-fasting glucose in WB 15% lower than serum or plasma
-venous blood 7mg/dl lower than capillary blood; peritoneal fluid gluc same with plasma -RT (20-25O)- glucose ↓7mg/dl/hr -Ref temp(4OC)- glucose ↓2mg/dl/hr -Frozen Temp (-20OC)- almost 0mg/dl/hr decrease I. CHEMICAL METHOD a. Oxidation Reduction Method 1. Alkaline Copper Reduction Method- -Reduction of cupric ions to cuprous ions forming cuprous oxide in hot alkaline sol. by glucose *Alkaline Copper Tartrate ---glucose, heat- Cuprous ions a. Folin Wu Method- Cuprous ions + Phosphomolybdate↓ Phosphomolybdic acid or Phosphomolybdenum blue b. Nelson Somogyi- Cuprous ions + Arsenomolybdate ↓ Arenomolybdic acid or Aresnomolybdenum blue c. Neocuproine Method (2,9 Dimethyl 1,10 Phenantroline Hydrochloride) Cuprous ion + Neocuproine↓ Cuprous-Neocuproine Comples (yellow or Yellow orange) d. Benedict’s Method (Modification of Folin-Wu)- detect and quanti reducing subs in body fluid -use citrate or tartrate as stabilizing agent 2. Alkaline Ferric Reduction Method (Hagedorn Jensen)- reduction of yellow ferricyanide to colorless ferrocyanide by glucose (Colorimetry) b. Condensation Method Ortho-toluidine (Dubowski Method) Glucose + Aromatic amines ---Glacial HAC, heat glycosylamine + schiff’s base II. ENZYMATIC METHOD- act on glucose but not on other sugard and not on other reducing subs 1. Glucose oxidase Method- measure B-D glucose a. Colorimetruc Glucose Oxidase Method (Saifer Gernstenfield Method) Glucose + O2 ---glucose oxidase Gluconic Acid + H2O2 H2O2 +Chromogenic subs ---peroxidase oxidized chromogenic subs + H2O b. Polarographic Glucose Oxidase- meas rate of O2 consumotions, prop to gluc conc. -conversion glucose quantified by consumption of O2 on oxygen-sensing electrode -H2O2 prevented from re-forming O2 by adding molybdate, iodide, catalase and ethanol Glucose +O2 ---glucose oxidase Gluconic Acid + H2O2 H2O2 + C2H5OH ---catalase CH3CHO + 2H2O H2O2 + 2H + 2 I ---molybdate I2 + 2H2O 2. Hexokinase Method- most specific glucose method; reference method; -plasma -- heparin, EDTA, fluoride, oxalate, or citrate; other sample urine, CSF, serous fluid Glucose + ATP ---Hexokinase Glucose-6-Phosphate + ADP G-6-P +NADP---G-6-PD 6Phosphogluconolactone +NADPH-reduced coenzyme meas. 3. Glucose Dehydrogenase Method- gluc red to prod Chrmophore meas by spectrophoto/ electric. -Mutarotase- shorten time to reach equilibrium; endcolor blue 4. Dextrostics (cellular strip)- imp in establishing correct insulin amount for next dose 5. Interstitial Glucose Measuring Device- for continuous monitoring of glucose level in diabetic px 6. Glycosylated Hgh (HbA1c) or Glycated Hgb- monitoring -represents a weighted average of glucose level, w/ youngest RBC contributing greater extent -spx EDTA whole blood; Method electrophoresis, immunoassay, HPLC, Affinity Chromatography -5.7-6.4% inc risk for diabetes; every 1% = 35mg/dl added to PG CHOLESTEROL DETERMINATION- can be measure w/o fasting; measured as a whole -Px prep-usual diet for 2 weeks, neither gaining nor losing weight I. CHEMICAL METHOD- Principle: Dehydration and oxidation of cholesterol to form a colored compound a. Lieberman Burchardt Reaction (Colorimetric) Endproduct= Cholestradienyl monosulfonic Acid (green color) Color Developer Micture (LB rgt)’ -Glacial acetic acid -con. H2SO4 -acetoc anhydride b. Salkowsk’s Reaction Endproduct= Cholestadieny Disulfonic Acid (red color) >General Methods: One Step Method- Colorimetry (Pearson, Stern and Mac Gavack) Two Step Method- Extraction + Colorimetry (Bloors) Three