Clinical-Chemistry.pdf

Desiccant – a drying agent or substance capable of INTRODUCTION TO CLINICAL CHEMISTRY absorbing moisture Desiccator – sealed chamber in which samples...

Desiccant – a drying agent or substance capable of INTRODUCTION TO CLINICAL CHEMISTRY absorbing moisture Desiccator – sealed chamber in which samples can be dried in the presence of a desiccant. SCOPE OF CLINICAL CHEMISTRY Lipemia – milky coloration of plasma caused by increased lipid accumulation (Triglycerides) Branch of laboratory pathology that deals with various bodily fluids, mainly blood, among others Meniscus – upper and lower curved surface of a solution or (urine, various fluids, etc.) liquid Osmotic pressure – force that moves water or another Involved in pre-analytic and analytic processes and solvent across a membrane separating a solution. The procedures that aids in proper disease diagnosis. movement is from lower to higher concentration. Integrates and utilizes basic units of measures Plasma – a yellow bodily fluid from whole blood that (laboratory mathematics) and various laboratory should be clear in appearance. supplies and machines. Reagent – a formulated manufactured chemical mixture that is added to a sample to conduct various laboratory OVERVIEW AND IMPORTANCE tests. The primary purpose of a clinical chemistry laboratory Sample – patient specimen that is well-prepared, ready to is to facilitate the correct performance of analytic be utilized for processing and analysis. procedures that yield accurate and precise Solute – substance that gets dissolved in a solvent information, aiding patient diagnosis and treatment. Solvent – liquid that dissolves a solute in a solution. The achievement of reliable results requires that the clinical laboratory scientist be able to correctly use Specimen – type of biologic fluid in which the important basic supplies and equipment and possess an analyte is measured (ex. Serum, CSF, etc.) understanding of fundamental concepts critical to Titer – amount of antibody that is found in a specimen as any analytic procedure. a result of exposure to an antigen ROLE OF MEDICAL TECHNOLOGISTS LABORATORY SAFETY AND REGULATIONS Perform routine and advance laboratory tests using UNIVERSAL PRECAUTIONS standard laboratory methods. To be able to integrate and apply problem-solving The universal precautions concept is basically an techniques and strategies to approach to infection control in which all human supervisory/administrative, technical, and research blood, tissue, and most fluids are handled as if known problems. to be infectious for the human immunodeficiency virus To conduct community-based studies/research (HIV), hepatitis B virus (HBV), and other bloodborne alongside other medical professionals pathogens. The standard also provides fairly detailed directions for decontamination and the safe handling of DEFINITION OF TERMS potentially infectious laboratory supplies and Aliquot – measured portion of a sample equipment, including practices for managing laundry and infectious wastes. Analyte – substance that is being measured (ex. Glucose, Cholesterol, Lipase, etc.) Employee information and training are covered Buffer – a solution or liquid that resists change in pH regarding recognition of hazards and risk of when an acid or base is added. This consists of a weak acid infection. and its conjugate base. There is also a requirement for HBV vaccination or Control – serum-based material with assigned values that formal declination within 10 days of assuming duties is considered acceptable to evaluate the accuracy and that present exposure. In the event of an actual correctness of a diagnostic assay. exposure, the standard outlines the procedure for post exposure medical evaluation, counseling, and recommended testing or postexposure prophylaxis.  PROPER HANDWASHING Know and comply with the established laboratory safe work practices. PERSONAL PROTECTIVE EQUIPMENT IS A MUST Have a positive attitude toward supervisors, coworkers, TREAT ALL SPECIMENS AS INFECTIOUS facilities, and safety training. DISPOSAL OF SHARPS AND NEEDLES PROPERLY Be alert and give prompt notification of unsafe IN CASES OF SPILLAGE, PROMPT AND STANDARD conditions or practices to the immediate supervisor CLEANING MUST BE DONE and ensure that unsafe conditions and practices are corrected. SYSTEMATIC WASTE COLLECTION AND DISPOSAL LABORATORY SAFETY AND REGULATIONS Engage in the conduct of safe work practices and use of PPE. Clinical laboratory personnel are exposed daily to a variety of real or potential hazards: electric shock, toxic vapors, compressed gases, flammable liquids, SIGNAGE AND LABELING