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CLINICAL CHEMISTRY 1 INTRODUCTION  ______________ types of absorption spectrophotometer Clinical Chemistry...

CLINICAL CHEMISTRY 1 INTRODUCTION  ______________ types of absorption spectrophotometer Clinical Chemistry  Designed to make one measurement at a time at 1  It is an applied science when analysis is performed on specified wavelength. body fluids or tissue specimens to provide important 2) ______________ information for the diagnosis or treatment of disease.  Splits the monochromatic light into ___ components:  Performance of different ________________________ that o One beam passes through the sample; other yields to accurate and precise information that will aid to through a reference solution or blank. diagnosis and treatment of patients. o Additional beam corrects for variation in light  Deals with ______________ of different analytes source intensity. o 2 TYPES: REAGENTS and REFERENCE MATERIALS a) Double-beam ______________ – uses 2 photodetectors for sample beam and reference beam. b) Double-beam ______________ – uses 1 photodetector and alternately passes the monochromatic light through the sample cuvet and then reference cuvette using a chopper. WET CHEMISTRY DRY CHEMISTRY PARTS OF SPECTROPHOTOMETER 1. Light source  Provides polychromatic light and must generate sufficient radiant energy or power to measure the analyte of interest.  2 TYPES: a. ______________ – emits radiation that changes in intensity; widely used in the lab o Examples: ______________: Entails freezing a material at _________or less ▪ ______________ – most common; gives both visible and then subjecting it to a high vacuum. Very low temperatures and infrared region cause the ice to sublimate to a vapor state. The solid non- ▪ ______________ – provide UV radiation sublimable material remains behind in a dried state. ▪ ______________ discharge lamps – produce continuous source of radiation which covers both UV and ANALYTICAL METHODS visible region  ______________ – transmitted via electromagnetic b. ______________ – emits limited radiation and wavelength waves that are characterized by their frequency and o Used in atomic absorption, molecular and fluorescent wavelength. spectroscopy o LASER is also used as light sources for spectroscopy  ______________ – distance between 2 successive peaks and expressed in nanometer (nm). o Examples: Mercury and sodium vapor lamps (UV and visible regions), _____________________(AAS) o ______________ – visible spectrum o 700nm – ______________ 2. Spectral distribution within the 3. range COLORIMETRY 4. The source of radiant production Photoelectric colorimetry 5. Stability of the radiant energy  The primary analytical utility of ______________ or 6. Temperature filter photometry is the isolation of discreet portions of Alternative Light Source: spectrum for purposes of measurement. 1. Mercury arc (visible and UV) a) Spectrophotometric measurement = measurement of light 2. Deuterium lamp (______________) intensity in a ______________ WAVELENGTH. 3. Hydrogen lamp (UV) b) ______________ measurement = measurement of 4. Xenon lamp (UV) ______________ without consideration of wavelength 5. Merst glower (IR) Spectrophotometry: 6. Globar (IR) Measures the light transmitted/ radiant energy (electromagnetic 2. Entrance Slit radiation) absorbed by a solution to determine the concentration Minimizes the unwanted or ______________ to prevent the of the substance in the solution. entrance of scattered light into the monochromator system Types of Spectrophotometers Stray light 1) Single-beam o Any wavelength outside the band transmitted by the monochromator 1 CLINICAL CHEMISTRY 1 o Does not originate from the polychromatic light source 12. Photomultiplier tube o Causes absorbance error MOST COMMON type which measures ______________ o Limits the maximum absorbance of the ______________ spectrophotometer can achieve With excellent sensitivity and has a rapid response. o Most ______________ ______________ at high- Detects low levels of light analyte concentration. Should never expose to room light because it will burn out. 3. Monochromator 13. Photoiodide Isolates specific or individual wavelength of light. Not as sensitive as PM but with excellent linearity 4. Prisms Measures light at a multitude of wavelengths Wedge-shaped pieces of glass, quartz, or sodium chloride