flexible Good optical properties Low cost For cryogenic experiments  GLASS: 5 GENERAL TYPES 1. High thermal resistant glass Borosilicate glass with low alkali content Resists heat, corrosion and thermal shock Most common are Pyrex, Kimax and Exax Corex - 6x stronger than borosilicate glass - Alumina-silicate glass - Better able to resist clouding due to to alkali or scratching - Ideal for higher temperature thermometers, graduated cylinders and centrifuge tubes. Vycor - recommended for use in application involving high temperature and drastic heat shock and extreme chemical treatment with acids and dilute alkalies - heatable to 900oC and withstand downshock from 900oC to ice water - ideal for ashing and ignition techniques 2. High silica glass - 96% silica content comparable to fused quartz in its thermal endurance, chemical stability and electrical characteristics - radiation resistant, good optical qualities and temperature capabilities - ideal for high precision analytical work and can also be used for optical reflectors and mirrors 3. Glass with high resistance to alkalies - boron-free glassware for strong alkali solutions and digestion with strong alkalies - less thermal resistant than borosilicate glass - known as soft glass 4. Low actinic glass - amber or red-colored to reduce the amount of light passing through the substance within the glassware - highly protective laboratory glassware for handling heat- labile substances in the 300-500nm range (bilirubin, carotene and vitamin A) 5. Standard flint glass - soda-lime glass which is composed of a mixture of oxides of Si, Ca and Na - Lowest in cost and readily fabricated - poorly resistant to high temperature and sudden changes in temperature - easy to melt and shape thus used as reagent bottles and disposable laboratory glasswares - release alkali into the pipetted liquid  Quick Quiz Which of the following is an aluminum-silicate glass that is at least six times stronger than borosilicate and is resistant to alkaline etching and scratching? a. Kimax b. Pyrex c. Corning boron free d. Corex The tinted glass which is ideal for delayed testing of light-sensitive substances is a. standard flint b. soda lime c. low actinic d. boron-free  PLASTICWARES 1. Polyolefins (polyethylenes, polypropylenes) Unique group of resins with relatively inert chemical properties Unaffected by acids (however, conc. sulfuric acid slowly attacks polyethylene at RT), alkalies, salt solutions and most aqueous solutions Aromatic, aliphatic and chlorinated hydrocarbons cause moderate swelling at RT Organic acids, essential oils and halogens slowly penetrate these plastics Polyolefins (polyethylenes, polypropylenes) Strong oxidizing agents attack this group at elevated temperatures only Polypropylene is more expensive than polyethylene therefore the latter is used in most disposable plastic wares Polypropylene is sterilizable however it absorbs pigment and tends to become discolored PLASTICWARES 2. Polycarbonate resin twice as strong as polypropylene (from –100oC to +160oC) Chemical resistance is not as wide as the polyolefins Unsuitable for use with bases as amines, ammonia and alkalies as well as oxidizing agents Glass-clear and shatterproof Ideal for centrifuge tubes and graduated cylinders 3. Tygon Nontoxic, clear plastic of modified PVC (polyvinylchloride) Used extensively for the manufacture of ACA tubings Flexible and used to handle most chemicals Can be steam-autoclaved or chemically sterilized Tubing is soft and flexible and quickly slips over tubulatures but gripping tightly on glass or metals 4. Teflon fluorocarbon resins Almost chemically inert with high corrosion resistance at extreme temperatures For cryogenic experiments and work at high temperatures over extended periods (-270oC to +255oC) Pure translucent white and inert Easy to clean and fast drying but it can be scratched and misshaped Quick Quiz The internal tubings of clinical analyzers should be made of a. Teflon fluorocarbon b. polycarbonate c. Tygon d. polyvinylchloride The advantages of this plastic is that it can be made into very thin sheets and can withstand extreme temperatures. a. Polyolefins b. Polycarbonate  PIPETS Two General Types 1. Volumetric or Transfer pipet Designed to deliver (TD) a fixed volume of liquid Consists of a cylindrical bulb joined at both ends to narrower glass tubing Calibration mark is etched around the upper suction tube Lower delivery tube is drawn out to a fine tip used for accurate measurements of aliquots of nonviscous samples, filtrates, controls, and standard solutions  OSTWALD-FOLIN PIPET Similar to volumetric pipet but have their bulb closer