CC1-TRANSES Lab Math Introduction PDF

INTRODUCTION-LAB MATH LDCU-BSMLS l CC1 l TRANSCRIBED BY HAGAR GRACE PATHOLOGY (Laboratory) Consequences of Inaccurate Procedure  O...

INTRODUCTION-LAB MATH LDCU-BSMLS l CC1 l TRANSCRIBED BY HAGAR GRACE PATHOLOGY (Laboratory) Consequences of Inaccurate Procedure  Oversight of a disease  Mismanagement of the patient Purpose & Functions To assist the Clinicians in Tests may be done: A. Confirming or rejecting a diagnosis A. Individually B. Providing guideline in patient management B. Panels C. Establishing a prognosis Groups of individual tests, D. Detecting disease through case finding and/or  Related to each other (belonging to one screening organ) E. Monitoring follow-up therapy  Or which demonstrate multi system involvement 2 Sections  Which screen for the specific cause among A. CLINICAL PATHOLOGY diseases with shared or common Deals with fluids & blood presentations B. ANATOMICAL PATHOLOGY Deals with tissues CLINICAL LABORATORY CALCULATIONS  Histopathology  Immunohistochemistry Metric System Clinical laboratory Sections  Most commonly used in scientific Sections Purpose measurements Hematology Cell counts  Decimal based system Blood Bank Blood transfusion Serology/Immunology Antigen-antibody  If solutions are not prepared accurately: reaction A. Experiments may fail Clinical microscopy Cell counts & B. A waste of time and money examinations of other body fluids  Biological experiments are particularly Microbiology Examinations for bacteria, sensitive to alterations in chemical fungi, virus, & parasites composition of solutions Special Chemistry Deals with cytogentics Clinical Chemistry Measurements of the 1. Enzymatic reactions are extremely sensitive to amount of chemicals alterations in pH, and salt concentrations 2. Incorrectly prepared media may inhibit bacterial growth 3. DNA migration in agarose gels can be altered CLINICAL CHEMISTRY by inexact calculations  One of the busiest sections of the clinical lab  Measurement of the amount of chemicals or Standard metric Units for expression quantitative analysis of metabolities in body weight gram fluids, particularly blood length meter  Clinicians request for the test volume liter time second MLS/ CLS are expected to: a) Accurately perform the test The metric system has the advantage that b) To ensure valid results fractional parts of measurements can be derived by multiplying the standard by some power of 10 MLS/ CLS must: a. Understand the principle of the tests b. Understand the physiology of the body system involved c. Know how to interpret the result d. Realize the medical use/relevance of the test All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  Concentrations  the amount or quantity of solute per volume or in a given quantity of solution/ solvent Solute  When it is dissolved in a solution Solvent  Dissolving matrix 2 Most Common Expression of Concentrations are based on: A. Molarity B. Concentration based on % 1. % w/v (weight/volume) solid/liquid 2. % v/v (volume/volume) liquid/liquid 3. % w/w 9weight/weight) w of solution Avogadro’s Number It is generally defined as 6.022x1023 molecules of a substance, and is the number of molecules in one mole of a substance = forms the basis of molarity system Gram-molecular weight (as depicted in the Periodic table of Elements) is the weight (in grams) of one mole of a substance MOLARITY Units of measurement of molarity: Moles/liter = mol/L or M 1 mole of a compound = gram molecular weight of that compound  Obtained by adding the atomic weights of the atoms comprising the compound  Solution components are almost always expressed as homogenous mixture of 2 or more solutions All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  MOLALITY  Expression of concentration that differs from molarity  Designates the amount of solute per 1000g (1kg) of solvent rather than final solution as used in molarity  Weight/weight measurement rather than weight/volume, and is thus independent of temperature variation, making it more accurate measurement than molarity B.Base or Salts  Equivalent weight is the quantity of substance Units of measurement of molality: moles/1000g that will react with one replaceable hydrogen  Less convenient to work with, and is not commonly used in clinical laboratory  Since most solutions used in clinical lab are aqueous (liquid) soutions, there is very little difference between molality & molarity Normality The number of equivalent weights (or equivalents) of solute per liter of solution Units of measurement= equivalents/Liter (expressed as N) NORMALITY Similar to molarity except that concentration is based on equivalent weight instead of molecular weight Equivalent weight  The mass of an element or compound that will combine with or replace 1 mole of hydrogen (H)  Dependent on the total charge of the positive ion, or the valence of the element Equivalent weight = molecular weight (of a compound) Valence Hydrates Water of hydration = water molecules attached 2 WAYS to each molecule of salt, in manufactured A. Acid Compound chemical compound  An equivalent is the quantity of substance Anhydrous = without water that contain one replaceable hydrogen Monohydrate = 1 H20 Pentahydrate = 5 H20 E.g HCl Equivalent weight = molecular weight Ex. Of prescribed hydrate of salt: copper sulfate 1 CuSO4 = 160g Thus, equivalent weight = molecular weight CuSO4. H2O= 178g CuSO4. 5 H2O = 250g All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  CONCENTRATION BASED ON % TEMPERATURE (3 scales) Dilutions  Weaker solution is made from stronger solution,  diluent, such as water, added to an aliquot of a stronger solution, to produce lesser concentration  A 1:10 dilution can also be expressed as = 1:10 or 1/10 which means 1 part conc.solution Kelvin + 9 parts of diluent  Used for very high or very low temp. 10 parts final solution  An absolute, thermodynamic temperature scale using as its null point absolute zero below which temperature do not exist All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  INTERNATIONAL SYSTEM OF UNITS  Systeme International d’ Unites (SI) adopted in 1960  Established, whereby all quantitative measurements could be expressed in standard units, although conventional Metric System are still frequently used today Serial Dilutions  Titering  Useful in serologic tests when estimates of the volume of antibody is necessary  “dilution fold” is constant in all tubes  Volume transferred is constant to each successive tubes Total volume = volume being transferred + volume of diluent already in the tube  The last volume to be transferred is discarded from the last tube Specific Gravity  Used when working with concentrated acids or base  The weight of 1 ml of any liquid  Method of measuring density  Ratio of mass/volume {g/ml or ml/g}  Concentrated commercial liquids : check labeks for specific gravity & percent purity (assay) All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  IONIC STRENGTH  First step in calculations is calculation of so called ionic strength, using following formula: ELEPHANT FORMULARY Abbreviate table of conversion values for some of the more commonly encountered tests that may be reported in SI units To convert blood glucose readings between the two units:  Divide a mg/dL figure by 18 (or multiply by RADIOISOTOPES 0.055) to get mmol/L  Multply a mmol/L figure by 18 (or divide by A. Half Life 0.055) to get mg/d Time required for a given amount of radioactivity How to convert meq/L to mg/L? to decrease to one-half its original value A. The amount of radioactivity decreases by a  You must multiply meq/L by mg/meq to get factor of 2 for every half-life period mg/L B. If an isotope has a half-life of 8 hours and an  ** mg/meq is simply the inverse of the activity of 16 mCi (millicuries), its activity will drop equivalent weight to 2 mCi in 24 hours CALCULATING pH B. 1 mCi Activity 3.7 x107 disintergration/second (1000 mCi=1 Ci) pH is only meaningful when applied to aqueous (water-based) solutions. To calculate the pH of an aqueous solution you need to know the concentration of the hydronium ion in moles per liter (molarity). The pH is then calculated using the expression: All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  According to the NBS (National Bureau of CALCULATING The Hydronium Ion Standards, Class A pipets are more accurate than Concentration From pH Class B pipets. In the clinical chemistry laboratory, the volumetric glassware must all be Class A. The hydronium ion concentration can be found from the ph by the reverse of the mathematical operation employed to find the pH UNIVERSAL INDICATOR COMPONENTS To Contain Pipets pH in living systems (20) These are also called rinse-out pipets. They contain Compartment pH an exact amount of liquid which must be Gastric acid 1 completely transferred for accurate measurement. Lysosomes 4.5 Granules of chromaffin 5.5 Examples: Micro-Folin, dual purpose, Sahli cells hemoglobin, Kirk Micro, White-Black lambda, Human Skin 5.5 transfer micro, measuring micro, and Lang-Levy. None of these met the Class A specification of NBS Urine 6.0  Calibrated by introducing the exact weight of Pure H2O at 37 deg. C 6.81 Hg equivalent to the aqueous volume desired Cytosol 7.2 Cerebrospinal fluid (CSF) 7.3 To Deliver-Blow-out Pipets They transfer or deliver an exact amount of the Blood 7.34- liquid and re not rinsed out. 7.45 Mitochondrial matrix 7.5 Examples are Ostwald Folin (recognized by its Pancreas secretions 8.1 bulb), serologic pipets, serological long tip, & serological large tip PIPETS/PIPETTES These pipets are readily identified by the two forested or etched bands near the mouthpiece. The diameter of the pipets is uniform and their Pipets volume graduations extend up to the delivery tip.  Used to transfer or measure aliquots of a liquid The last blown-out drop of the liquid is included in  Clinical laboratorian should be aware when the delivered volume. selecting type of pipet to use  “mouth pipeting” should not be done To Deliver- Non-Blow-out Pipets These pipets are filled and allowed to drain by Accessories gravity. The pipets must be held vertically and the A. Pipet aid tip placed against the side of the accepting B. Pipet bulb vessel. C. Pipet tips All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed Examples: 3. Pasteur (glass) & Disposable (plastic) transfer 1. Volumetric Pipets pipets  These are the most accurate  Used to transfer liquids from 1 receptacle to  They are class A pipets and can be used for another diluting standards, calibrators or quality  May be used to remove serum from a clot control materials. tube or plasma from all-coagulated tube  They hold and deliver specific volumes indicated at the upper end of te pipet 4. Semi-automated Pipets  After draining, the amount of transferred is  Single or multichanneled equal to the stated value  No pipetting bulb nor washing needed  A plunger or trigger is used to aspirate the 2. Mohr Pipets liquid into the pipet  Class A pipets  Graduations are made at uniform intervals but Techniques used: well away from the tapered delivery tip A. Air displacement techniques  Calibrated to deliver-in-between  Uses suction to draw up the fluid The listed accuracy is for the full volume; the B. Positive displacement smaller the volume used, the less accurate the  Uses a mechanical device such as piston volume delivered. Note that these pipets are to displace the liquid to be drawn up never blown out since they deliver volumes point  Like the ,movement of the barrel in to point hypodermic syringe  Use plastic tips usually polyproylene (retain < film)  Eg. Eppendorf pipets Electronic pipetters provide programmable application parameters: A. Pipetting mode  Blowout, manual, rinsing B. Fixed volume mode C. Dispensing mode  Provides repetitive dispensing of constant volume Micropipets  contain or deliver volumes ranging from 1- 500 Note; Tips of semiautomated pipettes are never uL wipe since the plastic tips are non-wettable  Also called lambda pipets; a lambda is equal to a volume of 1 uL. 5. Dispensers Used to add repeated volumes of reagent or Examples of micropipets: diluent to serial solutions or receptacles Consists of a reagent bottle to which a plunger 1. Lang-Levy pipet with a valve system is attached  The original micropipette  Filled to the constriction  Most accurate for micro volumes, but no longer commonly used today 2. Disposable capillary Micropipette  Consists of capillary tubing with a line marking a specific volume  Filled to the line by capillary action  Liquid is delivered by a positive pressure (blow-out) through a medicine dropper or an equivalent device  Calibrated to contain therefore it requires rinsing All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  INSTRUMENTATION-1 LDCU-BSMLS l CC1 l TRANSCRIBED BY HAGAR GRACE AUTOMATION Automated solutions are available for preanalytic, analytic, and postanalytic stages of laboratory testing. Preanalytic automation heavily dependent on uniquely identifying using bar code: = enhancing patient safety Benefits 1) reduce hands-on time 2) to meet scalable needs 3) to offset staff shortages Fully automated modular design 4) shorter turnaround time that is connected to an analytic and storage 5) fewer errors (postanalytic) component and eliminates any 6) focus on value-based testing handling after initial loading 7) reduces operator exposure to potentially hazardous biologic material and eliminates POSTANALYTIC repetitive stress injuries. Some systems include a refrigerator for sample Automated platforms significantly improve: storage and automatic disposal of samples at a) Reproducibility predetermined times. b) Accuracy c) Felxibility EMERGING TECHNOLOGIES : d) While lowwring costs AUTOMATED SPECIMEN INSPECTION potentially address two