Step Method- Saponification + Extraction + Colorimetry (Abell-Kenda) Four Step Method- Saponification+ Extraction + Colorimetry + Ppt. (Schoenheimer Sperry + Parekh + Jung) II. ENZYMATIC METHOD- don’t required preliminary extraction step Cholesterol Oxidase Reaction: commonly used/ routine test Cholesterol Ester + H2O --chol esterase cholesterol + fatty acid Cholesterol + O2—cholesterol oxidase cholest-4-en-3-one + H2O2 H2O2 + phenol + 4-aminoantipyrine –peroxidase quinoneimine dye CDC reference method (Abell, Levy and Brodle Method)- 1-Saponification, 2-extration, 3-colorimetry -use hexane extraction after hydrolysis w/ alcoholic KOH by rxn w/ Lieberman-Burchardt color reagent TRIGLYCERIDE MEASUREMENT –hydrolyze all fatty acid esters of TAG to produce glycerol I. CHEMICAL METHOD a. Colorimetric Method (Van Handel & Zilversmith) TAG—alcoholic KOHGlycerol + Fatty Acid Glycerol Oxidized by Periodic Acid Formaldehyde (HCHO) HCHO + Chromotropic Acid(+)Blue color compound b. Fluorometric Method (Hanstzch Condensation) TAG –alc. KOH Glycerol +FA Glycerol Oxidized by Periodic Acid Formaldehyde (HCHO) HCHO + Diacetyl Acetone + NH Diacetyl Lutidine Compound II. ENZYMATIC METHOD a. Glycerol Kinase Method- hydrolysis TAG to free FA and glycerol, then phosphorylation of glycerol to glyceropho *CDC reference method (Modified Van Handel and Zilversmith)-colorimetric pink end color Saponification- KOH (alkaline hydrolysis) Adsorption- Silicic acid Chrom (isolate TAG) Extraction- Chloroform Color Reaction Chromotropic acid +TAG= PINK Lipoprotein Methodologies: -differentiated based on electrophoresis and buoyant density (ultracentrifugation) -TC-HDL-C=non HLD-C 1. Ultracentrifugation (density gradient)- reference method for quantitationof lipoproteins (LPPs) -based on protein and TAG contents of lipoproteins; epressed in svedsverg(s) units -Lipid density 1.0g/mL while Protein density 1.4mg/L -Reagent: Potassium bromide solution w/ 1.063 density 2. Electrophoresis- Pattern: HDL, VLDL, LDL, Chylomicrons -preferred supporting medium: Agarose-gel-speed; sensitive; resolves LLPs classes -Lipid staining dyes: Oil Red O, Fat Red 7B or Sudan Black B -VLDL migrates w/ a-2 globulin (preB); Chylomicrons if present remain at origin 3. Chemical Precipitation- uses polyanions ) and divalent cations such as Mg, Ca, manganese. a. HDL- uses dextran sulphate (syntheric heparin) w/ magnesium (precipitants) CDC reference 3-step: ultracentrifugation, heparin manganese precipitation and Abell-Kendall assay b. LDL- EDTA plasma .. 4. Chromatographic method- utilizes Gel chromatography or affinity chromatography 5. Immunochemical methods0 uses AB specific to epitopes o apolipoproteins 6. Immunoassay or Immunonephelometry- Apolipoprotein assay -meas turbidity created by apolipoprotein-Ab complexes Friedewald Method (Indirect Method) Formula for LDL-Cholesterol (LDL-C)= Total Cholesterol –HDL-VLDL VLDL (mmol/L)= Plasma TAG/2.175 VLDL(mg/dL)= Plasma TAG/5 DeLong Method (Indirect Method) VLDL(mmol/L)= Plasma TAG/2.825 VLDL(mg/dL)= Plasma TAG/6.5 KINDEY FUNCTION TESTS 1. BUN Determination: Fluorid, citrate inhibit urease; thriosemicarbazide and ferric ions enhance color I. Chemical Method (Direct Method)- Diacetyl Monoxime Method Urea+DM Yellow diazine derivative II. Enzymatic Method (Indirect Method) a. Hydrolysis of Urea by Urease (Routine metho) Urea+Urease NH3 + CO2 -ammonia then treated with Berthelot rgt b. Coupled Urease/ Glutamate Dehydrogenase (GLD) method- UV enzymatic method Urea + Urease NH4 + CO2 NH4+2-oxoglutarate + NADH—GLD