to the delivery tip Used for measuring viscous fluids as blood or serum Has an etched ring near the mouthpiece (blowout pipet) Blown out only when the fluid has drained to the last drop in the delivery tip 2. Graduated or Measuring Pipets Plain, narrow tube drawn out to a tip Graduated uniformly along its length Two types which are calibrated for delivery are available Mohr pipet - calibrated between two marks on the stem Serological pipet - has graduation marks down on the tip and blown out to deliver the entire volume of the pipet if etched ring or double rings are evident near the mouth of the pipet Serological pipet Intended for the delivery of predetermined volumes Principally used for the measurements of reagents and are not generally considered accurate enough for measuring viscous samples and standards Serological pipets have larger orifice than the Mohr pipet and thus drain faster  MICROPIPETS Sahli (TC) Kirk (TC) Lang-Levy (TD/ TC) Overflow (TC) Capillary (TC) Sanz Pipet (TD=TC) Unopette (TC) Semiautomatic pipet Eppendorf (TD/TC) Quick Quiz The College of American Pathologists (CAP) requires that volumetric pipets and flasks be certified as a. Class A b. Class B c. Class C d. Class D “To deliver”(TD) pipettes are identified by a. two etched bands near the mouthpiece b. self-draining capacity c. dual purpose pipet labels  CHEMICALS The American Chemical Society (ACS) established the specifications of chemicals in the analytical grade category. The analytical reagent grade chemicals are of very high purity. They are labeled Analytical Reagent (AR) or ACS which state the actual purity of each lot. These include chemicals such as: spectrograde, nanograde, and High Performance Liquid Chromatography (HPLC) grade. The AR grade chemical are used in: a. most analytical laboratory procedures b. making up reagents for spectrophotometry, chromatography, trace metal analysis, atomic absorption spectrophotometry, fluorometry and standardization procedures. United States Pharmacopoeia (USP) and the National Formulary (NF) grade chemicals are not injurious to humans and are used to produced drugs. They are not pure enough for use in most chemical procedures. Less pure grade chemicals are referred to as purified and technical. The chemically pure (CP) or pure grade chemicals do not recommend for reagent preparations. Technical or commercial grade reagents are primarily used in manufacturing and should not be used in the clinical laboratory. The technical grade is the lowest quality and should not be used for analytical work.  STANDARDS A primary standard (PS) is a chemical that is the highest purity and can be measured directly to produce a substance of exact known concentration. The International Union of Pure and Applied Chemistry (IUPAC) requires that PS be at least 99.98% pure and that the working standards be 99.95% pure. Grade A - atomic weight standards Grade B – ultimate standards Grade C* – primary standards with >0.002% impurity Grade D* – working standards with >0.5% impurity Grade E – secondary standards derived from using Grade C standards *commercially available The National Bureau of Standards (NBS) now National Institute of Science and Technology (NIST) purified materials known as Standard Reference Materials (SRM) are used as primary standard materials in the clinical laboratory. They are relatively expensive and should be used as a reference for the comparison of commercially obtained standards and reagents. A secondary standard is a substance of lower purity whose concentration is determined by comparison to a primary standard.  Quick Quiz Regarded as the ultimate standard by IUPAC is a. Grade A b. Grade B c. Grade C d. Grade D What is the most common use of a standard in the clinical laboratory? a. to verify accuracy of results b. to standardize the testing c. to compare its value with the results obtained  WATER Reagent grade water (RGW) is water suitable for reagent and standard preparation. Most procedures use distilled water or deionized water. Distilled water is purified to remove almost all organic materials. Water may be distilled more than once and each distillation cycle will remove impurities. Deionized water is produced from distilled water using either an anion or cation exchange resin followed by replacement of the removed particles with hydroxyl or hydrogen ions respectively. The College of American Pathologists (CAP) and the National Committee for Clinical Laboratory Standards (NCCLS) issued minimum specifications for RGW. The NCCLS requirements for each type are tabulated below: Specifications Type I Type II Type III Bacterial content (maximum