MUST-KNOW - Clinical Chemistry PDF

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MUST TO KNOW IN CLINICAL CHEMISTRY(From CC by Rodriguez) Quality Control Practicability Method is easily repeated Reliability Maintain accuracy and precision Intralab/Interlab QC Daily monitoring of accuracy and precision Interlab/External QC Proficiency testing (Reference lab) Long-term accuracy Di...

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Practicability Reliability Intralab/Interlab QC Interlab/External QC

QC materials Bovine control materials Matrix effect

Precision study Nonlab. personnel SD CV Variance Inferential statistics T-test F-test Cumulative Sum Graph (CUSUM) Youden/Twin Plot Shewhart Levey-Jennings Chart Trend Shift Outliers

Kurtosis Precision Accuracy Random error (Imprecision; Indeterminate)

Systematic error (Inaccuracy/Determinate)

MUST TO KNOW IN CLINICAL CHEMISTRY (From CC by Rodriguez) Quality Control Method is easily repeated Maintain accuracy and precision Daily monitoring of accuracy and precision Proficiency testing (Reference lab) Long-term accuracy Difference of >2: not in agreement w/ other lab Available for a min. of 1 yr Preferred (Human: biohazard) Not for immunochem, dye-binding and bilirubin Improper product manufacturing Unpurified analyte Altered protein First step in method evaluation 29% of errors (lab results) Dispersion of values from the mean Index of precision Relative magnitude of variability (%) SD2 Measure of variability Compare means or SD of 2 groups of data Means of 2 groups of data SD of 2 groups of data V-mask Earliest indication of systematic errors (trend) Compare results obtained from diff. lab Graphic representation of the acceptable limits of variation Gradual loss of reliability Cause: Deterioration of reagents (Systematic error) Values: one side or either side of the mean Cause: Improper calibration (Systematic error) Values: far from the main set of values Highly deviating values Random or systematic errors Degree of flatness or sharpness Random error Systematic error Causes: -Mislabeling -Pipetting error -Improper mixing of sample and reagents -Voltage/Temperature fluctuation -Dirty optics Parameters: SD and CV Causes: -Improper calibration -Deterioration of reagents -Contaminated solution -Sample instability/unstable reagent blanks lec.mt 04 |Page | 1

Multirule Shewhart procedure Test method Reference method Analytical Run Physiologic Limit POCT Quality Assurance

Quality Patient Care Reference Range/ Interval Range/ Reference Values Wavelength

Spectrophotometric meas. Photometric measurement LASER Visible region UV

IR Stray light Diffraction gratings Prisms Nickel sulfate Cutoff filter Bandpass Alumina silica glass cuvet Quartz/plastic cuvet Borosilicate glass cuvet Photodetector Barrier layer cell/ photocell/ photovoltaic cell Phototube Photomultiplier tube

-Diminishing lamp power -Incorrect sample and reagent volume Parameter: Mean Control rules + Control chart Westgard: at least 40 samples Westgard: preferably 100 samples Control and patient specimens assayed, evaluated, and report together Referred to as absurd value Performed by nonlab personnel Tripod: Program development Assessment and monitoring Quality improvement Test request forms, clear instruction for patient prep., specimen handling… At least 120 individuals should be tested in each age and sex category Analytical Methods Distance bet 2 successive peaks (nm) Lower frequency = Longer wavelength (Ex. Red) Higher frequency = Shorter wavelength (Ex. Violet) Meas. light intensity in a narrower wavelength Meas. light intensity w/o consideration of wavelength Multiple wavelength (uses filter only) Light Amplification by Stimulated Emission of Radiation Light source for spectrophotometry Tungsten light bulb Mercury arc Deuterium lamp Mercury arc Xenon lamp Hydrogen lamp Merst glower Globar (Silicone carbide) Wavelength outside the band Most common cause of loss of linearity Most commonly used monochromator Cutting grooves Rotatable Prevents stray light Anti-stray light ½ peak transmittance Most commonly used cuvet UV Strong bases Converts transmitted light into photoelectric energy Simplest detector No external voltage For filter photometers Contains anode and cathode Req external voltage Most common type lec.mt 04 |Page | 2

Galvanometer/Ammeter Absorbance Double beam spectro.