radioactive material, corrosive substances, mechanical Appropriate signs to identify hazards are critical, not trauma, poisons, and the inherent risks of handling only to alert laboratory personnel to potential biological materials. hazards but also to identify specific hazards that may Each clinician should develop an understanding of the arise because of emergencies such as fire or explosion. risks associated with these hazards and must be Manufacturers of laboratory chemicals also provide “safety conscious” at all times. precautionary labeling information for users. All in-house prepared reagents and solutions should be labeled in a standard manner and include the chemical identity, concentration, hazard warning, OCCUPATIONAL SAFETY AND HEALTH ACT (OSHA) special handling, storage conditions, date prepared, expiration date (if applicable), and preparer’s initials. To provide all employees (clinical laboratory personnel included) with a safe work environment. Under this legislation, the Occupational Safety and Health SAFETY EQUIPMENT Administration (also known as OSHA) is authorized to Safety equipment has been developed specifically for conduct on-site inspections to determine whether an use in the clinical laboratory. employer is complying with the mandatory standards. The employer is required by law to have designated Safety is no longer only a moral obligation but also a safety equipment available, but it is also the federal law. responsibility of the employee to comply with all OSHA Standards include Bloodborne Pathogens, safety rules and to use safety equipment. Formaldehyde, Respiratory Protection, Air All laboratories are required to have safety showers, Contaminants, and Personal Protective Equipment. eyewash stations, and fire extinguishers and to periodically test and inspect the equipment for proper SAFETY AWARENESS FOR CLINICAL LABORATORY operation. PERSONNEL Other items that must be available for personnel include fire blankets, spill kits, and first aid supplies. EMPLOYER’S RESPONSIBILITIES Mechanical pipetting devices must be used for manipulating all types of liquids in the laboratory, Establish laboratory work methods and safety policies. including water. Provide supervision and guidance to employees. Mouth pipetting is strictly prohibited. Provide safety information, training, PPE, and medical surveillance to employees. FUME HOODS Provide and maintain equipment and laboratory facilities that are free of recognized hazards and Fume hoods are required to contain and expel noxious and adequate for the tasks required. hazardous fumes from chemical reagents. Fume hoods should be visually inspected for blockages.  After removing gloves, hands should be washed BIOSAFETY CABINETS (BSCs) thoroughly with soap and warm water, even if glove breakthrough or contamination is not suspected. BSCs remove particles that may be harmful to the employee who is working with potentially infectious The use of antimicrobial soap is not as important as biologic specimens. the physical action of washing the hands with water and any mild soap. The CDC and the National Institutes of Health have described four levels of biosafety, which consist of After any work with highly toxic or carcinogenic combinations of laboratory practices and techniques, chemicals, the face should also be washed. safety equipment, and laboratory facilities. LABORATORY HAZARDS BIOLOGICAL CHEMICAL STORAGE EQUIPMENT All blood samples and other body fluids should be Safety equipment is available for the storage and collected, transported, handled, and processed using handling of hazardous chemicals and compressed universal precautions (i.e., presumed to be infectious). gases. PPE, Handwashing → strict observance! Safety carriers should always be used to transport Spills: Alert everyone, Wear PPE, Do not touch broken glass bottles of acids, alkalis, or organic solvents in glass, Absorb spill with disposable towels or tissue, volumes larger than 500 mL. Clean with common aqueous detergent, Disinfect (10% Steel safety cabinets with self-closing doors are bleach), Rinse spill site with water, Dispose properly required for the storage of flammable liquids, and Bloodborne Pathogens: Exposure control plan by only specially designed, explosion-proof refrigerators employer and adopting universal precaution at all may be used to store flammable materials. times Only the amount of chemical needed for that day Airborne Pathogens: TB Exposure Control Program, should be available at the bench. Other specific pathogens, wearing of proper PPE such as N-95 mask for respiratory protection. UNIVERSAL PRECAUTION, WEARING OF PPE, AND