Glutamat+NAD+H2) c. Isotope Dilution Mass Spectromoetry (IDMS)- Reference method 2. Creatinine I. Chemical Method- Direct Jaffe Method: Red-orange tautomer of creatinine picrate is formed when creatinine is mexed with alkaline picrate rgt a. Folin Wu method- sensitive but nonspecific method b. Lloyd or Fuller’s earth Method- sensitive and specific Adsorbent: Lloyd’s reagent (Sodium aluminium silicate) Adsorbent: removes interference present Fuller’s earth rgt (Aluminum Mg silicate) in spx and elution done to separate crea Jaffe Reagent (Alkaline Picrate): Saturated Picric Acid; 10% NaOH II. Kinetic Jaffe Method- serum mixed with alkaline picrate sol, rate of change in absorbance measured bet 2points III. Enzymatic Method- routine method; specific than jaffe a. Creatinine Aminohydrolase- CK method –require large vol of pre-incubated sample: not widely used b. Creatinase Hydrogen Peroxide Method- Creatinase aka creatinine aminohydrolase Creatinine + H2O—creatinase Creatine + H2O—creatinasesarcosine + urea Sarcosine + H2O + O2—sacrosine oxidaseglycine + HCHO+ H2O2 H2O2+ phenol + 4-aminophenazone –peroxidase benzoquinonemine dye (Red) IV. Isotope Dilution Mass Spectrometry (IDMS)- reference method 3. Blood Uric Acid- I. Chemical Methods: Reduction-Oxidation (Redox) Reaction Uric acid + Phosphotungstic Acid –NaCN/NaCO3Tungsten Blue + Allantoin + CO2 *Sodium cyanide—Folin NewtonBenedict Brown *Sodium carbonate –Archibald Henry Caraway II. Enzymatic Method Uricase Method- routine, specific method Uric acid has absorbance of 293nm, allantoin do not Uric acid + O2 –uricase allantoim + CO2 + H2O III. Isotope Dilution Mass Spectroscopy (IDMS)- reference method Osmolality a. Direct Method: Freezing point osmometry*; Vapor pressure osmometry (Seebeck effect) ↑Osmo↓FP,VP b. Indirect Method: -use glocuse or urea in osmolality Serum Osmolality= 1.86X Na = Gluc(mg/dL)/18 + BUN(mg/dL)/2.8 *Osmolal gap- difference between measured amd calculated plasma osmolality LIVER FUNCTION TEST I. Test Measuring Hepatic Synthetic Function- quantitate severity of hepatic dysfunction (Albumin, Vit-K dep CF) A. Total Protein- RV: 6.5-8.3g/dL a. Kjeldahl Method- measure Nitrogen(15.1-16.8%) content of CHON; referenc method; 1gN= 6.45g CHON Serum + tungstic acid Protein-free filtrate (PFF) -reagent: H2SO4 (digesting agent) -Endproduct: Ammonia b. Biuret Method- widely used recommended by Int’l federation of clinical chemistry (IFCC) expert panel -required at least 2 peptide bonds and an alkaline medium -Principle: Cupric ions complex groups involved In peptide bond forming violet-colored chelate, proportional to number of peptide bonds present and represent total protein level @454nm -Reagents: Alkaline Copper Sulfate, Rochelle Salt (NaK Tartrate), NaOH and K iodide c. Folin-Clocateu (lowry) Method- highest analytical sensitivity -Priciple: Oxidation of phenolic compounds such as tyrosine, tryptophan, histidine deep blue color -Reagent: Phosphotungstic-molybdic acid or phenol reagent; Biuret rgt (color enhancer) d. Ultraviolet Absorption Method- absorbance of CHON @210nm due to abs. of peptide bonds @speci. waveL e. Serum Protein Electrophoresis (SPE)- migration of charged paticle in an electric field -important in ID or monoclonal spike of Ig and differentiating them from polyclonal hypergammaglobulinemia Normal SPE pattern: Albumin (1st band)- fastest band 53-65% Alpha 1-Globulin (2nd fastest)- glycoproteins, AAT, AAG, TBG; inc in nonspecific response to inflammation Alpha 2-globulin (3rd fastest)- haptoglobin, AMG, ceruloplasmin 