SEM01: Clinical Chemistry PDF

SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 QUALITY CONTROL Why do Quality Control: is a system of ensuring accuracy and precision 1. Validate the reliabil...

SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 QUALITY CONTROL Why do Quality Control: is a system of ensuring accuracy and precision 1. Validate the reliability of the test system. in the laboratory by including quality control a. Run after calibration of instrument reagents in every series of measurements. b. Run before and periodically during testing a process of ensuring that analytical results are 2. Evaluate condition of instruments and reagents correct by testing known samples that resemble 3. Evaluate the operator’s performance patient samples. 4. Evaluate environmental conditions that might involves the process of monitoring analytical impact the results. processes and detects analytical errors. it is a testing design to assess the “health” of an Characteristics of an Ideal QC Material: analytical method. 1. It resembles human sample. 2. Inexpensive and stable for long periods. PARAMETERS OF QUALITY CONTROL: 3. No communicable diseases. 1. Sensitivity 4. No matrix effects. 2. Specificity 5. With known analyte concentration. 3. Accuracy 6. Convenient packaging for easy dispensing and 4. Precision or reproducibility storage. 5. Practicability 7. Can be liquid or freeze - dried (lyophilized) 6. Reliability 7. Diagnostic sensitivity Types of Control: 8. Diagnostic specificity BUILT-IN CONTROLS – integrated to test kits KINDS OF QUALITY CONTROL: – usually used as a procedural control o confirms that the diluent has been 1. Intralab (Internal QC) applied successfully involves analyses of control samples together o confirms that the active ingredients in with the patient specimens. the strip are functional it is important for the daily monitoring of o not a confirmation that the sample has accuracy and precision of analytical methods. been applied – assesses the validity/performance of the kit, but detects both random and systematic errors. not the entire testing process 2. Interlab (External QC) TRADITIONAL CONTROLS it involves proficiency testing program which – mimics patient’s samples; known reactivity provides samples if unknown concentrations of – assesses integrity of the entire testing process analytes to participating laboratories. it is important in maintaining long-term accuracy TYPES: of the analytical methods. a. INTERNAL CONTROLS- comes with the kit; intended for kit use Sources of Control b. EXTERNAL CONTROLS Human-based Control - commercially prepared Bovine-based Control - In-house- testing documents must be kept as proof (validation and testing steps) Objectives of Quality Control: 1. To check the stability of the machine. Plotting a Quantitative Control Chart 2. To check the quality of reagents. - obtain control material 3. To check technical errors. -run each control 20 times over 30 days -calculate the mean and +/- 1 up to +/- 3 SD Characteristics of an Ideal QC Material: 1. It resembles human sample. Standard Deviation 2. Inexpensive and stable for long periods. - principle measure of variability used in 3. No communicable diseases. laboratory 4. No matrix effects. 5. With known analyte concentration. 6. Convenient packaging for easy dispensing and storage. 1|D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 a. Improper calibration b. Deterioration of reagents c. Contaminated solutions d. Sample instability/unstable reagent blanks e. Changes in standard materials f. Leaky ion selective electrode g. Diminishing lamp power Coefficient of Variation h. Incorrect instrument setting - is the SD expressed as a percentage of the i. Incorrect sample and reagent volume mean j. Poorly written procedures o CV is used to monitor precision o CV is used to comapare methods a. CONSTANT ERROR o CV ideally should be less than 5% - refers to a difference between the target value and the assay value. b. PROPORTIONAL/SLOPE/PERCENT ERROR - results to greater deviation from the target value due to higher sample concentration. It can be a SHIFT or TREND. 1. Levey- Jennings Chart - Graphical representation of data control points INTERPRETATION OF QUALITY CONTROL 1. TREND 2. SHIFT 3. WESTGARD MULTI-RULES a) Warning Rules b) Reject Rules (out-of-control) 4. OUTLIERS Statistics - is the science of gathering, analyzing, interpreting and presenting data. 1. Mean Analytical Variations/ Errors 2. Median 3. Mode 1. SYSTEMATIC ERROR 4. Standard deviation - error that occurs predictably once a pattern of 5. Coefficient of variation recognition has been established. 6. Variance 2. RANDOM/ INDETERMINATE ERROR 2. Cumulative Sum Graph (Cusum) - errors that occurs unpredictably with a basis of This plot will give the earliest indication of imprecision which results in varying differences between systematic errors (trend) and can be used with repeated measurements. the 13s rule. V-mask method – common method; requires Variations computer implementation Types: Very sensitive to small, persistent errors that 1. RANDOM ERROR: commonly occur in the modern, low calibration- a. Mislabeling frequency analyzer. b. Pipetting errors c. Improper mixing of sample and reagents 3. Youden Plot d. Voltage fluctuations Use to compare results obtained on a high and e. Temperature fluctuations low control serum from different laboratories. f. Dirty optics 2. SYSTEMATIC ERROR: 2|D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 TREND: Quality Control Chart 1. Gaussian Curve (Bell-Shaped Curve) Occurs when the data set can be accurately described by the SD and the mean. Obtained by plotting the values from multiple analyses of a sample. Occurs when data elements are centered around the mean with most elements close to the mean. A shift in the mean is an accuracy problem; an increase in SD is a precision problem. 5. Westgard Control Rules 4. Shewhart Levey-Jennings Chart Recognized that the use of simple and upper Also known as the Dot Chart lower control limits is not enough to identify Most widely used system in clinical lab. analytical problems. Allow the laboratorians to apply multiple rules Error detection rates can increase without without the aid of a computer. increasing the false rejection rate. Detects both random and systematic errors. CONTROL RULES: a. Trend – indicates a gradual loss of reliability in 12s - use as rejection or warning rule when one the rest system. Control moving in one direction 6 control result exceeds the mean ± 2SD; for consecutive times, heading towards an “out of control” screening purposes. value. 13s - reject a run when one control result exceeds the mean ± 3SD; determines CAUSES: random error. Deterioration of reagents 22s - reject a run when the last 2 Deterioration of the instrument light source control results (or 2 results from the same run) Gradual accumulation of debris in the exceed either the mean ± 2SD; determines sample/reagent tubing or on electrode surfaces. systematic errors. 