of the most significant preanalytic concerns: PREANALYTIC a) identifying sample identification errors b) sample integrity issues One of the earliest automated transport systems to be introduced—and still the most THE AUTOMATED CHEMISTRY ANALYZER: popular one today—is the PNEUMATIC TUBE. CORE COMPONENTS  rapidly transports 4- or 6-inch-diameter high- impact polycarbonate carriers Sampling  carriers are lined with foam to cushion a) cap-piercing technology contents and reduce the potential for b) aspiration and dispensing using a thin, breakage. stainless-steel probe  also designed to prevent hemolysis by  potential problem: formation of sample avoiding significant elevation of g forces clot that adheres to the probe during acceleration and deceleration. c) Reusable pipette probes- problem: subject to carryover. ** * to correct ANALYTIC  aspiration of wash solution between pipetting cycles. Standalone units  back-flush the probe using a wash solution. (with or without centrifugation) selectively target a group of preanalytic or postanalytic steps. Reagent Handling = targeted automation approach Automated analyzers are categorized as open or closed reagent systems. Example of a standalone automated unit is the AutomateTM 800 (Beckman Coulter Inc., Brea, Calif.); it can perform Open reagent system sample receipt, sorting, centrifugation, decapping, sample can accommodate reagents from third-party volume detection, and aliquotting functions).  provide users with greater flexibility and adapt easily to new methods and analytes. All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed Closed reagent system  The most common instrument used in clinical can only use the instrument manufacturer’s chemistry involves the principle of light o reagents absorption.  reagents may cost more,  reagent handling and identification are Colorimetry usually more tightly integrated with the  the constituent measured is colored, i.e., it analyzer operation absorbs light within the visible spectrum (350- 700nm light) & Mixing And Incubation  uses ordinary filters to screen light which will  Many unique approaches are used to mix strike the solution sample and reagent in automated systems.  since filter resolution is not sharp enough, what  They include magnetic stirring, rotating is measured is light intensity multiple paddles, forceful dispensing, the use of wavelength ultrasonic energy, and vigorous lateral displacement. 2 Basic Types of Colorimetry Detection,Data Processing and Real-Time Visual colorimetry Monitoring  color intensity of solution is matched against a standard solution  Signal processing converts an analog signal  relies much on human eye from the detector to a digital signal that is  example: Dubowski (Duboscq) colorimeter usable by all communication devices.  less precise,insensitive, subjective  Within the analyzer, the computer controls electromechanical processes so they are Photoelectric colorimetry uniform, repeatable, and in the correct  measurement of light intensity usi sequence: pipetting devices, moving cuvets photoelectric device or detector from one point to another, moving sample  independent of wavelength tubes, and dispensing reagents  Filter Photometry  User-friendly software can display  Spectrophotometry  Levey-Jennings QC charts  calibration curves, Standard Curve (straight line)  and online troubleshooting  used to define the limits of an assay  Real-time monitoring by vendors even during  involves measuring the absorbance of several off-hours standards & calibrating the results to establish the standard curve WORKCELL How to establish the STANDARD CURVE: 1. Determine range of values expected for both integrated groups of analyzers that can function normal & abnormal. as a single unit and can be monitored by a single 2. Assess whether these ranges can be technologist measured by the method you chose. Examples: chemistry analyzers, hematology cell 3. Prepare a number of standard solutions within counters, or coagulation instruments the ranges determined. 4. Assay the standards together with the “Virtual” Workcell samples the devices are not physically connected by a 5. Plot the Standard curve. track, but interfaced to one another through 6. Examine the curve for some limitations. common software known as middleware “Physical” Workcell Devices connected by a track and thus has a single sample loading and unloading station PRINCIPLES OF AUTOMATION  There are many instruments to choose from when doing a chemical analysis of blood samples.  They differ usually in their principles and applications. All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed Limitations in the Curve  ensures the accuracy of the results by eliminating interfering substances inherent Upper Limit of Detection with the sample or the reagent. identified when the upper portion of the curve  Blanks & Standards: reference materials (high concentration range) deviates from the straight line pattern While Controls Are used to test & check the accuracy TWO TYPES OF BLANKS 1.Reagent Blank  Contains only 1 reagent (no sample)  Used to correct for the absorbance rear=dings due to other materials in the reagents How to deal? Two ways of Reagent Blanking: a) report as "greater than (upper limit) A. Set the absorbance to zero b) Dilute the original sample & re-assay,then  Commonly used is distilled water compute w/ dilution factor  Take the absorbance of reagent blank & sample Lower Limit of Detection  corresponds to lower portion of the curve that B. “Zero” the instrument using a reagent blank deviates from the straight line.  Subtract the absorbance of the reagent  happens when 2 solutions of different blank from the sample concentrations give essentially same absorbance readings, but at the next highest 2.Sample Blank concentration in the series follows a linear  contains only the sample (no reagent) relationship between absorbance &  used to correct non-specific substances in the concentration sample  e.g., hemoglobin, bilirubin, lipids; or if the product being measured has the same absorbance as bilirubin or hemoglobin  a separate blank is needed for every sample Beer-Lambert-Bouguer’s Law Concentration of substance is inversely proportional to the log of the transmitted light or directly proportional to amount of light absorbed How to deal?  no way to measure the analyte under such prevailing conditions Where,  report result as “less than (lower limit)”  A = absorbance  Also take note of signs for reagents’  a = absorptivity of compound under standard deterioration  condition  Prepare new curve  b = light path of the solution  c = concentration of the compound  %T = percent transmittance. ABSORBANCE is proportional to the inverse log of transmittance Blanks  sets the disturbance to zero  commonly used is Distilled H20 All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  Among the possible causes of this deviatio from Beer’s Law are: 1) Absorbance of solution is too strong for the instrument to measure 2) More analyte present than the detecting agent Most spectrometers display absorbance on the vertical axis, and the commonly observed is from 0 Note: It is never valid to simply extend the line all (100% transmittance) to 2 (1% transmittance) the way to zero. Always determine the limits of the curve. ABSORPTIVITY ANALYTICAL METHODS & INSTRUMENTS  unique to one particular substance, depends on wavelength Spectrophotometry  specified absoptivity assured only if accuracy  monochromators, e.g., prisms & diffraction of wavelength is maintained gratings are used, instead of low-resolution filters.  