7-13% Beta-globulin (4th band)- transferrin, beta-lipoprotein, hemopexin, complement (C3, C4) 8-14% Gamma-globulin (5th band)- immunoglobulin and CRP 12-22% Abnormal Serum Electrophoretic Patterns: i. Gamma spike- multiple myeloma iv. a1-globulin flat curve0 juvenile cirrhosis (AAT deficien.) ii. beta-gamma bridging- hepatic cirrhosis v. Spikes of a1, a2, B globulin bands- inflammation iii. a2-globulin band spike- nephrotic syndrome f. Refractometry- alternative test to chem analysis of serum total protein; refractive index of solutes in serum g. Turbidimetric and Nephelometric Method- utilizes SSA and TCA h. Salt Fractionation- globulins can be separated from albumin by salting-out procedures using Na salts -Rgt: Sodium Sulfate Salts B. Prothrombin Time (Vit K Response Test)- diff intrahepatic disorder (prolong PT) from extrahepatic obs (NO PT) Albumin/Globulin Ratio- validate if globulin is higher than albumin -if globulin > than alb= inverted A/G ratio: cirrhosis, multiple myeloma, Walderstrom’s macroglobulinemia -RV: 1.3:1 to 3:1 II. Test Measuring Conjugation and Excretion Function *bili (mg/dL) x17.1 (mmol/L) A. Bilirubin Assay –unconjugated bilirubin reacts slowly, accelerants (Caffeine and Methanol) meas Total Bili -deletion of accelerants allow determination of direct-reacting or conjugated bilibun -Accelerators allow indirect bilirubin to react (solubilize) w/ the color reagent; read after 15min Principle: Van den Berg Reaction- diazotization of bilirubin to produce azobilirubin a. Evelyn and Malloy Method: Coupling Accelerator Methanol *PINK to PURPLE azobilirubin b. Jendrassik and Grof*: Coupling Accelerator Caffeine Sodium Benzoate Buffer: Sodium Acetate *PINK to BLUE azobilirubin B. Bromsulfonthalein (BSP) Dye Extraction Test –test for hepatocellular func and potency of bile duct a. Rosenthal White (Double Collection)- dose 2mg/kg BW; collect after 5min(50%) and 30min(0% dye retention) b. Mac Donald Method (Single Collection)- dose 5mg/kg; collect spx after 45min; NO= +/- 5% dye retention III. Test for Detoxification Function- involves enzymes and ammonia tests A. Enzyme Test-assess extent of liver damage & diff. hepatocellular (functional) from obstruct (mechan) disease. -enzymes secreted by liver: ALP, aminotransferases, 5’nucleotidase, GGT, OCT, LAP, LD B. Ammonia- from deamination of AA, converted by liver to uea RV: 19-60ug/dL (11-35mmol/L) ↑cirrhosis, hepatitis, Reye’s syndrome, chronic renal disease and acetaminophen poisoning ENYMES ^Alkaline Phosphatase^ 1. Electrophoresis- Liver, Bone most Anodal (neuraminidase,wheat germ lectin-separ.); intestinal ALP least anodal - High-resolution electrophoresis using polyacrylamide gel and isoelectric focusing resolve bands of ALP 2. Heat Fractination/Stability Test- performed @ 56OC for 10-15min -placental ALP (most heat stable); bone ALP most heat labile; Order decresing: Placenta, Intestinal, Liver, B 3. Chemical Inhibition Test- use diff conc of phenylalanine, synthetic urea and levamisole -P and I inhibited by phenylalanine rgt ; 3M urea inhibit Bone; Levamisole inhibit L and B ALP 4. Bowers and Mc Comb (Szasz modification)-most specific method IFCC recommended -continous-monitoring technique requiring a pH 10.15 and read @405nm p-nitrophenylphosphate ←ALP→p-nitrophenol + phosphate ion Method Substrate End Product 1. Bodansky 2. Shinowara Beta-glyceroPO4 InorganicPO4 + Glyverol 3. Jones 4. Reinhart 5. King and Armstrong Phenylphosphate Phenol 6. Bessy Lowry & Brock p-nitro phenyl PO4 (PNPP) p-nitrophenol or yellow