M_CC1L.pdf

Laboratory Safety and Basic Principles of Clinical Chemistry Valerie Jane Casandra Ty Villarin, RMT, MS MLS Faculty, Department of Medical Technology No part of this material may be distributed and reproduced. The file is for your own use only. Biologic Hazard...

Laboratory Safety and Basic Principles of Clinical Chemistry Valerie Jane Casandra Ty Villarin, RMT, MS MLS Faculty, Department of Medical Technology No part of this material may be distributed and reproduced. The file is for your own use only. Biologic Hazard Refers to a biological substance that poses a threat to the health of living organism, primarily humans. Bacteria, viruses, fungi, prions and other organisms and their toxins Chain of infection (transmission) No part of this material may be distributed and reproduced. The file is for your own use only. Biologic Hazard Chain of infection – necessary to prevent infection Requires continuous link between three elements (source, mode of transmission, susceptible host) Source – refers to the location of potentially harmful microorganisms (person, contaminated of object) MOT – direct contact, inhalation, ingestion, Host – individual who is at risk of infection No part of this material may be distributed and reproduced. The file is for your own use only. Types of Biological Hazards Pathogenic Microorganisms – Bacteria, viruses, fungi, prions Bioactive Substances – Toxins produced by microorganisms, plants, and animals Allergic Agents – Substances that can cause allergic reactions No part of this material may be distributed and reproduced. The file is for your own use only. Sources of Biological Hazards Medical and Clinical Settings – Hospitals, laboratories, and clinics where infectious agents are handled Natural Environment – Soil, water and air that may contain pathogenic organisms Biotechnology and Research – facilities involved in genetic engineering, pharmaceutical development, and biological research No part of this material may be distributed and reproduced. The file is for your own use only. Transmission of Biological Hazards Direct Contact – Physical contact with an infected person or contaminated surface Airborne Transmission – Inhalation of airborne pathogens (droplets) Vector – Borne Transmission – Spread through vectors (mosquitoes, ticks, fleas) Food and Water - Ingestion of contaminated food or water No part of this material may be distributed and reproduced. The file is for your own use only. Transmission Prevention Guidelines Wear appropriate PPE Change gloves between patients Wash hands after removing of gloves Dispose of biohazardous material in designated containers Properly dispose of sharps in puncture-resistant containers Do not recap needles Do not activate needle safety device using both hands No part of this material may be distributed and reproduced. The file is for your own use only. Transmission Prevention Guidelines Follow institutional protocol governing working during personal illness Maintain personal immunizations Decontaminate work areas and equipment Do not centrifuge uncapped tubes Do not eat, drink, smoke, or apply cosmetics in the work area No part of this material may be distributed and reproduced. The file is for your own use only. Biological Waste Disposal Equipment and supplies that are contaminated with blood and other body fluids must be disposed properly accordingly to its respective trash bins (color-coded) ü Black - non-infectious dry ü Green - non-infectious wet ü Yellow - infectious pathologic ü Yellow with black band - chemical waste and heavy metals ü Orange - radioactive waste ü Red - Sharps ü White or clear plastics - soiled linens ü Light Blue or Transparent w/ blue inscription - autoclaving ü HJ No part of this material may be distributed and reproduced. The file is for your own use only. Biological Waste Disposal For blood and other body fluid spill in the working area must be disinfected. The most common disinfectant is 1:10 dilution of Sodium hypochlorite (household bleach) prepared weekly and stored in a plastic (not a glass) ü HJ No part of this material may be distributed and reproduced. The file is for your own use only. Methods of Disposal 1. Autoclaving – (sterilization) uses high-pressure steam to sterilize waste, killing all pathogens (commonly used for laboratory waste) 2. Incineration – (Burning waste) involves burning biological waste at high temperatures, reducing it to ash 3. Chemical Disinfection – Often used for liquid waste 4. Landfill Disposal – (Burial) requires careful regulation to avoid contamination 5. Biological Treatment – (Composting) microorganisms break down the waste into harmless byproducts; more applicable to agriculture and food industry waste No part of this material may be distributed and reproduced. The file is for your own use only. Methods of Disposal No part of this material may be distributed and reproduced. The file is for your own use only. Sharp Hazards Sharps are a specific category of medical and laboratory waste that includes any items that can puncture or cut the skin Causing injuries and transmitting infections Ø Needles and syringes Ø Scalpel blades Ø Lancets Ø Broken Glass Ø Razors and Scissors No part of this material may be distributed and reproduced. The file is for your own use only. Sharp Hazards Hazards associated with sharps Needlestick injuries – can cause puncture wounds (bloodborne pathogens) Cuts and Lacerations – can lead to exposure to infectious materials and increase the risk of infection *All sharps must be disposed in puncture-resistant, leak-proof containers labelled with the biohazard symbol No part of this material may be distributed and reproduced. The file is for your own use only. Chemical Hazards Refers to the risks posed by chemicals that can cause harm to human health or the environment Toxic chemicals: Acute (immediate harm upon exposure) ; cyanide, carbon monoxide ; Chronic (long-term health effects) ; asbestos, benzene Flammable chemicals – easily ignite and cause fires (gasoline, ethanol, acetone) Corrosive chemicals – can cause severe damage to tissues and materials (sulfuric acid, hydrochloric acid) No part of this material may be distributed and reproduced. The file is for your own use only. Chemical Hazards Reactive chemicals – can undergo violent reactions under certain conditions (sodium, potassium, explosives) Carcinogens – known to cause cancer (formaldehyde, benzene) Mutagens and Teratogens – mutagens (cause genetic mutations) ; ethidium bromide ; teratogens (cause developmental