Introduction to Clinical Chemistry A.Y. 2014-2015 MT-IA 1A-MT Prepared by Group 3 De Guzman, Elisha Jhoanna De Lara, Harissa Katrina R. Deiparine, Jesmae Anril N. Diaz, Jorina P. Espina, Doxa Charisse L. Fajardo, Ma. Pamela Rica M.
Introduction to CLINICAL CHEMISTRY HISTORY: > 19th Century – Application of chemistry to medicine was more used on understanding the diseases rather than its cure/relief. >During the French Revolution Era, Fourcroy proposed that chemical laboratory to be located near the wards. >Justus Liebig, wrote the book of Clinical Chemistry, which was important in the development of the clinical chemistry since it introduced quantitative method of observation into physiological chemistry. - The use of the “Duboscq colorimeter” for color comparison in the analysis for creatinine in urine pushed the modern era of clinical chemistry. WHAT IS CLINICAL CHEMISTRY? > Also known as clinical pathology, clinical biochemistry, or medical biochemistry. > Roles in the Laboratory: The area of clinical pathology concerned with the analysis of bodily fluids. It uses chemical processes to measure levels of chemical components in bodily fluids. ROUTINE TESTS IN CLINICAL CHEMISTRY Glucose BUN [Blood Urea Nitrogen] Creatinine Lipids Profile Tests Cardiac Markers Liver Function Tests 1.) GLUCOSE > Definition: A simple sugar (monosaccharide) by product of carbohydrates. > Insulin -Insulin’s role in one’s body is to regulate the glucose present in one the blood, by storing these glucose into muscles or fats (glycogen) for use in obtaining energy (anabolic) . - Glucagon (produced by the alpha cells) opposes the insulin by breaking down glucose and releasing it to the bloodstream (catabolic) Metabolic Disorders caused by abnormally high or low blood glucose *Hypoglycemia – abnormally low blood glucose than the normal glucose level. *Hyperglycemia – abnormally high blood glucose than the normal glucose level. 27
* Diabetes – chronic metabolic disease caused by perennially high blood glucose. Types of Diabetes: 1. Type 1 – Beta Cells (producers of insulin) are destroyed by the immune system. -“Insulin Dependent”, can be genetically acquired. 2. Type 2 – The pancreas can still produce insulin, but it may not be enough or the immune system blocks it. -“Non-Insulin Dependent" 3. Gestational Diabetes – During pregnancy, the placenta secretes a hormone that blocks insulin, if the pancreas can’t match the amount of glucose, then it will cause Gestational Diabetes. > Tests for Blood Glucose: *All tests performed are usually done with venipuncture, with blood as the specimen. TEST USE SPECIMEN & COLLECTION - measures amount of - Patient must not eat for sugar in blood. 8-10 hrs before the test. Fast Blood Sugar Test Random Blood Sugar Test
Oral glucose tolerance test
- It is used to measure the body’s ability to use sugar after drinking a standard amount of glucose. - It is used to check if a pregnant woman acquired Gestational Diabetes.
- blood testing done anytime of the day. - Steps: 1. Get the FBS as baseline 2. Drink 75-100 g of glucose 3. Blood samples will be obtained again
VA
- Normal Value <100mg/dL g - hyperglycemia 100mg/dL- 1 - diabetes ≥126mg/dL = - Normal value <140mg/dL Amount of glucose intake 50g (1hr) 75g (2hrs)
100g (3hrs)
2-Hour PostPrandial Glucose Blood Sugar Test (2Hr PPBS) A1cTest for HbA1c - Glycosylated Hemoglobin test
- It is used to check how the body responds with sugar and starch after eating.
- Steps: 1. Get the FBS for baseline 2. Get another sample after 2 hrs after eating - It is to measure average - Test is usually done level of blood sugar with venipuncture with (glucose) over 3 months to blood as the specimen or monitor control with using finger stick for
Age Newborn-50yrs 50-60yrs 60yrs and above - Normal (no dia <5.7% - Pre-diabetes (h 5.7%-6.4% 28
diabetes. tests at home. - For this test we can also use electrophoresis, enzymatic assay (Diazyme A1c), ion-exchange chromatography or highperformance chromatography.
