Clinical Chemistry 1 Laboratory - Finals Week 1 - Glucose Measurement PDF

Clinical Chemistry 1 Laboratory Finals: Week 1: Glucose Measurement Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento OUTLINE TYPE OF SPECIMEN FOR GLUCOSE I....

Clinical Chemistry 1 Laboratory Finals: Week 1: Glucose Measurement Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento OUTLINE TYPE OF SPECIMEN FOR GLUCOSE I. Specimen collection and handling II. Types of specimens for glucose III. Methods for Glucose Analysis a. Chemical b. Enzymatic SPECIMEN COLLECTION AND HANDLING - Standard specimen: VENOUS PLASMA GLUCOSE Tube for plasma glucose: Gray top (sodium fluoride) Note: - Fasting glucose in whole blood is 10 to 15% (10 - FPG: any value higher than the NV, it will be pre- to 12%) LOWER compared to serum/plasma diabetic glucose. - GDM: Gestational Diabetes Mellitus – pregnant - Capillary blood glucose is 2 to 5 mg/dL HIGHER women who developed diabetes mellitus compared to venous blood glucose Note: if you use other colored tube, make sure to separate the serum first - Venous blood glucose is LOWER compared to arterial blood glucose. Deoxygenated blood – venous vein Oxygenated blood - artery - To convert whole blood glucose into serum/plasma level, multiply value by 1.15 - Plasma glucose is 5% LOWER compared to serum (old studies); essentially same values according to recent studies - Difference ng IVGTT sa OGTT is sa intravenous - Fasting hours: 8 to 10 hours (not longer than inaadminister yung glucose load 16 hours) - HBA1C – is the only chemistry test that utilizes a whole blood FBS: 6-8 hrs fasting Tube: EDTA (Ethylenediaminetetraacetic Lipid profile: 10-12 hrs fasting acid) Both: 10 hrs fasting Color: purple or lavender Old age: 6 hours fasting Guidelines for OGTT - Allowable delay (separation of cells from serum: - Intake of normal to high CHO diet (atleast 120g 30 to 60 minutes CHO/ day for 3 days) prior the test - Glycolysis lowers glucose by 5 to 7% (5 to 10 - Overnight fasting of at least 8 to 10 hours mg/dL) per hour. - Patient should be ambulatory, refrain from - Standard anticoagulant: exercise, eating, drinking (except water) and 2ng NaF/mL of whole blood (use with smoking. K+ oxalate) Ambulatory – patient that is capable 6 to 10 mg NaF/mL is used alone from moving one place to another - Glucose metabolism: 7 mg/dL at room temp - Adult glucose load is 75g: for children: 1.75 g/ and 2 mg/dL at ref temp - Finish drinking glucose within 5 minutes, - Renal threshold: 160 to 280 mg/dL (8.8 to 9.9 patient should NOT VOMIT (kasi kung oo, void mmol/L) ang test at uulit sila ng fasting) - CSF glucose concentration: 60 to 70% that of Procedure ng OGTT plasma, collect specimen 1-2 hours before 1. Sabihan ang patient na overnight fasting, 8-10 spinal tap hours, no drinking, smoking - 10% contamination with 5% dextrose (d5W 2. I-confirm sa patient kung ilang hrs sila nag will INCREASE GLUCOSE by 500 mg/dL or fasting, pag pasok, go na kayo sa OGTT. Kapag more. hindi, resched ang test kasi overfasting na ang Note: pag may swero ang patient at wala ng available patient and it will interfere with the result ng site, magdiscard ng 5-10 ml or ipa-stop yung swero ng 5 patient mins. 3. Then start na mag venipuncture 4. First specimen – fasting blood sugar 5. Bigyan na ng glucose load ang patient and instruct na ubusin within 5 minutes  Clinical Chemistry 1 Laboratory Finals: Week 1: Glucose Measurement Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento 6. After ng 1 hr interval, kukuha ng second blood extraction and ile-label sa collection tube is 1st hour OGTT. 7. After ulit ng 1 hr interval from the second extraction, mag eextract ulit and for the 3rd tube ang ilalagay is 2nd hour OGTT. 8. Sa ibang clinic hanggang 4th collection pero sa practice natin is hanggang 3rd collection lang. IVGTT - Indications of IVGTT Patients who are unable to tolerate large CHO load Patients with altered gastric physiology or GI d/o Patients with malabsorption syndrome METHODS FOR GLUOSE ANALYSIS - Methods: Janney – Isaacson (single dose); Exton Rose (Divided Oral dose or double dose) - Not recommended for routine use - Present lang si hgb sa whole blood sample - HPLC – high performance liquid chromatography principle - Life span ng RBC: 120 days  Clinical Chemistry 1 Laboratory Finals: Week 1: Glucose Measurement Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento A. Oxidation Reduction 2. Hagedorn Jensen/ Ferric Reduction - Principle: Inverse Colorimetry - It involves reduction of yellow ferricyanide to a colorless ferrocyanide by glucose - Disappearance of color measure at 400 nm - Reagent: hot alkaline solution of potassium ferricyanide - Used in AutoAnalyzers (Technicon) B. Condensation Method 1. Ortho-Toluidine (DUBOWSKI METHOD) - Principle: protein precipitation - Can be used for urine and CSF without protein precipitation. - Reagent: o-toluidine, glacial acetic acid, - Always avoid hemolysis 100 deg C heat - End color: green or bluish green measured at 630 nm. - Interfering substances: galactose and mannose - MTT: 3-(4, 5- dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide. - MTT is a sensitive and reliable indicator of the cellular metabolic activity.  