Detects less amount of light Can be rotated which allows only the desired wavelength to With lower dynamic range and higher noise compared to PM pass through an exit slit. tube 5. Diffraction Gratings Most useful as a simultaneously multichannel detector. Most used and better resolution than prism. Meter or ______________ Made by cutting grooves or slits into an aluminized surface of a Displays output of the detection system. flat piece of crown glass. Examples: galvanometer, ammeter, LED display 6. Filters Simple, least expensive, not precise but useful. Refers to the range of wavelength passing through the Made by placing a semi-transparent silver films on both sides of sample: ______________ a dielectric such as magnesium fluoride. o ______________ ______________ Produce monochromatic light based on the principle of ______________ constructive interference of waves. Defined as the proportion of incident light that is 7. Holographic gratings transmitted and is usually expressed as percentage: % A type of diffraction grating formed by an interference-fringe transmittance field of two laser beams whose standing-wave pattern is exposed Electromagnetic radiation is described as photons of energy to a polished substrate coated with photoresist. travelling waves described by: ______________ 8. Exit Slit Controls the width of light beam (bandpass). Wavelength (nm) Light o ______________ – also known as “width of light” → ______________ Red total range of wavelength transmitted ______________ Orange ______________ ______________ ______________ ______________ Yellow Accurate absorbance measurement requires a bandpass Design ______________ (TC) – DO NOT deliver the same volume PATIENT FACTORS THAT AFFECT TESTING when the liquid is transferred into a container ______________ (TD) – deliver the EXACT amount it holds; DIURNAL VARIATION  Cortisol: peaks at 4am to SHOULD NOT BE SHAKEN OR HIT AGAINST THE WALL of the 6am;lowest 8pm to container during draining. 12am  50% lower at 8PM than >Drainage Characteristics at 8am; increased with ______________ – a continuous ETCHED RING or two small, stress close, continuous rings located near the top of the pipet.  Best time to collect: ______________ – users allow the contents of the pipet to higher in the morning DRAIN BY GRAVITY. ACTH: lower at night; >Type increased with stress Measuring or graduated – capable of dispensing several Plasma renin activity: lower different volumes at night; higher standing than o ______________ – WITH GRADUATION MARKS to the tip and supine is a BLOWOUT pipet Aldosterone and insulin: o ______________ – WITHOUT GRADUATION MARKS to the tip lower at night and is a SELFDRAINING pipet. ▪The tip should not be allowed to touch the vessel GH and ACP: high in the while the pipet is draining. afternoon and evening ▪Micropipette – with a total holding volume of less Thyroxine: increased with than 1mL. exercise Prolactin: higher with stress, Transfer pipet – designed to dispense one volume without higher levels at 4 and 8am further subdivisions, bulblike enlargement in the pipet stem and 8 to 10 pm easily distinguishes the ______________ and ______________. Iron: peaks early to late o ______________ – dispense or transfer AQUEOUS SOLUTIONS morning, decreased up to and is SELFDRAINING 30% during the day  used for non-viscous fluids; has the greatest degree Serum iron is drawn in accuracy and precision successive days to avoid o ______________ – used with BIOLOGIC FLUIDS having a diurnal variation viscosity greater than water and is a BLOWOUT pipet. Calcium: 4% decreased in o ______________ – used to transfer solutions or biologic fluids supine without consideration of a specific volume. STRESS Increased ACTH, catecholamines, cortisol, Automatic pipet – most routinely used pipe in today’s clinical albumin, insulin, prolactin, laboratory lactate o Fixed volume – associated with only one volume Decreased HDL and o Variable – able to select different volumes however, only one respiratory alkalosis volume may be used at a time. POSTURE Increased albumin, o Fully automated/ self-operating, semiautomatic, completely cholesterol and calcium while manually operated standing General types: Decreased lipids due to 1. ______________ hemodilution Relies on a piston for suction creation to draw the sample 4 CLINICAL CHEMISTRY 1 ALCOHOL Decreased glucose; increase Objectives Tg