HANDWASHING PPE AND HYGIENE ARE REALLY IMPORTANT Safety glasses, goggles, visors, work shields protect the eyes and face from splashes and impact. CHEMICAL If any solution is accidentally splashed into the eye/s, thorough irrigation is required. Hazard Communication Standard - To ensure that health hazards are evaluated for all chemicals that Gloves and rubberized sleeves protect the hands and are produced and that this information is relayed to arms when using caustic chemicals. employees. Laboratory coats with knit-cuffed sleeves should be Safety Data Sheet - Major source of safety full length and buttoned and made of liquid=resistant information for employees who may use hazardous material. materials in their occupations. Proper footwear is required - closed shoes is a must- OSHA Laboratory Standard - Requires each laboratory wear. that uses hazardous chemicals to have a written Respirators may be required for various procedures in chemical hygiene plan. the clinical laboratory. (Ex. High-efficiency particulate Flammable/Combustible Chemicals (Ex. Acetone, air (HEPA) filters) Toluene, etc.) Training, maintenance, and written protocol for use of Corrosive Chemicals (Ex. Nitric acid, HCl etc.) - cause respirators are required according to the respiratory injury to the skin or eyes via direct contact, affects protection standard. the Respiratory/GI tract if inhaled or ingested. Hand washing is a crucial component of both Reactive Chemicals (Ex. Peroxide + Hydrogen, etc.) - infection control and chemical hygiene. spontaneously explode or ignite in certain conditions Carcinogenic Chemicals (Ex. Benzidine) - cancer- causing  Chemical Spills ELECTRICAL Use only explosion-rated (intrinsically wired) equipment in hazardous atmospheres Be careful when operating high-voltage equipment such as electrophoresis apparatuses Use only properly grounded equipment (three-prong plug). Check for frayed electrical cords Report ASAP any machine malfunctions Do not work on “live” electrical equipment Never operate electrical equipment with wet hands. RADIATION Know the exact location of the electrical control panel A radiation safety policy should include environmental for the electricity to your work area. and personnel protection. Use only approved extension cords in temporary All areas where radioactive materials are used or applications and do not overload circuits. stored must be posted with caution signs, and traffic Have ground, polarity, and leakage checks and other in these areas should be restricted to essential periodic preventive maintenance performed on outlets personnel only. and equipment. Radiation monitors must be evaluated regularly to detect degree of exposure for the laboratory employee. FIRE Fires have been divided into four classes based on the nature of the combustible material and requirements for extinguishment: Class A: ordinary combustible solid materials, such as paper, wood, plastic, and fabric Class B: flammable liquids/gases and combustible petroleum products OTHERS Class C: energized electrical equipment Compressed Gases Hazards - danger of fire, explosion, asphyxiation, or mechanical injuries. Class D: combustible/reactive metals, such as magnesium, sodium, and potassium Cryogenic Materials Hazards - hazards of pressure buildup, thermal burns, etc. (Ex. Liquid nitrogen, a cryogenic fluid - liquefied gas) Mechanical Hazards - hazards of equipment (centrifuge, autoclave, glasswares, etc.) Ergonomic Hazards - hazards due to required repeated manipulation of instruments, containers, and equipment. (Musculoskeletal injury due to lifting heavy objects, etc.) LABORATORY WASTE MANAGEMENT SEGREGATION  Other liquid wastes, including flammable solvents, must Needles should not be transported, recapped, bent, or be collected in approved containers and segregated broken by hand. into compatible classes. All biomedical waste must then be disposed of If practical, solvents such as xylene and acetone may according to one of the recommended procedures. be filtered or re-distilled for reuse. Highly pathogenic waste should undergo preliminary If recycling is not feasible, disposal arrangements treatment on-site. should be made by specifically trained personnel Potentially biohazardous material, such as blood or blood products and contaminated laboratory waste, STORAGE OF HAZARDOUS MATERIALS & CHEMICALS cannot be directly discarded. Only use appropriate containers for the storage of Contaminated combustible waste can be incinerated. waste materials. Contaminated non-combustible waste, such as Peroxide-forming chemicals should be stored away glassware, should be autoclaved before