Clinical Chemistry 1 Laboratory Finals: Week 1: Glucose Measurement Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento - Mg/dL – milligrams per deciliter - Mmol/L – millimole per liter - usually multiply - 500 mg/dL - patient is hyperglycemia (diabetes mellitus) - FPG – any values greater than 70 to 110 mg/dL, pre-diabetic or diabetic si patient  Clinical Chemistry 1 Laboratory Finals: Week 2: Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento LIPIDS - Lipids are lipoproteins are important indicators of coronary heart disease (CHD) risk, which is a major reason for their measurement in research, as well as in clinical practice - The lipid profile currently recommended for initial screening in adults, age 20 or older, includes high total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), Triacylglycerols (TAG) LDL – atherosclerosis (sakit sa puso) - Testing should be repeated at least once every 5 years. - Commonly referred to as fats. - Functions: Store energy (TAG) Cushion and shock absorber Integral part of the cell membrane (phospholipid) Part of hormone synthesis (cholesterol) FORMS OF LIPIDS 1. Fatty acids – building blocks of acid Saturated, unsaturated 2. Phospholipids – most abundant lipid in man Composition of Phospholipid - 2 Fatty acids + Phosphate group 3. Triglycerides – main storage form of lipid in man Composition of triglycerides – 3 fatty acids + glycerol 4. Cholesterol – part of the cell membrane, parent chain for cholesterol-based hormones (e.g. aldosterone, cortisol, sex hormones) 5. Fat-soluble vitamins – vitamins A, D, E and K BLOOD SAMPLING AND STORAGE POSTURE - Postural changes decrease TAG by almost 50% from upright to supine position - According to National Cholesterol Education Program (NCEP), patient should be seated for at least 5 minutes prior to venipuncture. PLASMA VS SERUM - Plasma or serum can be used when only cholesterol, TG, and HDL-C are measured, and LDL-C is calculated from these three measurements. LDL-C is not directly name-measure pag galing sa dugo. It is measured using Friedewald calculation. - Plasma is preferred when the lipoproteins are measured by ultracentrifugal or electrophoretic methods - EDTA is the preferred anticoagulant. When measuring the lipids both si plasma and serum can be used in determining HDL. STORAGE - For long term storage: -70 C or lower - For short term storage (up to month or two): -20 C TRIGLYCERIDES - Commonly referred to as Triacylglycerides Increased in: - Hyperlipoproteinemia types I, IIb, III, IV, V Genetic disorder that could affect lipid metabolism leading to abnormal increased in circulating triglycerides - Nephrotic syndrome Kidney disorder that causes the body to excrete to much protein (proteinuria) - Pancreatitis Inflammation ng pancreas  Clinical Chemistry 1 Laboratory Finals: Week 2: Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento - Hypothyroidism Low thyroid hormone level which can lead to increase TAG - Alcoholism Increased TAG Decreased in: - Malabsorption syndrome Condition that impairs the nutrients in the intestine such as celiac disease, Crohn’s disease - Hyperthyroidism Overactive thyroid speeds up the metabolism which can result to lower TAG - Brain infarction - Malnutrition SPECIMEN CONSIDERATIONS - Fasting requirement: 12-14 hours The fasting helps minimize the influence of recent food intake particularly TAG, LDL-C - Like total cholesterol, it increases at a rate of 2mg/dL/year between 50-60 years old - Phospholipids and glucose may cause interference in TAG measurement - Avoid alcohol consumption at least 2 days before the test Up to a month or two: -20 C LAB METHODOLOGIES Principle: hydrolysis of triglycerides and measurement of glycerol that is released in the reaction. A. COLORIMETRIC 1. COLORIMETRIC METHOD (VAN HANDEL AND ZILVERSMITH) - Formation of colored compounds that can be quantify and measure to the absorbance specific wavelength - The intensity is directly proportion to the concentration of analyte - This method is a specific colorimetry assay for determination of TAG and involves hydrolysis of TAG to release glycerol which is then reacted to chromogenic agent to produce a colored compound - Si TAG may GLYCEROL so yung GLYCEROL niya ang magrereact sa CHROMOGENIC COMPOUND/AGENT to produce the color BLUE PRODUCT - Keywords: TAG and BLUE COLORED PRODUCT 2. FLUOROMETRIC METHOD (HANTZSCH CONDENSATION) - TAG and alcoholic and free fatty acid - End products: yellow fluorescence - Sensitive - Glycerol is converted to fluorescent compound through enzymatic reaction  Clinical Chemistry 1 Laboratory Finals: Week 2: Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento B. ENZYMATIC METHODS - Now universally used for TAG analysis - Specific, rapid, and easy to use - Performed directly in plasma or serum - Not subject to interferences by phospholipids and glucose Major interference: endogenous glycerol (normally present in plasma) Remedy: use of blanking techniques either through a “single-cuvette blank” or “double-cuvette blank” - Enzymatic have become the preferred choice for TAG analysis providing accurate and reliable source GLYCEROL KINASE METHOD - It has certain enzymes na involved such as lipase, glycerol kinase, glycerol phosphate and dehydrogenase - Enzymatic approached in measuring TAG by quantifying glycerol release during TAG hydrolysis - It is specific and sensitive which serves as indirect indicator of TAG FORMER CDC REFERENCE METHOD - Modified Van Handel and Zilversmith - NEW CDC REFERENCE METHOD: Gas chromatography/ Mass Spectrometry (GC-MS) TAG: REFERENCE RANGES CHOLESTEROL  Clinical Chemistry 1 Laboratory Finals: Week 2: Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento Increased in: - Hyperlipoprotenemia types II-A, III, V - Familial hypercholesterolemia - Poorly controlled DM - Nephrotic syndrome - Biliary obstruction - Biliary cirrhosis - Alcoholism Decreased in: - Severe hepatocellular disease - Malabsorption syndrome - Hyperthyroidism - Severe burns - Malnutrition SPECIMEN CONSIDERATIONS - Fasting: at least 12 hours - May be assayed from NONFASTING samples - If analysis is delayed, can be refrigerated at 4 C for several days - Precise and accurate timing for color development must be observed - Avoid hemolyzed and icteric samples - Avoid water contamination LAB METHODOLOGIES Principle: dehydration and oxidation of cholesterol to form a colored compound A. Chemical methods B. Enzymatic methods 1. Cholesterol oxidase method 1. LIEBERMANN-BURCHARD REACTION - Principle: colorimetric - Reagent is composed of: Glacial acetic acid – oxidizing agent Concentrated Sulfuric acid – dehydrating agent and color developer Acetic anhydride – color stabilizer - End product: measure at 410 nm Cholestadienyl monosulfonic acid (green) 2. SALKOWSKI REACTION - End product: Cholestadienyl monosulfonic acid (red) - Measured at 500 nm A. CHEMICAL METHODS - Saponification Purpose: convert cholesterol esters into free cholesterol and fatty acids Converting TAG (fats and oils) into glycerol and fatty acids using alkaline - Extraction  Clinical Chemistry 1 Laboratory Finals: Week 2: Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento Purpose: separate free cholesterol from protein carriers Organic solvents - Precipitation Purpose: further isolation of cholesterol - Colorimetry Purpose: form a colored product which will then be measured spectrophotometrically 1. ONE-STEP METHOD - Direct colorimetric method Sample + color reagent Example: pearson, stern and mac gavack methods 2. TWO-STEP METHOD - Extraction and colorimetric method Sample -> extraction of lipids Lipids+ color reagent Examples: Bloor’s zeolite and chloroform methods 3. THREE STEP METHOD Saponification, extraction and colorimetry B. ENZYMATIC METHOD - Involves measurement of hydrogen peroxide produced - Advantages: rapid, uses smaller amounts of sx (uL) 7 do not require lipid extraction - Interferences: reducing and oxidizing agents may affect the results FORMER CDC REFERENCE METHOD – (Abell – kendall method) - Three – step chemical method for cholesterol determination NEW CDC REFERENCE METHOD – Gas Chromatography/ Mass spectrometry (GC-MS) TOTAL CHOLESTEROL: REFERENCE RANGES  Clinical Chemistry 1 Laboratory Finals: Week 2: Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento LIPOPROTEINS CLASSIFICATIONS OF LIPOPROTEINS 1. According to density: High density lipoproteins (HDL) Low density lipoproteins (LDL) Very low – density lipoprotein (VLDL) 2. According to rate of migration SPECIMENS CONSIDERATIONS - TC, TAG, HDL-c Can be used satisfactorily analyzed in frozen samples LDL-c concentrations can be estimated computed from these measured values - Preferred samples for LPP measurement: sample collected using serum separator tubes (SST) - preferred samples for ultracentrifuge or electrophoretic methods and research studies: EDTA plasma - specimen varies depending on the test na need magawa WHY EDTA IS THE PREFERRED ANTICOAGULANT? 1. Because samples can be cooled immediately to 4 C to prevent changes that can occur in the LPP at room temp 2. Heparin can alter the electrophoresis mobilities of the LPP 3. Citrate causes falsely low plasma lipid and LPP concentration LAB METHODOLOGIES 1. Electrophoresis – alkaline medium - Differentiates lipoproteins based on charge and mass in an alkaline medium - Preferred supporting medium: agarose gel - Lipid-staining dyes: oil red O fat red 7b  Clinical Chemistry 1 Laboratory Finals: Week 2: Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento Sudan black B Scharlach R (Sudan IV) 2. Ultracentrifugation - Reference method for quantitation of lipoprotein - Employs a density gradient - Differentiates lipoprotein based on density Increase protein = increase density Increased TAG = decreased density - Expressed in svedberg units - Reagent: Potassium bromide solution (density = 1.063) - Frozen samples are not recommended  Clinical Chemistry 1 Laboratory Finals: Week 2: Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento  CLINICAL CHEMISTRY LABORATORY S.Y. ‘23 - ‘24 | SEM 1 FINALS WEEK 9: NON-PROTEIN NITROGENOUS (NPN) SUBSTANCES (KIDNEY FUNCTION TESTS) Formula: 1.0 KIDNEYS 𝑈 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑚𝐿) 73 𝑐𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒 (𝑚𝐿/𝑚𝑖𝑛𝑢𝑡𝑒) = 𝑃 𝑥 𝑚𝑖𝑛𝑢𝑡𝑒𝑠 𝑥 1. 𝐴 Kidneys are paired, bean-shaped organs Nephron: functional unit of each kidney Where: Five basic parts namely: U = concentration of the analyte in urine ❖ Glomerulus P = concentration of the analyte in plasma ❖ Proximal convoluted tubule Volume = volume of urine in mL in 24 hours ❖ Loop of Henle Minutes = time required to collect urine (1440 minutes) ❖ Distal convoluted tubules 1.73 = constant value; average body surface of an ❖ Collecting duct adult individual Non-protein compounds: A = body surface of the patient ❖ Urea: 45% ❖ Amino acid: 20% Inulin Clearance ❖ Uric acid: 20% Reference method ❖ Creatinine: 5% However not routinely done because of the ❖ Creatine: 1-2% need for continuous IV infusion ❖ Ammonia: 0.2% Priming dose: 25 mL of 10% inulin solution Continuous infusion: 500 mL of 1.5% inulin Renal/Kidney Function Tests: solution It is divided into three groups mainly: Reference value: 1. Glomerular Filtration Tests ❖ Male: 127 mL/min 2. Tubular Reabsorption Tests ❖ Female: 118 mL/min 3. Tubular Secretion Tests Creatinine Clearance Provides an estimate of the amount of plasma 1.1 TEST FOR GLOMERULAR FILTRATION that must flowed through the kidney’s RATE glomeruli/minute Measures the completeness of a 24-hour urine collection Glomerular Filtration Rate Production and excretion of creatinine is This is a measure of the clearance of normal related directly to muscle mass molecules that are not bound to protein and Excretion of creatinine is not routinely affected are freely filtered by the glomeruli, neither by diet– 1.3 to 1.5 creatinine excreted/day reabsorbed nor secreted by the tubules Major limitation: accurate urine collection Considered the best overall indicator of the Reference values: level of kidney function ❖ Male: 85-125 mL/min 150 liters of glomerular filtrate is produced ❖ Female: 75-112 mL/min daily and GFR decreases by 1.0 mL/minute/year after 20 to 30 years Increased Creatinine Clearance Decreased Creatinine Clearance Clearance 1. High cardiac output 1. Impaired kidney This is the removal of the substance from 2. Pregnancy