nitrophenozide ion 7. Bowers and McComb PNPP p-nitrophenol or yellow nitrophenozide ion 8. Huggins and Talalay Phenolphthalein PO4 ALpa-naphtol 9. Moss Alpha naphthol PO4 Alpha- naphthol 10. Klein, Babson and Read Buffered phenolphthalein PO4 Free phenolphthalein -Refrigeration leads to inc ALP; ALP inhibited by phosphorous; Zinc component of ALP ^Acid Phosphatase^ Summary of ACP Methods Method Substrate End Product 1. Gutman and Gutman Phenyl PO4 Inorganic PO4 2. Shinowara PNPP p-nitrophenol 3. Babson, Read and Phillips Alpha naphthyl PO4-continuous Alpha-naphtol mon 4. Roy and Hillman Thymolphthalein MonoPO4* Free thymolphthalein *Tartrate-resistant Acid Phosphatase (TRAP)- present in certain chronic leukemia, esp Hairy Cell Leukemia *Postatic ACP used together w/ Prostate Specific Anitgen (PSA) to monitor recurrence of prostate cancer ^Aspartate Aminotransferase^ Karmen Method- pH 7.5; 340nm- use malate dehydrogenase (MD), monitor change in absorbance at 340nm Aspartate + a-ketoglutarate ←AST→ Oxaloacetate + Glumate Oxaloacetate + NADH + H ←MD→ Malate + NAD ^Alanine Aminotransferase^ -present in plasma, bile, CSF, saliva; require pyridoxal phosphate (vitB6) as coenzym Coupled Enzymatic Reaction: using pH 7.5 @340nm Alanine + a-ketoglutarate ←ALT→ Pyruvate + Glutamate Pyruvate + NADH + H ←LD→ Lactate + NAD AST/ SGOT ALT/ SGPT Major Organ Affected Heart Liver Substrate Aspartic a-ketoglutaric acid Alanine a-ketoglutaric acid End Product Glutamic Acid + Oxaloacetic Acid Glutamic Acid + Pyruvic Acid Color Developer 2,4 DNPH 2,4 DNPH Color Intensifier 0.4N NaOH 0.4N NaOH Method Reitman and Frankel Reitmand and Frankel *De Ritis Ratio (ALT:AST) > 1.0, seen in acute hepatitis ^Amylase/ Alpa-1-4-Glucohydrolase (AMS)^ Substrate for all methods Starch- polysaccharide, carbohydrate 1. Saccharogenic- reference method expressed in Somogyi units; -measure reducing sugars prod by hydrolysis of starch by the usual glucose method 2. Amyloclastic –measure amylase activity by following decreases in substrate conc (defradation of starch) 3. Chromogenic- meas amylase activity by increase in color intensity of soluble dye-substrate sol prod in rxn 4. Coupled-enzyme- measure amylase activity by a continuous-monitring technique ^Lipase/ Triacylglycerol Acylhydrolase (LPS)^ -uses Olive oil as substrate bec other esterases can hydrolyze TAG and syntheric diglycerides -Collipase (CHON from pancreas) and Bile salts- make assay more sensitive and specific 1. Cherry Crandal (reference method)- substrate 50%olive oil; End product Fatty Acid -Hydrolysis of olive oil after incubation for 24hrs @37OC and titration of FA using NaOH TAG (olive oil) +2H2O ←LPS→ 2-monoglyceride + 2 Fatty acids 2. Tiets and Flereck 3. Peroxidase coupling- most common; doesn’t use 50% olive oil ^Lactate Dehydrogenase^ -Lactate more specific substrate than pyruvate; LD1 prefer forward; LD5 reverse rnx 1. Wacker Method (forward/direct reaction)- reaction @ pH 8.8; most common Lactate + NAD –LD Pyruvate + NADH @340nm 2. Wrobleuski La Due (reverse/ indirect reaction)- reaction @ pH7.2; 2x faster; prefer for dry slide technology Pyruvate + NADH –LD Lactate + NAD 3. Wrobleuski Cabaud 4. Berger Broida ^Creatine Kinase (CK) 1. Tanzer-Gilbarg Assay (forward/direct method) –pH9.0 @340nm Creatine + ATP –CKCreatine PO4 + ADP + Phosphoenolpyruvate ←PK→ Pyruvate + ATP Pyruvate + NADH ←LD→ Lactate + NAD 2. Oliver-Rosalki (reverse/indirect method)-most common; faster; pH 6.8 @340nm Creatine PO4 + ADP –CPK Creatine + ATP + glucose ←hexokinase→ ADP+ glucose-6-PO4 