41s - reject a run when the last four (or b. Shift – represents a sudden change in the test any four) consecutive control results exceed performance. Controls on one side of the mean 6 either mean ± 1SD; determines systematic errors. consecutive times. R4s - reject a run if the range or difference Causes: between the highest and lowest control result Inaccurate calibration/recalibration within an analytical run exceeds 4SD. Change in reagent formulation/reagent lot 10x - reject a run when 10 consecutive Major instrument maintenance results are on the same side of the target mean; Failure in the sampling system/reagent dispense systematic error. system TERMINOLOGIES: c. Outlier – highly deviating values 1. DELTA CHECK Is the most commonly used patient based-QC SHIFT: technique. It requires computerization of test data so that current results can be compared with past results. 2. INTERFERENCE EXPERIMENTS Are used to measure systematic errors or inaccuracy caused by substances other than the analytes - lipids, bilirubin, hemoglobin 3. RECOVERY EXPERIMENTS 3|D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 Will show whether a method measures all the Beer’s Law analytes or only part of it – an estimate of It states that the concentration of the unknown systematic error. substance is directly proportional to the absorbed light and inversely proportional to the 4. POINT OF CARE TESTING amount of transmitted light. (POCT)/DECENTRALIZED TESTING FORMULA: Analytical testing performed outside the lab, 1. A = abc usually by nonlaboratorian personnel – blood 2. A = 2 – log% T glucose meters. 3. Au/As X Cs 5. PHYSIOLOGIC LIMIT (ADSURD VALUE) Parts of the Spectrophotometer Helps detect sample and volume error, method 2. ENTRANCE SLIT problem, or incorrect recording or transmission Minimizes stray light and prevents the entrance of results of scattered light into the monochromator system ANALYTICAL TECHNIQUES Stray light refers to any wavelength outside the AND INSTRUMENTATION band transmitted by the monochromator, which is the most common cause of loss linearity at A. Photometry/Spectrophotometry high analyte concentration Can be detected through the use of glass-cut 1. Single-beam spectrophotometer filters or solutions such as sodium nitrite, sodium Simplest type of absorption spectrophotometer bromide, acetone and methylene blue. Used to make one measurement at a time at one specified wavelength 3. MONOCHROMATOR 2. Double-beam spectrophotometer Isolates a specific wavelength of interest from Designed to compensate for possible variations the light source. in intensity of the light source by splitting the light beam from the lamp and directing one a. Interference filter portion to a reference cuvet and the other to the o Simple, least expensive, not precise but sample cuvet. useful o Based on constructive interference of Parts of the Spectrophotometer waves 1. LIGHT o Sharp cut of filter – isolates stray light Provides incident light for the system b. Prisms Spectrum of Light – measured in wavelength o Wedge-shaped made up of either Wavelength – refers to the distance between the aluminum, glass, sodium chloride or peaks of a light wave expressed in nanometers quartz a. 700nm c. Diffraction gratings o Most commonly used 2 types: o Bends light and form wave fronts. Lights a. Continuum source- emits radiation that that are in phase reinforce one another, changes in intensity if not it cancels out. b. Line source- emits limited radiation and wavelength 4. Exit slit Controls the width of bandpass – total range of Sources of Light: wavelengths transmitted. a) Xenon discharge lamp and Mercury arc lamp – UV and visible region 5. Sample Cell b) Deuterium and Hydrogen lamp – UV region Also known as cuvet/analytical cell, absorption c) Tungsten light bulb and tungsten iodide – cell Infrared and visible region Holds the solution of which the absorption is to d) Merst glower and globar (silicone carbide) – be measured Infrared region Should be free from scratches – it would scatter the light causing absorbance to decrease 4|D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 High alkali solution must not stay in cuvet longer than 30 minutes – alkali solution can dissolve b. Burner cuvet components leading to light scattering o A fuel gas (propane) with an oxidizing agent (compressed air) burned to produce the flame 6. Photodetector Converts transmitted radiant energy into an c. Monochromator system (filter) equivalent amount of electrical energy. o Allows only emitted light spectrum of specific element to strike the photodetector a. Photocell/Barrier layer cell/Photovoltaic cell/Selenide cell d. Photodetector: Photocell o Simpliest detector and least expensive o NO CUVET because the flame is the sample o Composed of selenium ion in silver holder o Generates electromotive force by electrons (no o Employs an internal standard – used to correct external voltage) interferences brought about by atomizer and o Output is not amplified (sufficient illumination) flame variations b. Phototube a. Lithium (IS) – red o Contains cathode and anode enclosed in a glass b. If lithium is measured, the IS is Cesium case c. Sodium – yellow o Similar to photocell but requires external d. Potassium – violet voltage. e. Calcium – orange f. Strontium –red c. Photomultiplier tube g. Copper II chloride, Arsenic, Antimony, Lead - o Most commonly used detector which detects blue and amplifies radiant energy h. Barium, Manganese (Manganese chloride) – o (200x sensitive) light green o Can detect smallest amount of light energy i. Copper, thalium, boron (boric acid) – green transmitted to the cuvet o Most sensitive and most rapid response to light Wavelength Calibration: energy transmission. 4. Atomic Absorption Spectrophotometry d. Phototransistors Components: o Uses a photosensitive negative-positive junction a. Light source – hallow cathode lamp diode to produce photocurrent b. Beam chopper – modulates the light source c. Nebulizer/atomizer – used to convert ion to Calibration of Spectrophotometer atoms d. Burner/cuvet A. SETTING TRANSMITTANCE AND e. Monochromator: Diffraction gratings ABSORBANCE LIMITS f. Photomultiplier tube 1. Zeroing – set transmittance to 0% g. Read-out device 2. Blanking – set transmittance to 100% o Advantages: More sensitive than FEP, B. Linearity Checks/Proportionality Check accurate, precise and very specific; does not – Determine the lowest and highest values that require an internal standard; reference method can be accurately measured by a particular method for calcium and magnesium – trace metals that – Use of neutral density filters. are not easily excited o Disadvantages: Inability of the flame to C. Wavelength Calibration: dissociate samples into free atoms 1. Holmium – 360 nm 2. Didymium – 600 nm B. LUMINESCENCE 3. Atomic Emission Spectrophotometry 1. Fluorometry Components: a. Light source – emits short wavelength