function plasma into urine over a fixed time. 3. Burns 2. Shock, dehydration 4. Carbon monoxide 3. Hemorrhage Expressed in milliliter/minute poisoning 4. Congestive heart failure Plasma concentration and clearance is inversely proportional–as clearance of a substance decline, its concentration in plasma Urea Clearance Increases This can demonstrate progression of renal disease or response to therapy In advanced renal failure, urea clearance approaches unity with GFR and is better predictor of GFR than creatinine clearance–as renal function declines, the fraction of urea reabsorbed declines progressively, whereas PPT | LECTURE | CC LAB PAGE 1  CLINICAL CHEMISTRY LABORATORY S.Y. ‘23 - ‘24 | SEM 1 FINALS WEEK 9: NON-PROTEIN NITROGENOUS (NPN) SUBSTANCES (KIDNEY FUNCTION TESTS) the tubular secretion of creatinine increases 1. Chemical Method (Direct Method) progressively Diacetyl Monoxime Method Urea + DAM → Yellow Diazine Cystatin C Derivative Produced at a constant rate by all nucleated cells. 2. Enzymatic Method (Indirect Method) Freely filtered at the glomerulus, not secreted by the renal tubules but reabsorbed. A. Hydrolysis of Urea by Ureases Since it is completely reabsorbed and Urea + Urease → NH3 + CO2 catabolized in PCR, the presence in urine Urease is prepared from jack beans. denotes damage of that tubule. (Serum level After urease reaction, the ammonia is an indirect estimate of GFR). produced can be treated with Advantage: Assess GFR among pediatric and Berthelot reagent. elderly patients and renal transplant patients. B. Coupled Urease/ Glutamate Dehydrogenase Specimen: serum or plasma (fasting is not (GLD) method- UV enzymatic method required). Urea + Urease → NH4 + CO2 Increased: acute and chronic renal failure, NH4 + 2 – oxoglutarate + NADH → diabetic nephropathy. (GLD) Glutamate + NAD + H2O Reference value: C. Isotope Dilution Mass Spectrometry (IDMS) ❖ Adults- 0.5-1.0mg/L >65 years old- Reference method 0.9-3.4mg/L BUN rises in response to renal Modified Cystatin C Equation: dysfunction. ❖ GFR(mL/min) = 84.69 x Cystatin Low BUN are not generally considered C(mg/mL) x 1.384 abnormal renal function. Serum urea levels drop in severe Beta-Trace Protein hepatic disease because of a decline Isolated primarily from CSF- plasma BTP in the capacity of the liver to generate originates from the brain and is freely filtered urea from ammonia. at the glomerulus, then is reabsorbed completely and catabolized by the proximal Increased BUN Decreased BUN tubule. Increased: renal disease (because of reduced 1. Chronic renal disease 1. Poor nutrition 2. Stress 2. Hepatic disease filtration in the presence of constant 3. Burns 3. Impaired absorption production). 4. High protein diet (celiac disease) 5. Dehydration 4. Preganancy 1.2 TESTS FOR RENAL BLOOD FLOW Creatinine Used to monitor renal function; an index of Blood Urea Nitrogen (BUN) overall renal function. This is the major end product of protein Used to evaluate fetal kidney maturity. (dietary) and amino acid catabolism– 45% of End product of muscle metabolism derived the total NPN. from creatine (a-methyl guanidoacetic acid). First metabolite to elevate in kidney diseases Produced by three amino acids such as and easily removed by dialysis. methionine, arginine and lysine. Good indicator of nitrogen intake and the state Partially secreted by the PCT and not affected of hydration. by protein diet. However, it is not easily To obtain the concentration of urea from BUN: removed by dialysis. 2.14 x BUN= urea (mg%). Reference value: Reference value: 8-23 mg/dL (2.9-8.2 mmol/L) ❖ Male: 0.9-1.3mg/dL (80-115 umol/L) BUN:Creatinine ratio: 10:1-20:1 ❖ Female: 0.6-1.1mg/dL (53-97 umol/L) Fasting sample is usually NOT REQUIRED. Fasting sample is NOT REQUIRED. Fluoride or citrate will both inhibit urease. Hemolyzed and icteric samples should be Thiosemicarbazide and ferric ions are added avoided. to enhance color development. Both serum and urine creatinine have been used as indices of renal function. PPT | LECTURE | CC LAB PAGE 2  CLINICAL CHEMISTRY LABORATORY S.Y. ‘23 - ‘24 | SEM 1 FINALS WEEK 9: NON-PROTEIN NITROGENOUS (NPN) SUBSTANCES (KIDNEY FUNCTION TESTS) Patients taking cephalosporin antibiotics may possible negative interference from have falsely increased results when the Jaffe bilirubin and catecholamines). reaction is used. A. Creatinine Aminohydrolase-CK Method: 1. Chemical Method–Direct Jaffe Method Requires a large volume of Principle: A red-orange tautomer of pre-incubated sample and is not creatinine picrate is formed when widely used creatinine is mixed with alkaline picrate reagent. Interferences: ascorbate, glucose, uric acid, cephalosphorin and a-keto acids: falsely increased Bilirubin and hemoglobin: falsely decreased A. Folin Wu Method: a sensitive but nonspecific method B. Lloyd or Fuller's Earth Method: B. Creatinase-Hydrogen Peroxide Method sensitive and specific method Has a potential to replace Jaffe Absorbent: Lloyd’s reagent (sodium method and without interference from aluminum silicate) acetoacetate or cephalosporins. The Fuller's earth reagent (aluminum Creatinase is also known as creatinine Mg silicate) act to remove aminohydrolase. interferences present in the specimen and elution techniques are then used to separate the creatinine from the adsorbent which is then made to react with the freshly prepared Jaffe reagent. Jaffe reagent (alkaline picrate): ❖ Saturated picric acid ❖ 10% NaOH 4. Isotope Dilution Mass Spectrometry (IDMS): reference method 2. Kinetic Jaffe Method Elevated plasma creatinine concentration is Popular, inexpensive, rapid and easy to associated with abnormal renal function, perform method. especially as it relates to glomerular function. Principle: Serum is mixed with alkaline Plasma creatinine is a relatively insensitive picrate solution and the rate of change marker and may not be measurably increased in absorbance is measured between until renal function has deteriorated more than two points. 