Glucose-6-PO4 + NADP ←G-6-PD→ 6-phosphogluconaye + NADPH 3. Electrophoresis- reference method *Adenosine monophosphate (AMP)- added to reverse method to inhibit AK (Adenylate kinase)-it hydrol ADP *imidazole- buffer; urate and cysteine- potent inhibitor of CK; CK light and pH sensitive ^Sodium and Potassium^- use heparinized blood 1. Emission Flame Photometry 2. Ion Selective Electrode (Valinomycin gel) 3. Atomic Absorption Spectrophotometry 4. Colorimetry (Lockhead and Purcell) ^Chloride^ 1. Mecurimetric Titration (Schales and Schales) -Diphenylcarbazone –indicator; HgCl2 (blue violet)=end p 2. Spectrophotometric Methods Mercuric Throcyanate (Whiterhorn Titration Method) =Reddish complex Ferric Perchlorate =colored complex 3. Coulometric Amperometric Titration –Cotlove Chloridometer- Sweat Chloride (cystic fibrosis) 4. Ion Selective Electrode-using ion exchange membrane (tri-n-octylpropylammonium chloride decanol); common ^Calcium^ -serum 1. Precipitation and Redoc Titration: Clark Collip ppt- endproduct (purple color) Ferro Ham Chloranilic Acid ppt- endproduct: Chloranilic acid (purple color) 2. Ortho-Cresolpthalein Complexone Dyes (Coloremetric Method) Dye: Arzeno III 3. EDTA Titration Method (Bachra, Dawer and Sobel) 4. Ion Selective Electrode (Liquid membrane) 5. Atomic Absorption Spectrophotometry-reference method 6. Emissio Flame Photometry ^Inorganic Phosphorus^- require fasting, high CHO diet dec level; only inorganic phosphate is measured -affected by circadian rhythm- high level late morning, low in evening 1. Fiske Subbarow Method (Ammonium Molybdate method)- most common -Reducing agent: *Pictol (Amino Naphthol Sulforic Acid); elon, senidine -Endproduct: Ammonium molybdate complex (unstable); Reduced form blue color det bet 600 to 700nm ^Magnesium^ 1. Colorimetric Methods a. Calmagite Method= (+) Reddish-violet complex b. Formazen Dye Method= (+) Colored complex c. Magnesium Thymol Blue Method= (+) Colored complex 2. Atomic Absorption Spectrophotometry- reference method 3. Dye-Lake Method- Titan Yellow Dye (Clayton Yellow or Thiazole Yellow) ^Bicarbonate^ -spx blood anaerobically collected 1. Ion selective electrode (using pCO2 electrode) 2. Enzymatic (Phosphoenolpyruvate carboxylase and dehydrogenase) *Cystic Fibrosis- Sweat Inducer: Pilocarpine] Diagnostic test:Sweat-teest Coulometry (↑Sodium and Cl) Reference Method: Gibson and Cooke Pilocarpine Iontophoresis >50mg sweat sample collected w/in 30min (+) Result: >65 mmol/L sweat electrolytes (RV: 5-40mmol/L) *Iron 1. Colorimetry (HCl and ferrozine) –(+) Blue Color 2. Anodic Stripping Voltammetry- 1st separation form transferrin by acidification, ^Blood Gas^- spx Arterial Blood; Anticoagulant: 0.05mL heparin/mL blood 1. Gasometer 2. Electrodes a. Van Slyke a. pH (potentiometry) b. Natelson i. Silver-silver chloride electrode- reference electrode i. mercury- to produce vacuum ii. Calomel electrode (Hg2CL2)- reference electrode ii. caprylic alcohol- anti-foam reagent iii. Gas electrode- most common, used for pH iii. Lactic acid b. pO2 Clark electrode- polarography-amperometry iv. NaOH and NaHSO3 c. pCO2 Severinghaus electrode –potentiometry
Thyroid Function Test:
1. TRH Stimulation Test (Thryrotropin Releasing Hormone)- measure relationship bet TRH and TSH secretions -differentiate euthyroid and hyperthyroid Px w/ both undetected TSH; detect thyroid hormone resistance synd. -↑1O hypothyroidism; ↓hyperthyroidism 2. TSH Test- most important thyroid function test- best from detecting clinically significant thyroid dysfunction -detect 1O thyroid disorfers; differentiate 1O from 2Ohypothyroidism ↑1O hypothyroidism, hashimoto’s thyroiditis. TSH Ab; ↓1O hyperthyroidism, 2O and 3O hypothyroidism 3. Radioactive Iodine Uptake (RAIU)- measure ability of thyroid gland to trap iodine; ..hyperthyroidism -high uptake =metabolically active; high uptake + TSH deficiency= autonomous thyroid activity 4. Thyroglobulin (Tg) assay- normally used as postoperative marker thyroid cancer, ↑untreated and metastatic differentiated thyroid cancer, nodular goiter and hyperthyroidism ↓infants w/ goitorous hypothyroidism and thyrotoxicoxis factitia Method: double-Ab RIA, ELISA, IRMA, immunochemiluminescent assay (ICMA) 5. Reverse T3 (rT3)- formed by removal one iodine from inner ring of T4; enproduct of T4 metabolism -identifies px w/ euthyroid sick syndrome (↑rT3) 6. Free Thyroxine Index (FTI or T7)- indirectly assess level of free T4 in blood;↑hyperthyroidism; ↓hypothyroid 7. Total 3 (TT3), Free T3 (FT3) and FreeT4 (FT4)- FT4 used to differentiate drug induced TSH elev and hypothyroid -TT3 or FT3 confirm hyperthyroidism; direct/reference method: Equilibrium dialysis (FT4) 8. T3 Uptake- measure number available binding sites of thyroxine-binding proteins, a test for TBG -reflects serum level of TBG, inversely related to TBG- ↓T3 uptake ↑TBG, vice versa ↑hyperthydoisim, ↓hypothyroidism 9. Thyroxine binding globuline (TBG)- confirm results of FT3 and FT4, or abnormalities in relation to TT4 and THBR -hyperthydoism (↑T4 + NO TBG); euthyrdoism (↑T4 and TBG); hypothyroidism ↓TBG 10. Fine-needle aspiration- most accurate tool in evaluation of thyroid nodules 11. Recombinant Human TSH- test pc w/ thyroid cancer 12. Tanned Erythrocyte Hemagglutination- measure antithyroglobulin Ab 13. Serum Calcitonin- tumor marker for detecting thyroid metastasis in medullary thyroid carcinoma 14. Pentagastrin (Pg) Stimulation Test- diagnose MTC ^Pheochromocytoma^: Pharmacologic Tests: a. Clonidine Tests- diff. pheochromocytoma (not suppressed) to neurogenic hypertension (50% ↓catecholamine) b. Glucagon Stimulation Test- for indiv w/ normal blood pressure and when catecholamines only modestly elev. ^Neuroblastoma^ Spx: 24hr urine and blood (plasma) a. Chromatography- HPLC or GC-MS (VMA and metanephrines) b. Radioimmunoassay- sensitive screening test for total plasma catecholamines >2000pg/mL plasma catecholamines- diagnostic for pheochromocytoma -Urine preservation: 10mL 6N HCl ^Hormonal Assay^ 1. Whole blood- LH, testosterone 2. Plasma- EDTA (ACTH, ADH, PTH) and Heparin (Catecholamines, cortisol, dopamine, FSH) 3. Serum- aldosterone, androstenedione, DHEA, estrogen, FSH, GH, HCG, progesterone 4. Urine- for measurement of estriol -Boric acidin a concentration of 1g/dL urine elements such as estriol and estrogen for up to 7days -for catecholamines, VMA, 5-HIAA collections, 10mL 6N HCl in 3-4L container -HCl establishes a pH of approximately <3.0, good for chemical testing a. Classical Assay -Bioassay- - Competitive Protein Binding (CPB) b. Immunologic Assays -Radioimmunoassay (RIA) -Immunoradiometric (IRMA) -Enzyme-Linked Immunosorbent Assay (ELISA) -Enzyme Multiplied Immunosorbent Technique (EMIT)-for TDM -Immunometric- for TSH c. Fluorescent Technique- FPIA d. High Performance Liquid Chromatography (HPLC) e. Colorimetry i. Porter-Silber Method- for 17-OHCS ii. Zimmerman Reaction- measure those steroid w. 17-keto structure iii. Pisano Method- for quantitating metanephrines and normetanephrines iv. Kober Reaction- for estrogen (H2SO4 + hydroquinone = (+) reddish-brown color