Double-beam in space Double-beam in time Dydimium filter Holmium oxide filter Reagent blank Sample blank FEP

Cesium and Lithium Lithium AAS

Atomizer (nebulizer) Chopper Lanthanum/Strontium chloride Volumetric (Titrimetric) Turbidimetry

Nephelometry

Electrophoresis Iontophoresis Zone electrophoresis Endosmosis Cellulose acetate Agarose gel Polyacrylamide gel Electrophoretic mobility

Most sensitive UV and visible region Meter or read-out device A = abc (a = absorptivity; b = length of light (1cm); c = concentration) A = 2 – log%T Splits monochromatic light into two components: One beam  sample One beam  reference soln or blank (corrects for variation in light source intensity) 2 photodetectors (sample beam and reference beam) 1 photodetector Monochromatic light  sample cuvet and reference cuvet 600 nm 360 nm Color of reagents Optical interference (Hgb) Meas. light emitted by a single atom burned in a flame Principle: Excitation Lt. source and cuvette: Flame For excited ions (Na+, K+) Internal standards (FEP) Correct variations in flame Preferred internal std Potent antidepressant Meas. light absorbed by atoms dissociated by heat Principle: Dissociation (unionized, unexcited, ground state) Lt. source: Hollow-cathode lamp For unexcited trace metals (Ca++ and Mg++) More sensitive than FEP Convert ions  atoms Modulate the light source Complex with phosphate Avoid calcium interference Unknown sample is made to react with a known solution in the presence of an indicator Light blocked Meas. abundant large particles (Proteins) Depend on specimen concentration and particle size Meas. amt of Ag-Ab complexes Scattered light Depends on wavelength and particle size Migration of charged particles in an electric field Migration of small charged ions Migration of charged macromolecules Movement of buffer ions and solvent relative to the fixed support Ex: gamma globulins Molecular size Electrical charge Charge and molecular size 20 fractions (ex. isoenzymes) Directly proportional to net charge Inversely proportional to molecular size & viscosity of the supporting medium lec.mt 04 |Page | 3

Isoelectric focusing

Densitometry Capillary electrophoresis Southern blot Northern blot Western blot Chromatography Paper chromatography TLC Retention factor (Rf) value

Gas chromatography

Gas Solid chromatography Gas Liquid chromatography Mass Spectrometry GC-MS MS/MS HPLC Hydrophilic gel

Hydrophobic gel

Ion exchange chromatography Partition chromatography Affinity chromatography Adsorption chromatography Fluorometry/Molecular Luminescence Spectro.

Quenching

Molecules migrate through a pH gradient pH = pI For isoenzymes: same size, different charge Scan & quantitate electrophoretic pattern Electro-osmotic flow DNA RNA Proteins Separation by specific differences in physical-chemical characteristics of the different constituents Fractionation of sugar and amino acid Sorbent: Whatman paper Screening: Drugs Relative distance of migration from the point of application Rf = Distance leading edge of component moves Total distance solvent front moves Separation of steroids, barbiturates, blood, alcohol, and lipids Volatile compounds Specimens  vaporized Mobile phase: Inert gases Differences in absorption at the solid phase surfaces Differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase Fragmentation and ionization Gold standard for drug testing Detect 20 inborn errors of metabolism from a single blood spot Most widely used liquid chromatography Fractionation of drugs, hormones, lipids, carbohydrates and proteins Gel filtration Separation of enzymes, antibodies and proteins Ex: Dextran and agarose Gel permeation Separation of triglyceride and fatty acid Ex: Sephadex Separation depends on the sign and ionic charge density Based on relative solubility in an organic solvent (nonpolar) and an aqueous solvent (polar) For lipoproteins, CHO and glycated hemoglobins Based on differences between the adsorption and desorption of solutes at the surfaces of a solid particle Det. amt. of lt. emitted by a molecule after excitation by electromagnetic radiation Lt. sources: Mercury arc and Xenon lamp (UV) Lt. detector: Photomultiplier tubes 2 monochromators: Primary filter – selects wavelength absorbed by the solution to be measured Secondary filter – prevents incident light from striking the photodetector Sensitivity: 1000x than spectro Major disadvantage of fluorometry pH and temperature changes, chemical contaminants, UVL changes lec.mt 04 |Page | 4

Borosilicate glasswares Boron-free/Soft glasswares Corex (Corning)