50%. In this reaction, the carbonyl oxygen of creatinine may attack the I-carbon Increased Serum Creatinine Decreased Serum Creatinine of picric to form covalent adducts. 1. Impaired renal function 1. Decreased muscle Interferences: a-keto acids and 2. Chronic nephritis mass cephalosporins: falsely increased. 3. Congestive heart failure 2. Advanced and severe liver disease 3. Prenanancy 3. Enzymatic Method 4. Inadequate dietary Used to eliminate nonspecificity of the protein Jaffe reaction; this is more specific than the Jaffe test. Interference by glucose and other Jaffe chromogens in creatinine measurement does not occur with enzymatic methods. However, enzymatic methods have certain interferences of their own (e.g. PPT | LECTURE | CC LAB PAGE 3  CLINICAL CHEMISTRY LABORATORY S.Y. ‘23 - ‘24 | SEM 1 FINALS WEEK 9: NON-PROTEIN NITROGENOUS (NPN) SUBSTANCES (KIDNEY FUNCTION TESTS) Blood Uric Acid 2. Enzymatic Method Freely filtered, partially reabsorbed and Uricase Method: A specific method secreted in the renal tubules. and uric acid has a UV absorbance Major product of purine catabolism. peak at 293 nm. Formed from xanthine by the action of Principle: The enzyme uricase xanthine oxidase in the liver and intestine. oxidizes uric acid to form allantoin. About 1 gram of uric acid is excreted normally. The resultant product (allantoin) has Reference value: no absorption at this wavelength. The ❖ Male: 3.5-7.2 mg/dL (0.21-0.43 decrease in the absorbance is mmol/L) proportional to the concentration of ❖ Female: 2.6-6.0 mg/dL (0.16-0.36 uric acid present in the sample. mmol/L) Disease Correlation: A. Hyperuricemia Gout Disease Correlation: Increased nuclear metabolism 1. Azotemia: Chronic renal disease elevated concentrations of nitrogenous Lesch-nyhan syndrome (inborn errors substances like urea and creatinine in the of purine metabolism) blood. Why does Lesch-Nyhan Syndrome Pre-renal: diminished glomerular filtration with contribute to hyperuricemia? normal renal function. ❖ Increased dietary intake Renal: damaged within the kidneys. ❖ Ethanol consumption Lab result: BUN >100mg/dL, creatinine 20 B. Hypouricemia mg/dL, BUA 12 mg/dL Fanconi’s syndrome– renal type Post renal: usually the result of urinary tract aminoaciduria obstruction. Urea is higher than creatinine due Wilson’s disease to back-diffusion of urea into the circulation. Hodgkin’s disease 2. Uremia: Kidneys fail to eliminate waste products of Blood Uric Acid metabolism. Characterized by anemia, uremic Fasting may NOT BE REQUIRED, but for frost (dirty skin), generalized edema, foul diagnostic purposes, fasting sample is breath and sweat is urine-like. preferred. High plasma uric acid is observed in newborns which eventually decreases in the succeeding years. Uric acid is stable in both serum and urine for 3 days at room temperature. Potassium oxalate (anticoagulant) cannot be used because ascorbic acid and bilirubin are the major interferences. 1. Chemical Methods Principle: Reduction-Oxidation (Redox) Reaction Lag Phase: incubation period after the addition of an alkali to inactivate non-uric acid reactants. PPT | LECTURE | CC LAB PAGE 4  CLINICAL CHEMISTRY LABORATORY S.Y. ‘23 - ‘24 | SEM 1 FINALS WEEK 9: NON-PROTEIN NITROGENOUS (NPN) SUBSTANCES (KIDNEY FUNCTION TESTS) Urine osmolality is due primarily to 1.3 TESTS FOR MEASURING TUBULAR urea; serum osmolality is primarily due FUNCTION to sodium and chloride. Proteins and lipids do not contribute Excretion Test to osmolality and normal ratio of 1. Para-Amino Hippurate Test (Diodrast Test) urine:serum osmolality is 1:1 Measures renal plasma flow and Reference value: requires clearance of the dye. ❖ Serum- 275-295 mOsm/kg Reference value: 600-700mL/minute. ❖ 24-hour urine- 300-900 2. Phenolsulfonthalein Dye Test mOsm/kg Measures the excretion of dye proportional to renal tubular mass. Direct Method: Freezing point osmometry (popular Dose of 6mg of PSP administered IV. method) Reference value: 1200mL blood flow/ Vapor pressure Osmometry (Seebeck Effect) minute. wherein increase in osmolality decreases the freezing point and vapor pressure. Concentration Test Reflects the function of the collecting tubules Indirect Method: Formula for Computing Serum and the loops of Henle. Osmolality Can detect renal damage that is not yet severe To use glucose or urea in osmolality, enough to cause elevated plasma urea and calculations must be converted from mg to creatinine levels. molar units. Three most prevalent solutes excreted: urea, chloride and sodium. Specimen: first morning urine. 1. Specific Gravity (SG) Simplest test of renal concentrating Conversion Factors: ability. 1. BUN "Fixation" of SG at 1.010 indicates Conventional unit: 8-23 mg/dL severe loss of concentrating ability of SI unit: 2.9-8.2 mmol/L the kidneys. Conversion factor: 0.357 Specific gravity of 1.010 is equal to SG of ultrafiltration in Bowman's space. 2. Serum or Plasma Creatinine In AUBF, we have isosthenuria which is Adult: SG = 1.010; hypersthenuria wherein SG ❖ Conventional unit: 0.6- 1.2 is >1.010 or hyposthenuria where SG mg/dL is 1.050) ❖ SI unit: 27-53 umol/L Reference value: 1.005-1.030 Conversion factor: 88.40 Why can't we consider 1.000 a normal SG of urine? 