and GGT Check the ____________________________ RECENT FOOD INTAKE Increase glucose, insulin, Check the ____________________________ gastrin, Tg, sodium, uric acid, Check ____________________________ iron, LD, Ca2+ Dcreased chloride, Characteristics of an Ideal QC Material phosphate, potassium 1) Resembles human sample DAY TO DAY VARIATION Bilirubin, CK, steroid 2) Inexpensive and stable for long periods hormones, Tg 3) No communicable disease. FASTING >72 hours: Increase plasma 4) No matrix effects/known matrix effects. Tg decreased glucose 5) With known analyte concentrations. >42 hours: Increased bilirubin 6) Convenient packaging for easy dispensing and storage. VARIATIONS / TYPES OF ERRORS QUALITY CONTROL Random Error A system of ensuring accuracy and precision in the laboratory Non-repeating, occurs once by reagents (quality control) in every series of measurement The basis for varying differences between repeated Process of ensuring that analytical results are correct by testing measurement. known samples (control solution) that resembles patient It is due to instrument, operator, and environmental conditions samples. o Pipetting errors, mislabeling of samples, temperature Involves the process of monitoring the characteristics of the fluctuations, improper mixing of sample and reagent. analytical processes and detects analytical errors during testing > Affects the reproducibility or precision. It is one component of the quality assurance system. ______________ ______________ Repeating, can be predicted Often related to calibration problems, deterioration of reagents Terms to Remember and control materials, unstable and inadequate reagent blanks, 1) ______________ – ability of an analytical method to measure contaminated solutions, failing instrumentation and poorly the ______________ CONCENTRATION of the analyte of written procedures. interest. It is a measure of the agreement between the measured 2) ______________ – ability of an analytical method to measure quantity and the true value. ONLY the ______________ ______________. > Affects the ______________. 3______________ – NEARNESS OR CLOSENESS of the assayed ______________ ______________ value to the true or target value. Refers to a difference between the target value and the assay 4______________ or Reproducibility – ability to give REPEATED value. RESULTS on the same sample that agree with one another. Independent of sample concentration. 5) ______________ – ability of analytical method to maintain Proportional/ Slope/ Percent Error accuracy and precision over extended period of time during Results in greater deviation from the target value due to higher which equipment, reagents and personnel change. sample concentration. 6) Diagnostic Sensitivity Clerical Error Ability of the test to detect proportion of individual with that The highest frequency of clerical errors with the use of disease who test positively with the test. handwritten labels and request forms. Indicates the ability of the test to generate more true-positive results and few false-negative. STAGES OF ERROR 7) Diagnostic Specificity PRE-ANALYTICAL POST ANALYTICAL Ability of the test to detect proportion of individual with that Improper patient Unavailable or delayed disease who test negatively with the test. preparation laboratory results. It reflects the ability of the method to detect true negatives Mislabeled specimen Incomplete laboratory with very few false-positive Incorrect order of draw results. Kinds of Quality Control Incorrect patient Wrong transcription of the 1) Intralab (Internal QC) identification patient’s data and laboratory Involves the analysis of control samples together with patient Wrong specimen container results. specimens. Incorrect anticoagulant to blood ratio. > Routinely done in the laboratory. Improper mixing of sample Important for the daily monitoring of accuracy and precision of and additives analytical methods. Incorrect specimen Detects both ______________ _____________. preservation 2) Interlab (External QC) Incorrect used of tubes for Involves proficiency testing programs that periodically provide blood collection. samples of unknown concentration of analytes to participating Mishandled specimen laboratories. (transport and storage) Important in maintaining ______________ ______________ Missed or incorrectly of the analytical method. interpreted laboratory Difference of >2 in the results indicates that a laboratory is not requests. in agreement with the rest of the laboratories included in the program. 