abnormalities) ; thalidomide No part of this material may be distributed and reproduced. The file is for your own use only. Chemical Hazards *ADD ACID TO WATER When skin or eye contact occurs , the best first aid is to flush the area immediately with water for at least 15 minutes and then seek medical attention No part of this material may be distributed and reproduced. The file is for your own use only. Routes of Exposure Inhalation Ingestion Skin contact Injection No part of this material may be distributed and reproduced. The file is for your own use only. Radioactive Hazards The amount of radioactivity present in most medical situations is very small and represents little danger The effects of radiation are related to the length of exposure and are cumulative Exposure to radiation is dependent on the combination in time, distance, and shielding Exposure to radiation during pregnancy presents danger to the fetus No part of this material may be distributed and reproduced. The file is for your own use only. Electrical Hazard Refer to dangerous conditions where a person can encounter energized equipment or conductors (electric shock, burns) Electrical equipment is closely monitored by designated hospital personnel Equipment that has become wet should be unplugged and allow to dry completely before reusing Equipment should be unplugged before cleaning No part of this material may be distributed and reproduced. The file is for your own use only. Types of Electrical Hazards Electric shock Electrical burns Arc flash Arc blast Fire hazard Static electricity No part of this material may be distributed and reproduced. The file is for your own use only. Fire/Explosive Hazards Initial steps to follow when a fire is discovered are identified by the code word RACE Rescue – rescue anyone in immediate danger Alarm – activate the institutional fire alarm system Contain – close all doors to potentially affected areas Extinguish/Evacuate – extinguish the fire, if possible, or evacuate, closing the door No part of this material may be distributed and reproduced. The file is for your own use only. National Fire Protection Association (NFPA) No part of this material may be distributed and reproduced. The file is for your own use only. Fire Extinguisher No part of this material may be distributed and reproduced. The file is for your own use only. Fire Extinguisher No part of this material may be distributed and reproduced. The file is for your own use only. Physical Hazards Environmental factors that can cause harm to human body without necessarily touching it Injuries, health issues, accidents No part of this material may be distributed and reproduced. The file is for your own use only. Physical Hazards General precautions that should be observed are the following: ü Avoid running in rooms and hallways ü Be alert for wet floors ü Bend the knees when lifting heavy objects or patients ü Keep long hair tied back and remove dangling jewelry to avoid contact with equipment and patients ü Wear comfortable, closed-toe shoes with non-skid soles that provide maximum support ü Maintain a clean, organized work area No part of this material may be distributed and reproduced. The file is for your own use only. Basic Principles Water Specifications Critical reagent in clinical chemistry laboratories (solutions, reagents, diluent) Quality of water directly impacts the accuracy and reliability of clinical tests Specific standards and specifications for laboratory water are essential to ensure consistent and reliable results Water – most frequently used reagent in the laboratory Distilled water – purified by distillation Deionized water – purified by ion exchange RO water – reverse osmosis; pumps water across semipermeable membrane No part of this material may be distributed and reproduced. The file is for your own use only. Process for water purification 1. Distillation 2. Deionization 3. Reverse Osmosis 4. Ultrafiltration 5. Ultraviolet oxidation No part of this material may be distributed and reproduced. The file is for your own use only. Reagent Grade Water Type I (ultrapure) – requiring minimum interference; for preparation of standard solutions, buffer, and in analytical techniques (HPLC and mass spectrometry) Type II – preparation of reagents and solutions, general laboratory procedures Type III – glassware washing No part of this material may be distributed and reproduced. The file is for your own use only. Solution Properties Solute – dissolved in liquid Analytes – biologic solutes Solvent – liquid in which solute is dissolved Solution– solute + solvent No part of this material may be distributed and reproduced. The file is for your own use only. Colligative Properties Osmotic pressure – the pressure required to stop the flow of solvent into the solution through a semipermeable membrane Vapor pressure – liquid solvent is in equilibrium with the water vapor Freezing point – temperature at which a vapor pressures of a solid and liquid phases are the same Boiling point – temperature ate which the vapor pressure of the solvent reaches one atmosphere No part of this material may be distributed and reproduced. The file is for your own use only. Redox Potential Oxidation – reduction potential – measure of the ability of a solution to accept or donate electrons Reducing agents – donate electrons Oxidizing agents – accept electrons No part of this material may be distributed and reproduced. The file is for your own use only. Conductivity Conductivity – how electricity passes through a solution Resistivity – resistance to the passage of electrical current No part of this material may be distributed and reproduced. The file is for your own use only. Clinical Laboratory Apparatus and Supplies Valerie Jane Casandra Ty Villarin, RMT, MS MLS Faculty, Department of Medical Technology No part of this material may be distributed and reproduced. The file is for your own use only. Thermometers/Temperature Celsius ℃ - Predominant Fahrenheit and Kelvin – also used No part of this material may be distributed and reproduced. The file is for your own use only. Thermometers/Temperature All analytic reactions occur at an optimal temperature Reactions that are temperature dependent use some type of heating/cooling cell, heating/cooling block, water/ice bath Laboratory refrigerator temperatures are often critical and need periodic verification No part of this material may be distributed and reproduced. The file is for your own use only. 2 types of thermometers 1. Liquid-in-glass thermometers use a colored liquid (red or other colored material) encased in plastic or glass 20C – 400C continuous line of liquid; free from separation or bubbles calibrated against an NIST – certified or NIST – traceable thermometer for critical laboratory applications n No part of this material may be distributed and reproduced. The file is for your own use only. 2 types of thermometers 2. Electronic thermometer or thermistor temperature-sensitive resistors can be calibrated against SRM thermometers provided by NIST fast-reading and accurate ideal for applications requiring immediate temperature adjustments No part of this material may be distributed and reproduced. The file is for your own use only. Glassware Categories: Kimax/Pyrex Corex High silica Low actinic Flint No part of this material may be distributed and reproduced. The file is for your own use only. Precision and Accuracy Class A: stamped with letter “A” high-precision laboratory applications Class B: twice the tolerance limits of Class A suitable for educational and general laboratory purposes No part of this material may be distributed and reproduced. The file is for your own use only. Volume Designations To Contain (TC) : to contain a specified volume of liquid When liquid is transferred out, it does not deliver the exact contained volume due to residual liquid To Deliver (TD) : to deliver the specified volume of liquid accurate delivery of the contained volume, considering the liquid that remains in the vessel No part of this material may be distributed and reproduced. The file is for your own use only. Plasticware Replace glassware Unique high resistance to corrosion and breakage Inexpensive Disposable No part of this material may be distributed and reproduced. The file is for your own use only. Plasticware Polystyerene Polyethylene Polypropylene Tygon Teflon Polycarbonate Polyvinyl chloride No part of this material may be distributed and reproduced. The file is for your own use only. Laboratory Vessels Flasks, beakers, and graduated cylinders Volumetric and Erlenmeyer flasks – containers used in the clinical laboratory Volumetric flask – calibrated; hold 1 exact volume of liquid (TC) Erlenmeyer flasks and Griffin beakers – hold different volumes rather than one exact amount No part of this material may be distributed and reproduced. The file is for your own use only. Laboratory Vessels Graduated cylinders – long, cylindrical tubes usually held upright by an octagonal or circular base No part of this material may be distributed and reproduced. The file is for your own use only. Pipettes Glass or plastic Used to transfer liquids Reusable or disposable 20ml or less Larger volumes – automated pipetting devices No part of this material may be distributed and reproduced. The file is for your own use only. Pipette Classification No part of this material may be distributed and reproduced. The file is for your own use only. No part of this material may be distributed and reproduced. The file is for your own use only. Serologic and Mohr Pipette Serological pipette – have a graduation marking that extend to the tip Blow-out pipettes – any residual liquid remaining in the tip after dispensing should be expelled Usually indicated by an etched or colored ring near the top of the pipette 1 to 50 ml No part of this material may be distributed and reproduced. The file is for your own use only. Serologic and Mohr Pipette Mohr pipette - have graduation markings that stop before the tip They are calibrated to deliver the volume between two specified points (zero;final) 1 to 25 ml Does not require blowing out No part of this material may be distributed and reproduced. The file is for your own use only. Bacteriologic Pipette Precise handling and transfer of microbial cultures and samples They are designed to maintain sterility and facilitate accurate handling of microbial materials No part of this material may be distributed and reproduced. The file is for your own use only. Micropipette 0.1 ul to 1000 ul (1ml) Adjustable Air-displacement micropipettes and Positive- displacement micropipettes Attach disposable tip 1st stop (aspiration) 2nd stop (dispense) No part of this material may be distributed and reproduced. The file is for your own use only. Volumetric Pipette Measure and transfer a single accurate volume of liquid Deliver a specific volume with high precision To deliver (TD) Class A Volumetric pipette Class B Volumetric pipette No part of this material may be distributed and reproduced. The file is for your own use only. Pasteur Pipette AKA dropper or dropping pipette Small volumes of liquids Louis Pasteur Simple and quick liquid handling No part of this material may be distributed and reproduced. The file is for your own use only. Syringe No part of this material may be distributed and reproduced. The file is for your own use only. Syringe No part of this material may be distributed and reproduced. The file is for your own use only. Balances No part of this material may be distributed and reproduced. The file is for your own use only. Analytical Balance Measure small masses with very high accuracy and precision 0.0001 grams (resolution) Enclosed in a draft shield Single pan Frequent calibration and maintenance No part of this material may be distributed and reproduced. The file is for your own use only. Top-Loading Balance Less precise; sufficient accuracy for laboratory applications 0.1 grams to 0.001 grams Open pan (do not require a draft shield) Routine weighing More cost-effective Easier to maintain No part of this material may be distributed and reproduced. The file is for your own use only. Centrifuge No part of this material may be distributed and reproduced. The file is for your own use only. Centrifuge Used to separate components of a mixture (spinning) Utilizes the principles of centrifugal force Density, size and shape General Purpose Centrifuges Refrigerated Centrifuges Ultracentrifuges Gen No part of this material may be distributed and reproduced. The file is for your own use only. Components of Centrifuge 1. Rotor – central component (samples are placed for spinning) Fixed-angle, swinging bucket rotors, vertical rotors 2. Speed control – rotation per minute (rpm) and duration (time) of the spin 3. Temperature control – some centrifuge offer temperature control features (maintains integrity) 4. Safety features – lid locks and imbalance detection No part of this material may be distributed and reproduced. The file is for your own use only. Types of Centrifuge 1. General Purpose Centrifuges – routine laboratory applications 2. Refrigerated Centrifuges – Equipped with cooling mechanisms to maintain low temperatures (suitable for samples sensitive to heat) 3. Ultracentrifuges – Capable of reaching extreme high speeds (separation of very small particles or 4. molecules) Ref No part of this material may be distributed and reproduced. The file is for your own use only. Operation of Centrifuge 1. Loading – samples are loaded into centrifuge tubes or rotor buckets 2. Centrifugation – spins rapidly (heavier particles- settle ate the bottom; lighter particles-move to the top); based on their density and size 3. Collection – extracted using pipettes or decanting methods No part of this material may be distributed and reproduced. The file is for your own use only. Order of Draw No part of this material may be distributed and reproduced. The file is for your own use only. Order of Draw No part of this material may be distributed and reproduced. The file is for your own use only. Evacuated Tubes No part of this material may be distributed and reproduced. The file is for your own use only. Spectrophotometer No part of this material may be distributed and reproduced. The file is for your own use only. Spectrophotometer Analytical instrument used to measure the intensity of light as a function of its wavelength For quantitative analysis of substances Provides highly accurate and precise measurements of light absorbance Concentration of substances and identifying molecular structures No part of this material may be distributed and reproduced. The file is for your own use only. Principle of Operation Light source – emits a broad spectrum of light, typically ranging from UV to Vis and sometimes into the IR region Deuterium lamps (UV); Tungsten or Halogen lamps (Vis) No part of this material may be distributed and reproduced. The file is for your own use only. Principle of Operation Monochromator– isolates specific wavelengths of light (prism or diffraction gratings) Allows only the desired wavelength to pass through to the sample No part of this material may be distributed and reproduced. The file is for your own use only. Principle of Operation Sample holder - usually a cuvette (placed in the path of the isolated light beam) Quartz, glass, plastic (transparent) No part of this material may be distributed and reproduced. The file is for your own use only. Principle of Operation Detector – photodiode or photomultiplier tube Measures the intensity of the transmitted light (inversely related to the amount of light absorbed by the sample) No part of this material may be distributed and reproduced. The file is for your own use only. Principle of Operation Readout and Data Analysis – instrument converts the detected light intensity into a digital signal, displaying the absorbance (or transmittance) on a readout screen Beer – Lambert Law No part of this material may be distributed and reproduced. The file is for your own use only. Beer – Lamber Law AKA Beer’s Law (fundamental principle in spectrometry) The concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light No part of this material may be distributed and reproduced. The file is for your own use only. Beer – Lamber Law No part of this material may be distributed and reproduced. The file is for your own use only. Types of Spectrophotometer UV – Visible – Measures light absorbance in UV and Vis regions (190-1100nm) ; used for analyzing nucleic acids, proteins, biological molecules IR Spectrophotometer – Measures absorbance in the IR region (NIR:700 – 2500nm; MIR: 2500-25,000nm; FIR: 25,000nm – 1mm) Atomic Absorption Spectrophotometer (AAS) – Measures the concentration of metal ions in solutions by detecting the absorption of light by free atoms No part of this material may be distributed and reproduced. The file is for your own use only.  Quality Management Valerie Jane Casandra Ty Villarin, RMT, MS MLS Faculty, Department of Medical Technology No part of this material may be distributed and reproduced. The file is for your own use only. Quality Assurance (QA) Ensuring the accuracy, reliability, and consistency of laboratory test results Critical for patient diagnosis, treatment, and monitoring Includes the pre – analytic, analytic, and post – analytic phases Pre-analytic phase – involves all the steps taken before the actual analysis of the specimen Analytic phase – involves the actual testing of the specimen Post-analytic phase – involves all the steps taken after the analysis to ensure the accurate reporting and interpretation of results No part of this material may be distributed and reproduced. The file is for your own use only. Quality Control (QC) Part of quality assurance Focusing on monitoring and maintaining the accuracy and precision of laboratory tests Help ensure that test results are reliable and valid 2 major type: 1. External QC – monitors primarily the accuracy of laboratory tests 2. Internal QC – monitors the day-to-day performance of laboratory tests, namely precision No part of this material may be distributed and reproduced. The file is for your own use only. Errors 1. Analytical errors – usually systematic errors Erroneously calibrated pipettor Deteriorating reagent Improperly calibrated instrument 2. Personnel or operator errors – usually random errors that usually affect only a few analyses Mislabeling the specimen Analytical result is assigned to a wrong specimen Wrong number entry on test result No part of this material may be distributed and reproduced. The file is for your own use only. Accuracy and Precision 1. Accuracy Nearness to the true value Method is reflected by its ability to reproduce the values of reference samples of unknown concentration 2. Precision (expressed in terms of SD) Reproducibility of a laboratory determination when it is run repeatedly under identical conditions No part of this material may be distributed and reproduced. The file is for your own use only. Standard Deviation Measure of the amount of variation or dispersion in a set of values No part of this material may be distributed and reproduced. The file is for your own use only. Standard Deviation Sample data set of tests scores: 85, 90, 78, 92, 88 Calculate the mean: find the sample mean = 85+90+78+92+88/5 = 433/5 = 86.6 Calculate each deviation from the mean and square it Find the deviation of each score from the mean and square the result (85-86.6)2 = (-1.6)2 = 2.56 (90-86.6)2 = (3.4)2 = 11.56 (78-86.6)2 = (-8.6)2 = 73.96 (92-86.6)2 = (5.4)2 = 29.16 (88-86.6)2 = (1.4)2 = 1.96 No part of this material may be distributed and