Chemistry Sample Collection and Handling PDF

IOJ CHEMISTRY by Larry Broussard and Mary Muslow * Sample Collection and Handling 1. Serum (r ed top) for most chemistry tes...

IOJ CHEMISTRY by Larry Broussard and Mary Muslow * Sample Collection and Handling 1. Serum (r ed top) for most chemistry tests 2. Plasma - H eparin better th an oxalate, EDTA, citrate REMEMBER! 3. H ep arin (on ice) - ammonia, blood ga ses, lactic acid Hemolysis... as the cell bursts, 4. Sodium fluoride (gray top) a. Glucose (slows glycolysis) help (c)KLAMP the leak! b. NOT for BUN (inhihits urease) K x+,t.. 5. Acid Phosphatase must be stabilized or L W♦ tpH A Aldolase (ALD +) 6. EDTA Acid Phosphatase (ACP ♦) a. NOT for Na+ or K+ (EDTA contains Na+ and K+) M Mg++,+.. b. Not for Ca++ since calcium is ch elated in E DTA ( will cau,.-,e a false t ) p P04 ,t.. ,t.. Hemoglobin = Protein Electrophoresis May Sb.ow Extra Band ,t.Fe++ But, Bilirubin t or ♦ (Depending on hemoglobin and bilirubin concentrations) Carbohydrates : Glucose 2. Sp ecin1en c ollection and handling I. Digestion , metabolism, r egulation a. Glucose levels decr ease a. End product of car bohydrate approximately 10 mg/dL per hour digestion in the intestine in whole blood (7%) b. Blood glucose is maintained at a b. R efrigerated serum is fairly stable fairly constant level b y hormones c. Sodium fluoride (anticoagulant) (Insulin t ; all other s ,I.; see table on slows glycolysis (gray top tube) page 102) d. Fasting reference range for serum c. Provides ener gy for life p rocesses or plasma is 70-99 mg/dL e. Arterial and capilla ry values are 2- 3 mg/dL higher f. "Normal" CSF values are two- thirds (approximately 60-65%) of plasma l evels Relationship Between CSF and Plasma Glucose LevelJJ "Original contribution by Melanie S. Chapman 102 HORMONAL ACTIVITY AFFECTING SERUM GLUCOSE LEVELS HORMONE SOURCE ACTION Insulin beta cells of Islets of t serum glucose Langerhens of pancreas Stimulates glucose uptake by cells Glucagon alpha cells of ,t. glucose pancreas Stimulates glycogenolysis (breakdown of glycogen ➔ glucose) ACTH anterior pituitary ,t. glucose Insulin antagonist Growth Hormone anterior pituitary ,t. glucose Insulin antagonist Acromegaly = hyperglycemia Cortisol adrenal cortex ,t. glucose Stimulates gluconeogenesis (glucose from non-carbohydrate sources) Human Placental placenta ,t. glucose Lactogen Insulin antagonist Epinephrine adrenal medulla ,t. glucose Stimulates glycogenolysis Pheochromocytoma-tumor of adrenal medulla ➔ hyperglycemia T3 &T4 thyroid gland ,t. glucose Stimulates glycogenolysis REMEMBER! REMEMBER! Fido the GAG CHET Diabetic Dog Glucagon Cause:+ or No Insulin Production ACTH Growth Hormone SYMPTOMS: Tired 1,1-l ~)§) Polyuria Cortisol + Thirst & Hunger Human Placental Lactogen Weight Loss Poor Wound Healing Epinephrine T 3&T4 58@3 All cause an increase in serum glucose LAB TESTS: + Glucose (serum & urine), + AlC May Have + Cholesterol Diabetic Acidosis: t' Na+ + K+ Hormones affectmgtflllll t' Cl- gl.ucose - those that it vs. + Ketones (blood & urine) i.n.sulin wbich it  103 TEST INTERPRETATION TO DIAGNOSE DIABETES MELLITUS TEST STAGE Fasting plasma Casual plasma glucose Oral glucose tolerance A1c glucose (FPG) test (OGTT) * Normal FPG 100 mg/dL CHO ~ 100 mg/dL Diet from Notional Cholesterol Education Prag-am 106 ❖ Bran ched chain k etoaciduria (maple syrup urine disease) - branched chain aJni.no acids J. in blood and urine b. Renal-normal plasma level but Specimen Collection for decreased r enal threshold or Li.pid Analysis reahsorption ❖ Cystinuria.+ cystine, lysine, ornitbine, arginine in urine Calculate LDL and VLDL METHODS 1. Scr eening Tests (Initial Diagnosis) a. Thin la yer chromatography with ninhydrin Lipid Cut-OHAction Levels b. U rine color tests c. Guthrie bacterial inhibition assays F or PKU: positive when phenylalanine Cardiac Risk Factors m etabolites in blood overcome 1. Positive Risk Factors inhibition in agar and B. subtillis grows. a. Age >45 years men, >55 years women 2. Quantitative Tests b. Family history of premature CHD a. fon-excbange chromatography c. Current cigarette smoking b. High performance liquid d. H ypertension systolic hp >120 chromatography (HPLC) e. HDLc 35 mgldL ❖ Tritdycerides < 150 mgldL 1. Most proteins are synthesized a nd ❖ LDLc < 100 mgldl catabolized in the liver c. Follow-up testing if levels outside 2. Basic unit - amino acids linked together goals by amide bonds to form protein ❖ 12 hour fasting sample ❖ Lipoprotein analysis 3. Prot ein breakdown in the body cl. Begin treatment b ased on LDLc produces urea and ammonia ; urea r esults ( see page 105) and other risk prod uced in liver and eliminated in factors (see above) urine b y kidneys METHODS : SERUM TOTAL PROTEIN Amino Acids 1. Kjeldabl GENERAL a. Reference method 1. Arninoacidurias b. Principle - measures nitrogen a. Overflow-plasma level above renal c,o ntent threshold as result of metabolic c. Acid digestion converts nitrogen in disorder protein to ammonium ion (NH4+) ❖ PKU - enzyme defi.ciencies cause which i s mea sured l- of phenylalanine in hlood and d. Difficult to p erform , infrequently j phenyl compounds in urine used  107 2. Biuret reaction 6. Relative concentration of each band a. Used most frequently determined by densitometry b. Depends on presence of z2 peptide bonds which react to form a purple 7. Specimen collection and handling complex with copper salts in a. Plasma samples (mistaken as serum) alkaline solution result in fibrinogen peak migrating METHODS : URINE AND CSF TOTAL PROTEIN between gamma and beta fractions b. Recollect and repeat to verify that 1. Dye - Coomassie brilliant blue peaks not fibrinogen 2. Turbi dimetric methods CLINICAL SIGNIFICANCE a. Acid (sulfosalicylic acid or 1. Plasma proteins - albumin and trichloroacetic acid) precipitates globulins protein a. Liver produces albumin, alpha-I, b. Measured spectrophotometrically or alpha-2 and beta globulins visually b. RE system produces gamma METHODS : SPECIFIC SERUM PROTEINS globulin (antibodies secreted by plasma cells) 1. Dye-binding methods for albumin a. Bromcresol green (BCG) 2. Prealbumin b. HABA (2- [4' - H ydroxyazobenzene) a. Appears as a faint band on serum - benzoic acid) electrophoresis b. Used clinically to assess nut ritional 2. ImmumochemicaI methods for specific status proteins other than allmmin MAJOR SERUM PROTEIN ELECTROPHORESIS FRACTIONS PROTEIN ELECTROPHORESIS 1. Albumin 1. Direction of migration of proteins in an a. Largest plasma protein fraction (52- electrical field determined by 62%) surface charge of protein b. Regulator of osmotic p ressure a. Protein at pH higher than its c. Transport protein because of ease isoelectric point is negatively of binding with blood components charged and mii:,rrates toward anode d. Causes oft values (positive charge) ❖ t synthesis (liver impairment) b. Albumin (smallest M. W.) has largest ❖ t associated with edema, number of free negative charges and decreased osmotic pressure migrates most rapidly traveling ❖ Ma/absorption or malnutrition grea test distance from application ❖ Nepbrotic syndrome (renal loss) point ❖ Severe burns c. Urine protein electrophoresis same e. ,t. values generally have no clinical a s serum except it must be significance (hemoconcentration, concentrated before application dehydration) 2. Electroendosmosis causes gamma 2. Alpha-I-globulins globulins to migrate toward the cathode a. Alpha-1-antitrypsin (AAT) even though they ar e slightly negatively ❖ + in acute phase and pregnancy ❖ t associated with emphysema in charged ( due to electrical charge on support medium) neonates 3. At pH 8.6, in order of migration, the 5 major bands are albumin, alpha-!, alpha-2, beta and gamma 4. Support media include cellulose acetate, agarose gel, and starch gel 5. Stains include Amido Black, P onceau S and Coomassie Brilliant Blue Basic Tests for Protein 108 b. Alph a fetoprotein (AFP) b. ,t- in: ❖ ,t- in amniotic fluid and maternal ❖ Elevated beta lipoprotein (LDL) serum in neural tube defects ❖ Iron deficiency anemia (spina bifida) ❖ liver cancer marker 5. Gamma globulin ❖ t in maternal senun during a. ,t- in: p1·egnancy associated with Down's ❖ Chronic inflammation syndrome ❖ Cirrhosis or viral hepatitis ❖ Collagen diseases 3. Alpha-2-globulins ❖ Pai-aproteins (monoclonal b ands, a. Haptoglobin gammopathies) ❖ Binds free hemoglobin b. t in congenital or acquired ❖ ,t- in acute phase and nephrotic immuno-deficien cy syndrome ❖ 't seen in transfusion reactions ❖ t i.J1 hemolysis and liver disease b. Ceruloplasmin ❖ Transports copper REMEMBER! ❖ ,t- iu acute phase and pregnancy Protein ❖ t Wilson's disease Electrophoresis 4. Beta globu]in a. Carrier proteins for iron Abbie Albumin is attracted to (transferri11) and lipids (lipoproteins) Andy Anode because of his _ , _..__..ositive attitude. 0 Some Common Protein Electrophoresis Patterns Beta Gamma Bridge =.E N "EE " E :, ,L -a." -a." co"s E < < < " 0 " 0 + Normal + + Monoclonal Gammopathy normal = normal= '® = Point of Application \ ', N ' ".c ' E- < --L....l.'-lo~-_;::....,_;;...._-,c;. + + Hypogammaglo bulinemia normal = Graphs Adapted with Permission from Helena Labo,atories' Electrophoresis Reference Chart