high- Components: energy excitation light a. Nebulizer (Atomizer) o Mercury-vapor lamps – filter fluorometer o Delivers a fine spray of sample containing the o Xenon-arc lamps - spectrofluorometer metallic ion to the burner b. Attenuator – controls the light intensity 5|D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 c. Primary filter – selects the wavelength that is b. Nephelometry best absorbed by the sample d. Sample holder Principle: Measurement of the amount of light e. Secondary filter – passes longer wavelengths scattered in a particulate solution of fluorescent light, preventing incident light from striking Application: Measurement of antigen-antibody the photodetector reactions f. Photodetector: photomultiplier tube – measures fluorescence light and incidence light 4. LASER Applications (Light Amplification by o Advantages: 1000X more sensitive than stimulated emission of radiation) spectrophotometry o Disadvantages: Quenching interference – pH Principle: The interaction of radiant energy and and temperature changes, chemical suitably excited atoms or molecules leads to contaminants, UV light changes which reduces stimulated emission of radiation intensity of fluorescence Application: Differential analysis of WBC 2. Chemiluminescence Principle: C. ELECTROANALYTICAL TECHNIQUES Process of exciting molecules by chemical or electrochemical 1. Potentiometry means and measuring the light emitted as molecules return to their ground state Involves direct measurement of electrical Requires no excitation radiation and no potential (voltage) due to the activity of free ions monochromator needed – Follows the Nernst equation a. Involves oxidation reactions of luminol, acridinium ester and dioxetane a. Ion Selective Electrode (ISE) o Advantages: subpicomolar detection limits, o Concentration of ions in a solution is calculated speed, ease of use, simple instrumentation from the measured potential difference between o Application: Immunoassays the reference and indicator electrodes 3. Turbidity and Nephelometry Theories of Light a. Reference electrode Scatters: o Consists of a metal and its salt in contact with a 1. Rayleigh Theory – If the wavelength of light is solution containing the same anion much larger than the diameter of the particle, the light scatter is o Calomel (Hg/HgCl2) electrode – mercurous chloride in direct contact with metallic mercury in symmetric around the particle. an electrolyte solution of KCl 2. Mie Theory – If the wavelength of light is much o Ag/AgCl electrode – consists of a silver smaller than the diameter of the particle, the light electrode immersed in a solution of potassium scatters forward owing to destructive out-of-phase back chloride that has been saturated with silver scatter chloride 3. Rayleigh-Debye Theory – If the wavelength of light is approximately the same as the particle size, more light scatters in the forward direction than in other b. ISE Membranes a. Turbidimetry o Sodium – glass aluminum silicate o Potassium – Valinomycin gel o Calcium and lithium - Organic liquid membrane Principle: Measurement of the reduction in light ion exchanger transmission caused by particle formation or of the amount of light blocked by a suspension of 2. Amperometry particles Application: Measurement of the concentration Measurement of the current flow produced by an of antigen-antibody complexes, prealbumin and other serum proteins, detect bacterial growth in oxidation reaction broth cultures, antibiotic sensitivity from cultures Application: Used in the determination of pO2 and detection of clot formation (Clarke electrode) glucose and peroxidase. 6|D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 3. Cuolometry If the buffer is more acidic than the isoelectric point (pI) of the ampholyte, it binds H+ ions, Measurement of the amount of electricity in becomes positively charged, and migrates coulombs at a fixed potential toward the cathode Electrochemical titration in which the titrant is If the buffer is more basic than the pI, the electrochemically generated and the endpoint is ampholyte loses H+ ions, becomes negatively detected by amperometry charged, and migrates toward the anode Follows Faraday’s Law Charged particles migrate toward the opposite Application: Serum and sweat chloride analysis charged electrode 4. Voltammetry d. Sample Measurement of current after which a potential e. Detecting system is applied to an electrochemical cell Allows sample to be preconcentrated, thus Densitometer – a specialized colorimeter utilizing minimal analyte designed to measure the absorbance of the Anodic stripping voltammetry – for lead and iron stain on a support medium. testing Factors that affect the Rate of Migration: 5. Electrophoresis a. Net charge of the particle Migration of charged solutes or particles in an b. Size and shape of the particle electrical field c. Strength of the electric field Iontophoresis – migration of small charged d. Chemical and physical properties of particles the supporting medium Zone electrophoresis – migration of charged e. Electrophoretic temperature macromolecules The rate of migration is directly proportional to Procedure: the net charge of the particle and inversely proportional to its size and the viscosity of the I. The sample is soaked in hydrated buffer support for approximately 5 minutes II. The support is put into the Five Components: electrophoresis chamber, which was previously filled with the buffer a. Driving force (electrical power) III. Electrophoresis is carried by applying a constant voltage or constant current for a specific time b. Support Medium IV. The support is then removed and placed in a fixative or rapidly dried o Paper to prevent diffusion of the sample o Cellulose acetate V. This is followed by staining the o separates by molecular size zones with appropriate dye o Agarose gel VI. The uptake of dye b the sample is o Polyacrylamide gel proportional to sample concentration Involves separation of protein on the basis of charge and molecular size f. Isoelectric Focusing Separates serum proteins into 20 or more Charged proteins migrate through a support fractions rather than the usual 5 fractions medium that has a continuous gradient pH separated by cellulose acetate or agarose gradient is created by adding acid to the anodic Widely used to study isoenzymes area of the electrolyte cell and adding base on o Starch gel the cathode area. Separates proteins on the basis of Molecules move towards the medium at its surface charge and molecular size optimum pH. When pH is reached, the molecule c. Buffer loses its charge and so movement stops. Two buffer properties that affect the charge of Application: measurement of isoenzymes of ampholytes: pH and ionic strength ACP, CK and ALP and oligoclonal bands in CSF 7|D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 7. Capillary Electrophoresis Packed columns – larger specimen or sample capacity, useful in purification work