Vycor (Corning) Flint glass

TD: To deliver TC: To contain Blowout Self-draining Transfer pipet

Graduated or measuring pipet

Micropipettes

Air displacement pipet Positive displacement pipet Dispenser/Dilutor pipet Distilled H2O Mercury Acid dichromate (H2SO4 + K2Cr2O4) Continuous flow analyzer

Centrifugal analyzer

Instrumentation For heating and sterilization Ex: Pyrex and Kimax High resistance to alkali Special alumina-silicate glass Strengthened chemically than thermally 6x stronger than borosilicate For high thermal, drastic heat and shock Can be heated to 900OC Soda-lime glass + Calcium, Silicon, Sodium oxides Easy to melt For making disposable glasswares Exact amount Does not disperse the exact volume w/ etched rings on top of pipet w/ o etched rings Drain by gravity Volumetric: for non-viscous fluid; self-draining Ostwald folin: for viscous fluid; w/ etched ring Pasteur: w/o consideration of a specific volume Automatic macro-/micropipets Serological: w/ graduations to the tip (blowout) Mohr: w/o graduations to the tip (self-draining) Bacteriologic Ball, Kolmer and Kahn Micropipettes: 400mg/dL Bilirubin: 25.2 mg/dL Interfere with: "TACGu” Total Protein Albumin Cholesterol Glucose Preferred position Patient should be seated/supine at least 20 mins before blood collection to prevent hemodilution or hemoconcentration Vasoconstriction  Reduced plasma volume Increased: “ECA” Enzymes Calcium Albumin Hemoconcentration lec.mt 04 |Page | 7

Increased: “P(u)BLIC” Proteins BUN Lipids Iron Calcium Standing  Supine Hemodilution Decreased: “TLC” Triglycerides Lipoproteins Cholesterol Prolonged standing Increased: K+ (muscles) Prolonged bedrest Decreased: Albumin (Fluid retention) Tourniquet Recommended: 1 minute application Prolonged tourniquet app. Hemoconcentration Anaerobiosis Increased: “C2LEA2K” Calcium Cholesterol Lactate Enzymes Ammonia Albumin K+ Tobacco smoking (Nicotine) Increased: “TUNG2C3” Triglycerides Urea Nonesterified fatty acid Glucose GH Catecholamines Cortisol Cholesterol Increased: “THUG” Alcohol ingestion Triglycerides Hypoglycemia (chronic alcoholism) Uric acid/Urates GGT Increases by 100-200μg/L/cigar Ammonia Stress (anxiety) Increased: “LAGIC” Lactate Albumin Glucose Insulin Cholesterol Drugs Medications affecting plasma volume can affect protein, BUN, iron, calcium Hepatotoxic drugs: increased liver function enzymes Diuretics: decreased sodium and potassium Diurnal variation "CA3PI2TG” Cortisol ACTH ACP lec.mt 04 |Page | 8

Sleeping patients Unconscious patients Venipuncture Tourniquet

Needle

After blood collection BP cuff as tourniquet Benzalkonium chloride (Zephiran) IV line on both arms

IV fluid contamination

Renin blood level Basal state collection Lancet Incision (Skin puncture) 1.5-2.4mm Arterialized capillary blood Flea Indwelling umbilical artery 1000-3000 RCF for 10 mins Hemolysis

Aldosterone Prolactin Iron Insulin Thyroxine GH Specimen Collection and Handling Must be awakened before blood collection Ask nurse or relative Identification bracelet Median Cubital (1st)  Cephalic (2nd)  Basilic (3rd) Velcro or Seraket type 3-4 inches above the site Not exceed 1 minute Bevel up 15-30O angle Length: 1 or 1.5 inch (Butterfly needle: ½ to ¾ inch) Cotton  site Apply pressure for 3-5 minutes Inflate to 60 mmHg Disinfectant for ethanol testing Dilution – 1:750 Discontinue IV for 2 minutes Collect sample below the IV site Initial sample (5mL)  discard Increased: Glucose (10% contam. w/ 5% dextrose  increased bld glucose by 500 mg/dL) Chloride Potassium Sodium Decreased: Urea Creatinine Collected after a 3-day diet, from a peripheral vein Early morning blood collection 12 hours after the last ingestion of food 1.75mm: preferred length to avoid penetrating the bone 500 mg/dL  nonketotic hyperosmolar coma Screening: 1hr GCT (50g) – bet. 24 and 28 weeks of gestation Confirmatory: 3-hr GTT (100g) Infants: at risk for respiratory distress syndrome, hypocalcemia, hyperbilirubinemia After giving birth, evaluate 6-12 weeks postpartum Converts to DM w/in 10 years in 30-40% of cases FBS = ≥95 mg/dL 1-Hr = ≥ 180 mg/dL 2-Hr = ≥ 155 mg/dL 3-Hr = ≥ 140 mg/dL GDM = 2 plasma values of the above glucose levels are exceeded FBS = 100-125 mg/dL FBS = 0.5 g/dL Hgb) Dextrostics OGTT