3. Uric Acid Conventional: 2. Osmolality ❖ Males: 4-8.5 mg/dL This is affected only by the number of ❖ Females: 2.7-7.3 mg/dL solutes present, thus more accurate Conversion Factor: 0.059 than SG in assessing renal tubular function. Also useful for assessing water deficit or excess. PPT | LECTURE | CC LAB PAGE 5  Clinical Chemistry 1 Laboratory Finals Week 4: Liver Function Test, Bilirubin and Urobilinogen Test Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento LIVER - Yellowish discoloration of skin, mucous - Chief Metabolic Organ membrane and sclera of the eyes - 80% of the liver must be destroyed to diminish it is caused by the increase bilirubin in liver function the blood - Severe loss of hepatic function may result to - Visible indication of hyperbilirubinemia changes in the functions of: - Icterus – yellowish discoloration of serum Synthesis – protein and clotting factor, - Clinically evident when bilirubin levels exceeds 2 cholesterol or 3 mg/dL Storage – glucose, vitamins and Classification of jaundice minerals 1. Pre hepatic jaundice – before bilirubin reaches Conjugation – convert bilirubin into a the liver, excessive breakdown of rbc water soluble (hemolytic/ unconjugated Excretion and secretion – remove toxins hyperbilirubinemia) Detoxification and drug metabolism – - Mechanism: increased amount of bilirubin neutralizes alcohols being presented to the liver, such as in acute BILIRUBIN of chronic hemolytic anemias - Principal pigment in bile - E.g HDN, HTR - Major metabolite of heme or major product of - Lab results: metabolic breakdown B1 markedly increase - Approximately 250-350 mg of bilirubin is B2 normal produced daily in healthy adults, about 85% of (-) urine bilirubin which is derived from turnover of senescent red Increase urobilinogen blood cells Dark stool - Other sources: 2. Hepatic jaundice – the problem lies within the Myoglobin liver (affects the liver to conjugate) Cytochromes - Mechanism: intrinsic liver defect or disease Catalases Impaired cellular uptake COMPARISON BETWEEN CONJUGATED Defective conjugation BILIRUBIN AND UNCONJUGATED BILIRUBIN Abnormal secretion pf bilirubin by liver cell - Ex: alcoholic cirrhosis, hepatitis - Lab results: B1 increases B2 increases Increased urobilinogen (+) urine bilirubin 3. Post hepatic jaundice (conjugated/ obstructive hyperbilirubinemia) – occurs after the liver and typically obstruction in the bile ducts which prevents the bile from draining in the intestine B1: initial breakdown of hgb, it travels to the liver bound - Mechanism: failure of bile to flow to the to albumin where it undergoes a process called intestine due to physical obstructions conjugation to become water soluble para maging B2. - Lab results for post – hepatic jaundice B2: after the bilirubin conjugates it becomes water B2 markedly increase soluble or also known as unconjugated bilirubin. This B1 normal form is excreted in bile to the intestine (+) urine bilirubin JAUNDICE Decrease urobilinogen - Comes from French word jaune: yellow Pale- colored stool (alcoholic stool)  Clinical Chemistry 1 Laboratory Finals Week 4: Liver Function Test, Bilirubin and Urobilinogen Test Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento DERANGEMENTS OF BILIRUBIN METABOLISM this usually happens in newborns with 1. Gilbert syndrome (unconjugated severe hyperbilirubinemia hyperbilirubinemia) – reduced activity fat soluble kaya nag c-cross sa blood - Bilirubin transport deficit brain barrier ng infant - Mutation of UGT1A1 gene leading to lower - Floppiness (hypotomia) enzymatic activity - Lethargy (opisthotonus) - Possible defect in transport protein - the danger of kernicterus is certainly at levels - Affected individuals may have no symptoms but exceeding 20 mg/dL mild icterus - treatments: phototherapy or exchange Increased B1 transfusion in extreme cases 2. Crigler-Najjar syndrome (unconjugated CAUSES OF UNCOJUGATED hyperbilirubinemia) HYPERBILIRUBINEMIA - Bilirubin conjugation deficit 1. hemolysis – increase breakdown ng RBC, - More severe and dangerous form of excessive bilirubin hyperbilirubinemia due to multiple mutations in 2. Gilberts syndrome the UDPGT gene 3. Crigler najjar syndrome - Infants are treated by means of phototheraphy 4. Hepatic diseases (hepatitis) – liver damage, - Type1: complete deficiency of enzyme UDPGT reduced liver function Kernicterus bilirubin in brain 5. Fasting – reduced in uptake of bilirubin, - Type 2: partial deficiency of enzyme UDPGT prolonged fasting increases unconjugated Managed with medications bilirubin 3. Dubin-johnson (conjugated CAUSES OF CONJUGATED hyperbilirubinemia) HYPERBILIRUBINEMIA 1. dubin Johnson - Bilirubin excretion deficit 2. rotor syndrome - Impaired excretion of B2 in the canaliculi due 3. biliary obstruction – blockage in the bile duct to an inherited deficiency of the canalicular 4. septicemia multidrug resistance/ Multi specific organic 5. total parenteral nutrition anionic transporter/ ATP binding cassette protein 6. Androgens - Blocked B2 returns to the blood and becomes 7. Spasms conjugated to albumin forming delta bilirubin 8. Pancreatic cancer - Distinguishing feature: intense dark 9. Parasitism pigmentation of the liver due to accumulation of lipofuscin pigment I. BILIRUBIN ASSAYS SPECIMEN CONSIDERATIONS 4. Rotor syndrome (conjugated hyperbilirubinemia) - Avoid hemolysis and lipemia - less common and relatively benign with excellent Fasting sample is preferred because prognosis lipemia will increase mesh with bilirubin - same as Dubin Johnson except that liver biopsy concentration shows no dark staining granules 5. Lucey – Driscoll syndrome (unconjugated Hemolyze samples decrease the hyperbilirubinemia) reaction of bilirubin with diazo reagent - Caused by a circulating inhibitor to bilirubin - protect specimen from light and process as conjugation soon as possible (or within two to three hours) KERNICTERUS Photoisomeration converts B1 to B2 - severe unconjugated hyperbilirubinemia - Visible icterisia ossurs at > 25mg/L characterized by deposition of bilirubin in the 1. Diazo reactions brain, causing severe motor dysfunction and principle: reaction of bilirubin with the sulfanilic retardation acid solution to form a colored product neurological conditions that occur in azobilirubin unconjugated indirect B1, accumulate the toxic levels in the brain  Clinical Chemistry 1 Laboratory Finals Week 4: Liver Function Test, Bilirubin