Separation is performed in narrow-bore fuse silica capillaries d. Detectors Sample molecules are separated by electro- osmotic flow Flame ionization detector – widely used and Cations migrate fastest because both EOF and capable of detecting almost all organic electrophoretic attraction are toward the cathode compounds Neutral molecules are all carried by the EOF but Thermal conductivity are not separated from each other Anions move slowest because, although they e. Recorders are carried to the cathode by the EOF, they are attracted to the anode and repelled by the 2. Liquid Chromatography cathode Uses lower temperatures for separation, thereby D. Chromatography achieving better separation of thermolabile compound Techniques used to separate complex mixtures on the basis of different physical interactions Forms: between the individual compounds and the stationary phase of the system a. Paper Composition: Solvent move up through the paper by capillary action and the fractions move up at different a. Mobile phase (gas/liquid) – carries the complex rates mixture Sorbent (stationary phase) – Whatman paper b. Stationary phase (solid/liquid) – through which the b. Thin-layer Chromatography mobile phase flows The stationary phase is a thin layer sorbent c. Column holding the stationary phase (silica gel) coated on a glass plate or a plastic sheet d. Eluates (separated components) The mobile phase (solvent) is place in one edge of the plate which migrates up the thin layer 1. Gas Chromatography – Used to separate mixture of compounds that are naturally volatile or can easily be c. High-Performance Liquid Chromatography converted into a volatile form. (HPLC) Components: Uses pressure for fast separation of thermolabile substances like drugs and hormones a. Gas cylinder (mobile phase) Analysis times are usually shorter Reproducibility is greatly improved Must be chemically inert (helium, hydrogen, Application: Separation of amino acids, nitrogen) proteins and nucleic acids b. Sample injector Separation Techniques in Liquid Chromatography: Hypodermic syringe or automated sample a. Adsorption Each injection part is heated to very high temp Also known as liquid-solid chromatography c. Column Based on the competition between the sample and the mobile phase for adsorptive sites on the Capillary columns – generally have higher solid stationary phase (silica or alumina) efficiency and better detection 8|D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 b. Partition Laboratory Equipment and Reagent Preparation Also known as liquid-liquid chromatography A. Centrifuge Separation of solute is based on relative solubility between a liquid mobile phase and a Used to separate substances of different mass liquid stationary phase coated on a solid support or density Application: Separation of therapeutic drugs and metabolites 1. Horizontal head c. Steric/Size Exclusion Swinging bucket type; the centrifuge tubes are held in a vertical position when not moving, but Separation of molecules based on difference in are horizontal when the centrifuge is fully in size and shape motion As solutes travel through, the small molecules Recommended for serum separator tubes can enter the pores, whereas the largest ones cannot and will elute first from the column 2. Angle Head d. Affinity Chromatography Has a fixed 25 -52 angle at which the tubes are held during centrifugation Uses immobilized biochemical ligands as the stationary phase 3. Ultracentrifuge Uses the so-called lock-and-key binding that is widely present in biological systems Generates the highest speeds In order to reduce the heat produced by the E. Mass Spectrometry friction generated by high centrifugal speeds, ultracentrifuges are refrigerated Based on the fragmentation and ionization of Recommended for lipoprotein analysis since molecules using a suitable source of energy refrigeration enhances separation Definitive identification and quantitation of samples or compounds eluting from GC or B. Pipet HPLC columns Application: Measurement of drugs of abuse in According to Design: urine toxicology confirmations; analysis of low levels and mixed polarity analytes such as 1. To contain – holds the particular volume but does Vitamin D, testosterone, and not dispense the exact volume immunosuppressant drug 2. To deliver – will dispense the exact volume F. Osmometry indicated Measure of solute particles in a solution in terms According to Drainage Characteristics: of thei colligative properties 1. Blow-out – has a continuous etched ring located As the osmolality of the solution increases: near the top of the pipet; exact volume is obtained when the last drop is blown out a. Osmotic pressure increases b. Boiling point is elevated 2. Self-draining – no etched rings; liquid is allowed c. Freezing point is depressed to drain by gravity d. Vapor pressure is depressed According to Purpose: Freezing point osmometry – most commonly used method for measuring the changes in 1. Transfer pipet colligative properties of a solution; based on the principle that addition of solute molecules lowers Volumetric – designed to transfer the temperature at which a solution freezes nonviscous or aqueous solutions and is 9|D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 self_x0002_draining; has the greatest degree of accuracy and precision Ostwald-Folin – viscous fluid; blow-out pipet Pasteur pipet 2. Graduated/Measuring pipet Mohr pipet – without graduations to the tip; self-draining Serological pipet – with graduations to the tip; blow-out Bacteriological pipet Instrumentation Pipet Classification I. Calibration Marks/Design 1. TD pipets 2. TC pipets II. Drainage Characteristics 1. Blow-out pipets 2. Self-draining pipets III. Types C. Chemicals Used for Reagent Preparation 1. Transfer pipets 1. Analytical Reagent Grade 2. Graduated or measuring pipets For quantitative and qualitative analyses Mechanical or Automatic Pipets 2. Ultrapure Reagent 1. Air displacement Type of reagent that has been put through Relies on piston for suction to draw the sample additional purification steps into a disposable tip. 3. Chemically Pure or Pure Grade 2. Positive Displacement Limitations of this type are not usually stated Operates by moving the piston in the pipet tip or and is not recommended for research and barrel, much like a hypodermic syringe. analytical chemistry Does not require a different tip for each use. 4. Technical or Commercial Grade 3. Dispenser/Dilutor Pipet Used primarily in manufacturing and should never be Obtain a liquid from a common reservoir and used in clinical laboratory setting dispense it repeatedly – combines sampling and dispensing. 