IVGTT

Requirements for OGTT

Glucose load

HbA1c

WB = 15% lower than in serum or plasma VB = 7 mg/dL lower than capillary and arterial blood 60-70% of the plasma glucose Same with plasma glucose Fasting: 2 mg/dL/decade Postprandial: 4 mg/dL/decade Glucose challenge: 8-13 mg/dL/decade Separate serum/plasma from the cells Glycolysis at room temperature Glycolysis at refrigerated temperature Cupric  Cuprous  Cuprous oxide Cuprous ions + phosphomolybdate  phosphomolybdenum blue Cuprous ions + arsenomolybdate  arsenomolybdenum blue Cuprous ions + neocuproine  Cuprous-neocuproine complex (yellow) Reducing substances in blood and urine Ferricyanide ---(Glucose)--> Ferrocyanide (Yellow) (Colorless) Schiff’s base Measures beta-D-glucose (65%) Converts alpha-D-glucose (35%) to beta-D-glucose (65%) Absorbance at 340nm Consumption of oxygen on an oxygen-sensing electrode O2 consumption α glucose concentration Most specific method Reference method Uses G-6-PD Most specific enzyme rgt for glucose testing False-decreased Bilirubin Uric acid Ascorbate Major interfering substance in hexokinase method (false-decreased) Cellular strip Strip w/ glucose oxidase, peroxidase and chromogen Janney-Isaacson method (Single dose) = most common Exton Rose (Double dose) Drink the glucose load within 5 mins For patients with gastrointestinal disorders (malabsorption) Glucose: 0.5 g/kg body weight Given w/in 3 mins 1st blood collection: after 5 mins of IV glucose Ambulatory Fasting: 8-14 hours Unrestricted diet of 150g CHO/day for 3 days Do not smoke or drink alcohol 75 g = adult (WHO std) 100 g = pregnant 1.75 g glucose/kg BW = children 2-3 months Glucose = beta-chain of HbA1 lec.mt 04 |Page | 15

IDA and older RBCs RBC lifespan disorders Fructosamine (Glycosylated albumin/ plasma protein ketoamine) Galactosemia

Essential fructosuria Hereditary fructose intolerance Fructose-1,6-biphosphate deficiency Glycogen Storage Disease

Ia = Von Gierke II = Pompe III = Cori Forbes IV = Andersen V = McArdle VI = Hers VII = Tarui XII = Fanconi-Bickel CSF glucose < 0.5 C-peptide 5:1 to 15:1 D-xylose absorption test Gerhardt’s ferric chloride test Nitroprusside test Acetest tablets Ketostix KetoSite assay Normal Values (Carbohydrates)