being from light and heat with tightly secured caps and discarded. labeled with dates of receipt and opening. Special attention should be given to the discarding of Unwanted lecture bottles should be removed from the syringes, needles, and broken glass that could also laboratory when they are no longer needed as they inflict accidental cuts or punctures. present a genuine concern for long-term storage and management. ACCIDENT DOCUMENTATION AND INVESTIGATION Any accidents involving personal injuries, even minor ones, should be reported immediately to a supervisor. TREATMENT & DISPOSAL OF HAZARDOUS MATERIALS Manifestation of occupational illnesses and exposures The safe handling and disposal of chemicals and to hazardous substances should also be reported. other materials require a thorough knowledge of their The investigation report should include information on properties and hazards. the injured person, a description of what happened, Generators of hazardous wastes have a moral and the cause of the accident (environmental or personal), legal responsibility, to protect both the individual and other contributing factors, witnesses, the nature of the the environment when disposing of waste. injury, and actions to be taken to prevent a recurrence. There are four basic waste disposal techniques: flushing down the drain to the sewer system, incineration, landfill burial, and recycling. SPECIMEN COLLECTION AND PROCESSING SPECIMEN DISPOSAL OF MEDICAL WASTE The approved methods for treatment and disposition BLOOD of medical waste are incineration, steam sterilization, phlebotomy, or venipuncture, is the act of obtaining a burial, thermal inactivation, chemical disinfection, or blood sample from a vein using a needle attached to a encapsulation in a solid matrix. syringe or a stoppered evacuated tube. All biomedical waste should be placed in a bag marked these tubes come in different volume sizes: from with the biohazard symbol and then placed into a pediatric sizes (≈150 μl) to larger 7 ml tubes. The leakproof container that is puncture resistant and most frequent site for venipuncture is the antecubital equipped with a solid, tight-fitting lid. vein of the arm. All containers must be clearly marked with the word a tourniquet made of pliable rubber tubing or a strip biohazard or its symbol. with hook and loop tape (velcro) at the end is wrapped All sharp instruments, such as needles, blades, and around the arm, causing cessation of blood flow and glass objects, should be placed into special puncture dilation of the veins, making them easier to detect. resistant containers before placing them inside the bag and container.  Sites adjacent to IV therapy should be avoided; however, if both arms are involved in IV therapy and the IV cannot be discontinued for a short time, a site below the IV site should be sought. The initial sample drawn (5 mL) should be discarded because it is most likely contaminated with IV fluid and only subsequent sample tubes should be used for analytic purposes. Analytic testing of blood involves the use of whole blood, serum, or plasma. Whole blood, as the name implies, uses both the liquid portion of the blood called plasma and the cellular components (red blood cells, white blood cells, and platelets). This requires blood collection into a vessel containing an anticoagulant. Complete mixing of the blood immediately following venipuncture is necessary to ensure the anticoagulant can adequately inhibit the blood’s clotting factors. As whole blood sits, the cells fall toward the bottom, leaving a clear yellow supernatant on top called plasma. If a tube does not contain an anticoagulant, the blood’s clotting factors are active to form a clot incorporating the cells. The clot is encapsulated by the large protein The gauge of the needle is inversely related to the size fibrinogen. of the needle; the larger the number, the smaller the needle bore and length. The remaining liquid is called serum rather than plasma. An intravenous (iv) infusion set, sometimes referred to as a butterfly because of the appearance of the setup, The major difference between plasma and serum is may be used whenever the veins are fragile, small, or that serum does not contain fibrinogen (i.e., there is hard to reach or find. less protein in serum than plasma) and some potassium is released from platelets (serum potassium The butterfly is attached to a piece of tubing, which is is slightly higher in serum than in plasma). then attached to either a hub or a tube. Because of potential needle sticks, this practice may be It is important that serum samples be allowed to discouraged. completely clot (20 minutes) before being centrifuged.  