5. United States Pharmacopoeia (USP) and National Formulary (NF) Approved for human consumption but not applicable for laboratory analysis 10 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 D. Grades of Reagent Water For mixing, it uses acceleration and deceleration of the rotor to transfer the reagents 1. Type I Reagent Water and sample from one chamber to another. Major advantage: Batch analysis uExample: Used for test methods requiring minimum Cobas-Bio (Roche) interference Used for procedures that require maximum 3. Discrete Analyzer water purity for accuracy and precision Most popular and versatile analyzer 2. Type II Reagent Water Employs a variety of syringe pipets to aspirate and dispense sample and reagents – positive Acceptable for preparation of reagents and liquid- displacement pipets quality control materials Each sample and reagent mixture is handled separately in its own reaction vessel. 3. Type III Reagent Water Capable of running multiple tests-one-sample- at-a-time (sequential testing). Used for washing glasswares Also have random access capability For mixing, magnetic driven teflon stirring bar, stirring paddle, ultrasonic energy, forceful AUTOMATION dispensing For dry slide technology, the spreading layer permits a rapid uniform spreading layer over the reagent layer. Principles of Automation: Reflectance photometry – measurement of light reflected from solid surfaces. 1. Single channel 2. Multi-channel Terminologies: 3. Random access 4. Batch analysis 1. Sequential testing 5. Sequential analysis 6. Open reagent system Multiple tests analyzed one after another on 7. Closed reagent system a given specimen. Three Basic Approaches to Automation 2. Batch testing 1. Continuous Flow Analyzer All samples are loaded at the same time, and a single test is conducted on each Liquids are pumped through a system of sample. continuous tubing. Samples flow through a common reaction vessel 3. Parallel testing or pathway. Air bubbles at regular intervals serve as cleaning More than one test is analyzed concurrently media. on a given clinical specimen. Mixture of sample and reagent takes place by using a glass coil inserted into the flow path. 4. Random access testing A heating bath maintains the required temperature of the reaction – color development Any test can be performed on any sample in Example: Simultaneous Multiple Analyzer any sequence. 2. Centrifugal Analyzer 5. Open reagent system Uses the force generated by centrifugation to A system in which reagents other than the transfer specimen and reagents. instrument manufacturer’s reagents can be Liquids are placed in separate cuvets for used. measurement at the perimeter of a spinning The operator may change the parameters – rotor. it provides flexibility. 11 | D P D  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 6. Closed reagent system Total protein, ammonia, potassium, cholesterol, lactate, calcium and enzymes A system where the operator can only used the manufacturer’s reagents. 6. Smoking (nicotine) Lesser imprecision May not introduce other tests that are not Affects the concentration of cathecolamines, performed by the machine. cortisol, glucose, GH, cholesterol, triglyceride and urea. Manual Steps to Automation: Ammonia = increases by 100-200μg/L/1cigar 1. Identifying the sample and patient 7. Alcohol ingestion 2. Measuring and adding reagents 3. Mixing the sample and reagents Affects TAG, urate, gamma glutamyl transferase 4. Incubating the sample mixture glucose (chronic alcoholism)- decreased 5. Calibrating the assay 6. Measuring and reading the sample reaction 8. Stress 7. Recording, analyzing and storing sample data. 9. Drugs Types of Reaction Vessels: SPECIMEN COLLECTION AND HANDLING 1. Tubing 2. Sealed plastic bag Proper patient identification is the first step in 3. Teflon or plastic rotors sample collection. 4. Plastic cuvets 5. Multiplayer thin film slides. 1. Arterial puncture – radial, brachial, femoral PATIENT PREPARATION 0.05ml heparin/ml blood Factors contributing to the variation of results: 1. Venipuncture 1. Exercise Median cubital vein – best site Tourniquet application 3-4 inches above the site AST, CPK, LDH, aldolase - skeletal muscle and not longer than one minute. enzymes If blood pressure cuff is used, 60mmHg inflation. ALT, ACP, GH, LH, prolactin, steroids Traces of alcohol may cause hemolysis. Ammonia, lactate, fatty acid, pyruvate, creatinine Order of Draw (CLSI formerly NCCLS) 2. Fasting 1. Blood culture bottle 3. Diet 2. Light blue stopper 3. Serum tube – w/ or w/o clot activator or gel 4. Posture/Position separator 4. Green stopper Changes in position result to efflux of filterable 5. Lavander stopper substances from the intravascular space to the 6. Gray stopper (NaF & K oxalate/iodoacetate & interstitial fluid spaces. heparin) Total protein, albumin, calcium, cholesterol, TAG, bilirubin, enzymes, hormones Complications of Venipuncture: (aldosterone, cathecolamines, ADH) Prolonged standing - increased potassium 1. Hemoconcentration 2. Failure of blood to enter the syringe/vacutainer 5. Tourniquet application 3. Syncope (fainting) 4. Thrombosis Prolonged application results to 5. Thrombophlebitis hemoconcentration and anaerobiosis 6. Serum Hepatitis and AIDS 12 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 3. Capillary Puncture (Skin puncture) 4. Flouride 1.75mm - length of the lancet Forms weakly dissociated calcium component. Depth of incision should be less than 2.0 mm for uInhibits the glycolytic enzyme. infants and children and less than 2.5 mm for Concentration: 10mg/mL of blood 5. Heparin adults (ideal universal anticoagulant) Lateral plantar heel surface - ideal for NB and Acts as anti-thrombin and anti-thromboplastin infants Concentration: 0.2mg/mL of blood Reasons for rapid separation of blood: CARBOHYDRATES 1. To prevent glycolysis. They are hydrates of aldehyde or ketone 2. To prevent lipolysis. derivatives 3. Certain substances are unstable. The presence of a double bond and a negative 4. To prevent shift of electrolytes. charge in the enol anion makes glucose an active 5. To prevent hemolysis. reducing substance. o Increased levels of ACP, transaminases, LD, K+ , Mg+ , P+ , TP, Fe+ , albumin, bilirubin 6. Interferes with colorimetric reactions. 7. Inhibits lipase enzyme. Ten Common Errors in Specimen Collection: 1. Misidentification of patient 2. Mislabeling of specimen 3. Short draws/wrong anticoagulant to blood ratio 4. Mixing problem/clots 5. Wrong tubes/wrong anticoagulant 6. Hemolysis/ lipemia 7. Hemoconcentration 8. Exposure to light/extreme temperature 9. Improperly timed specimens/delayed transport 10. Processing errors: incomplete centrifugation, in- correct log-in, improper storage Anticoagulants 1. Oxalate Combines with calcium to form an insoluble salt Temperature of >80 – calcium carbonate u Concentration: 1-2mg/mL of blood 2. Citrate Combines with calcium in a non-ionized form. Concentration: 3.2-3.8g/dL in a ration of 1 part to 9 parts of blood 3. EDTA Combines with calcium in a process called chelation. Concentration: 1-2mg/mL of blood 13 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 Hormones that Promote Hyperglycemia: 1. Cortisol and corticosteroids Decreases intestinal entry of glucose into the cell. 2. Cathecolamines Inhibits insulin secretion. 3. Growth hormone (Somatotrophic) Decreases intestinal entry of glucose. 4. Thyroid Hormones (T3 and T4) Promotes intestinal absorption of glucose. 5. Adrenocorticotropic hormone (ACTH) Stimulates release of cortisol. 6. Somatostatin Inhibits insulin and glucagon HYPERGLYCEMIA inc. in plasma glucose diagnosis by glucose tolerance test and postprandial glucose testing. MICROALBUMIN TEST FBS = >/ 126mg/dl , NV: FPG: 70-110mg/dl Are useful to assist in diagnosis at an early stage of DIABETES MELLITUS glomerular dysfunction and before the development of proteinuria ( >0.5g/day). Is a group of metabolic disorder characterized by 140 mg/dL hyperplasia/nesidioblastosis, factitial plasma glucose – requires 3-hour GTT hypoglycemia from insulin. Sulfonylurea , severe exercise , ketotic hypoglycemia Compensated coexistent Drugs/ disease 15 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 1-hour = significant decrease (but still > 120mg/dL) 2-hour = < 120 mg/dL 3-hour = fasting level Requirements (OGTT): 1. Patient should be ambulatory. 2. Fasting of 8-16 hours. 3. Unrestricted diet of 150 gms/day for 3 days prior to testing. 4. The patient should not smoke and drink alcohol prior to testing. 5. Glucose load (OGTT) 75 gms 100 gms 1.75 gms glucose/kg BW PROCEDURE: 1. Extract FBS ( 0 hour sample). 2. Give glucose load ( 5-15 mins). 3. Collect 30-minute blood sample. 4. Collect 1st hour, 2nd hour, and 3rd hour blood samples respectively. Results: Urine - negative c. Dextrostics FBS - 95 mg/dL ( 5.2 mmol/L) Cellular impregnated with glucose oxidase, 30 min - 150 mg/dL (8.3 mmol/L) peroxidase and chromogen. 1 hour - 130 mg/dL (7.2 mmol/L) Important for establishing correct insulin amount 2 hour - 105 mg/dL ( 5.8 mmol/L) for next dose 3 hour - 100 mg/dL ( 5.5 mmol/L) Samples for Analysis: FBS - is a result of a fasting process 2-Hour - is a result of insulin and glucagon secretion 1. Random Blood Sugar 2. Fasting Blood Sugar Three Possible OGTT Results: Gives the best indication of overall glucose homeostasis 1. Normal (Non-diabetic) 3. 2-Hour Post Prandial Blood Sugar FBS - 126 mg/dL ( > 7.0 mmol/L) Kinds of Glucose Tolerance Tests: OGTT - 2-hour glucose value of > 200 mg/d 3. Impaired Glucose Tolerance 1. Oral Glucose Tolerance Test FBS - > 110 mg/dL but < 126 mg/dL 30 mins = 30-60 mg/dL above fasting OGTT- > 140 mg/dL but < 200 mg/dL 1-hour = 20-50 mg/dL above fasting Glucose levels do not meet the criteria for 2-hour = 5-15 mg/dL above fasting diabetes but are too high to be considered 3-hour = fasting level normal. Interpretations (ADA): 2 hours after a glucose load, blood glucose 2. Intravenous GTT should return to the fasting level. Possible results from Nondiabetic persons: Persistent elevation at 2 hours is abnormal. Serum/plasma = 70-110 mg/dL 5 minutes = max. of 250 mg/dL 16 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 A modest 2-hour increment and normal 3-hour - Mixture of glycine, Na+ Nitroprusside, disodium level suggest impaired glucose metabolism w/o PO4 and lactose. overt diabetes. - (+) purple color of acetoacetate or acetone A very sharp rise followed by decline to - ß-hydroxybutyrate does not react w/ subnormal levels may occur in hyperthyroidism nitroprusside. and alcoholic liver disease. - (+) purple = 5 mg/dL (0.5mmol/L) - urine Glucose level increases by 6mg/dL/decade over - = 10 mg/dL (1 mmol/L) - serum age 30. If glycosuria occurs w/o hyperglycemia, the 4. Ketostix (reagent strip) patient must be evaluated for abnormal renal A modification of the nitroprusside test tubular function. (+) result – at least 50mg/L of acetoacetate within 15 seconds. 5. Glycosylated Hemoglobin (HbA1c) Also called glycated hemoglobin 5. ß-hydroxybutyrate test (Ketosite assay) It reflects the average blood glucose level over NAD, diaphorase, nitroblue tetrazolium the previous 2-3 months. (+) purple color 2 factors: average glucose concentration, RBC NV = 0.21-2.81 mg/dL (0.02-0.27 mmol/L) life span 3-6% indicates normal glycosylation. 18-20% Specific Gravity indicates prolonged hyperglycemia SG increases by 0.004 units/1% change in Sample for testing: EDTA whole blood glucose concentration. Methods: Electrophoresis, Colorimetry, Ion Capture, Immunoassay LIPIDS Preferred Method: Affinity Chromatography They are commonly referred to as fats, composed mostly of carbon-hydrogen bonds. 6. Fructosamine They are sources of fuel and provide stability to Also known as glycated albumin cell membrane. A method of monitoring short-term glucose 1. Phospholipids control - 2 to 3 weeks. 2. Cholesterol May be useful for monitoring individuals w/ 3. Triglyceride chronic hemolytic anemia and hemoglobin 4. Fatty Acid variants (Hb S or Hb C) – shortened RBC life 5. Fat Soluble vitamins – ADE span. Reference values: 205-285µmol/ Phospholipids Most abundant forms derived from phosphatidic GLYCOGEN STORAGE DISEASE: acid found on the surface of lipid layers. Deficiency of a specific enzyme involved in the Are amphipathic - contain polar hydrophilic head metabolism of glycogen. groups and nonpolar hydrophobic fatty acid side Type I – Von Gierke = Gluc-6-PO4 chain. Type II - Pompe = 1,4-Glucosidase Similar structure w/ TAG except that they have Type IIIa - Cori Forbes = De brancher two fatty acids. Type IV - Andersen = Brancher Alter fluid surface tension- lung surfactant Type V - Mc Ardle = Muscle Phosphorylase Type VI - Hers = Liver Phosphorylase Kinds: Type VII - Tarui = Phosphofructokinase 1. Lecithin or Phosphatidyl Choline – 70% Type XI - Fanconi-Bickel = Gluc-transporter 2 2. Spingomyelin - 20% 3. Cephalin - 10% Tests for Ketones: Phosphatidyl Ethanolamine 1. Gerhardt’s test Phosphatidyl Serine - FeCl3 + acetoacetic acid = (+) red color lysolecithin + Inositol Phosphatide 2. Nitroprusside test CHOLESTEROL - Na+ Nitroprusside + acetoacetic acid (+) purple An unsaturated steroid alcohol whose transport color and excretion is promoted by estrogen. 3. Acetest tablet 17 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 Are amphipathic; found on the surface of lipids. Hydrolysis of TAG will yield FA and Not catabolized by most cells – does not serve converted to energy – excellent insulator as a source of fuel Fasting requirement: 12-14 hours Forms: Reference values: 1. Cholesterol Ester - 70% < 150 mg/dL - normal Plasma/serum 150-199 mg/dL - borderline high Lecithin Cholesterol Acyl Transferase 200- 499 mg/dL - high TAG catalyzes the esterification of cholesterol by >500 mg/dL - very high (acute and promoting the transfer of fatty acids from lecithin recurrent pancreatitis) to cholesterol which results in the formation of lysolecithin and CE Current CDC reference method Modified Van Handel and Zilversmith 2. Free Cholesterol - 30% It involves chloroform extraction, and the Plasma/serum/RBC extract is treated with silicic acid(adsorption), glycerol is released by alc. Chemical Reactions: KOH, then oxidized with Na periodate. The 1. Liebermann Burchardt Reaction formaldehyde is measured by the H2SO4 2. Salkowski Reaction solution of chromotropic acid. (+) result: pink colored product Precautions: 1. Avoid hemolyzed blood. Fatty Acid 2. Avoid icteric samples. Linear-chain of carbon-hydrogen bonds. 3. Avoid water contamination. Mainly derived from hydrolysis of TAG in the 4. Precise and accurate timing for development must be adipose tissues. observed Major constituents of TAG and phospholipids. General Methods: Important sources of energy. 1. One- Step method 2. Two-Step Method Only a small amount is present in plasma, 3. Three-Step Method most bound to albumin. 4. Four-Step Method Lipoproteins CURRENT CDC REFERENCE METHOD Large molecular molecular complexes of Abell, Levy and Brodie Method lipids with specialized proteins known as Hydrolysis with alc. KOH, hexane extraction and apolipoproteins. colorimetry with Liebermann Burchardt reagent. Transport lipids (chole and TAG) to sites of energy storage and utilization. Apolipoproteins helps to keep the lipids in solution during blood circulation. interact with specific cell-surface receptors. 1. Chylomicrons Largest but the least dense (0.95 g/mL) 90 % TAG, 1-2% CHON Transports exogenous (dietary TAG) to muscle and depot. Triglyceride (Neutral Fat) Apo B-48, Apo C, Apo E Comprises one molecule of glycerol and 3 FA 2. Very Low Density LPP (Pre-ß LPP) Do not contain charged or hydrophilic 65% TAG, 13% chole, 6-10% CHON groups-very hydrophobic and water Transports endogenous TAG to muscles insoluble. and fat depot Main storage lipid in man (adipose tissues). Apo B-100, Apo C, Apo E 18 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 3. High Density LPP ( Alpha LPP) Deficiency of enzyme (spingomyelinase) Smallest but the most dense LPP (1.063-1.21) responsible for the removal of phosphorylcholine Transports chole from the tissues to the liver. from sphingomyelin. Vehicle for reverse chole transport HDL2 is more cardioprotective 4. Tangier Disease 30% phospholipid, 15% CE, 45-50% CHON Markedly decreased almost deficient HDL CDC reference method: ultracentrifugation, Increase HDL catabolism. precipitation and Abell-kendall assay. Apo A-I, II; Apo C 5. Lipoprotein Lipase Deficiency Inability to clear chylomicrons creating Reference values: < 40 mg/dL - low chylomicronemia syndrome (TAG- > 60 mg/dL - high 10,000mg/dL) Abdominal pain and pancreatitis 4. Low Density LPP ( Beta LPP) Do not develop premature CHD Most abundant LPP – 50% of the total LPP Major end product of VLDL catabolism Frederickson Classification Transports dietary chole to peripheral tissues a. Type I – Familial LPP Deficiency Most atherogenic LPP; target of therapy Refrigerator test: (+); clear plasma 50% CE, 18-22% CHON Electrophoresis: normal Apo B-100, Apo E Reference values: < 100 mg/dL b. Type 2a - Familial Hypercholesterolemia 100-129 mg/dL - above optimal RT: (-); clear plasma 130-159 mg/dL - borderline Electrophoresis: increased β band 160-189 mg/dL - high > 190 mg/dL - very high c. Type 2b - Familial Combined Hyperlipidemia RT: (-); cloudy plasma o LDL- Chole = TC-HDL-VLDL Electrophoresis: increased pre-β and β bands o Friedewald Formula: o VLDL= Plasma TAG = mmol/L d. Type 3 - Familial Dysbetalipoproteinemia o 2.175 RT: (-); occasionally cloudy plasma o VLDL = Plasma TAG = mg/dL Electrophoresis: “broad β” band = ↑ pre-β band o 5.0 e. Type 4 - Familial Hypertriglyceridemia o De Long: RT: (-); cloudy plasma – “fluffy” TAG-rich VLDL o VLDL= Plasma TAG = mmol/L particles o 2.825 Electrophoresis: increased α-2 band o VLDL= Plasma TAG = mg/dL o 6.5 f. Type 5 – Familial Hyperlipoproteinemia RT: (+); cloudy plasma Disorders Associated with Lipids and LPPs: Electrophoresis: increased α-2 band 1. Dysbetalipoproteinemia (Type III) Plasma VLDL rich in cholesterol and PROTEINS chylomicron remnants. The presence of E2/2 (rare form of apo E) MEDICAL AND BIOLOGICAL IMPORTANCE VLDL-Chole:TAG = 0.689 1. Proteins are involved in the transport of substances in the body. 2. Abetalipoproteinemia (Basses-Kornzweig) 2. Enzymes which catalyze chemical reactions in Deficiency of VLDL, LDL and chylomicrons the body are proteins. Defective apo-B synthesis 3. Proteins are involved in defence function. They Low levels of Cholesterol and TAG act against bacterial or viral infection. Defects in absorption of vitamins ADEK 4. Hormones are proteins. They control many biochemical events. 3. Niemann-Pick Disease (lipid storage disease) 5. Some proteins have a role in contraction of Accumulations of spingomyelin in the bone muscles. marrow, spleen and lymph nodes. 6. Proteins are involved in the gene expression. They control gene expression and translation. 19 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 7. Proteins serve as nutrients. Proteins are also Examples: Human plasma albumin, Trypsin, involved in storage functions. Chymotrypsin, pepsin, insulin, soyabean, trypsin 8. Proteins act as buffers. inhibitor and ribonuclease. 9. Proteins function as anti-vitamins. 10. Proteins are infective agents. 2. Conjugated proteins: They are proteins 11. Some toxins are proteins. containing non-protein part attached to the 12. Some proteins provide structural strength and protein part an elasticity system. 13. Some proteins are components of structures of tissues. CHEMICAL NATURE OF PROTEINS All proteins are polymers of amino acids. The amino acids in proteins are united through Based on MEDICAL BIOCHEMISTRY “Peptide” linkage. Sometimes proteins are also called polypeptides because they contain many peptide bonds. PROPERTIES OF PROTEINS 1. Proteins have high molecular weight. 2. Proteins are colloidal in nature. 3. Proteins have large particle sizes. 4. Different kinds of proteins are soluble in different solvents. 5. Proteins differ in their shape. 6. Some proteins yield amino acids only on hydrolysis whereas others produce amino acids plus other types of molecules. Based on ACTS 7. Charge properties GLYCOPROTEINS Charge of a protein depends on the MUCOPROTEINS surroundings like amino acids. So, by changing LIPOPROTEINS the pH of surroundings the charge of protein can METALLOPROTEINS be altered. This property is used for separation NUCLEOPROTEINS of proteins. 8. Proteins act as buffers Since proteins are 3. Derived proteins: proteins are formed from amphoteric substances, they act as buffers. simple and conjugated proteins. CLASSIFICATION OF PROTEINS There are two classes of derived proteins. There is no single universally satisfactory system of I. Primary derived proteins: They are protein classification so far. formed from natural proteins by the 1. One system classifies proteins according to their action of heat or alcohol etc. The composition or structure. peptide bonds are not hydrolysed. They 2. One system classifies them according to are synonymous with denatured solubility. proteins. 3. One system classifies them according to their Example: Coagulated proteins like shape. cooked-egg albumin. 4. Classification of proteins based on their function II. Secondary derived proteins: They are is also found in literature. formed from partial hydrolysis of proteins. Examples: Proteoses, Classification of Proteins peptone, gelatin, and peptides. Based on their Composition Proteins classification according to their Three major classes according to their structure. SOLUBILITY 1. Albumins: Soluble in water and salt solutions. 1. Simple proteins: Simple proteins are made up Examples:Albumin of plasma, egg albumin and of amino acids only. lactalbumin of milk. 20 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 2. Globulins: Sparingly soluble in water but soluble in salt solutions. Examples:Globulins of plasma, ovoglobulins of egg, lactoglobulin of milk. 3. Glutelins: Soluble in dilute acids and alkalies. Examples:Glutenin of wheat, oryzenin of rice, zein of maize. 4. Protamins:Soluble in ammonia and water. Examples:Salmine from salmon fish, sturine of sturgeon. 5. Histones:Soluble in water and dilute acids. Example:Histones present in chromatin. 6. Prolamines:Soluble in dilute alcohol and insoluble in water and alcohol. Prealbumin Examples:Gliadin of wheat, zein of corn. migrates ahead of albumin 7. Sclero proteins:Insoluble in water and dilute has a half-life of only 2 days acids and alkalis. rich in tryptophan and contain 0.5% CHO Examples:Collagen, elastin and keratin. and has considerable B-pleated sheet conformation Classification of proteins based on SHAPE Proteins are divided into two classes based on used to detect malnutrition and individual’s response to dietary supplementation their shape. 1. Globular proteins:Polypeptide chain(s) of these used as landmark to confirm that the proteins are folded into compact globular specimen is really CSF- it crosses more (Spherical) shape. easily into the CSF than other protein Examples: Haemoglobin, myoglobin, albumin, increased : alcoholism, chronic renal failure, lysozyme, chymotrypsin. steroid treatment 2. Fibrous proteins:Polypeptide chains are decreased : poor nutrition extended along one axis. Reference Value: 18-45mg/dl (CF: 10 - Examples: α-keratin, β-keratin, collagen, mg/L) troponin and elastin ALBUMIN Forces that stabilize these aggregates the protein present in highest concentration in (assembles of monomers) are: serum 1. Hydrogen bonding synthesized in the liver 2. Electrostatic interactions general transport protein (binds various 3. Hydrophobic interactions substances in the blood) 4. Vander waals interactions maintains osmotic pressure and indicator of 5. Disulfide bridges (in some proteins) nutritional status Examples: serves as circulating reservoir of amino acids 1) Haemoglobin consist of 4 polypeptide sensitive and highly prognostic marker in cases chains. of cystic fibrosis 2) Hexokinase contains 2 subunits. a negative “Acute Phase Reactant”- 3) Pryuvate dehydrogenase contains 72 lowest plasma levels are seen in active subunits. nephritic/nephrotic syndrome Reference Value : 3.5 – 5.0 g/L (CF: 10 – g/L) PLASMA PROTEINS ANALBUMINEMIA- absence of albumin BISALBUMINEMIA – presence of 2 bands in the albumin region HYPOALBUMINEMIA – decreased levels of albumin, typical of nephritic syndrome Decreased: 1. liver disorders 2. Gastrointestinal disease 21 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 3. malabsorption SPECIMEN: maternal serum, amniotic fluid, 4. muscle wasting disease serum (for cancer screening) 5. severe burns 6. renal diseases INCREASED: hepatoma, spina bifida, neural Increased: tube defects, atresia of the GIT, Fetal distress, 1. dehydration ataxia telangiectasia, tyrosinosis, HDN and 2. excessive albumin administration Anencephaly. DECREASED: Down Syndrome and Edward's syndrome REFERENCE VALUE: 5mg/ml (adult and children sera) GLOBULIN a group of protein consist of alpha1, alpha2, beta ALPHA-1-ANTITRYPSIN (AAT) and gamma fractions a glycoprotein and an acute phase reactant elevated concentration of globulin in early a major inhibitor of protease activity (prevents cirrhosis will balance loss of albumin self-destruction of tissues) deficiency is associated with emphysematous Reference values: 2.3 – 3.5 g/dL (23- 35 g/L) pulmonary disease and juvenile Hepatic cirrhosis A/G RATIO : normal value – 1.5 - 3.5 increased: inflammation, pregnancy and Albumin greater 1.5 – 3.5x compared to globulin contraceptive use Abnormal = A/G ratio becomes inverted = Reference values: 145 – 270 mg/dl globulin ˃ albumin (seen in MM) o (CF: 0.01 – g/L) ALPHA-1-ACID GLYCOPROTEIN (AAG) it has low pH (2.7), negatively charged even in acid solution has greatest affinity for progesterone, binds quinidine (cardio active drug) ALPHA-FETOPROTEIN (AFP) also provide useful diagnostic tool in neonates a glycol protein normally present in fetus, with bacterial infections migrates between albumin and alpha-1 globulin increased: pregnancy, cancer, pneumonia, band rheumatoid arthritis(RA), Cell proliferation synthesized initially by the yolk sac and then by reference value: 55-140mg/dl (CF: 0.01 – g/L) the fetal parenchymal cells of the liver it peaks in the fetus about 13 weeks gestation Ceruloplasmin AFP screening is done between 15 and 20 a copper-binding alpha 2 glycoprotein that has weeks gestational age when the maternal level enzymatic activities. increases gradually. synthesized in the liver, where 6-8 atoms of detectable in the maternal blood up to the 7 or 8 copper are attached month of pregnancy imparts blue color to protein maternal serum increased in the presence of measurement is based on its copper oxidase twins (transmitted across the Placenta) activity elevated serum levels postnatal occur only with indicator for Wilson’s disease (0.1g/L) conditions of abnormal cell multiplication (cancer) 22 | D PD  SEM01: CLINICAL CHEMISTRY MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW 1ST SEMESTER A.Y. 2024 – 2025 decreased: wilson’s dse., malnutrition, TRANSFERRIN (SIDEROPHILIN) malabsorption, nephritic dse, Menkes’ kinky-hair major component of the beta-2 globulin fraction syndrome (electrophoresis) reference values: 18-45 mg/dl (CF: 10mg/L) a glycoprotein, synthesized in the liver and also a negative APR transports iron to its storage sites, CSF contains small amount prevents loss of iron through the kidney in IDA, its concentration is normal or increased deficiency may result in accumulation of iron in apoferritin or in histiocytes or precipitates in tissues as hemosiderin HAPTOGLOBIN INCREASED: hemochromatosis (bronze-skin), IDA an alpha 2-glycoprotein, an acute phase reactant, DECREASED: liver dse, malnutrition, nephritic synthesized in the hepatocytes syndrome has 2 heavy chains and 2 light chains linked by REFERENCE VALUES: 215-365mg/dl – Male 250- disulfide bonds in analogy to the basic structure 380mg/dl – female (CF: 0.01-g/L) of immunoglobulins binds free hemoglobin by its alpha chains prevents loss of hemoglobin and its constituent iron into urine a useful measu