1% increase in HbA1c = 35 mg/dL increase in plasma glucose 18-20% = prolonged hyperglycemia 7% = cutoff Specimen: EDTA whole blood Test: Affinity chromatography (preferred) High HbA1c Low HbA1c 2-3 weeks Useful for patients w/ hemolytic anemias and Hgb variants Not used in cases of low albumin Specimen: Serum Congenital deficiency of 1 of 3 enzymes in galactose metabolism Galactose-1-phosphate uridyl transferase (most common) Galactokinase Uridine diphosphate galactose-4-epimerase Autosomal recessive Fructokinase deficiency Defective fructose-1,6-biphosphate aldolase B activity Failure of hepatic glucose generation by gluconeogenic precursors such as lactate and glycerol Autosomal recessive Defective glycogen metabolism Test: IVGTT (Type I GSD) Glucose-6-Phosphatase deficiency (most common worldwide) Alpha-1,4-glucosidase deficiency (most common in the Philippines) Debrancher enzyme deficiency Brancher enzyme deficiency Muscle phosphorylase deficiency Liver phosphorylase deficiency Phosphofructokinase deficiency Glucose transporter 2 deficiency Collect blood glucose at least 60 mins (to 2 hrs) before the lumbar puncture (Because of the lag in CSF glucose equilibrium time) Normal CSF : serum glucose ratio Formed during conversion of pro-insulin to insulin Normal C-peptide : insulin ratio Differentiate pancreatic insufficiency from malabsorption (low blood or urine xylose) Acetoacetate 10x more sensitive to acetoacetate than to acetone Acetoacetate and acetone Detects acetoacetate better than acetone Detects beta-hydroxybutyrate but not widely used RBS = anabolism Excessive tissue destruction Anabolism > catabolism Growth and repair processes Transports thyroxine and retinol (Vit. A) Landmark to confirm that the specimen is really CSF Maintains osmotic pressure Negative acute phase reactant Acute phase reactant Major inhibitor of protease activity 90% of alpha1-globulin band Gestational marker Tumor marker: hepatic and gonodal cancers Screening test for fetal conditions (Spx: maternal serum) Amniotic fluid: confirmatory test Increased: Hepatoma, spina bifida, neural tube defects Decreased: Down Syndrome (Trisomy 21) Low pI (2.7) Negatively charged even in acid solution Acute phase reactant Binds and inactivates PSA Increased: Alzheimer’s disease, AMI, infection, malignancy, burns Acute phase reactant Binds free hemoglobin (alpha chain) Copper binding (6-8 atoms of copper are attached to it) Has enzymatic activities lec.mt 04 |Page | 21

Alpha2-macroglobulin

Group-specific component (Gc)-globulin (bet. alpha1 and alpha2) Hemopexin (beta) Beta2-microglobulin Transferrin/Siderophilin (beta)

Complement (beta) Fibrinogen (bet. beta and gamma) CRP (gamma)

Immunoglobulins (gamma) Myoglobin Troponins TnT (Tropomyosin-binding subunit) TnI (Inhibitory subunit or Actin-binding unit)

TnC Glomerular proteinuria Tubular proteinuria Overload proteinuria

Postrenal proteinuria Microalbuminuria

CSF Oligoclonal banding

Decreased: Wilson’s disease (copper  skin, liver, brain, cornea [KayserFleisher rings]) Larges major nonimmunoglobulin protein Increased: Nephrotic syndrome (10x) Forms a complex w/ PSA Affinity w/ vitamin D and actin

Binds free heme HLA Filtered by glomeruli but reabsorbed Negative acute phase reactant Major component of beta2-globulin fraction Pseudoparaproteinemia in severe IDA Increased: Hemochromatosis (bronze-skin), IDA C3: major Acute phase reactant Between beta and gamma globulins General scavenger molecule Undetectable in healthy individuals hsCRP: warning test to persons at risk of CAD Synthesized by the plasma cells IgG>IgA>IgM>IgD>IgE Marker: Ischemic muscle cells, chest pain (angina), AMI Most important marker for AMI Specific for heart muscle Det. unstable angina (angina at rest) Only found in the myocardium Greater cardiac specificity than TnT Highly specific for AMI 13x more abundant in the myocardium than CK-MB Very sensitive indicator of even minor amount of cardiac necrosis Binds calcium ions and regulate muscle contractions Most common and serious type Often called albuminuria Defective reabsorption Slightly increased albumin excretion Hemoglobinuria Myoglobinuria Bence-Jones proteinuria Urinary tract infection, bleeding, malignancy Type 1 DM Albumin excretion ≥30 mg/g creatinine (cutoff: DM) but ≤300 mg/g creatinine Microalbuminuria: 2 out of 3 specimens submitted are w/ abnormal findings (w/in 6 months) 2 or more IgG bands in the gamma region: Multiple sclerosis Encephalitis Neurosyphilis Guillain-Barre syndrome Neoplastic disorders lec.mt 04 |Page | 22

Serum Oligoclonal banding

Alkaptonuria Homocystinuria

MSUD

PKU Normal Values (Proteins)

T...