reproduced. The file is for your own use only. Standard Deviation Sum the squared deviations Sum these squared deviations 2.56+11.56+73.96+29.16+1.96 = 119.2 Divide by the number of data points minus one (n-1) For a sample, we divide by n-1, where n is the number of data points: 119.2/5-1 = 119.2/4 = 29.8 Take the square root Finally, take the square root of this value to obtain the sample SD: s= √29.8 = 5.46 No part of this material may be distributed and reproduced. The file is for your own use only. Variance Expectation of the squared deviation of a random variable from its mean (measures how far a set of numbers are spread out from their average value) No part of this material may be distributed and reproduced. The file is for your own use only. Variance Calculate the Mean (86.6) Calculate each deviation from the mean and square it (85-86.6)2 = (-1.6)2 = 2.56 (90-86.6)2 = (3.4)2 = 11.56 (78-86.6)2 = (-8.6)2 = 73.96 (92-86.6)2 = (5.4)2 = 29.16 (88-86.6)2 = (1.4)2 = 1.96 No part of this material may be distributed and reproduced. The file is for your own use only. Variance Sum the squared deviations 2.56+11.56+73.96+29.16+1.96 = 119.2 Divide by the number of data points minus one (n-1) 119.2/5-1 =119.2/4 = 29.8 No part of this material may be distributed and reproduced. The file is for your own use only. Coefficient of Variation (CV) Normalized measure of the dispersion of the probability distribution Ratio of the standard deviation to the mean No part of this material may be distributed and reproduced. The file is for your own use only. Coefficient of Variation (CV) The sample mean = 86.6 The sample standard deviation = 5.46 Calculate CV CV = 5.46/86.6 x 100% = 6.30% No part of this material may be distributed and reproduced. The file is for your own use only. Descriptive Statistics: Measures of centers, Spread, and Shape No part of this material may be distributed and reproduced. The file is for your own use only. Measures of Center Three most used descriptions of the center of a date set: Mean Median Mode 1. Mean – commonly used and often called the average 2. Median – middles point and often used with skewed data (calculation is not significantly affected by outliers) 3. Mode – rarely used as a measure of data’s center (often used to describe data that seem to have 2 centers) No part of this material may be distributed and reproduced. The file is for your own use only. Mean Data set is calculated by summing all the values and then dividing by the number of values Formula for the mean is different depending on whether you are dealing with a population or a sample No part of this material may be distributed and reproduced. The file is for your own use only. Mean Example calculation: using the previous data set: 85, 90, 78, 92, 88 85+90+78+92+88/5 =433/5 =86.6 No part of this material may be distributed and reproduced. The file is for your own use only. Median Middle value of data set when it is arranged in ascending or descending order If the data set has an odd number of observations, the median is the middle number Example calculation: Previous data set: 85, 90, 78, 92, 88 Arrange the data: 78, 85, 88, 90, 92 Determine the middle position: 5 observations (5+1)/2 = 3 *the median is the 3rd value in the ordered list, which is 88 No part of this material may be distributed and reproduced. The file is for your own use only. Median If the data set has an even number of observations, the median is the average of the two middle numbers Example calculation: Data set: 78,85,90,92 Arrange the data: 78,85,90,92 Determine the middle position: 4 observation (4/2 = 2 ; (4/2)+1 = 3) *the median is the average of the second and third values in the ordered list: 85+90/2 = 175/2 = 87.5 No part of this material may be distributed and reproduced. The file is for your own use only. Mode Value that appears most frequently in data set Data set may have one mode, more than one mode, or no mode at all Types of Mode: Unimodal – data set with one mode Bimodal – data set with two mode Multimodal – data set with more than two modes No mode – data set where no value repeats No part of this material may be distributed and reproduced. The file is for your own use only. Mode Steps to find the mode 1. Count the Frequency – determine the frequency of each value in the data set 2. Identify the most frequent value – the value with the highest frequency is the mode Example Calculation: 85,90,78,92,88,90,85,90 Count the frequency 85 – 2 times 90 – 3 times 78 – 1 time 92 – 1 time 88 – 1 time No part of this material may be distributed and reproduced. The file is for your own use only. Mode Identify the most frequent value: 85 – 2 times 90 – 3 times 78 – 1 time 92 – 1 time 88 – 1 time Example with no mode: 85,90,78,92,88 Each value appears only once Example with multiple modes: 85,90,78,92,88,85,90 85 – 2 times 90 – 2 times 78 – 1 time 92 – 1 time 88 – 1 time *Bimodal No part of this material may be distributed and reproduced. The file is for your own use only. Control Specimens Standard – reference of the unknown, to achieve accuracy Control – to check for precision Blank – to achieve accuracy by setting the reading to zero Unknown – sample/specimen No part of this material may be distributed and reproduced. The file is for your own use only. Control Specimens Characteristics of control specimens: 1. Should behave like the real specimen 2. Should be available in sufficient quantity to last for a minimum of 1 year 3. Should be stable over a period of 1 year 4. Should be available in convenient vial volumes 5. Should vary minimally in concentration and composition from vial to vial 6. Should include clinically normal, high, and low abnormal ranges 7. Should be preferably lyophilized require reconstitution before use Sources: 1. Pooled control sera 2. Assayed commercial control 3. Unassayed commercial control No part of this material may be distributed and reproduced. The file is for your own use only. Precision Monitoring Techniques Levey-Jennings QC chart The control results are plotted on the ordinate versus time on the abscissa Random error shows a wider range of scatter of points on the control chart, while systematic error can be seen when the points drift or shift on one side of the central solid line Solid line indicates the mean of the control, dotted lines are the control limits (usually +/- 2SD) No part of this material may be distributed and reproduced. The file is for your own use only. Levey-Jennings QC chart Components of L-J QC chart 1. X-axis – represents time or the sequence of the control measurements 2. Y-axis – represents the measurement values of the quality control sample 3. Center Line – represents the mean (target) value of the control measurements 4. Control Limits – usually set at ±1, ±2, and ±3 standard deviations (SD) from the mean ±1 SD: warning limits ±2 SD: Action limits ±3 SD: Critical action limits No part of this material may be distributed and reproduced. The file is for your own use only. Levey-Jennings QC chart Creating a Levy-Jennings QC chart 1. Collect data: Obtain a series of control measurements over time 2. Calculate the Mean and SD 3. Determine Control Limits: ±1, ±2, and ±3 SD from the mean 4. Plot the Data: Plot the control measurements on the Y-axis against time or sequence on the X-axis ; Draw the mean line and the control limits (±1, ±2, and ±3 SD) No part of this material may be distributed and reproduced. The file is for your own use only. Levey-Jennings QC chart Interpretation of the Chart Within ±1 SD – indicates that the process is well within control Between ±1 SD and ±2 SD – indicates a warning, process may need to be monitored more closely Between ±2 SD and ±3 SD – indicates an action level, process may require adjustment Beyond ±3 SD – indicates a critical action level, process likely needs immediate attention No part of this material may be distributed and reproduced. The file is for your own use only. Levey-Jennings QC chart Example Calculation and Plot Control measurement data set: 98,102,100,101,99,103,97,104,96,100 Calculate the Mean: No part of this material may be distributed and reproduced. The file is for your own use only. Levey-Jennings QC chart Calculate the SD No part of this material may be distributed and reproduced. The file is for your own use only. Levey-Jennings QC chart Determine Control Limits No part of this material may be distributed and reproduced. The file is for your own use only. Levey-Jennings QC chart Plot the Data 106 104 102 100 98 96 94 92 1 2 3 4 5 6 7 8 9 10 No part of this material may be distributed and reproduced. The file is for your own use only.  Westgard QC Chart Developed by James O. Westgard Used extensively in clinical laboratories to monitor the performance of analytical tests and ensure the accuracy and reliability of laboratory results Designed to detect both random errors and systemic errors No part of this material may be distributed and reproduced. The file is for your own use only. Westgard QC Chart Components of Westgard QC chart 1. X-axis – represents time or the sequence of measurements 2. Y-axis – represents the measurement values of the quality control sample 3. Center Line – represents the mean (target) value of the control measurements 4. Control Limits 1s Control Limits: ±1 SD from the mean 2s Control Limits: ±2 SD from the mean 3s Control Limits: ±3 SD from the mean 2s Warning Limits: indicates a warning zone before reaching the 2s control limits R4s/R6s/R10s Rules: These are additional rules used to detect trends or shifts in data points over time No part of this material may be distributed and reproduced. The file is for your own use only. Westgard QC Chart Rules in Westgard QC chart 1. 1s,2s,3s Control Limits These limits help identify when a measurement falls outside expected variability (1s), indicating a potential issue 2. 2s Warning Limits These are typically set closer to the mean than the 2s control limits and serve as an early warning for potential issues 3. R4s, R6s, R10s Rules These rules are used to detect systematic shifts or trends in the QC data R4s – one control measurement falls beyond ±2s R6s – 2 consecutive control measurements fall beyond ±1s R10s – 9 consecutive control measurements fall on the same side of the mean No part of this material may be distributed and reproduced. The file is for your own use only. Westgard QC Chart Example: Suppose we have a series of daily control measurements for a specific analyte in a clinical laboratory 98,102,100,101,99,103,97,104,96,100 No part of this material may be distributed and reproduced. The file is for your own use only. Westgard QC Chart No part of this material may be distributed and reproduced. The file is for your own use only. Westgard QC Chart 106 104 102 100 98 96 94 92 1 2 3 4 5 6 7 8 9 10 No part of this material may be distributed and reproduced. The file is for your own use only. Westgard QC Chart Interpretation Points falling within the 1s control limits indicate normal variation Points beyond the 1s control limits but within the 2s control limits may indicate a need for closer monitoring Points beyond the 2s control limits indicate a significant deviation that requires investigation and corrective action No part of this material may be distributed and reproduced. The file is for your own use only. Indicators of Systemic error 1. Trend – because of reagent, increasing values passing through mean, reject on the 6th test 2. Shift – because of standard, stable on one side of the mean, reject on the 6th test Sensitivity – ability to measure minute concentration Specificity – ability to react with only one unknown No part of this material may be distributed and reproduced. The file is for your own use only. Pipetting Techniques Valerie Jane Casandra Ty Villarin, RMT, MS MLS Faculty, Department of Medical Technology Pipette Pipette (pipet) – laboratory equipment Pipetting is a fundamental laboratory technique used for transferring precise volumes of liquids Based on volume/capacity to dispense Micropipettes – dispense between 1- 1000 ul Macropipettes – dispense greater volume Types of Pipettes Air Displacement Pipette Used for aqueous solutions (include single-channel and multi- channel pipettes) Aspirating: When the plunger is pressed to the first stop, the piston inside the pipette moves downward, displacing air from the tip Upon releasing the plunger, a vacuum is created, drawing liquid into the tip up to the calibrated volume Dispensing: Pressing the plunger to the first stop dispenses the aspirated liquid from the tip Pressing the plunger to the second stop expels any residual liquid to ensure complete delivery Air Displacement Pipette Types: 1. Single- Channel Pipettes 2. Multi-Channel Pipettes 3. Adjustable-Volume Pipettes 4. Fixed-Volume Pipettes Positive Displacement Pipettes Used for viscous, volatile, or high-density liquids (have a direct piston mechanism that makes them more accurate for challenging liquids Aspirating: The piston is in direct contact with the liquid When the plunger is depressed, the piston moves down, displacing an equivalent volume of liquid into the capillary tip Dispensing: As the plunger is pressed again, the piston pushes the liquid out of the tip, ensuring complete delivery without the risk of residual liquid or air bubbles Positive Displacement Pipettes Types: 1. Manual Positive Displacement Pipettes 2. Electronic Positive Displacement Pipettes Pipettes and Color Coding Guidelines Select the right pipette and tip: Use a pipette appropriate for the volume you intend to transfer Choose the correct tip that fits the pipette securely to avoid leaks or inaccuracies Pre-rinse the pipette tip: Aspirate and dispense the liquid 2-3 times to condition the tip, especially when using new tips Proper tip attachment: Attach the tip firmly by pressing it onto the pipette Guidelines Aspirating technique: Press the plunger to the first stop before immersing the tip in the liquid Immerse the tip just below the surface of the liquid to avoid drawing air Release the plunger slowly and steadily to aspirate the liquid, ensuring no air bubbles are drawn into the tip Dispersing technique: Touch the tip to the side of the receiving container to prevent splashing Press the plunger smoothly to the first stop to dispense the liquid Press the plunger to the second stop to expel any remaining liquid in the tip Maintenance and Calibration 1. Regular Calibration Calibrate pipettes regularly according to the manufacturer’s instructions to ensure accuracy 2. Clean Pipettes Clean pipettes regularly to prevent contamination (follow manufacturer guidelines for cleaning procedure) 3. Proper Storage Store pipettes vertically on a stand to prevent damage to internal components and to avoid contamination Maintenance and Calibration 1. Regular Calibration Calibrate pipettes regularly according to the manufacturer’s instructions to ensure accuracy 2. Clean Pipettes Clean pipettes regularly to prevent contamination (follow manufacturer guidelines for cleaning procedure) 3. Proper Storage Store pipettes vertically on a stand to prevent damage to internal components and to avoid contamination Preparation of Solutions and Dilutions Valerie Jane Casandra Ty Villarin, RMT, MS MLS Faculty, Department of Medical Technology No part of this material may be distributed and reproduced. The file is for your own use only. Solutions Solutions - uniform homogenous mixture of two or more substances (solute and solvent) Standard solution - very precise solution (3-4 significant figures) ; used in quantitative analysis or an analytical procedure Saturated solution – contains the maximum amount of a particular solute that will dissolve at that temperature Supersaturated solution – a solution that contains more solute than equilibrium condition allow No part of this material may be distributed and reproduced. The file is for your own use only. Preparing Solutions Solutions of known concentration can be prepared in several ways depending on the nature of the analyte and/or the concentration required: Weighing out a solid material of known purity, dissolving it in a suitable solvent and diluting to the required volume Weighing out a liquid of known purity, dissolving it in a suitable solvent and diluting to the required volume Diluting a solution previously prepared in the laboratory Diluting solution from a chemical supplier No part of this material may be distributed and reproduced. The file is for your own use only. Weight Measurements Measuring mass using a top-loading balance 1. Turn on balance (0.0g) 2. Place weighing vessel on the balance pan (weighing paper, weigh boat) 3. Press tare button so that display reads 0.0g 4. Gently add the substance being weighed to the weight sample 5. Record mass No part of this material may be distributed and reproduced. The file is for your own use only. Weight Measurements Measuring mass using an analytical balance 1. Turn on balance (0.0000g) 2. Check the level indicator and do not lean on the table while weighing 3. Place weighing vessel on the balance pan 4. Close the sliding door and wait for stability light indicator 5. Press tare button 6. Gently add the substance being weighed 7. Record mass No part of this material may be distributed and reproduced. The file is for your own use only. Units of Measure No part of this material may be distributed and reproduced. The file is for your own use only. Units of Measure No part of this material may be distributed and reproduced. The file is for your own use only. Reagents Ready - to - use form (kit) Specific type of chemical or compound that is added to a system to cause a chemical reaction or to test if a reaction occurs High purity Stored under controlled conditions to maintain their purity and effectiveness No part of this material may be distributed and reproduced. The file is for your own use only. Chemicals Analytic chemicals exist in varying grades of purity, analytic reagent (AR), ultrapure, chemically pure (PR), United States Pharmacopeia (USP), National Formulary (NF), technical or commercial grade MSDS (guidelines for safe handling and storage No part of this material may be distributed and reproduced. The file is for your own use only. Concentration Percent solution – expressed as the amount of solute per 100 total units of solution 3 expressions: weight / weight (w/w) volume / weight (v/w) weight / volume (w/v) No part of this material may be distributed and reproduced. The file is for your own use only. Concentration Weight per weight (% w/w) – refers to the number of grams of solute per 100 g of solution Volume per volume (% v/v) – used for liquid solutes and milliliters of solutes in 100 mL of solution ; recommended that g/dL be used Weight per volume (%w/v) – most used percent solution in CL; number of grams of solutes in 100 mL of solution No part of this material may be distributed and reproduced. The file is for your own use only. Molarity (M) Expressed as the number of moles per 1 L of solution 1 mole of substance equals its grams molecular weight (gmw) moles/liter No part of this material may be distributed and reproduced. The file is for your own use only. Molarity (M) No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of M No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of M You dissolve 5.85 grams of Sodium chloride (NaCl) in enough water to make 0.500 liters of solution. Calculate the M of the NaCl solution. 1. Calculate the molar mass of NaCl Na: 22.99g/mol + Cl 35.45g/mol = 58.44g/mol 2. Calculate the number of moles NaCl n = 5.85g/58.44g/mol = 0.100 mol 3. Calculate the molarity M = 0.100 mol/0.500 L = 0.200 M No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of M You dissolve 10 grams of Potassium chloride (KCl) in enough water to make 0.750 liters of solution. Calculate the M of the KCl solution. 1. Calculate the molar mass of KCl K: 39.10g/mol + Cl 35.45g/mol = 58.44g/mol 2. Calculate the number of moles NaCl n = 10.0g/74.55g/mol = 0.134 mol 3. Calculate the molarity M = 0.134 mol/0.750 L = 0.179 M No part of this material may be distributed and reproduced. The file is for your own use only. Molality (m) Represents the amount of solute per 1kg of solvent Can be easily distinguished from M because molality is always expressed in terms of moles per kilogram (w/w) moles/kilogram (mol/kg) Not influenced by temperature or pressure (based on mass rather than volume) No part of this material may be distributed and reproduced. The file is for your own use only. Molality (m) Represents the amount of solute per 1kg of solvent Can be easily distinguished from M because molality is always expressed in terms of moles per kilogram (w/w) moles/kilogram (mol/kg) Not influenced by temperature or pressure (based on mass rather than volume) No part of this material may be distributed and reproduced. The file is for your own use only. Molality (m) No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of m No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of m You dissolve 10.0 grams of Sodium chloride (NaCl) in 200 grams (0.200kg) of water. Calculate the m of the NaCl solution. 1. Calculate the molar mass of NaCl Na: 22.99g/mol + Cl 35.45g/mol = 58.44g/mol 2. Calculate the number of moles NaCl n = 10.0g/58.44g/mol = 0.171 mol 3. Measure the mass of the solvent (water) msolvent = 0.200kg 4. Calculate the molality m = 0.171 mol/0.200kg = 0.855 mol/kg No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of m You dissolve 15.0 grams of glucose (C6H12O6) in 250 grams (0.250kg) of water. Calculate the molality of the glucose solution. 1. Calculate the Molar Mass of Glucose Molar mass of C6H12O6: 6 x 12.01 g/mol + 12 x 1.01 g/mol + 6 x 16.00 g/mol = 180.18 g/mol No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of m 2. Calculate the number of moles of glucose n = mass of glucose / molar mass of glucose n = 15.0g / 180.18 g/mol n = 0.0832 mol No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of m 3. Measure the Mass of solvent (water) 250 g = 0.250 kg 4. Calculate the Molality (m) 0.0832 mol / 0.250 kg = 0.333 mol / kg No part of this material may be distributed and reproduced. The file is for your own use only. Normality (N) Often used in chemical titrations and chemical reagent classification Number of gram equivalent weights per 1 L solution Equivalent weight (gmw) of a substance divided by its valence Valence is the number of units that can combine with or replace 1 mole of hydrogen ions and hydroxyl ions Equal to or greater than M of the compound No part of this material may be distributed and reproduced. The file is for your own use only. Normality (N) No part of this material may be distributed and reproduced. The file is for your own use only.  Normality (N) The equivalent factor (f) depends on the type of reaction: For acids: The number of H+ ions provided by one molecule of the acid For bases: The number of OH- ions provided by one molecule of the base For redox reactions: The number of electrons exchanged per molecule of the substance For precipitation reactions: The number of ions that participate in the reaction No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of N Calculate the number of equivalents neq neq = n x f Where n is the number of moles of the solute Calculate the N N = neq / V solution No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of N You dissolve 4.9g of sulfuric acid (H2SO4) in enough water to make 250 mL of solution. Calculate the normality of the H2SO4 solution. Determine the Molar Mass of H2SO4 Molar mass of H2SO4 2 x 1.01 g/mol + 32.07 g/mol + 4 x 16.00 g/mol = 98.09 g/mol No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of N Calculate the number of Moles of H2SO4 H2SO4 provides 2 H+ ions per molecule f=2 Calculate the number of equivalents (neq) neq = n x f 0.05 mol x 2 = 0.10 eq No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of N Calculate the Normality Volume of solution (Vsolution) – 250 mL = 0.250 L N = 0.10 / 0.250 L = 0.40 N No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of N You dissolve 8.0 g of Sodium hydroxide (NaOH) in enough water to make 500 mL of solution. Calculate the normality of the NaOH solution. Calculate the Molar mass of NaOH Na 22.99 g/mol + O 16.00 g/mol + 1.01 g /mol = 40.00 g/mol No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of N Calculate the number of moles of NaOH n = 8.0g / 40.00 g/mol = 0.20 mol f=1 Calculate the neq neq = 0.20 mol x 1 = 0.20eq No part of this material may be distributed and reproduced. The file is for your own use only. Calculation of N Convert the volume of solution to L 500 mL / 1000 L = 0.500 L Calculate the N N = 0.20 eq / 0.500 L = 0.40 N No part of this material may be distributed and reproduced. The file is for your own use only. Preparing Dilute Solutions from Concentrated The equation C1V1 = C2V2 is commonly used in dilution calculations. It expresses the relationship between the concentrations and volumes of solution before and after dilution. No part of this material may be distributed and reproduced. The file is for your own use only. Preparing Dilute Solutions from Concentrated No part of this material may be distributed and reproduced. The file is for your own use only. Calculation Suppose you have 50 mL of a 2.0M NaCl solution, and you want to dilute it to a concentration of 0.5M. How much water should you add to achieve this concentration? Identify the known quantities: C1 = 2.0M V1 = 50 mL C2 = 0.5M V2 = ? No part of this material may be distributed and reproduced. The file is for your own use only. Calculation Plug in the known values: 2.0M x 50 mL = 0.5M x V2 Solve for V2: V2 = 2.0 M X 50 ML / 0.5 M V2 = 100 MML V2 = 200 ML No part of this material may be distributed and reproduced. The file is for your own use only. Calculation Calculate the amount of water to add: Since the final volume of V2 is 200mL and you started with 50mL of the solution, the amount of water to add is: Vwater = V2 – V1 = 200 mL – 50 mL = 150 mL No part of this material may be distributed and reproduced. The file is for your own use only. No part of this material may be distributed and reproduced. The file is for your own use only.