and Urobilinogen Test Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento - Historically first performed on urine samples Most urobilinogen is reabsorbed and only, but van den Bergh found that diazo reexcreted by the liver a minor fraction reaction may be applied on serum samples but may be excreted in the urine only in the presence of accelerators Some urobilinogen in the stool are (solubilizers) converted into stercobilin - The third fraction, delta bilirubin will react as - Absence in the urine or stool denotes complete conjugated bilirubin biliary obstruction Delta bilirubin is conjugated bilirubin - Specimen for testing: tightly bound to albumin 2-hour urine sample it is elevated in obstructive jaundice 2-4 pm sample is preferred because of A. Evelyn and Malloy method alkaline tide - Coupling accelerator: 50% methanol Freshly collected stool for fecal - Final reaction: (+) pink to purple azobilirubin urobilinogen (560 nm) Avoid photoexposure - Uses pH 1.2 - Method for testing: EHRLICH REACTION B. Jendrassik and Grof method - Principle: reaction of Ehrlich’s reagent (PDAB: - Most sensitive and most widely used para dimethyl amino benzaldehyde) to form a - more sensitive than Evelyn Malloy red color which is read spectrophotometrically - popular technique for the discrete analyzer - Reporting of results: ehrlich unit - not falsely elevated by hemolysis - Reference range (urine): 0.3-1 ehrlich units (2 - coupling accelerator: caffeine sodium benzoate hr) - Buffer: sodium acetate (less sensitive to pH NOTES TO REMEMBER change) - unconjugated bilirubin reacts SLOWLY - Ascorbic acid: terminates the initial reaction and Therefore, accelerants such as caffeine destroys the excess diazo reaction or methanol are used to measure both - final reaction: (+) pink to blue azobilirubin (600 direct and indirect bilirubin (total nm) bilirubin) C. Modified Jendrassik and Grof Method without accelerants only direct acting - Candidate reference method for total bilirubin bilirubin will be measured - coupling accelerator: caffeine benzoate - unconjugated bilirubin - the fraction that - reliable and well established produce a color only after addition of alcohol - Reference range: - conjugated bilirubin - the fraction that produce a color in aqueous solution AMMONIA DETERMINATION - from amino acid to nucleic acid metabolism - Also produce from glutamine - Toxic compound metabolized exclusively by the liver - unique enzyme believer responsible for converting toxic ammonia to non-toxic urea: ornithine carbamoyl transferase (OCT) - clinical significance: Hepatic failure Reye’s syndrome – rare; affects children - Increase levels: II. UROBILINOGEN ASSAYS - Reye’s syndrome - colorless and product of bilirubin metabolism - Cirrhosis that is oxidized by intestinal bacteria to the - Hepatic, brown pigment urobilin - Acetaminophen poisoning - after formation in the intestine: - Idiopathic hyperammonemia  Clinical Chemistry 1 Laboratory Finals Week 4: Liver Function Test, Bilirubin and Urobilinogen Test Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento SPECIMEN CONSIDERATIONS - Increased in: - Smoking is a source of contamination malignancy - Avoid prolonged standing because ammonia Multiple myeloma level will rise Waldenstrom macroglobulinemia - Preferred specimen: arterial blood - Decreased in: - specimen requirements: Hepatic cirrhosis Heparinized or EDTA plasma (fasting) glomerulonephritis kept at 4°C and transported on ice Nephrotic syndrome Nonhemolyzed Starvation Must be tested within 20 minutes 1. Kjeldahl Method (indirect method) REYE’S SYNDROME - The ultimate reference method for - group of disorders caused by infectious, determining protein concentration metabolic, toxic or drug induced disease But not routinely performed/ used - Precise cause is unknown but studies found - Quantifies protein by its nitrogen content strong epidemiological association between - Proteins have an average nitrogen content of ingestion of aspirin during a viral syndrome and 16% (multiply by 6.25) subsequent development of Reye syndrome. - Digestion of nitrogen containing compounds to - An acute illness, characterized by non- release ammonium ions using concentrated inflammatory encephalopathy and fatty sulfuric acid (H2SO4) degeneration of the liver - Ammonia measured as ammonia gas, via ASSESSMENT FOR LIVER FUNCTION titration as a base, or via nesslerization (uses I. Test that evaluate hepatic synthetic of dimercuric potassium iodides to form a function colored complex with ammonia in alkali) - useful to quantify and determine the severity of - End product: ammonia hepatic dysfunction - Reagent: sulfuric acid - includes: 2. Biuret method (direct method) Total protein determination - Recommended by the IFCC (International Albumin test Federation of Clinical Chemist) Albumin/globulin ratio (A/G ratio - Also used by the chemical analyzer in which prothrombin time test protein can be measured down to 10 or 15 A. Total Protein Determination mg/dL - Protein: - Principle: cupric ions with peptide bonds in an 1. Enzymatic activity (increase catalyst) alkaline medium producing a Violet colored 2. Defense/ immunity (by immunoglobulin) complex that is proportional to the to the number 3. Transport substances (hgb – carries oxygen of peptide bonds present and reflects the total to the blood, ferritin – iron storage) protein level at 545 nm - Important in assessing: - Requires at least two peptide bonds and then Nutritional status alkaline medium presence of severe diseases involving - Sensitivity: 10 – 15 mg/dL the liver, kidney and bone marrow - REAGENTS: - About 10% higher in ambulatory individuals 1. Copper sulfate – main reagent (cupric ion - Total plasma protein is 0.2 to 0.4 g/dL higher complexes with peptide bond) than Serum 2. Potassium sodium tartrate (rochelle's - Transudates have a total protein of 3.0 g/dL 3. Potassium iodide - prevents auto reduction Fasting is not required in total protein of copper Specimen: serum (it does not have 4. Sodium hydroxide - alkalinizes the solution fibrinogen and anticoagulant) NOTE: Avoid hemolyzed and icteric sample - Avoid hemolysis - hemoglobin is absorbed at the (can cause elevation in TPD) same wavelength as the biuret reagent - Reference value: 6-8 g/dL  Clinical Chemistry 1 Laboratory Finals Week 4: Liver Function Test, Bilirubin and Urobilinogen Test Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento 3. Other methods ✓ Alpha 1-globulin – A1 antitrypsin 3.1 Refractive index – alternative test to which inhibits proteolytic chemical analysis enzymes 3.2 Specific gravity (ex. Hemoglobin ✓ concentration determination via copper sulfate method) absorption at 280 nm 3.3 turbidimetry (ex. TCA, SSA) 3.4 Folin – Ciocalteu method - oxidation of ✓ Alpha 2-globulin – haptoglobin, phenolic compounds such as tyrosine ceruloplasmin (transporting using folin – ciocalteu reagent (phenol) copper to one place to another), to give a deep blue color most of the time mga Highest analytical sensitivity transporters ng copper Main reagent: phenol or phosphotungstic molybdic acid 3.5 Coomassie Brilliant Blue Dye – detects down to 1ug of protein 3.6 Lowry assay – used the Biuret method ✓ Beta-globulin – transferrin (iron followed by the phenol reagent to transport) and complement enhance color formation protein (involved in immune 3.7 Ninhydrin - reacts with primary amines system) to produce a Violet color. Widely used for detection of peptides and amino acids after paper chromatography 3.8 Immunologic - quantitation of specific proteins by nephelometry 4. Serum protein electrophoresis (SPE) - Electrophoresis – applying electrical charge ✓ Gamma-globulin – - Introduced by Tiselius immunoglobulins or antibodies, - Principle: migration of charged particles in an primarily responsible for the electrical field immune response in the body - separates protein according to their electrical charge - Application: Identification of monoclonal spikes of Note: pre-albumin, if present, migrates ahead of albumin immunoglobulins SEQUENCE IF PRESENT SI PRE-ALBUMIN: Differentiating them from polyclonal Pre-Albumin → albumin → A1 →A2 → Beta → hypergammaglobulinemia gamma - (+) ions – CATIONS SEQUENCE IF HINDI PRESENT SI PRE-ALBUMIN: - (-) ions – ANIONS albumin → A1 →A2 → Beta → gamma - At pH of 8.6, proteins are negatively charged WHY WE SPE IS IMPORTANT? - (+) electrode – ANODE 1. Easily testing or determination - (-) electrode – CATHODE 2. Diagnose certain conditions - Migrations of serum proteins: 3. Monitoring therapy CATHODE → ANODE Fastest → slowest: ✓ Albumin – major protein in the body and involve in transporting substances  Clinical Chemistry 1 Laboratory Finals Week 4: Liver Function Test, Bilirubin and Urobilinogen Test Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento - Regions are stained using 1. Bromphenol blue 2. Ponceau S 3. Amido black 10B 4. Lissamine green 5. Coomassie brilliant blue - The bands are scanned by densitometer after staining to quantify the amount of proteins present on each band. A. ELECTROPHORETIC PATTERNS - Gamma spike ▪ Monoclonal gammopathy (multiple myeloma increase IgM, waldenstrom macroglobulinemia increase IgM) - Alpha-1-globulin flat curve ▪ Emphysema and juvenile cirrhosis (alpha1-antitrypsin deficiency) - Alpha-2-macroglobulin band spike ▪ Nephrotic syndrome; accompanied by decrease albumin - Spikes of A1, A2, and beta globulin bands ▪ Inflammation  Clinical Chemistry 1 Laboratory Finals Week 4: Liver Function Test, Bilirubin and Urobilinogen Test Lecturer: Ms. Jalocon and Ms. Delos Reyes Section: MED223A By: Micka Sheldine Zhaine Adviento - Beta-gamma region bridging ▪ Conversion factor: conventional →SI: ▪ Hepatic cirrhosis (increased IgA) x10 - Small spikes in the beta region MUST KNOW!!! ▪ Iron-deficiency anemia - Analbuminemia B. ALBUMIN DTERMINATION ▪ Hereditary absence of albumin - Albumin concentration is inversely proportional ▪ Inability to synthesize albumin to the severity of the liver disease - Bisalbuminemia - Albumin can be measured by direct methods ▪ albumin that has unusual molecular based on its dye binding property characteristics - Dye binding methods: ▪ presence of two albumin bands ▪ Bromcresol purple: most specific dye instead of a single band in ▪ Bromcresol green: most commonly used electrophoresis ▪ Methyl orange ▪ Associated with the presence of ▪ Hydroxyaminobenzoic acid (HABA) therapeutic drugs in serum (increase - Increased in: penicillin = bisalbuminemia) ▪ Severe dehydration (relative increase) D. PROTHROMBIN TIME/ VITAMIN K ▪ Prolonged tourniquet application RESPONSE TEST (artifactual) - differentiates intrahepatic disorder from - Decreased in: extrahepatic obstructive liver disease ▪ Liver disease  decreased synthesis ▪ intrahepatic – prolonged PT ▪ Nephrotic syndrome  increased ▪ extrahepatic – normal PT excretion (20-30 g/day) - Remember that the liver is responsible for the ▪ Malabsorption syndrome  decreased synthesis of vitamin K dependent clotting factor absorption - vitamin K is administered IM, 10 mg daily for 1-3 ▪ Muscle wasting disease  increased days destruction ▪ If PT is prolonged: intrahepatic disorder ▪ Malnutrition  decreased dietary intake ▪ If PT is normal: extrahepatic disorder ▪ Thyrotoxicosis  increased catabolism ▪ Burns  increased destruction in catabolism C. ALBUMIN/ GLOBULIN RATION - Measured to determine if globulin is higher than albumin in serum - Globulin (antibodies): ▪ Not analyzed ▪ Pag increase si globulin, matic na may infection or inflammation si patient ▪ Computed value → TSP - albumin= globulin - Reference value: 1.3-3:1 (normally, Albumin > Globulin) - Increased in: ▪ Chronic inflammation ▪ Multiple myeloma ▪ Waldenstrom macroglobulinemia - If globulin is A