allows measurement of light intensity in a much narrow wavelength range  Suitable for both colored or colorless solutions  both visible & UV spectrophotometers, depending on the absorbance peak of the analyte A solution transmits light corresponding in wavelength to its color, and usually absorbs light Band pass- line BC of wavelengths complimentary to its color:  smaller spectral bandwidth, the higher the absorptivity, the better the analysis Wavelength Color- Complimentary Absorbed Color 350-430 Violet Yellow-blue BEER’S LAW 430-475 Blue Yellow 475-495 Green-blue Orange  may only be applied in accurate quantitative 495-505 Blue-Green Red analysis by light absorption, if the following 505-555 Green Purple requisites are met: 555-575 Yellow-Green Violet 1) Incident radiation on the substance of interest 575-600 Yellow Blue is monochromatic 600-650 Orange Green-blue 2) Solvent absorption is insignificant compared 650-700 Red Blue-Green to solute absorption 3) solute concentration is within “linear limits”  based on the measurements of radian energy 4) a chemical reaction does not occur between absorbed or transmitted under controlled the molecule of interest and another solute or conditions solvent molecule (“quenching” phenomenon) - due to inertness. Electromagnetic radiation (EMR)  Flow of energy through space at the speed of Deviations from Beer’s Law light. 1. Simultaneous absorption at multiple  EMR exists as Maxwell's waves and as streams wavelength of particles called photons 2. absorption of light by other species 3. transmission of light by other mechanisms  Photons of radiant energy are exchanged whenever electrically charged subatomic To measure background interference: particles interact. - Allen's Correction or Dual Wavelength method  When these electrons move from one orbit to another, some energy is either absorbed or released.  The wavelength of light is the distance between successive peaks. All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed Frequency  Tungsten and halogen quartz lamps are good number of waves that pass an observation point in sources of radiant energy. a unit of time  For ultraviolet range, hydrogen lamp is widely used. Wavelength is inversely related to frequency and  Function of the entrance slit is to reduce stray energy, that is, the shorter the wavelength the light and prevent scattered light from entering higher the frequency and energy and vice versa the monochromator.  Types of monochromators indude prisms, Photometer diffraction gratings, and interference filters. often used in its generic sense as any instrument that measures light intensity Prisms wedge-shaped pieces of glass, quartz, or sodium Spectrophotometer chloride Measures the absorption of monochromatic light produced by a grating monochromator Diffraction gratings made by cutting tiny grooves or slits into an Flame photometer aluminized surface of a flat piece of crown glass Measures the light emitted by single atoms burned in a flame Cuvettes made of soft glass, borosilicate glass, quartz, or Atomic Absorption photometer plastic Measures the light absorbed by atoms dissociated by heat Photodetectors indude barrier-layer cell, phototube, Fluorometer photomultiplier tube (PMT), and a variety of Measures the light of a specific wavelength semiconductor photodetectors. emitted by a molecule after it has been excited by electromagnetic radiation or a given energy  All of these devices use photosensitive materials in their cathodes that release Cathode electrons when they are exposed to light In an electrolytic cell, this is the half-cell where energy reduction takes place;  In a double-beam system, monochromatic I Cathode = negative electrode that attracts cations from either a single or two identical monochromators pass through both a  When the pH-sensitive glass electrode is not reference and a sample compartment actively in use, it Should be kept in the medium recommended by the manufacturer  The intensity of these two light beams is then  The main advantages of fluorometric over measured by one or two photodetectors. spectroscopic methods of analysis are  Increased specificity and increased  In a double-beam in time spectrophotometer, sensitivity the light beam is split with a rotating chopper that altemnately presents a mirror and an *Basic components of a spectrophotometer opening. consist of the exciter lamp, the entrance slit, the monochromator , the analytical cell or cuvette,  When performing spectrophotometer quality and photodetector control checks, the holmium oxide glass filter is used to assess Wavelength accuracy Another feature that distinguishes a double-beam from a single-beam spectrophotometer is that the double-beam compares sample and reagent blank absorbances simultaneously Flame Emission Spectroscopy (FES)  Most applications of FES have been the determination of trace metals All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  Aflame photometer must have an Atomizer as the emitted light is longer than that of the a component. exciting light. a) the sample solution is nebulized (converted Fluorescence into a fine aerosol) and introduced into the light emission from a single excited state is flame where it is desolvated, vaporized, and atomized, all in rapid succession. If the excitation energy comes from a chemical or b) atoms and molecules are raised to excited electrochemical reaction and not from states via thermal collisions with the photolumination: constituents of the partially burned flame gases. Chem iluminescence c) Upon their return to a lower or ground involves the oxidation of an organic compound electronic state, the excited atoms and (luminal, isoluminol, acridinium ester, or luciferin) molecules emit radiation characteri of the by an oxidant. sample components. d) The emitted radiation passes through a Fluorescence os the more common of these monochromator that isolates the specific processes in clinical laboratory application. wavelength for the desired analysis. e) A photodetector measures the radiant power Basic components: a light source, an excitation of the selected radiation, which is then (primary) monochromator, a cuvette, an emission amplified and sent to a readout device, (secondary) monochromator, and a meter, recorder, or microcomputer system. photodetector. An advantage of fluorescence is its extremely high sensitivity, which is approximately 100 to 1000 times that of absorption measurements. Spectrophotofluorometer  Fluorometers are designed so that the path of the exciting light is at a right angle to the path of the emitted light...  