- Diabetes >6.5%
2.) BUN [Blood Urea Nitrogen] > Urea/Urea Nitrogen - is the chief component of the NPN (non-protein nitrogen) material in the blood. - A waste product of protein metabolism > Liver – site of urea formation. > Normal Value of Urea: Urea nitrogen, serum Urea, serum Urea Nitrogen, urine Adult (7-18 mg/dL)
2.5 – 6.4 mmol urea/L
Adult (5-39 mg/dL)
2.5 – 6.4 mmol/L
12-20g/24 hrs
0.428 -0.714 mmol urea/24 hr
> Method: 1. Addition of enzyme, Urease, to the whole blood, serum, or plasma during incubation - Urease hydrolyzes urea in the sample and the ammonium ion produced in the reaction is quantified. 2. Analysis of reaction through Classical and Indirect Method. > Importance of Urea Tests: a. helps us see the rough estimate of renal function b. evaluates renal function c. assesses hydration status d. determine nitrogen balance e. aid in diagnosis of renal disease f. verify adequacy of dialysis > Uremia – abnormal or high urea nitrogen in the blood due to impaired kidney function since urea is not removed from the blood and excreted in the urine. 3.) CREATININE > Creatinine in the blood results in the spontaneous formation of creatinine from creatine (is synthesized primarily in the liver and then transported to other tissue where it serves as high energy source to drive metabolic reactions) and creatine phosphate. Its formation and release into the body fluids occur at a constant rate and have a direct relationship to muscle mass. 29
Creatinine concentration varies with age and gender and is measured in the blood and in the timed urine specimen How is creatinine produced? - Creatinine is made from creatine, serves as high energy source to drive metabolic reactions. - Creatinine Phosphate loses phosphoric acid and creatine loses to water to form creatinine. Specimen: Serum or heparinized plasma > Methods: 1. Jaffe reaction - The oldest clinical chemistry method in use. - Creatinine reacts with alkaline picrate to form an orange-red solution that is measured in spectrophotometer - To improve the specifity of the reaction and to eliminate the interference from the many non-creatinine in blood, acidification step is added. - Noncreatinine Jaffe-reacting choromogens include proteins, glucose,ascorbic acid and pyruvate. > Normal Values:
2.) Serum Creatinine o blood test that is performed as a part of a physical examination if a person has blood work done. o Serum creatinine concentration is relatively constant and is somewhat higher in males than in females. Glomerular Filtration Rate o measures of a kidney function. o The quantity of glomerular filtrate per unit time in all nephrons of both kidneys. o Various substances in plasma and urine can be used to estimate the GFR. o Inulin has historically been the gold standard for measuring GFR. > Clinical Significance: - Creatinine in the blood results from creatinine originating in the muscles of the body. - Creatinine is freely filtered by the glomeruli of the kidney but it is not reabsorbed under normal circumstances. - The concentration of creatinine is not affected by dietary intake, degree dehydration in the body or protein metabolism. 4.) LIPID PROFILE TEST 30
> Lipids - a class of biochemical compounds of fats, oils, hormones, and certain components of membranes that are grouped together because they do not interact appreciably with water. > Lipid Profile Testing - a collection of tests carried out in order to assess the levels of different types of cholesterols in the blood stream. Its objective is to assess the risk of coronary heart disease that these different types of cholesterol pose to the patient under examination. Preferred lipid testing specimen: A serum collected in a serum separator evacuated tube from a patient who has been fasting for 12-15 hours. if analysis must be delayed, the serum can be refrigerated at 4°C for several days. Acute illness, high fever, starvation or recent surgery lowers the blood cholesterol and triglyceride levels. Patients should stop taking medications that may affect the accuracy of the test and avoid alcohol intake for 24 hours. Parameter
Cholesterol
Triglycerides