Plasma samples also require centrifugation but do not URINE need to allow for clotting time and their use can Urine is the next most common fluid for decrease turnaround time for reporting results. determination. Most quantitative analyses of urine require a timed sample (usually 24 hours); a complete sample (all urine must be collected in the specified time) can be difficult because many timed samples are collected by the patient in an outpatient situation. OTHERS Other body fluids analyzed by the clinical chemistry laboratory include paracentesis fluids (pleural, pericardial, and peritoneal), and amniotic fluids. The color and characteristics of the fluid before centrifugation should be noted for these samples. CENTRIFUGATION OF BLOOD SAMPLE TUBES Before centrifugation, a laboratorian should also verify that the sample is designated for clinical Centrifugation of the sample accelerates the process chemistry analysis only because a single fluid sample of separating the plasma and cells. may be shared among several departments (i.e., Specimens should be centrifuged for approximately 10 hematology or microbiology) and centrifugation could minutes at an RCF of 1,000g to 2,000g but should invalidate certain tests in those areas. avoid mechanical destruction of red cells that can result in hemoglobin release, called hemolysis. COLLECTION AND LABELING ARTERIAL BLOOD Proper patient identification is the first step in sample Arterial blood samples measure blood gases (partial collection. pressures of oxygen and carbon dioxide) and pH. The importance of using the proper collection tube, Syringes are used instead of evacuated tubes because avoiding prolonged tourniquet application, drawing of the pressure in an arterial blood vessel. tubes in the proper order, and proper labeling of tubes The radial, brachial, and femoral arteries are the cannot be stressed strongly enough. primary arterial sites. Prolonged tourniquet application causes a stasis of Arterial punctures are more difficult to perform blood flow and an increase in hemoconcentration and because of inherent arterial pressure, difficulty in anything bound to proteins or the cells. stopping bleeding afterward, and the undesirable Having patients open and close their fist during development of a hematoma, which cuts off the blood phlebotomy is of no value and may cause an increase supply to the surrounding tissue. in potassium and, therefore, should be avoided. ARTERIAL PUNCTURE IS ONLY DONE BY PHYSICIANS & TRAINED MEDICAL PERSONNEL Handling, Transport Processing, CEREBROSPINAL FLUID Storage, and Preservation CSF is an ultrafiltrate of the plasma and will, ordinarily, reflect the values seen in the plasma. For SAMPLE PROCESSING (HANDLING) glucose and protein analysis (total and specific proteins), it is recommended that a blood sample be When samples arrive in the laboratory, they are analyzed concurrently with the analysis of those processed. analytes in the CSF. In the clinical chemistry laboratory, this means This will assist in determining the clinical utility of the correctly matching the blood collection tube(s) with values obtained on the CSF sample. the appropriate analyte request and patient identification labels.  This is a particularly sensitive area of preanalytic error. Barcode labels on primary sample tubes are a popular means to detect errors and to minimize clerical errors at this point of the processing. SAMPLE PROCESSING (TRANSPORT AND STORAGE) Once processed, the laboratory scientist should note the presence of any serum or plasma characteristics such as hemolysis and icterus (increased bilirubin pigment) or the presence of turbidity often associated with lipemia (increased lipids). Samples should be analyzed within 4 hours, and kept away from areas of rapid airflow, light, and heat. If testing is to occur after a later time (such as in PRE-ANALYTICAL send-out cases to other labs), samples should be appropriately stored. PRE-ANALYTICAL VARIABLES For most, this means refrigeration at 4°C for 8 hours. 1. Assay selection based on patient indication SAMPLE PROCESSING (PRESERVATION) 2. Implementation of assay selection 3. Patient identification and preparation Many analytes are stable at refrigerator temperature (4°C), with the exception of alkaline phosphatase 4. Specimen collection equipment and technique (increases) and lactate dehydrogenase (decreases as a 5. Specimen transport, preparation, and storage result of temperature labile fractions four and five). 