To prevent emitted fluorescent light from reaching the detector Reflectance Spectroscopy Atomic Absorption Spectrophotometry  based on the ability of a spectrophotometer  are now almost limited to analysis of metals to measure the reflectance of materials such as lead in specialized toxicology  provides a reference standard for the laboratories. comparison of the colour of different samples Most atomic absorption spectrophotometers  A reflectance spectrophotometer is similar to incorporate a beam chopper and a tuned a standard UV/Visible spectrophotometer. amplifier to avoid errors caused by measurement of light of specific wavelength emitted by analyte.  It quantitatively measures the color and intensity of reflected light  The reflected light maintains the same Molecular Luminescence Spectroscopy (Fluometry) wavelength, although different wavelengths are absorbed and reflected to different Luminescence (Bioluminescence) degrees. based on an energy exchange process that  A standard example is which, when occurs When certain compounds absorb illuminated with white light absorbs much of electromagnetic radiation, become excited, and the blue light, and reflects back the red light, return to an energy level higher than or equal to giving its characteristic color their original leve!  Deoxygenated hemoglobin absorbs a higher degree of red light, appearing bluer when  Because some energy is lost before emission illuminated with white light from the excited state by collision with the solvent or other molecules, the wavelength of All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  Turbidimetry Nephelometry & Turbidity  is the measurement of the reduction in light transmission caused by particle formation.  Two useful methods available for measuring Light transmitted in the forward direction is the concentration of a solution that contains detected. particles too large for absorption  The amount of light absorbed by a suspension spectroscopy are nephelometry and of particles depends on the specimen turbidimetry. concentration and on the particle size.  These methods may be suitable fo quantitative assays using antigen-antibody Many clinical applications exist for turbidimetry. complexes or measuring the amount of proteins in fluids Various microbiology analyzers measure turbidity  When a collimated light beam strikes a of samples (1) to detect bacteria; growth in broth particle in suspension, portion of the light are cultures absorbed, reflected, scattered, and transmitted  Tubidimetry is routinely used (2) to measure antibiotic sensitivity from such Nephelometry cultures. measurement of the light scattered by a (3) In coagulation analyzers, turbidimetric particulate solution measurements detect clot formation in the sample cuvettes.  "Three types of light scatters occur based on the relative size of the light wavelength  Turbidimetric assays have long been available (Gauldie, 1981). in clinical chemistry (4) to quantify protein  "If the wavelength (A) of light is much larger concentration in biological fluids, such as than the diameter (d) of the particle, where urine and cerebrospinal fluid (cSF). d > 10 A, the light scatters forward owing to the destructive out-of-phase back-scatter, as ------END---- described by the Mie theory.  If the wavelength of light is approximately the Psalm 94:18-19 same as the particle size, more light scatters in When | said, *My foot is slipping,” Your unfailing the forward direction than in other directions, love,"LORD supported me. When anxiety was as defined by the Rayleigh-Dabye Theory great within me. Your consolation brought me joy  A common application of nephelometry is the measurement of antigen-antibody reactions  A typical nephelometer consists of a light source, a collimator, a monochromator, a sample cuvette, stray light trap, and a photodetector.  For macromolecules with size close to or larger than the light wavelength, measurement of the forward light scatter increases the sensitivity of nephelometry  Light sources: Mercury-arc lamp, a tungsten- filament lamp, a light-emitting diode, and a laser All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  INSTRUMENTATION-2 LDCU-BSMLS l CC1 l TRANSCRIBED BY HAGAR GRACE  They are called colligative properties of the REFRACTOMETRY solution because they can be related to each other and to the osmolality  Refractometry is based on light refraction  When light passes from one medium into As the osmolality of a solution increases: another, the light beam changes its direction 1. Osmotic pressure increases at the boundary surface if its speed in the 2. Boiling point is elevated second medium is different from that in the 3. Freezing point is depressed first. The ability of a substance to bend lights is 4. Vapor pressure is depressed called refractivity.  Osmometry is based on measuring changes in  The refractivity of a liquid depends on the the colligative properties of solutions that 1. wavelength of the incident light occur owing to variation in particle variation 2. The temeprature 3. The nature of the liquid medium Freezing point depression osmometry 4. Concentration of the solute dissolved in the most common used method for measuring the medium changes in colligative properties of solution  If the first three factors are held constant, the refractivity of a solution is an indirect FLOW CYTOMETRY measurement of the total solute concentration  Flow cytometry measures some of the properties of cells suspended in a moving fluid  Refractometry has been applied to various medium. As cells pass single-file through a measurements, for example: sensing point where they are intercepted 1. Total serum protein concentration typically by an argon laser beam. 2. Specific gravity of urine 3. Column effluent of high-performance liquid  An analogue signal from the photomultiplier chromatography(HPLC) analysis tube is converted to a digital signal that the computer can use for quantitation A typical laser-based flow cytometer includes a: 1. Cell transportation system 2. A laser light source 3. A flow chamber 4. Monochromic filters 5. Lenses 6. Dichroic mirrors 7. Photomultiplier tubes 8. Computer for data analysis OSMOMETRY  Analysis of simultaneous signals from forward and right-angle light scatter allows fir  Osmometry is the measurement of the separation of granulocytes, monocytes, and osmolality of an aqueous solution such as the lymphocytes based on their size and serum, plasma, or urine granularity  As osmolality active particles are added to a Electronic gating solution causing its osmolality to increase, four And analysis of the simultaneous output from the other properties of the solution are also fluorescent channels help delineate the desired affected cell subpopulations depending on label uptake  These properties are:  Flow cytometers may be designed as cell 1. Osmotic pressure sorters to physically sort the cells from the 2. Boiling point liquid suspension 3. Freezing point 4. Vapor pressure All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  In cell sorters, the cells of interest are identified Particles measuring between 2 and 20 fL are with electronic gating and are given an counted as platelets whereas those measuring electronic charge greater than 36 fL are counted as erythrocytes and particles greater than 35 fL are also recorded  Due to speed at which droplets containing as leukocytes the desired cells can be electronically identified, they can be electrically charged COULTER with a voltage pulse while still in the flow 2-20 fL platelets stream before entering an electrical field and Greater than 36 fL erythrocytes deflected into suitable collection containers Greater than 35 fL leukocytes for further analysis  The unwanted cells are not charged and not ELECTROCHEMISTRY deflected upon passing through the field Electrochemistry involves the measurement of the  Fluorochromes with a diversity of excitation current or voltage generated by the activity of and emission wavelengths and their specific ion species conjugation to monoclonal antibodies have contributed to the widespread application of Analytical electrochemistry for the clinical flow cytometry to immunophenotyping in laboratory includes leukemia, lymphoma, and monitoring of 1. Potentiometry immune status 2. Cuolometry 3. amperometry  Flow cytometry can also be used in cell cycle analysis by staining the cells with a fluorescent The measurement of potential voltage between dye such as propidium iodide that binds to two electrodes in a solution forms the basis for a DNA and by quantifying the number of cells in variety of procedures for measuring analyte different stages of the cell cycle concentration. Electrical potentials are produced at the interface between a metal and ions of that metal in a solution Such potentials also exists when different concentrations of an ion are separated by a membrane semipermeable to that ion. To measure the electrode potential, a constant- coltage source is needed as the reference potential Reference electrode electrode with a constant voltage Concentrations of ions in a solution can be calculated from the measured cell potential CONDUCTOMETRY difference between the two electrodes Ion-Selective Electrode Conductometry is the measurement of electrical An ion-selective electrode (ISE) is an current between two nonpolarized electrodes with electrochemical transducer capable of a known potential. The current is directly responding to one given ion. An ISE is very sensitive porportional to the solution conductivity and elective for the ion it measures With an increase in the conductivity, there is an When measuring k+ with an ion-selective increase in the current flow. The solution is inversely electrode by means of liquid ion-exchange proportional to its impedance (resistance) membrane, the antibiotic Valinomycin is incorporated into the membrane Electrical impedance is used primarily in the hematology laboratory to enumerate leukocytes, pH Electrode. Glass electrodes were the first and erythrocytes, and platelets are still the most common electrode for measuring hydrogen ion activity (pH or negative log of the In a typical electrical impedance instrument by hydrogen ion concentration) Coulter, aspirated blood is divided into two separate volumes for measurements All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  COULOMETRY AND AMPEROMETRY ELECTROPHORESIS Coulometry Electrophoresis is the separation of charged  An electrochemical titration in which the compounds based on their electrical charge titrant is electrochemically generated and the endpoint is detected by amperometry When a voltage is applied to a salt solution  Coulemetry is based on Faraday’s law, which (usually sodium chloride), an electrical current is related electrical charge (Q), current (I), and produced by the flow of ions: cations towards the time (t), according to the ff. Equation: cathode and anions towards the anode. Q = It Conductivity of a solution increases with its total ionic concentration. Amperometry  Measurement of the electrical current at a The net charge of a compound, in turn, depends single applied potential on the solution pH. Electrophoresis separations  The most widely used oxygen-sensing often require high voltages (50) electrodes use an amperometric or current- sensing electrolytic cell as the indicator system Applications of Electrophoresis 1. Serum proteins Voltammetric techniques 2. Hemoglobins  Are used to measure solution composition 3. Isoenzymes based on the current-potential relationship in an electrochemical cell when the potential is Isoenzymes varied  Include creatine kinase (CK), lactate  Detection limits can be down to the parts-per- dehydrogenase (LD), and alkaline billion range phosphatase (AP)  Several analytes can be measured simultaneously in a single voltammetric study Proteins  Polymers of amino acids that can be anions or cations depending on the pH environment DENSITOMETRY  At a specific pH, a protein will have a net charge of zero when the positive charge and Densitometry is basically an absorbance the negative charge of its amino acids cancel measurement each other out  At this pH value, known as the protein’s Densitometer isoelectric point (pi), the protein is isoelectric measures the absorbance of the stain on a support medium This pH gradient is created by adding acid to the anodic area of the electrolyte cell and adding The basic componnets of a densitometer include: base to the cathode area 1. Light source 2. Monochromator A solution of ampholytes (mixtures of small 3. Movable carriage to scan the medium over amphoteric ions with different pH) is placed the entire area between the two electrodes 4. Optical system 5. Photodetector The advantage of isoelectric focusing techniques lies in their ability to resolve mixtures of proteins Signals detected by the photodetector are related to the absorbance of the sample stain on the support, which is proportional to the specimen concentration All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  The compounds interacting more strongly with the stationary phase are retained longer in the medium than those that favor the mobile phase Chromatographic techniques may be classidied according to their mobile phase: 1. gas chromatography 2. liquid chromatography Gas chromatography (GC)  Useful for compounds that are naturally volatile or can be easily converted into a volatile  Has been widely used method for decades owing to its high resolution, low detection limits, accuracy, and short analytical time  Applications include various organic molecules including many drugs Retention of a compound in GC is determined by its vapor pressure and volatility which, in turn, depend on its interaction with the stationary Migration of DNA fragments in agarose gel phase electrophoresis via traditional method (A: 0.8% gel) and the modified method (B: 2% + 0.8% gel). Lane Two types of stationary phases commonly used in 1-3: 1kb DNA marker, ┣DNA HindIII plus ΦX174 GC DNA-HaeIII marker and DNA analytical marker, 1. Solid absorbent (gas-solid chromatography respectively. {GSC} 2. Liquid coated on solid supports (gas-liquid chromatography) {GLC} CAPILLARY ELECTROPHORESIS In GSC, the same material (usually alumina, silica, Consists of: or activated carbon) acts as both the stationary 1. fused silica capillary phase and the support phase 2. Two electrolyte buffer reservoirs 3. A high-voltage power supply GLC uses liquid phase such as polymers, 4. A detector linked to a data acquisition unit hydrocarbons, fluorocarbons, liquid crystals, and molten organic salts to coat the solid support Mechanism/Principle material When a high voltage is applied across the capillary ends, the sample molecules across the The use of fused silica capillary columns in which capillary ends, the sample molecules are the stationary phase is chemically bonded onto separated by electro-osmotic flow, a bulk flow the inner surface of the column has become very resulting from excess positive ions at the inner popular with chromatographers capillary surface moving toward the cathode Advanatage of this type of column:  Positive ions in the specimen emerge early at 1. The stationary phase does not leave the solid the capillary outlet because the electro- phase and bleed into the detector somotic flow and the ions movement are in 2. A uniform monomolecular layer of the stationary the same direction phase is obtained through the bonding procedure  Negative ion moved toward the capillary but at slower rate CHROMATOGRAPHY A separation method based on the different interactions of the specimen compounds with the mobile phase and with the stationary phase, as the compounds travel through a support medium , All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed The carrier gas is usually helium, hydrogen, or Mechanism: exchange of sample ions and nitrogen. This serves as the mobile phase that mobile-phase ions with the charged group of the moves the sample through the column stationary phase 4. Affinity Chromatography  Uses immobilized biochemical ligands as the stationary phase to separate a few solutes from other unretained solutes  Mechanism: uses the so-called lock and key binding that is widely present in biological systems 5. Size-exclusion Chromatography  Separates molecules according to the difference in their size. The support material has a certain range of pore size LIQUID CHROMATOGRAPHY  Mechanism: as solutes travel through, the small molecules can enter the pores, whereas GC as a separation technique has some the larger ones cannot and will elute first from restrictions that make liquid chromatography a the column suitable alternative High Performance Liquid Chromatography 1. Many organic compounds are too unstable or Uses gradient type of elution technique are insufficiently volatile to be assayed by GC without prior chemical derivation MASS SPECTROMETRY Liquid chromatography techniques 2. use lower temperature for separation, thereby achieving  Based on fragmentation and ionization of better separation of thermolabile compounds molecules using a suitable source of energy  The resulting fragment masses and their These two factors are allowed in liquid relative abundance yield the characteristic chromatography than in gas chromatography mass spectrum of the parent molecule  Before a compund can be detected and The mobile phase can be removed, and the quantified by mass spectrometry it must be sample can be processed further or reanalyzed isolated by another method: GC= greater under different conditions specificity & sensitivity for the combination Five Commonly Used Separation techniques in Composed of: Liquid Chromatography: 1. Inlet unit 2. Ion source 1. In adsorption (liquid-solid) chromatography, the 3. Mass analyzer compounds are adsorbed to a solid support, such 4. Ion detector as silica or alumina 5. Data unit  First type developed but not widely used owing to strong retention of many One commonly used source of ions is a beam of compounds by the supports (difficult to elute electrons produced by a heated filament from the column) Electron-impact ionization 2. Partition (liquid-liquid) chromatography Process of bombarding the sample with electrons separates compunds based on their partition between a liquid mobile phase and a liquid Iron-trap Mass Spectrometer stationary phase coated on a solid support More modern form of mass analyzers, now in  Partition chromatography includes normal widespread use phase liquid chromatography which uses a polar liquid stationary phase, and reverse- phase liquid chromatography, which uses a nonpolar stationary phase 3. Ion exchange chromatography Uses column packing that have charge bearing functional groups attached to a polymer matrix All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  drugs in patient's blood, serum, plasma, urine, or saliva Immunocytochemical investigations of fixed cells are used to enhance diagnostic accuracy in haematology and oncology. Immunohistochemical staining widely used in the diagnosis of abnormal cells such as those found in cancerous tumors SCINTILLATION COUNTER  Scintillations are flashes of light that occur when gamma rays or charged particles interact with matter  Chemicals are used to convert their energy into light energy  If gamma rays or ionizing particles are absorbed in a scintillator, or some energy absorbed by the scilllilator is emitted as a pulse of visible light or near - UV radiation  Two types exist: 1. Crystal Scintillator Used to detect gamma region 2. Liquid Scintillator Primarily used to count radionuclides that emit Figure 2. Morphology and immunocytochemical staining beta particles reaction of neoplastic mast cells at the time of leukemic progression. A. B As assessed by Wright-Giemsa-staining, the bone marrow smear was found to contain a significant IMMUNOCHEMISTRY number of immature mast cells, leading to the diagnosis of mast cell leukemia. As assessed by immunocytochemistry,  offers simple, rapid, robust yet sensitive, and these cells were found to stain positive for tryptase (G) KIT easily automated methods for routine (D), histidine decarboxylase (HDC) (E), and CD63 (F) analyses in clinical laboratories.  based on highly specific binding between an antigen and an antibody.  an epitope (immunodeterminant region) on the antigen surface is recognized by the antibody's binding site.  The type of antibody and its affinity and avidity for the antigen determines assay sensitivity and specificity. Depending on the assay format, immunoassays can be qualitative or quantitative.  They can be used for the detection of antibodies or antigens specific for bacterial, viral, and parasitic diseases as well as for the diagnosis of autoimmune diseases.  Immunoassays can measure low levels of disease biomarkers and therapeutic or illicit All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed Hematoxylin and eosin (H&E stain showing dimorphic reagent layer(s) to a photocell while some is picture of syncytiotrophoblast and cytotrophoblast in abscrbed. Choriocarcinonsa Human Chorionic Gonadotrophi  The amount of reflected light, which is immunohistochemical stain showing positive staining in indirectly proportional to colour intensity, is Choriocarcinoma components H&E stain showing sheets of used to determine the concentration of the monomorphic intermediate trophoblasts and find material analyte. in placental site trophoblastic tumor; d immunohistochemistry (HC): HPL immunostain showing positive staining in intermediate trophoblasts of placental se trophoblastic tumor; e IHC: Inhibin immunostain showing positive staining in intermediate trophoblasts of placental se trophoblastic tumor, HC: B-HCG immunostain showing negative staining in intermediate trophoblasts of placentals trophoblastic tumor DRY CHEMISTRY Dry chemical methods utilize reagent slides that are composed of several layers which may BIOSENSORS include: 1-spreading layer 2-scavenger layer  Miniaturized (micromachined) sensing 3-reagent layer(s) device/ analyzers (including near-bedside 4-plastic or support layer testing)  Developments are towards: Examples of these analyzers indude bed-side a) Improvements in patient care glucometers, Cholesterol and Triglyceride analyzer, b) Convenience hemoglobin analyzer, blood group reader. c) Cost d) Turnaround time Advantages: 1. The storage requirements for reagents are minimal since no wet reagents are required. 2. No pipetting steps are needed as the manufacturing company prepares the slides. 3. No sample dilution is required and 10 or 11 μl of sample per test is used. Principle of dry chemical analyzer:  Spreading layer is for adding sample or control or standard.  Scavenger layer allows selected components to filter through and penetrate to the reaction layer(s), which in tum activate the Biosensor comprises a biologically sensitive dehydrated reagents. material (a biocatalyst) in contact with a suitable transducing system that converts the biochemical  Reagent layer(s) contains lyophilized or dry signal into an electrical signal. enzymes, and buffers necessary for the analysis of a specific analyte in the sample. Four types of transducers most widely used in biosensor technology. These are based on  A chemical reaction is initiated to produce a changes in: colour. a. electrochemical properties (potentiometric, amperometric, or conductometric)  Light is passed from beneath the support or b. mass (piezoelectric) plastic layer and is directed through the c. heat (calorimetric) reagent layer (s). d. optical properties (luminescent, fluorescent, reflective)  As the light hits the white spreading layer, some of the light reflects back through the All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  LAB-on-a CHIP a) Improvement in technology b) automation c) miniaturization (micromachining, microfabrication) = resulted to production of device called μ-TAS, which may be used as a probe Lab-on-a-Chip devices for point of care applications usually comprise a number of passive or active structures which are arranged such that a distinct application can be carried cut with the chip at hand. Depending on the application such chips will contain means of introducing a sample liquid and/or reagents, potentially reagent storage on- chip, means of mixing, separating, or filtering of the fluids and structures for analytical readouts. Typical structures for storage of used reagents and wasteliquids also need to be provided All things work together for good to those who love God- Rom. 8:28 Bishop 8th Ed  QUALITY ASSURANCE PROGRAM LDCU-BSMLS l CC1 l TRANSCRIBED BY HAGAR GRACE 2. Internal quality assessment QUALITY  Set of procedures undertaken for continuously and concurrently assessing laboratory work  Providing systems for ensuring that products and emergent results and services meet the customers needs  Decision levels for accepting results as reliable  Degree of congruence between expectation enough for release and realization  It controls the laboratory output  " Quality is an ongoing activity , not a goal to  Main objective is ensure day to day be reached “ consistency  “ A continuous cycle of plan , do , check , act ( Deming cycle ) Differences between IQC & EQA Expectations of quality laboratory services Feature IQC EQA Quality = fitness for purpose Concurrent & Retrospective  Right product =correct, efficacious Nature continuous & periodic  Right value= accurate Laboratory Independent  Consistent = precise Performed by staff agency  On time = timeliness Release of Ensure inter-  Pleasing = visual attributes reliable results laboratory  Not harmful = Safe Objective on day to day comparability  Courteous personnel basis  Complaints are swiftly addressed  Economical  Clean....... It can go on and on. ASPECTS OF PROPER LABORATORY OPERATION MAINTENANCE OF GOOD ACCURACY AND QUALITYASSURANCE PROGRAM PRECISION  Adequate calibration  Sum total of all activities done to ensure the desired target/ product is met METHODS OF ERROR DETECTION  covers all matters that individually or  N and abN control collectively influence the quality of the  abN results checked against px's condition product ACTION TO BE TAKEN WHEN ANALYSES 60 OUT OF REWARDS OF QAP CONTROL  Quality product , reliable service  investigate, hold result until solved, repeated  Establish proper diagnosis rapidly when error is > 10%  Better health care for the patient  Good laboratory reputation and confidence PARTICIPATION IN EQuAS  Motivation to work better  Satisfy accreditation/licensing requirements EQPT MAINTENANCE  Prevention of legal suits and associated  done at reg intervals, acc. to manufacturer's complications recommendation  Equipment reliability TWO COMPONENTS OF QAP Quality assurance = Internal quality control + TRAINING AND EDUCATION external quality assessment  qualified and trained personnel  regular continuing education process 1. External quality assessment  A k.a proficiency testing DOCUMENTATION  System of objectively assessing a lab  req'd by inspection and accreditation performance by an outside agency agencies  Retrospective and periodic assessment  aids in detecting trends  Main objective is to est