Method/Routine Enzymatic Assay - Most common method used to determine cholesterol - The common lipid panel, including measurements of total, LDL, HDL cholesterol, together with triglycerides can be completed routinely using chemistry analyzers. Enzymatic reagents are mild compared with the acid reagent and better suited for automated chemistry analyzers. - The enzyme, cholesterol esterase, hydrolyzes cholesteryl esters to free cholesterol. The free cholesterol is reacted by the second enzyme, cholesterol oxidase producing hydrogen peroxide, which oxidizes various compounds to form a colored product which is measured photometrically. - The intensity of the resulting color, proportional to the amount of cholesterol, can be measured by a spectrophotometer. -Measurement of serum triglycerides in conjunction with cholesterol is useful in detecting certain genetic and other types of metabolic disorders, as well as in characterizing risk of CVDs. The triglyceride value is also commonly used in the estimation of LDL cholesterol by the Friedewald equation. Routine: -The appearance of plasma or serum can be observed after a minimum of 12-hour fast. -Plasma – clear → triglyceride level < 200 mg/dL Plasma – hazy and turbid → triglyceride level > 300 mg/dL Plasma – opaque and milky (lipemic) → 31
Ad
- Desirab 199mg/dL or
- Borderli 200-239mg
- Higher R 240mg/dL greater
Adult Values: - Desirable 149 mg/dL
- Borderline 150-199 mg
- Higher Risk 200-499 mg
MALE: 40-160 mg/
High-Density Lipoprotein (HDL) Cholesterol (HDL parin po ito )
Low-Density Lipoprotein (LDL) Cholesterol
triglyceride level > 600 mg/dL -Triglyceride methods are usually enzymatic. In most automated enzymatic methods, hydrolysis of the triglyceride present in the sample is usually achieved by lipase. The resulting glycerol is assayed by various coupled-enzyme methods. - The measurement of HDL cholesterol has assumed progressively greater importance in the NCEP treatment guidelines. In the earliest guidelines, HDL cholesterol was measured as a risk factor but otherwise was not considered in treatment decisions. Because the risk associated with HDL cholesterol is expressed over a relatively small concentration range, accuracy in the measurement is especially important. Routine: -Chemical Precipitation -involves a two-step procedure with manual pretreatment. A precipitation reagent added to serum or plasma aggregated non-HDL lipoproteins, which were sedimented by centrifugation, at forces of approximately1500g (gravity) with lengthy centrifugation times of 10to 30 minutes or higher forces of 10,000 to 15,000g, decreasing centrifugation times to 3 minutes. - LDL cholesterol, well validated as a treatable risk factor for CHD, is the primary basis for treatment decisions in the NCEP clinical guidelines. Routine: - Beta-quantification combines ultracentrifugation and chemical precipitation. Ultracentrifugation of serum at the native density of 1.006 g/L is used to float VLDL and any chylomicrons for separation. The fractions are recovered by pipetting.
5.) CARDIAC MARKERS > Cardiac marker tests identify blood chemicals associated with myocardial infarction (MI), commonly known as a heart attack. > Collection of specimen: *Homocysteine tests require the patient to fast. 32
FEMALE: 35-135 mg/
- Desirable: 40mg/dL or - Higher Risk: 39 mg/dL o
Adult Values (2
-Desirable if p has CHD 99 mg/dL or - Desirable 129mg/dL o - Borderline 130-159mg/ - Higher Risk 160mg/dL o greater
*Blood Collection after the onset of Myocardial Infarction: 1.) On admission 3.) 6-8 hours 2.) 2-4 hours 4.) 12 hours *if earlier specimens are found negative but high MI suspection collected again after 24 hours > Current tests used for acute MI and myocardial injury are: a.) Myoglobin b.) Troponins c.) CK isoenzyme (CK-MB) TESTS
Myoglobin
Creatinine Clearance USES - Heme protein found in striated skeletal and cardiac muscles. -released rapidly after tissue injury and may be elevated as early as one hour after myocardial injury -Can be measured by: >Latex agglutination >Enzyme-linked immunosorbent assay (ELISA)
VALUES NORMAL CHANGES IN VALUES VALUE - Adult men -rise in 30-90 concentration: mg/Dl 2-6 hours period - Adult women after MI symptoms < 50 mg/dL -return to normal: 12–24 hours.
>Immunonephelometry >Fluoroimmunoassay >Spot test using Troponins
immunochromatography - “gold standard” of cardiac markers - is a complex of 3 proteins that form the thin filaments of muscle fibers and regulate muscle contraction: 1. Troponin T (TnT) 2. Troponin I (TnI); 3. Troponin C (TnC)
Troponin T: < 0.1 ng/mL - rise in concentration:
-rise in concentration: 2-4 hours and peak at 10–24 hours after MI - return to normal: withing 5-14 days. Troponin I: - rise: < 1.5 ng/mL 2-4 hours and peak at 10–24 hours after MI - return to normal: 33
within 5-10 days.