6. Monitoring of specimen condition Samples may be frozen at -20°C and stored for longer periods without deleterious effects on the results. PATIENT IDENTIFICATION AND PREPARATION Repeated cycles of freezing and thawing, like those The preanalytic considerations start with proper that occur in so-called frost-free freezers, should be patient identification, which is absolutely essential avoided. before any blood specimen is collected. Appropriate specimen tubes and containers must SPECIMEN VARIABLES be identified and prepared before specimen collection takes place (including other laboratory SAMPLE VARIABLES materials needed for the procedure.) Sample variables include physiologic considerations, Once collected, the specimen must be correctly proper patient preparation, and problems in labeled and accompanied by accurate collection, transportation, processing, and storage. information, needed for result interpretation. Physiologic variation refers to changes that occur SPECIMEN CONSIDERATIONS within the body, such as cyclic changes (diurnal or circadian variation) or those resulting from exercise, The process of specimen collection, handling, and diet, stress, gender, age, underlying medical conditions processing remains one of the primary areas of (e.g., fever, asthma, and obesity), drugs, or posture. preanalytical error. Most samples are drawn on patients who are fasting Careful attention to each phase is necessary to (usually overnight for at least 8 hours). ensure proper subsequent testing and reporting of meaningful results. All accreditation agencies require laboratories to clearly define and delineate the procedures used  for proper collection, transport, and processing of ANTICOAGULANTS AND patient samples and the steps used to minimize PRESERVATIVES and detect any errors, along with the documentation of the resolution of any errors. The best practice in avoiding many of the preanalytic errors is to analyze the sample as quickly as possible. The CLSI guidelines advocate samples be kept at room temperature and analyzed in less than 30 minutes. Consideration should be given to the additional sources of preanalytical errors for samples that are to be analyzed on multi-analyte instruments. ORDER OF DRAW 7. Patient outcome 1. Yellow Top - Blood Culture 2. Light Blue Top - Citrate 3. Red Top - Serum 4. Green Top - Heparin 5. Lavender Top - EDTA 6. Gray Top - Sodium Fluoride/ Potassium Oxalate ANALYTICAL CONTENT OF LABORATORY RESULTS/REPORTS ANALYTICAL VARIABLES 1. Laboratory staff competence 2. Assay and instrument selection 3. Assay and instrument validation, including linearity, accuracy, precision, analytical measurement range (AMR), and specificity 4. Internal quality control 5. External quality assessment ANALYTICAL VARIABLES IN A GLANCE Analytical variables occur during actual testing of the specimen. Performance of tests in the laboratory is rigorously controlled, with quality control procedures in place that markedly reduce errors in the analytic phase of testing. Differences in the methods for performing tests in different laboratories may yield slight differences in reported results or normal ranges, so check reference ranges given for the test. POST-ANALYTICAL POST-ANALYTICAL VARIABLES 1. Accurate transcription and filing of results 2. Content and format of laboratory report, narrative report 3. Reference interval (RI) and therapeutic range 4. Timeliness in communicating critical values 5. Patient and physician satisfaction 6. Turnaround time; cost analysis; physician application of laboratory results  UNITS OF MEASURE AND UNIT CONVERSIONS All nonzero digits are significant. ○ For example, the value 575.8 has four significant UNITS OF MEASURE figures. Any meaningful quantitative laboratory result consists All zeros that are found between nonzero digits are of two components: the first component represents the significant. number related to the actual test value, and the second is a label identifying the units. ALWAYS WRITE THE UNIT. ○ Thus, the number 30,009, with three 0s between the 3 and 9, has a total of five significant figures. The Système International d’Unités (SI) is the unit preferred in scientific literature and clinical laboratories Leading zeros (to the left of the first nonzero digit) are and is the only system employed in many countries. not significant. Lab Results usually contain CONVENTIONAL UNITS and SI ○ A value such as 0.0019, for example, has two UNITS significant figures because the 0s before the 1 are placeholders and are not significant. Trailing zeros to the right of the decimal place are significant. ○ Example: 95.000 has a total of five significant figures, since the 0s after the decimal place have been measured to be zeros, indicating they are as significant as any other nonzero digit. LOGARITHMS The base 10 logarithm (log) of a positive number N greater than zero is equal to the exponent to which 10 must be raised to produce N. Therefore, it can be stated that N equals 10x, and the log of N is equal to x. The number N is the antilogarithm (antilog) of x. SIGNIFICANT FIGURES The minimum number of digits needed to express a particular value in scientific notation without loss of accuracy.  