5 CLINICAL CHEMISTRY 1 STATISTICS Is the science of gathering, analyzing, interpreting, and presenting data. 1) ______________ / Average A measure of central tendency. It is associated with symmetrical or normal distribution 2) ______________ ______________ A measure of the dispersion of values from the mean. It helps describe the normal curve. Errors observed using LJ Chart A measure of the distribution range. a) ______________ > Inversely proportional to precision. Formed by control values that either increase or decrease for 3) ______________ ______________ six consecutive days. A percentile expression of the mean Main cause: ______________ ______________ Index of precision b) ______________ Also known as ______________ ______________ Formed by control values that distribute themselves on one 4) ______________ side or either side of the mean for six consecutive days. Is called the standard deviation squared Shift in the reference range is due to transient instrument Measure of variability differences. > Detects significant differences among groups of data. Main cause: ______________ ______________ c) ______________ Terminologies Are control values that are far from the main set of values 1) ______________ They are highly deviating values Value of the observation that divides the observations into two Cause by ______________ ______________. groups, each containing equal numbers of observation. NOTES: It is a midpoint of distribution ______________ confidence limit (+/- 2SD) – acceptable range. ______________ ______________ -refers to the degree of flatness or sharpness 2______________ – is the most ______________ observation in the peak of a set values having a gaussian distribution. 3) Inferential Statistics – used to COMPARE the MEANS OR STANDARD DEVIATIONS of two groups of data. WESTGARD RULES 4) ______________ – used to determine whether there is CONTROL RULES statistically significant difference between the means of two It recognized that the use of simple upper and lower control groups of data. limits are not enough to identify analytical problems. 5) ______________ – determine whether there is statistically Westgard used the term control rule to indicate if the analytical significant difference between the standard deviations of two process is out of control. groups of data. Control Rules UALITY CONTROL CHART a) ______________ – used as ______________ when one ______________ ______________ control result exceeds the mean +/- 2SD: for screening purposes. ______________ curve b) 13s – rejects a run when one control result exceeds the mean it occurs when the data set can be accurately described by the +/- 3SD. SD and mean c) 22s – rejects a run when the last 2 control results (or 2 results it is obtained by plotting the values from multiple analysis of a sample. from the same run) exceed the mean +/- 2SD. It occurs when the data elements are centered around the d) 41s – rejects a run when the last four (or any four) consecutive mean with most elements close to the mean. control results exceed either mean +/- 1SD. It focuses on the distribution of errors from the analytical e) R4s – rejects a run if the range or difference between the method rather than the values from a healthy or patient highest and lowest control result within an analytical run exceeds population. 4SD. f) 10x – rejects a run when ten consecutive results are on the same side of the target mean. ______________ Levey-Jennings Chart (LJ Chart) Most widely used system in clinical laboratory Allows the laboratorians to apply multiple rules without the aid of a computer A graphic representation of the acceptable limits of variation in the results of an analytical method. 6 CLINICAL CHEMISTRY 1 1) _______________ – as et of control and patient specimens assayed, evaluated, and reported together. 2) ______________ Is the most used patient based-QC technique It requires computerization of test data The difference between two consecutive measurements of the same analytes on the same individual. > Check for the PREVIOUS RESULT. 3) Point of care testing (POCT) Analytical testing performed outside the confines of the central laboratory, usually by non laboratorians personnel Use of portable whole blood glucose meters for the management of patients with diabetes – most used POCT Other names: near-patient testing, decentralized testing, bedside testing, alternate site testing 4) Reference limit/ Reference Interval/ Reference value A value obtained by observation or measurement of a particular type of quantity on a reference individual. Usual values for a healthy population that represents 95% central tendency. Five factors when establishing reference intervals: 1. The composition of reference population. 2. The criteria of excluding and including individuals from the reference population. 3. The physiologic and environmental conditions of the reference population. 4. Specimen collection procedure, preparation for testing 5. Analytical method used. 5) ______________ - comparison of an instrument measurement or reading to a known physical constant 6) ______________ - highly purified substances with known concentration Laboratory Computations 1. Equivalent weight 𝐴𝑡𝑜𝑚𝑖𝑐 𝑤𝑒𝑖𝑔ℎ𝑡 𝑉𝑎𝑙𝑒𝑛𝑐𝑒 2. Milliequivalent weight 𝐴𝑡𝑜𝑚𝑖𝑐 𝑤𝑒𝑖𝑔ℎ𝑡 𝑉𝑎𝑙𝑒𝑛𝑐𝑒 𝑥 1000 3. Mole 𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 4. Millimole 𝑚𝑖𝑙𝑙𝑖𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 5. MG% to MEQ/L 𝑚𝑔% 𝑥 10 𝑥𝑣𝑎𝑙𝑒𝑛𝑐𝑒 𝑎𝑡𝑜𝑚𝑖𝑐 𝑤𝑒𝑖𝑔ℎ𝑡 6. MEQ/L to MG% 𝑚𝐸𝑞 𝑥 𝑎𝑡𝑜𝑚𝑖𝑐 𝑤𝑒𝑖𝑔ℎ𝑡 10 𝑥 𝑣𝑎𝑙𝑒𝑛𝑐𝑒 7. PERCENT SOLUTIONS 𝑤 𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑒 % = 𝑥 100 𝑤 𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑤 𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑒 % = 𝑥 100 𝑣 𝑚𝑙 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑣 𝑚𝑙 𝑠𝑜𝑙𝑢𝑡𝑒 % = 𝑥 100 𝑣 𝑚𝑙 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 8. NORMALITY 𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑒𝑞. 𝑤𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛) N=Valence x Molarity 9. Molarity 𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑀𝑊 𝑠𝑜𝑙𝑢𝑡𝑒 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛) M=N/V TERMINOLOGIES 10. Molality 𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑀𝑊 𝑠𝑜𝑙𝑢𝑡𝑒 𝑥 𝑘𝑔 𝑠𝑜𝑙𝑣𝑒𝑛𝑡) 7 CLINICAL CHEMISTRY 1 11. Dilution 𝑣𝑜𝑙𝑢𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 Oligosaccharides >2 to 140 mg/dl, proceed to Step 2. If not, STOP.  Peritoneal fluid glucose is the same as plasma glucose  STEP 2  Whole blood gives approximately 10-15% LOWER 4. Collect fasting plasma (8 hour). glucose levels than serum or plasma.  Venous blood glucose is 7mg/dL LOWER than 5. Give 100 gram glucose. capillary blood glucose due to tissue metabolism 6. Collect plasma after 1, 2, and 3 hr.  Capillary blood glucose is the same with arterial blood glucose. INTRAVENOUS GLUCOSE TOLERANCE TEST  Glucose is metabolized at room temperature at a  Used for diabetics with gastrointestinal (GIT) disorders rate of ______________.  0.5 g of glucose/kg body weight (given within 3 minutes) is administered INTRAVENOUSLY  At 4°C, glucose decreases by approximately  Collect blood after 5 minutes of IV glucose ______________. administration  The rate of metabolism is higher with bacterial  Indications for the use of IVGTT: contamination or leukocytosis.  Unable to tolerate large carbohydrate in the diet  In serum specimens without bacterial contamination  Presence of altered gastric physiology or leukocytosis, results are clinically acceptable for up  Previous operation or surgery of the gastrointestinal to 90 minutes before separation of serum from cells. tract  Presence of chronic malabsorption syndrome  Direct methods for measuring β- hydroxybutyrate are now replacing the strips and tablets for urine TESTS INVOLVED ketone testing. Specimens for carbohydrate analysis:  Because β-hydroxybutyrate levels are high in  Whole blood diabetic ketoacidosis (DKA) and fall with treatment, whereas acetoacetic acid and acetone levels rise  Serous fluid with treatment, urinary ketone strips are not useful 10 CLINICAL CHEMISTRY 1 for monitoring therapy. o After PFF preparation, non- glucose reducing substances are adsorbed by barium sulfate  Calculation of the anion gap is employed instead to c. NEOCUPROINE METHOD monitor recovery from DKA. METHODOLOGIES CHEMICAL METHODS (oxidation- reduction d. BENEDICT’S METHOD methods) o Modification of Folin Wu o Used to detect and quantify reducing 1. Copper reduction substances in body fluids 2. Folin Wu Method o Uses CITRATE and TARTRATE as stabilizing agent o Negative result: BLUE 3. Nelson Somogyi o Positive result: GREEN→YELLOW→BRICK RED PPT 4. Neocuproine method 5. Benedict’s method e. CLINITEST TABLET 6. Clinitest Method o Detects ALL reducing sugars 7. Ferric reduction (Hagedorn- Jensen) o uses URINE as sample o Watch out for “______________” 8. Condensation method (Ortho- phenomenon Toluidine)  occurs when glucose level is VERY HIGH  Remedy: use TWO instead of five ENZYMATIC METHODS drops 1. Glucose Oxidase CLINITEST: BENEDICT'S: 2. Hexokinase-G6PD Copper sulfate Copper sulfate Sodium citrate Sodium carbonate ISOTOPE DILUTION GAS Sodium hydroxide Sodium citrate CHROMATOGRAPHY/MASS SPECTROMETRY Sodium carbonate CHEMICAL METHODS 1. COPPER REDUCTION METHOD 2. FERRIC REDUCTION  OLDEST METHOD  Principle: Glucose and other reducing sugars  a.k.a. ______________ ______________ METHOD convert cupric to cuprous ions in the presence of heat  Principle: INVERSE COLORIMETRY and alkali  Reduction of yellow ferricyanide to colorless ferrocyanide by reducing sugars  Disappearance of color is measured at 400 nm  Employed in AUTOANALYZERS a. FOLIN WU METHOD o SENSITIVE but not specific o MAJOR DISADVANTAGE: o non-glucose reducing substance also reacts with the tests 3. CONDENSATION METHOD  Principle: The aldehyde group of glucose condenses with aromatic amines in hot acetic acid solution to form colored derivatives b. NELSON SOMOGYI METHOD  ORTHO-TOLUIDINE METHOD o a.k.a. ______________ METHOD o SENSITIVE and SPECIFIC o MOST SPECIFIC NON- ENZYMATIC METHOD for o glucose measurement o MAJOR DISADVANTAGES:  carcinogenic and teratogenic 11 CLINICAL CHEMISTRY 1 formalin, hemoglobin, tetracycline, l- cysteine, l-dopa, dopamine, methyldopa, and citric acid can cause FALSELY DECREASED results o Presence of bleach and detergents, can  Measures only GLUCOSE and not other reducing ENZYMATIC METHODS sugars cause FALSELY INCREASED results  Three enzyme systems are commonly used to  Polarographic Glucose Oxidase Method measure glucose: 1. Glucose Dehydrogenase 2. Glucose Oxidase a. Colorimetric b. Polarographic 3. Hexokinase NOTES TO REMEMBER:  Glucose exists either as alpha- glucose or beta- glucose. o Oxygen depletion is measured and is o Alpha-glucose = 35% proportional to the amount of glucose o Beta-glucose = 65% present. o Alpha glucose is converted into beta o H2O2 is prevented from re-forming O2 glucose using the enzyme by adding molybdate, iodide, catalase ______________. and ethanol 1. GLUCOSE DEHYDROGENASE METHOD NOTE:  Glucose is measured spectrophotometrically or via a  Glucose oxidase is not the reference method change in electrical current  Glucose oxidase is very specific, it only measures beta- glucose. A. Spectrophotometric  Glucose oxidase is affected by reducing and oxidizing agents. 3. HEXOKINASE METHOD  REFERENCE METHOD/ GOLD STANDARD TEST  MOST COMMONLY USED METHOD to determine serum glucose levels 2. GLUCOSE OXIDASE METHOD  Measures both alpha & beta D-glucose  Glucose concentration is proportional to the rate of  Most specific enzyme reacting with only ß -D- production of nicotinamide adenine dinucleotide glucose phosphate (NADPH)  Colorimetric Glucose Oxidase Method  Advantages o More accurate than the glucose oxidase mtd  Not affected by ascorbic acid or o a.k.a. ______________ ______________ uric acid Method o May be performed on serum or plasma o The coupled reaction involved in glucose collected using heparin, oxidase method is known as EDTA, fluoride, oxalate, or citrate ______________ ______________ o High concentrations of uric acid, ascorbic o Other samples may be used: urine, CSF, acid, bilirubin, glutathione, creatinine, and serous fluids 12 CLINICAL CHEMISTRY 1  Disadvantages patients. o Gross hemolysis and icterisia may cause a FALSE  Procedure DECREASE in results. o Patient must eat a complete  NONSPECIFIC- it reacts with any meal (75 grams of glucose) sugars with six carbon units OR a solution containing 75 (fructose, galactose, glucose) g of glucose is administered o RBCs contain glucose-6- o Specimen for plasma glucose phosphate and measurement is drawn 2 intracellular enzymes that hours later generate NADH causing  Reference value must be POSITIVE INTERFERENCE. 200 mg/dL and is samples require a confirmed on a subsequent day by either an increased random or serum blank fasting glucose level, the patient is correction diagnosed with diabetes (subtraction of the reaction rate with 4. Glucose Tolerance Test (GTT) hexokinase  Multiple blood and urine glucose test omitted from the  Also referred to as CHALLENGE TEST reagent) LABORATORY DETERMINATION FOR GLUCOSE  Used to diagnose Gestational Diabetes Mellitus  used to determine how well the body 1. Random Blood Glucose (RBG) metabolizes glucose over a required  MONITORING TEST for blood glucose period of time  For determination of glucose  Based on the principle that, a normal at anytime of the day individual when given a glucose  Requested during: challenge is capable of converting it to glycogen (for storage) and blood a. Insulin Shock glucose returns to normal after 2 hours. b. Hyperglycemic Ketonic Coma  Diabetic patients will remove glucose c. EMERGENCY CASES from the circulation at a slower rate due to insulin deficiency 2. Fasting Blood Glucose (FBG)  SCREENING TEST for serum glucose in 5. GLYCOSYLATED HEMOGLOBIN (GLYCATED the diagnosis of Diabetes Mellitus HEMOGLOBIN) (HbA1C)  NPO (non-per-orem) for 8-10 hours  Now the PREFERRED TEST FOR THE ASSESSMENT OF GLYCEMIC CONTROL 3. 2-Hour Post Prandial Blood Glucose  for monitoring of LONG TERM GLUCOSE  Measures how well the body CONTROL (______________) metabolizes glucose o Sample of choice:  Based on the principle that glucose level n blood drawn 2 hours after a o non fasting meal returns to normal in normal WHOLE BLOOD drawn in EDTA individuals while tube remains significantly increased in diabetic  Reference range: ______________ 13 CLINICAL CHEMISTRY 1  Results > 6.5% on more than two  WHOLE BLOOD CAPILLARY GLUCOSE occasions is indicative of Diabetes  Uses a GLUCOMETER Mellitus  Hematocrit affects POCT  For every 1% change in HbA1C value, glucose measurements. 35 mg/dL is added to plasma glucose level o High hematocrit = lower glucose  Disadvantage: Falsely low values observed o RBC glucose in hemolytic anemias (sickle cell, concentration is lower hemoglobinopathies, etc.) than plasma concentration 6. FRUCTOSAMINE (GLYCATED ALBUMIN/ GLYCOSYLATED ALBUMIN)  NOT used to diagnose diabetes or hypoglycemic  MOST WIDELY USED TO ASSESS disorders ______________ GLYCEMIC CONTROL  For confirmation, laboratory measures  Used in monitoring glucose level at a of plasma glucose are required because shorter time interval (______________) of higher accuracy INTERPRETATION OF RESULTS INBORN ERROR OF METABOLISM 1. FOR FASTING BLOOD GLUCOSE 1. Galactosemia Non Diabetic: < 100mg/dL  Congenital deficiency of one of the three enzymes involved in Impaired FBS: > 100mg/dl but < 126 mg/dL the conversion of galactose into Diabetes Mellitus: > 126 mg/dL glucose. 2. FOR ORAL GLUCOSE TOLERANCE TEST o Galactose-1-phosphate Normal OGTT (2hr Glucose): < 140 mg/dL uridyl transferase Impaired OGTT (2hr Glucose): 140-199 mg/dL o Galactokinase Diabetes Mellitus (2hr Glucose): > 200 mg/dL o Uridine diphosphate galactose 4-epimerase  DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS  Since galactose cannot be o RBG: > 200 mg/dL with symptoms of converted into glucose, in DM accumulates in the blood and o FBG: > 126 mg/dL urine o 2-hr Post Prandial: > 200 mg/dL 2. Essential fructosuria o HbA1c : > 6.5%  a genetic disorder charaterized by accumulation of fructose in blood and urine due to lack of fructokinase MISCELLANEOUS TEST FOR GLUCOSE  Anion Gap - used to monitor recovery from DKA 3. Hereditary fructose intolerance  C-peptide - used to identify the cause of hypoglycemia  inborn error of fructose metabolism  insulinoma = ↑ C-peptide and ↑ insulin caused by a deficiency of the enzyme  injected or exogenous insulin = ↓ C- aldolase B peptide and ↑ insulin 4. Fructose-1,6-biphosphate deficiency POINT OF CARE TESTING 5. Glycogen storage diseases (GSD)  Due to inherited deficiencies of  Specimen: enzymes that control the synthesis or breakdown of glycogen 14 CLINICAL CHEMISTRY 1  Glycogen storage diseases that primarily affect the liver usually manifest with hypoglycemia and hepatomegaly, whereas those affecting muscle commonly cause muscle cramps, weakness, fatigue, and exercise intolerance. TYPES OF GSD SYNONYMS ENZYME DEFICIENCY ______________ la Glucose-6-Phosphatase ______________ II 1,4- Glucosidase ______________ Illa Amylo-1,6-glucosidase ______________ IV Glycogen branching enzyme ______________ V Muscle Phosphorylase ______________ VI Liver Phosphorylase ______________ VII Phosphofructokinase ______________ XI Glucose transporter-2 15