- Creatine kinase is an enzyme responsible for transferring a phosphate group from ATP to creatine. Creatine Kinase But it is not cardiacMB specific. - but the MB isoenzyme is cardiac-specific (CK-2); excellent sensitivity after MI onset - an amino acid in the blood - Laboratory testing for plasma homocysteine levels can improve the assessment of cardiovascular disease risk. Homocysteine - The only cardiac marker test that requires FASTING. - High levels of homocysteine are the result of a lack of certain B vitamins, inheritance, or dietary excess and have been implicated in vascular-wall injury.
10–13 units/L
- abnormal/rise: 4-6 hours and peaks at 10–24 hours after MI - Returns to normal: within 72 hours.
*Normal Fasting Level: 5–15 micromol/L - Hyperhomocysteinemia Concentrations: Moderate - 16-30 micromol/L Intermediate - 31-100 micromol/L Severe - < 100 micromol/L
6.) LIVER TESTING Bilirubin taken from the non-contaminating heme portion of hemoglobin, released from the breakdown of red blood cells. Specimen: serum or plasma must be protected from light must be use within 2-3 hrs can be stored in a dark refrigerator for 1 week or in a freezer for 3 months without significant loss of albumin. > Methods: -Diazo Reaction first described by Ehrlich in 1883 with the use of urine. used diazotized sulfanillic acid to produce azobilirubin dye, color: red-purple 34
most commonly used in determining assays of serum biliburin modified by van der Bergh and Muller to be used on serum specimens. uses accelerators(solubilizer)
> Procedures: - Malloy – Evelyn Procedure -diazotized sulfanilic acid reaction forms two molecules of azobiliburin. developed the first clinically useful methodology for the quantitation of bilirubin. uses 50% methanol solution as an accelerator. -Jendrassik-Grof Method for Total and Conjugated Bilirubin Determination - diazo reagent and an accelerator is used to determine the total bilirubin value. Thus, determining conjugated, unconjugated & delta bilirubin. - uses caffeine-benzoate-acetate as an accelerator. - conjugated bilirubin - polar compound that is found in the plasma Population Type of Biliburin Reference Range Adults
Conjugated bilirubin
0.0–0.2 mg/dL (0–3 mol/L)
Unconjugated bilirubin
0.2–0.8 mg/dL (3–14 mol/L)
Total bilirubin
0.2–1.0 mg/dL (3–17 mol/L) - unconjugated bilirubin – nonpolar compound that is found in the plasma bound to the albumin. - delta blirubin – conjugated bilirubin found in albumin. (significant in hepatic obstruction) > Normal Values: c. Proteins > a large polypeptide (a chain of amino acids) > make up antibodies (aka immunoglobulins, w/c are synthesized in plasma cells) , a major component of our immune system. >Different Classification of Protein Acc. to Function: a) Enzymes - catalyzed chemical reactions b) Hormones – chemical messengers that control actions of specific cells/organs. 35
c) Structural Proteins – structure of cells and tissues in muscle, tendons, and cone matrix d) Storage proteins – energy source >Plasma Proteins (albumins and globulin) are more frequently analyzed for protein. in a blood panel, 4 measurements of determination is given total protein, Albumin, Globulin and Alubimin/Globulin Ratio > Methods: Biuret Method o To determine Total protein in the body. Hypoproteinemia – total protein level less than the reference interval. Indicative of renal disease and liver damage. o Cupric ions piled with the groups involved in the peptide bond. In an alkaline medium, & in presence of two peptide bonds, a violetcolored chelate is formed. o The Biuret reagent also contains sodium-potassium tartrate which complexes cupric ions to prevent their precipitation in the alkaline solution and potassium iodide which acts as an antioxidant o measured photometrically, darker the solution, the higher the concentration of protein Dye-binding Method o For determining Albumin (binds various substances in the blood) - Indicative of Liver damage when Albumin level is low (Hypoalbuminemia). o based on the ability of most proteins in serum to bind to dyes such as bromphenol blue. o Coomassie brilliant blue 250 binding to protein causing a gift in the absorbance maximum of the dye from 465-595 nm. the increase in absorbance at 595 is used to determine protein concentration. o used to stain protein bands after electrophoresis *when abnormality is found in total proteins or albumin; separates proteins on the basis of their electric charge densities. >Normal Values: Serum = 6.5 – 8.3 g/dL Albumin = 3.5 – 5.5 g/dL d. Enzymes >Methods: measurement of catalytic activity -Detects increase in product concentration, decrease in subtrate concentration, decrease in coenzyme concentration, or increase in the concentration of an altered coenzyme. -Performed in zero-order kinetics
36