PERCENT SOLUTIONS MOLARITY A percent solution is determined in the same manner regardless of whether weight/weight, volume/volume, or weight/volume units are used. Percent implies “parts per 100,” which is represented as percent (%) and is independent of the molecular weight of a substance. (Mole = g/mw) Molarity (M) is routinely expressed in units of moles per liter (mol/L) or sometimes millimoles per milliliter (mmol/mL). Remember that 1 mol of a substance is equal to the gmw of that substance. The final units will be moles per liter (mol/L) or millimoles per milliliter (mmol/mL). While molarity is given in these examples, the approach for molality is the same except that one molal is expressed as one mole of solute per kilogram of solvent. For water, one kilogram is proportional to one liter, so molarity and molality are equivalent. NORMALITY Normality (N) is expressed as the number of equivalent weights per liter (Eq/L) or milliequivalents per milliliter (mmol/mL). Equivalent weight is equal to gmw divided by the valence (V). Normality has often been used in acid–base calculations because an equivalent weight of a substance is also equal to its combining weight. DILUTIONS A dilution represents the ratio of concentrated or stock SERIAL DILUTIONS material to the total final volume of a solution and A serial dilution may be defined as multiple progressive consists of the volume or weight of the concentrate plus dilutions ranging from more concentrated solutions to less the volume of the diluent, with the concentration units concentrated solutions. remaining the same. Serial dilutions are extremely useful when the volume of This ratio of concentrated or stock solution to the total concentrate or diluent is in short supply and needs to be solution volume equals the dilution factor. minimized or a number of dilutions are required, such as in The relationship of the dilution factor to concentration determining a titer. is an inverse one; thus, the dilution factor increases as the The serial dilution is initially made in the same manner concentration decreases. as a simple dilution. To determine the dilution factor, simply take the concentration needed and divide by the stock concentration, leaving it in a reduced-fraction form.  Subsequent dilutions will then be made from each ONE-POINT CALCULATION (CALIBRATION) preceding dilution. Refers to the calculation of the comparison of the known standard/calibrator concentration and its corresponding absorbance to the absorbance of the unknown value according to the following ratio: pH GRAPHING AND BEER’S LAW A lowercase p in front of certain letters or abbreviations BEER’S LAW operationally means the “negative logarithm of” or The Beer-Lambert law (Beer’s law) mathematically “inverse log of” that substance. establishes the relationship between concentration and In keeping with this convention, the term pH represents absorbance in many photometric determinations. the negative or inverse log of the hydrogen ion A = abc concentration. Where A is absorbance; a is the absorptivity constant for Mathematically, pH is expressed as: a particular compound at a given wavelength under specified conditions of temperature, pH, and so on; b is the length of the light path; and c is the concentration. Assays measuring absorbance generally obtain the concentration results by using a Beer’s law graph, known as a standard graph or curve. This graph is made by plotting absorbance versus the Where [H+] equals the concentration of hydrogen ions in concentration of known standards. moles per liter. Because most photometric assays set the initial The pH scale ranges from 0 to 14 and is a convenient absorbance to zero (0) using a reagent blank, the initial way to express hydrogen ion concentration. data points are 0,0. Graphs should be labeled properly and the HENDERSON-HASSELBALCH EQUATION concentration units must be given. Henderson-Hasselbalch equation mathematically The horizontal axis is referred to as the x-axis, whereas describes the dissociation characteristics of weak acids the vertical line is the y-axis. (pKa) and bases (pKb) and the effect on pH: pOH Measure of hydroxide ion (OH-) Mathematically expressed as: