Chemistry Theory.docx

Table of Contents {#table-of-contents.TOCHeading} ================= [Urinalysis 5](#urinalysis) [Urinalysis Overview 5](#urinalysis-overview) [Specimen Collection 5](#specimen-collection) [Types of Specimens 5](#types-of-specimens) [Random Specimens 5](#random-specimens) [First Morning Specimen 5](#first-morning-specimen) [Timed Specimen 5](#timed-specimen) [24hr Urine Specimen 6](#hr-urine-specimen) [Clean Catch Specimen 6](#clean-catch-specimen) [Catheter Specimen 6](#catheter-specimen) [Suprapubic Specimen 6](#suprapubic-specimen) [Urinalysis Requisition 6](#urinalysis-requisition) [Specimen Transportation and storage 7](#specimen-transportation-and-storage) [Transportation 7](#transportation) [Storage: REFRIGERATION 7](#storage-refrigeration) [Storage: Preservatives 7](#storage-preservatives) [SPecimen Preservation 7](#specimen-preservation) [Boric Acid 7](#boric-acid) [Other preservatives 8](#other-preservatives) [Specimen Requirements/acceptability 8](#specimen-requirementsacceptability) [Criteria for specimen collection 8](#criteria-for-specimen-collection) [Delayed Specimens 9](#delayed-specimens) [Examination of urine specimens 9](#examination-of-urine-specimens) [physical examination 9](#physical-examination) [Urinalysis: dipstick analysis 11](#urinalysis-dipstick-analysis) [Chemical examination 11](#chemical-examination) [Reagent Test Strips 11](#reagent-test-strips) [Reagent Strip Result Interpretation 12](#reagent-strip-result-interpretation) [pH 12](#ph) [Protein 12](#protein) [Glucose and Other Reducing Substances 13](#glucose-and-other-reducing-substances) [Ketones 15](#ketones) [Occult Blood 16](#occult-blood) [Bilirubin and Urobilinogen 17](#bilirubin-and-urobilinogen) [Nitrite 19](#nitrite) [LEUKOCYTES 20](#leukocytes) [Microscopy 20](#microscopy) [Identifying the need for MICROSCOPIC samples 21](#identifying-the-need-for-microscopic-samples) [Low Power 21](#low-power) [High Power 21](#high-power) [Point of Care Systems 22](#point-of-care-systems) [POCT (point of care testing) overview 22](#poct-point-of-care-testing-overview) [Characteristics of POCT DEVICES 22](#characteristics-of-poct-devices) [ADVANTAGES of POCT 23](#advantages-of-poct) [Disadvantages of POCT 23](#disadvantages-of-poct) [Functional Context of POCT 23](#functional-context-of-poct) [Technological Context os POCT 23](#technological-context-os-poct) [Accuracy rEQUIREMENTS of POCT 23](#accuracy-requirements-of-poct) [POCT Staff 24](#poct-staff) [CLIA 24](#clia) [Laboratory Informatics 24](#laboratory-informatics) [POCT Tests 25](#poct-tests) [Glucometers 25](#glucometers) [Non-Instrument BAsed POCT 25](#non-instrument-based-poct) [Automation 27](#automation) [Overview 27](#overview) [Factors that Drive Automation 27](#factors-that-drive-automation) [Benefits of Automation 27](#benefits-of-automation) [Automation Analyzer Types 27](#automation-analyzer-types) [Sequential vs. Discrete 27](#sequential-vs.-discrete) [Wet Chem vs. Dry Chem 27](#wet-chem-vs.-dry-chem) [Specimen SEPARATION to Recording 28](#specimen-separation-to-recording) [Common Chemistry Tests and Ranges 28](#common-chemistry-tests-and-ranges) [CUSTOMIZABLE Panels 28](#customizable-panels) [Special Considerations 29](#special-considerations) [Analytical Techniques 30](#analytical-techniques) [Spectrophotometry and Photometry 30](#spectrophotometry-and-photometry) [Components of a Spectrophotometer 30](#components-of-a-spectrophotometer) [Radiant Energy 31](#radiant-energy) [Quality Assurance in Spectroscopy 31](#quality-assurance-in-spectroscopy) [Wavelength accuracy 31](#wavelength-accuracy) [Reflectometry 32](#reflectometry) [Reflectometer 32](#reflectometer) [Reflectance vs. Concentration 33](#reflectance-vs.-concentration) [Fluorometry 33](#fluorometry) [Instrumentation 33](#instrumentation) [TURBIDIMETRY 33](#turbidimetry) [Nephelometry 34](#nephelometry) [Chemiluminescence 34](#chemiluminescence) [Electrochemistry 34](#electrochemistry) [Potentiometry 35](#potentiometry) [Reference Electrodes 35](#reference-electrodes) [Ion Selective Electrode 35](#ion-selective-electrode) [pH Electrode 35](#ph-electrode) [Amperometry 36](#amperometry) [Amperometry and Chloride 36](#amperometry-and-chloride) [PO2 Gas-Sensing Electrode 36](#po2-gas-sensing-electrode) [Osmometry 37](#osmometry) [Osmolality 38](#osmolality) [Techniques 38](#techniques) [Quality COntrol 40](#quality-control) [Lean vs. Six Sigma 40](#lean-vs.-six-sigma) [Sigma Metrics 40](#sigma-metrics) [Sigma and QC 40](#sigma-and-qc) [Method EVALUATION 42](#method-evaluation) [Method Selection 42](#method-selection) [Estimation of Inaccuracy 43](#estimation-of-inaccuracy) [Carryover 44](#carryover) [Reference Intervals 44](#reference-intervals) [Diagnostic EFFICIENCY 44](#diagnostic-efficiency) [Chemistry QC 46](#chemistry-qc) [QC Statistics 46](#qc-statistics) [Standard Deviation 46](#standard-deviation) [Confidence Intervals 46](#confidence-intervals) [Coefficient of Variation 47](#coefficient-of-variation) [Determination of Control Range 47](#determination-of-control-range) [Control Solution Assessment 47](#control-solution-assessment) [Method for Establishing Range 47](#method-for-establishing-range) [Calculating ACCEPTABLE Limits 47](#calculating-acceptable-limits) [CONTROl Implementation 47](#control-implementation) [Steps 48](#steps) [Sources of Variance and Error 48](#sources-of-variance-and-error) [Levey-Jennings Charts 48](#levey-jennings-charts) [Quality Control Multi-Rules 50](#quality-control-multi-rules) [Chromatography 52](#chromatography) [Chromatography Overview 52](#chromatography-overview) [Chromatograms 52](#chromatograms) [Methods of Separation 53](#methods-of-separation) [ion Exchange Chromatography 54](#ion-exchange-chromatography) [Partition Chromatography 54](#partition-chromatography) [ADSORPTION Chromatography 55](#adsorption-chromatography) [Size-Exclusion Chromatography 55](#size-exclusion-chromatography) [HPLC -- High Pressure Liquid Chromatography 55](#hplc-high-pressure-liquid-chromatography) [Gas CHROMATOGRAPHY 55](#gas-chromatography) [COmponents 55](#components) [Process 56](#process) [Analyte Identification 56](#analyte-identification) [Liquid Chromatography Advantages 58](#liquid-chromatography-advantages) [Mass Spectrometry 58](#mass-spectrometry) [Clinical Applications 58](#clinical-applications) [Sample Introduction and Ionization 59](#sample-introduction-and-ionization) [Atomic Mass Spectrophotometry Steps 59](#atomic-mass-spectrophotometry-steps) [Components of Mass Spec 60](#components-of-mass-spec) [Liquid Chromatography-Mass Spectrometry 61](#liquid-chromatography-mass-spectrometry) [MALDI-TOF and SELDI-TOF 61](#maldi-tof-and-seldi-tof) Urinalysis ========== Urinalysis Overview ------------------- **Urinalysis** is the testing of urine with procedures commonly performed in an expeditious, reliable, accurate, safe, and cost-effective manner and includes: Macroscopic evaluation (colour, clarity) Physical measurements (volume, specific gravity) Chemical reagent or strip testing Microscopic examination **Reasons For Urinalysis Testing** To aid in the diagnosis of disease To screen a population for asymptomatic, congenital or hereditary diseases To monitor progress of disease Monitor effectiveness or complications of therapy All patient and lab specimens are treated as infectious and handled according to "Standard Precautions." Specimen Collection ------------------- When instructing patients, emphasize hand washing and general cleanliness Instruct patients to secure the lid of the specimen to prevent leakage Types of Specimens ------------------ ### Random Specimens Collected at any time Time of voiding must be recorded on specimen contained Necessary as **diurnal variation** can occur (results may differ at different times of day Several hours of urinary continence before collection can be necessary to provide a specimen suitable for analysis ### First Morning Specimen Usually collected immediately on the patient arising from sleep in the morning Considered the best specimen as urine will be more concentrated meaning chemical changes we want to look for will happen in the body and the urine results will reflect it when it has been sitting in the bladder for an extended period of time This is also called overnight, eight hour or early morning specimen ### Timed Specimen Collected at a specific time. Some substance like urobilinogen demonstrate diurnal variation and is best evaluated between 2 and 4 pm This is used when looking for something specific like a drug ### 24hr Urine Specimen Necessary to measure total amount of solutes excreted in 24 hour period Eliminates diurnal variation and gives complete picture of the urine throughout the day Lowest concentrations of catecholamines, 17-hydroxysteroids, and electrolytes occur in the early morning where highest concentrations occur at noon or shortly thereafter ### Clean Catch Specimen Also referred to as **midstream** or **clean-voided** midstream. Usually best specimen for obtaining noncontaminated specimens Good for routine urinalysis examination and micro samples Patient cleans genital area before urinating so there is no bacterial contamination Up to 90% of rejected clean catch urines occur due to collection issues ### Catheter Specimen Involves insertion of sterile tubing into the bladder Useful if patient is having difficulty urinating Involves risk of contamination which can lead to bacterial infection for the patient. Thorough cleaning of the area is required ### Suprapubic Specimen Involves insertion of a needle directly into the distended bladder Technique avoids vaginal and urethral contamination and is useful for infants and small children Not a common practice Urinalysis Requisition ---------------------- Requisitions should include: Date and time of collection Specialized collection circumstances (catheter, clean catch, first morning specimen) If specimen was refrigerated before transporting Time received in lab and time analysis was performed Tests requested Form should also include an are for noting any specific situations that might influence the results of the analysis (medications, vitamins, antibiotics, menstrual blood, strenuous exercise, and any other pertinent clinical information Specimen Transportation and storage ----------------------------------- ### Transportation Long transportation times are sometimes required for samples where one lab cannot test the specimen for any number of reasons If this happens, the sample can be preserved, refrigerated, frozen, etc. to assist in maintaining sample integrity during transport In some cases an agar film (attached to a plastic support) is dipped into the urine and placed within a closed container to be sent with the urine sample so that the next lab to receive the sample may subculture from the agar. ### Storage: REFRIGERATION If the specimen cannot be transported and analyzed immediately, it should be refrigerated 2-8 degrees after collection Reduces contamination risk ### Storage: Preservatives If the specimen cannot be refrigerated, a preservative should be used. Especially when transporting the sample to another laboratory for analysis where temperature cannot always be controlled SPecimen Preservation --------------------- Preservation is done to avoid changes that happen due to decomposition If urine can be maintained at a **low pH and high SG**, the sample will deteriorate slower Preservation is necessary when the urine is going to need to be transported to a different lab or if testing cannot be done within an appropriate time period Specimens begin to decompose within 20-30 minutes of collection primarily because the action of urea splitting bacteria producing ammonia Refrigerated specimens with no chemical preservative should not be analyzed after 48 hours of collection You may freeze specimens ### Boric Acid Most commonly used preservative Works by using buffered boric acid preservatives to reduce harmful effects of preservatives on organisms. Designed to maintain the specimen in a state equivalent to refrigeration by **deterring the proliferation of organisms** that would result in a false positive culture or bacterial growth Grey stoppered tube Commonly used by smaller labs that send out microbiology samples to larger labs NOT the same as blood tube that has sodium fluoride and potassium oxalate Works as well as refrigeration. Does not need to be refrigerated if preserved ### Other preservatives HCL Antioxidant and keeps constituents in solution Chlorohexidine Red/yellow conical evacuated tube by BD Prevents bacterial overgrowth Formalin Fixation of formed elements Unpreserved urine for urine pregnancy tests Specimen Requirements/acceptability ----------------------------------- Due to the trueness of urinalysis results depending on quality of specimens, proper refrigeration and transportation is key. Although random specimens may be used for chemical analysis using test strips, the preferred urine specimen (especially for micro) is a well-mixed first morning specimen **tested within 2 hours of collection.** First morning specimens maximize the recovery of sediment elements as they are the most concentrated ### Criteria for specimen collection Should arrive promptly at the lab and undergo analysis as soon as possible Generally accepted that standing for 2 hours at room temp, the chemical composition of urine changes and formed elements begin to deteriorate Urine constituents, such as bilirubin and urobilinogen are unstable (and sensitive to light) so sample should be shielded from it if possible Bacteria can alter glucose concentration and pH changes can occur so samples should be refrigerated to prevent bacterial growth Casts, erythrocytes and leukocytes are especially susceptible to lysis in samples with a low SG (\7.0). Samples should be analyzed quickly to ensure lysis does not occur Minimum sufficient quantity for both macroscopic and microscopic is usually 12mL (50mL is preferred) but may vary with collection container Samples from infants may necessitate smaller volumes Specimen collection containers should she sterile, leak proof, and disposable Sample should be free of contamination from feces, bathroom tissue, or other foreign materials If these criteria are not met, the ordering physician should ne notified of the specimen's unsuitability and another specimen collection may be requested. ### Delayed Specimens If a urine cannot be analyzed within the **2 hours after collection** the specimen must be refrigerated Specimen must be brought back to room temp prior to analysis as enzymatic reactions on the test strips may be slowed if analyzed while cold Delays and associated temperature history should be documented if urine specimens are not tested promptly Examination of urine specimens ------------------------------ ### physical examination **Colour** Normal urine colour varies (pale yellow to dark amber) which depends on the amount of pigment present such as urochrome (urobilin) Some urines may have an abnormal colour which may be due to medication, diet, or disease Pale to colourless Dilute, diuretics (coffee, alcohol) Red Blood cells Hemoglobinuria Myoglobinuria Brown or black RBC or heme pigments Age pH Yellow-green Products of bilirubin metabolism (obstructive jaundice) **Clarity** Clarity reflects the degree of urine transparency. Can be clear, hazy or cloudy Urines may become cloudy due to a number of reasons: Leukocytes Epithelial cells Amorphous crystals -- products of metabolism Bacterial growth Mucous RBCs Fat Cloudy urine does not indicate disease and clear urine does not indicate health **Foam** Presence of foam indicates high amounts of proteins present This is not reported **Concentration** Reflects amount of solutes present in urine Used to assess renal function SG and osmolality are *non-specific* tests used to determine concentration **Specific Gravity** Ratio of weight of volume of urine to the weight of the same volume of distilled water at a constant temperature Dependent on number *and* weight of particles present Used to measure concentrating or diluting ability of the kidneys **Normal range: 1.003-1.035** (always above 1, which is water's SG) SG and Diabetes: Mellitus: high SG due to deficiency in insulin and therefor more glucose which exceeds normal renal threshold. Glucose molecules are very dense Insipidus: very low SG due to deficiency of ADH (vasopressin) **Osmolality** Another way to measure kidney function Better than SG because small amounts of glucose and protein will not affect it, unlike SG test **Normal range:** 500-800 mOsm/kg May be anywhere from 40-1400 mOsm/kg if there is disease. Terminal renal failure urine may stay around 285 mOsm/kg which is the same as plasma and the glomerular filtrate (indicates that the kidney is unable to dilute or concentrate the urine) Osmolality requires more time, expense and equipment than SG tests Not preferred for that reason **Note**: There is usually a relationship between SG and osmolality (in the absence of renal disease). The **osmolic gap** is when SP and osmolality don't quite match up. Can happen in patients with alcoholism Urinalysis: dipstick analysis ============================= Chemical examination -------------------- Chemical examination is necessary to diagnose many diseases and screen for others. Urinalysis is a routine, cost-effective test. This is more commonly done using a reagent strip. ### Reagent Test Strips - Consists of plastic support that has one or more chemical tests sites available in many configurations - Sites also known as **dry reagent chemistry reaction sites** **Common tests:** - pH - protein - glucose - ketones - blood - bilirubin - urobilinogen - nitrite - leukocyte esterase - specific gravity #### Ensuring good test results with reagent strips - Store strips in original containers (follow manufacturer's recommendations) - Keep container tightly closed - Exposure to light and room humidity can affect strips - Remove only a few strips at a time and close container immediately - Avoid combining reagent strips from different containers - Avoid touching chemical test sites on the reagent strip - Sample being tested should be brought to room temperature - In the event the testing is delayed and samples have been refrigerated to preserve sample integrity #### Controls - Negative and positive controls will be run each time the analyzer is used by you for the day - Semi-qualitative or qualitative controls - Positive: - Designed to yield weakly positive results so control material is sensitive to early deterioration of the system Reagent Strip Result Interpretation ----------------------------------- ### pH - kidneys must vary pH of urine to maintain constant pH of the blood to compensate for diet and products of metabolism - If there is excess of acid in the body, more will be excreted into the urine - **Normal range:** 6 - **Possible ranges:** 4.6-8.0 - pH meter may be used if more precise measurement required #### Issues with pH test - ![](media/image2.png)Excess urine left on the strip may cause a **run over effect** - When the acid buffer from the reagent in the protein area runs into the pH area - Causes **false negative** ### Protein - **Mucoprotein:** kidney will always excrete small amounts of it. If pt has kidney disease there is an increase in mucoproteins - Presence of increased proteins in urine can be an important indicator of renal disease - Physical exercise and fever can cause increase in protein levels - Low molecular weight protein (mucoprotein) secreted in small amounts and large mw proteins (albumin) is not unless there is kidney disease - Strip is sensitive for albumin - Gamma globulins, Hgb, Tamm-Horsfall and Bence Jones are much less readily detected - It is important to correlate the protein result with the SG - Trace protein in dilute urine is much more significant #### Issues with Protein Test False Positives: - Alkaline pH - Affects acid buffer of the reagent and colour may change without protein - Leaving dipstick in urine too long - Washed out buffer - Some cleaning components - Blood - High bilirubin - Reacts as a colour, not a real chemical reaction - Would be good to follow up with SSA test in this instance False Negatives: - Dilute urines - Proteins other than albumin present ### Glucose and Other Reducing Substances - ![](media/image4.jpeg)Presence of significant amounts of glucose in urine is called **glycosuria** or **glucosuria**. Amount of glucose is dependent on: - Blood glucose level -- not usually seen until plasma is about 9mmol/L - Glomerular filtration - Degree of tubular reabsorption - If glomerulus becomes more permeable, it lets substances out that should not be leaving the system. Same with tubular reabsorption issues. You get glucose spilling out in the urine - Positive test: - Should be based on other screening tests including specific gravity, ketones, and albumin - Strip will be more sensitive with dilute urines (lower SG) - If you had two tests but different glucose readings, you would believe the more dilute one as you would want to trust that the presence of anything at all in a dilute urine indicates more certainly that there is glucose in the urine #### Issues with Glucose test Enzymatic Reaction - Test needs to be run at room temperature as the reaction for the test is enzymatic - If the sample is refrigerated, enzyme reaction could be slower False Positives: - Oxidizing cleaning agents - Those with peroxide, bleach - Elevated glucose may tend to be even further elevated with increased urobilinogen - Increased sensitivity at low SG because not many other things in the sample - Improper storage - Exposure to air can cause false positive result False Negatives: - Temp too cold - Elevated SG and pH (synergistic) - High concentrations of vitamin C - Vitamin C can cause interference in al most every test - Sodium fluoride preservative (would lose enzyme activity) - High ketones (4+) may decrease sensitivity of the test #### Screening for reducing substances - Other sugars that can be found in the urine are reducing substances such as: - Galactose - Lactose - Fructose - Maltose - Procedures that are based on the ability of glucose to reduce copper will detect these sugars if present - Sucrose (dimer of glucose and fructose) is *not* a reducing sugar #### Clinitest - "Clinitest" is a brand name of a copper reduction test used to screen for reducing substances - In strongly alkaline solutions and in the presence of heat, reducing substance sugars will reduce cupric ions to cupric oxide - This test should be included in routine urinalysis of all pediatric patients as there are some genetic diseases that can be screened this way - Early detection of metabolic defects such as galactosemia - Has been largely replaced with neonatal screening at birth - tablet reacts in urines, heats up, and then alkaline medium that comes with it gets copper reduction. - Blue = negative. Gets more positive with each subsequent reaction seen above False positives for Clinitest: High doses of vitamin c Formalin used as preservative Some drugs False Negatives for Clinitest: Urines of high protein Proteins lower the surface tension of the urine and thus increase boiling time of the reaction X-ray contrast media -- CT scan dyes ### Ketones ![](media/image6.jpeg)Ketones or ketone bodies are formed during catabolism of fatty acids if there aren't enough carbs to break down Intermediate product is **acetyl CoA** Which enters the Krebs cycle when there is a balance between fat and carb degradation Acetyl CoA combines with oxaloacetate to yield citrate. When carbs are not available, all the oxaloacetate will be used to form glucose. There will be none left to join with acetyl CoA so then forms ketones Three possible ketones that are produced: Acetone (2%) Acetoacetic acid (diacetic acid 20%) The one that gets detected by dipstick B-hydroxybutyrate (78%) Cannot be detected Reagent strips contain nitroprusside and an alkaline buffer that reacts with diacetic acid in urine to form a maroon colour Some strips may be sensitive to acetone #### Issues with ketone test False Positives: Highly pigmented urines If red colour in urine, the test will read it as a positive test High SG and low pH Some drugs False Negatives: Improper storage Conversion of acetoacetate to acetone. Urines must be tested when freshly voided or immediately refrigerated ### Occult Blood occult blood means hidden blood or blood not visible to the naked eye (macroscopically) Could happen if there is hemolysis **Hematuria:** presence of blood or intact RBCs in the urine. Cells will lyse in high alkaline or low specific gravity environment Microscopic may show empty RBC membranes called "ghost cells" Gross hematuria is visible macroscopically **Hemoglobinuria:** presence of free Hgb (not bound to RBCs) in the urine as a result of intravascular hemolysis (hemolytic anemia or transfusion rxn, PNH (paroxysmal nocturnal hemoglobinuria), PCH (Paroxysmal cold hemoglobinuria)). This condition without hematuria occurs as a result of hemoglobinemia and therefore, has primarily nothing to do with the kidneys (happens *before* it gets to the kidneys) even though it may result in kidney damage **Myoglobinuria:** myoglobin is the heme protein of striated muscle. Helps in movement and supply of O~2~ to tissue. Injury to skeletal or cardiac muscle (heart attack, MVC, etc.) can cause this Reagent strips are able to detect intact RBCs as well as free Hgb and myoglobin Intact RBCs result in a "spotted" pad #### Issues with Occult Blood Tests False Positives: Bleach, bromides, iodides (oxidation of the chromogen) May give false positive with high bacterial content (peroxidase activity) Betadine -- common skin disinfectant Blood from hemorrhoids or menstruation False Negatives: Failure to mix sample High levels of ascorbic acid Preserved in formalin (kills enzymes) ### ![](media/image8.jpeg)Bilirubin and Urobilinogen urochrome is the "urine colour" which essentially comes from the amount of urobilinogen in the urine Bilirubin is formed from breakdown of Hgb from RBCs in reticuloendothelial system Bound to albumin to be transported to the liver Insoluble (cannot be excreted in the urine so it goes through a process which makes it soluble) Normally *no* detected bilirubin in the urine Can indicate jaundice (obstructive or hepatic) Before entering liver, enters intestine where bacteria converts it (oxidation) to urobilinogen Urobilinogen is formed from bilirubin and can be excreted into the stool, reabsorbed by the blood stream and passed back through the kidney Normal amounts expected in urine Decreased or absent urobilinogen **cannot be detected on the strips** It can occur (but not be detected in certain liver diseases and in pt's taking broad-spectrum Abs ![](media/image10.png)Increased RBC breakdown = increased bilirubin. Thus the body may have difficulty breaking down the excess and it gets passed through the kidneys #### Screening tests for Bilirubin Can be detected in urine before other clinical symptoms are present or recognizable Yellow/amber but turns green if exposed to light Urines must be tested fresh or kept away from light #### Issues with Bilirubin and Urobilinogen Tests False positive for bilirubin: Waiting too long to read (over 30 seconds) Some metabolites and some drugs False negative results for bilirubin: Large amounts of Vit C Elevated levels of nitrite will lower the result Exposure to light and room temperatures False positives for urobilinogen: High levels of bilirubin Some pigments and drugs (mostly red) The excipients in the drugs can cause interference in the test, not necessarily the medical ingredients False negatives for urobilinogen: High nitrite Formalin Improperly stores samples Oxidation of urobilinogen and urobilin ### Nitrite indirect method for the early detection of significant asymptomatic bacteriuria Any colour change at all is a positive result We form nitrates in the urine, but nitrites are due to the presence of bacteria, thus it could indicate a UTI. The organisms produce enzymes that reduce urinary nitrate to nitrite Organisms that can cause urinary tract infections include: *Escheriachia coli* *Entrobacter* *Citrobacter* *Klebsiella* *Proteus* First morning sample or a sample incubated 4 hours in the bladder is best #### Issues with Nitrite Test False positives: Allowing to stand could cause organisms to grow Red urines False negatives: High: Vit C SG Urobilinogen Negative test is not an indication that there is *no* bacteria. Tests could be negative due to: Non nitrite producer Not in bladder long enough No nitrate in diet (low protein) Produced nitrogen gas -- further reduction of nitrite Interference ### LEUKOCYTES ![](media/image12.jpeg)Increased neuts (pyuria = pus in urine) usually indicates UTI Demonstrated with positive leukocyte esterase test Number reflected on the macroscopic examination may not necessarily correspond to the microscopic (some could lyse, be at bottom of sample, etc.) #### Issues with Leukocyte Esterase Test False positives: Strong detergents (bleach) Contamination with vaginal discharge Some preservatives (formalin) False negatives: High SG (WBC crenate and don't release enzyme) Urines with high SG containing high glucose and/or protein Drugs and chemicals Vit C Oxalic acid Gentamicin Tetracycline Microscopy ---------- Must use Kohler Most urine sediment examinations are done using wet mount slides and bright field microscopy Some institutions use supravital stains such as Sternheimer- Malbin Contains crystal violet and safranin Microscope slides/viewing devices Commercially available Disposable Standardized Calibrated chambers Use of plain glass microscope slides and coverslips is not encouraged as they do not yield standardized results \~12mL of sample is best for analysis In urinalysis, if there is too much light, some structures will be missed You must sure that the fine adjustment is constantly being used since it is a wet prep and your elements will be on more than one focal plane ### Identifying the need for MICROSCOPIC samples When a urine requires microscopic analysis, it will have elements that would be visible under the microscope Protein RBCs WBCs Nitrites #### cENTRIFUGATION rEQUIREMENTS Centrifugation time: 5 min 1.5 (1500RPM) -- Cambrian Centrifugation Speed -- Applies to all labs Centrifuge to provide relative centrifugal force (RCF) of 400xg 400 x g RCF = 1.118 x 10~~^-5^ r x N^2^ R = radius in cm (from the center of spindle to the bottom of the tube) N = rotations per minute ### Low Power Check for crystals, elements that are only present in a few fields and casts Enumerate casts here but you may have to switch to the high power objective to classify the cast type ### High Power Check for all other elements and grade accordingly such as cells, bacteria, crystals, and a parasite Pay attention to which elements are quantitative and which are qualitative Point of Care Systems ===================== POCT (point of care testing) overview ------------------------------------- - Diagnostic tests performed **at the point of care in any healthcare institution** - Geographical context for POCT based on testing location - There is not dedicated space for testing equipment - Testing equipment is portable - Where you can find POCT: - Emergency dept - Operating room - Radiology - A physician's office lab - Ambulatory care settings - Patient's home - POCT results do not go onto the patient's medical records, it is more for a quick test just to get a baseline of a patient's health (blood glucose, etc.) - Paramedics use POC tests for in the field to determine various parameters of patient health so they can know the best way to treat and transport the patient - Can use several methodologies: - Reflectance spectrometry - Electrochemistry (potentiometry) - Immunoturbidity - Spectrometry - Fluorescence - Flow-through immunoassay Examples: - Glucose (highest vol POC test) - Blood chemistry (blood gases, electrolytes) - Coagulation - Cardiac markers - HbA1c (home test) ### Characteristics of POCT DEVICES - Portable - Consumable reagent cartridges - Generate results within minutes - Requires minimum operating steps - Have the capability to perform tests on *whole blood specimens* - Requires ambient temperature storage for reagents - Must be durable and reliable over a long period of time - Produce results that are in close agreement with the central lab ### ADVANTAGES of POCT - Reduced turnaround time - Improved patient management due to speed and efficiency - Less trauma (older patients, pediatric patients, etc. will have an easier time with a finger prick than if a tech is taking vials of blood from them) - Smaller blood spec. required - More convenience for the patient - Reduction in length od hospital stay or no hospital stay ### Disadvantages of POCT - Inventory harder to control - Not necessarily as tightly controlled as reagents (for example) for a laboratory instrument. The operator, if not lab personnel, may not be trained in the storage of test related inventory - Maintenance of QC and QA - Proper integration of data into the patient's medical record - Interfacing - Due to lower volume of tests, proficiency of the testing personnel may be of concern - For example, if a test is not frequently performed, the operator may forget the procedure Functional Context of POCT -------------------------- - Rapid turnaround times (often immediate results) - Testing that is carried out and reported without referring to a laboratory - Provides diagnostic data without delay Technological Context os POCT ----------------------------- - Most often conducted with: - Small, portable, handheld devices - Manual kits - **Portability** of the test equipment/methods is what provides the advantages over lab tests - Although used most often outside the central lab, they may also be used inside the lab Accuracy rEQUIREMENTS of POCT ----------------------------- - Point of Care tests are held to the same standards that labs are held to in regard to the accuracy of the tests. This is to ensure everything works and the users of the tests are competent - Accuracy requirements include: - Comparisons to central lab (should be in close agreement) - Daily QC (just like any other kind of lab analysis) - Proficiency testing -- usual operators must perform PT - Management of user records POCT Staff ---------- Director: - PhD, MD, or DO (doctor of osteopathic medicine), laboratory scientist, or pathologist - Responsibilities include making decisions regarding policy, administration, finances, and technical aspects of POCT POCT Coordinator: - Responsible for implementing and coordinating POCT, facilitating compliance with procedures, policies, and regulations - Essentially someone's name must be the signoff for training and other documentation so this would be that person Administrators: - Designated contacts or trainers in non-lab depts - Responsibilities include facilitating efficient POCT program, serve as a link between the coordinator and staff, help ease training - User can be nurses, physicians, respiratory therapists, paramedics ### CLIA - CLIA stands for Clinical Laboratory Improvement Amendments of 1988 - Regulations under CLIA are applicable to **U.S. facilities** (not in Canada) - Rough equivalent for Canada would be **IQMH** (Institute for Quality Management Healthcare) - In the states, lab testing systems are assigned "complexity" categories - If a test is deemed "CLIA-waived" it means that it is a simple test with low risk for incorrect results - POCT systems would fall under that category Laboratory Informatics ---------------------- - LIS used for: - Collection of pt info - Generation of test results - Assembly of data output - Production of ancillary reports - Storage of data - Transcription errors are more likely with POCT that are not connected to LIS - Having POCT device connected to LIS reduces risk for errors because data is delivered directly to the patient's electronic file and there is no manual entry of data needed POCT Tests ---------- ### Glucometers - Based on electrochemical (biosensor) measurements but affected by variability of hemoglobin concentration - Accu-check in lab uses bi-amperometry - Glucose oxidase on strip - Blood sample added - Glucose to gluconic acid and H~2~O - H~2~O~2~ converted to 2H^+^ + O~2~ +2e^-^ - The amount of electrons is proportional to glucose but must use another electrode to subtract "interferences" ### Non-Instrument BAsed POCT - Manual rapid test methods can be used for certain tests like those for pregnancy, occult blood, infectious mononucleosis and COVID - Non-instrument-based tests can apply the principles of: - Competitive and non-competitive immunoassay - Enzymatic assay - Chemical reactions with a visually read endpoint - Since this testing is non-instrument-based, results must be manually entered into the computer system - Usually test whole blood, urine, feces, saliva, or even throat swabs can be used depending what the testing is Pregnancy Testing - Designed to detect small concentrations of human chorionic gonadotropin (hCG) present in urine - Many pregnancy test kits contain a monoclonal Ab directed against beta subunit of the hCG (beta hCG) to increase the specificity of the rxn - Beta-hCG can be directed typically by the 12^th^ day after a missed period - Reported as hCG positive or negative -- **qualitative** - There are other methods that measure beta-hCG level in serum, which is far more sensitive (able to detect lower concentrations) -- **quantitative** Fecal Occult Blood Test - Used as a screening test to detect blood in stool samples - Test kits include cards that fecal samples are applied to - Two "windows" so you can apply sample from two separate areas from the whole sample - An indicator liquid added to the card and a colour change can be observed (typically blue) - There is also a positive and neg control area on the card to ensure all cards work - Can be done within minutes or can have longer incubation time (48h) - There are interfering substances: - Avoid red meat, lamb, raw fruits/veggies 3 days before collection - These can give false negatives Automation ========== Overview -------- - Estimated that labs generate 80% of information physicians rely on to make crucial decisions - Automation eliminates errors that could impact patient health - Essentially everything but sample collection can be done with automation ### Factors that Drive Automation - Aging population - More tests required - Need accurate tests quickly - POCT and better automation is the answer to this need ### Benefits of Automation - Reduced medical errors - Reduced sample volume - Increased accuracy and precision - Improved safety (stopper removal, piercing) - Faster TATs - Helping with staff shortages Patient Benefits - Workflow standardization - Reduced errors - Frees up techs to work on more complicated cases Automation Analyzer Types ------------------------- ### Sequential vs. Discrete - **Discrete:** have largely replaced sequential analyzers. Can do all tests required on one patient sample before moving to the next - **Sequential (Sequential Multichannel Automated Chemistry -- SMAC):** All patient samples go through the same tests regardless of whether they need it or not. The samples are split into several channels and everyone's samples go through the instrument together, one test at a time. Essentially a screening tool, but not always necessary ### Wet Chem vs. Dry Chem **Wet Chem:** chemicals in solution that get added to the sample to see reaction **Dry Chem:** Usually slides that have substrate in which reactions can take place on it (you don't need much plumbing or waste removal for used samples like you need for wet chem) Specimen SEPARATION to Recording -------------------------------- Separation -- remove interferant (protein) often by ratio of reagent to sample or use correction factors Reaction times-end point or rate reactions Analysis time shortened Interfering chromogens negated Measuring device-principle of spectrophotometry, ISE, chemiluminescence, etc. Recording mechanism is computer based modules End point: Take reading as reagent goes in and then one reading done again at the end. Light absorption tells you the concentration Rate Reaction Time reading of electrodes (ex. See how long colour changes take, etc.) Common Chemistry Tests and Ranges --------------------------------- +-----------------------------------+-----------------------------------+ | Test | Reference Range | +===================================+===================================+ | Glucose | Fasting 4.0 -- 6.0 mmol/L | +-----------------------------------+-----------------------------------+ | Urea | 2.1 -- 7.1 mmol/L | +-----------------------------------+-----------------------------------+ | Creatinine | Male: 53-97 umol/L | | | | | | Female: 44-71 umol/L | | | | | | Child: 0-53 umol/L | +-----------------------------------+-----------------------------------+ | Sodium | 135-145 mmol/L | +-----------------------------------+-----------------------------------+ | Potassium | 3.5 -- 5.1 mmol/L | +-----------------------------------+-----------------------------------+ | Chloride | 98-107 mmol/L | +-----------------------------------+-----------------------------------+ | Total Protein | 60-80 g/L | +-----------------------------------+-----------------------------------+ | Albumin | 35-55 g/L | +-----------------------------------+-----------------------------------+ CUSTOMIZABLE Panels ------------------- Renal function tests Urea and creatinine Electrolytes Na, K, Cl Liver Function Tests AST, ALT, ALP, could include LDH Drug Screens VBG, Acetaminophen, ethanol. Salicylates Tox Screen Urine for multiple drugs Cardiac Pack Troponin, maybe coagulation Lipid Cholesterol, triglyceride, HDL, LDL Special Considerations ---------------------- Some tests need to be protected from light Bilirubin Some need to be centrifuged with strict time limits Lactic acid Ammonia Some are volatile Ethanol Ketones Some are glycolic Glucose Some can't be refrigerated LDH Some can't be tested after a period of time Blood gases Some need to be sent out and have special protocols either patient prep/collection or preservation Analytical Techniques ===================== Spectrophotometry and Photometry -------------------------------- - Photometric instruments measure light intensity without consideration of wavelength - Most instruments use filters (photometers), prisms, or gratings (spectrophotometers) to select (isolate) a narrow range of incidence wavelength - **Beer's Law**: concentration of a substance is directly proportional to the right amount of light absorbed or inversely proportional to logarithm of transmitted light - **Spectrophotometric Instruments**: measure light transmitted by a solution to determine concentration of light-absorbing substance in solution to determine concentration of light-absorbing substance in solution ### Components of a Spectrophotometer #### Light source - Tungsten or deuterium/mercury arc lamp (UV) - Certain types of glass block UV light #### Monochromator - Wavelength isolator, filters, prisms, diffraction - Sometimes monochromators absorb more than we want them to - Different varieties: - Filters - Prisms - Grating monochromators - Holographic gratings - Quality of selectors is described by **nominal wavelength**, effective bandwidths and bandpass - Nominal wavelength: wavelength in nanometers at peak transmittance - Spectral bandwidth: range of wavelengths halfway between baseline and peak - Bandpass: total range of wavelengths transmitted - *Photo: spectral percent transmittance characteristics of a wavelength selector* #### Sample cell - Plastic or quartz (UV) scratches/dust/fingerprints will scatter light #### Photodetectors - Converts [radiant energy](#radiant-energy) into equivalent amount of electrical energy - Photocell, phototube, photomultiplier tube **Photomultiplier Tubes** - Photomultiplier tubes can multiply light by 10^6^ times - Commonly used when radiant power is low which is characteristic of very low analyte concentrations - Similar to phototube but output signal is amplified up to approximately a million-fold - Highly sensitive to UV and visible radiation - Very fast response time **Signal Processors and Readout** - Processing of signal received from transducer accomplished by: - Device that amplifies signal - Rectifies altering current - Directs current - Alters phase of signal - Filters unwanted components - Does math ### Radiant Energy - Radiant energy that passes through an object will be partially absorbed and transmitted - Absorbance (A) is the amount of light absorbed. It cannot be measured directly by a spectrophotometer - Mathematically derived from %T A=2-log%T - Amount of light absorbed at a particular wavelength depends on molecular and ion types of present - Varies with concentration, pH or temperature - Deviations from linearity are typically observed at high absorbances (around 2.0 absorbance units) Quality Assurance in Spectroscopy --------------------------------- - For optimal performance photometric parameters must be monitored ### Wavelength accuracy - Photometer is measuring to the wavelength it is set to - Assessed easily using special glass type optical filters like **didymium** and **holmium oxide** - Didymium has broad spectrum absorption peak around 600nm - Holmium oxide has multiple absorption peaks with sharp peak at 360nm - Wavelength accuracy failures are usually a problem with monochromator - Specific absorption peaks ensure us that what we set it to is what is read - ![](media/image14.jpeg)Linearity *(photo right: deviation from linearity)* - Straight line for Beer's Law - Coloured solutions may be diluted and used to check - Deviation from linearity can be caused by problems with light source, monochromator, or detector - If deviation occurs: dilute the sample - Less than expected absorbance - Indication of stray light or of band pass that is wider than specified - Stray light is caused by scratches, fingerprints or other impurities in the glass Reflectometry ------------- Clinical Applications: - Urine Dipstick analysis - Dry chemistry ### Reflectometer - Filter photometer that measures quantity of light reflected by a liquid sample that has been dispensed on a non-polished surface - Tungsten-quartz halide lamp serves as source of polychromatic radiation - Light passes through slit and directed to surface - Filter or filter wheel isolated wavelength of interest - Photodiodes detect reflected radiant energy - Computer converts non-linear reflectance signals into direct readout concentration units - *Photo: diagram of a typical reflectometer.* - *A = polychromatic light source, B = monochromator, C = slit, D = diffuse reflectance surface, E = lens, F = photodetector, G = readout device* ### Reflectance vs. Concentration - Relationship is non-linear - Ideal reflectance of a pure **white** standard is **one**, signifying that all light is **reflected** - Ideal reflectance of a pure **black** sample is **zero**, signifying that all light is **absorbed** Fluorometry ----------- - ![](media/image16.jpeg)Overview: - Filter fluorometers measure concentrations of solutions that contain fluorescing molecules - Source emits shot-wavelength high-energy excitation light - Mechanical attenuator controls light intensity - Advantages and Disadvantages - Advantages: greater specificity and sensitivity, 1000x more sensitive than absorption techniques and has increased specificity (since optimal wavelengths are chosen for absorption/excitation) - Disadvantages: sensitive to environmental changes like chemical contamination, humidity, stray light, and UV light (can decrease fluorescence or **quenching**) - **Source should be 90° from detection** ### Instrumentation - Conventional design is in place to detector at 90° angle from polychromatic light source - Advantage is that light reflected/fluorescence from cuvette surface enters monochromator - Uses one or two grating monochromators - The use of two gets rid of all the light you do not want - Uses PMT -- photomultiplier tube due to low intensity - Cuvettes or cells can be rectangular or cylindrical - Baffles are used to reduce the amount of scattered radiation reaching detector - Important not to leave fingerprints TURBIDIMETRY ------------ - Measurement of the reduction in light transmission caused by particle formation - Light transmitted in **forward direction** is detected - Sensitivity compared to nephelometry can be attained by using low wavelengths and high-quality specs Clinical application: - Used in micro to detect bacterial growth in broth cultures - Can be used in coagulation studies to detect clot formation - Used in chemistry to quantify protein concentration in fluids such as urine or CSF Nephelometry ------------ - Related to turbidimetry except the difference is that this method measures **scattered light** - Different angle of measurement Chemiluminescence ----------------- - Part of clinical energy generated produces excited intermediates that decay to a ground state with emission of photons - Unlike fluorescence, no excitation radiation or monochromator are required - Oxidation reactions of luminol, acridinium esters, and dioxetanes Electrochemistry ---------------- - [Ion-Selective Electrodes (ISE)](#ion-selective-electrode) - Designed to be sensitive toward individual ions (Na, K, Cl, etc.) - [pH electrodes](#ph-electrode) - Gas-Sensing Electrodes - Senses O~2~ and CO~2~ - Separated from solution by thin, gas-permeable membrane - Enzyme electrodes - An ISE covered by immobilized enzymes that can catalyze a specific chemical reaction. When the reaction happens it measures what is coming off of it - Colourimetric Chloridometers and Anodic Stripping Voltammetry - Voltammetry = looking for specific metal (example: lead poisoning) - Chloride ISEs have largely replaced colorimetric titrations - Anodic stripping voltammetry was widely used for analysis of lead ### Potentiometry - Measurement of potential voltage between 2 electrodes in solution - Electrical potentials are produced at the interface between a metal and ions of that metal in solution - To measure electrode potential, constant-voltage source is needed as the reference potential (fluctuations cause issues in measurement) - Electrode with constant voltage is the reference electrode - Measuring electrode is the indicator electrode - Concentration of ions can be calculated by measuring the potential difference between two electrodes - Measured cell potential is related to molar concentration by the Nernst Equation - Equation is useful for predicting electrochemical cell potential given the concentration of oxidized and reduced species for a given electrode system ### Reference Electrodes - Calomel (solution of mercurous chloride) - Silver/silver chloride electrode - Used in different applications - Both provide many of the analytical qualities necessary for reliable measurements ### Ion Selective Electrode - Membrane based electrochemical transducer capable of responding to a specific ion - Potential difference or electron flow is created by transferring the ion to be measured from the sample solution to the membrane phase - Membranes made of plasticizers, organic solvents, inert polymers and ionophores - Not reactive with anything else so they can be specific to the ion you want to measure - Ionophore: molecule that increases the permeability of a membrane to a specific ion Advantages - No reagent prep - No standard curve prep - Direct measurement - Cost effective (can use them over and over) - Fast - Precise - Sensitive - Easy to maintain (make sure they don't dry out) - Easily adapted to automation ### pH Electrode - ![](media/image18.png)Glass electrodes are the most common - Measures hydrogen ion activity - Inside electrode is chloride ion buffer with known hydrogen concentration - Internal silver/silver chloride electrode serves as reference electrode Theory behind pH electrode - Sodium ions drift out - Specimens with hydrogen ions replace sodium - Results in net increase in external membrane potential difference - Chloride ions in the buffer respond by migrating to internal glass layer - Potential difference Is referenced to the external reference electrode and the difference or change is displayed as a numerical value for pH #### PCO~2~ Electrode - pH electrode with a plastic "jacket" - Plastic jacket filled with sodium bicarbonate buffer has a gas permeable membrane (Teflon or silicone) across its opening - When whole blood containing CO~2~ comes into contact with membrane, the CO~2~ from blood passes through and mixes with buffer - Chemical reaction occurs and results in change of pH: ![](media/image20.png) Amperometry ----------- - Measurement of the current flow produced by an oxidation-reduction reaction - Used for PO~2~ electrode and can be used for chloride ### Amperometry and Chloride - Chloride titrator includes a pair of silver electrodes that serve as the indicator electrodes - When all of the chloride in the sample has been consumed, silver ions appear in excess PO2 Gas-Sensing Electrode ------------------------- - The Clark PO2 electrode consists of a gas-permeable membrane - Usually polypropylene -- allows dissolved oxygen to pass through - Membrane prevents other substances from passing through that may interfere with electrode - When oxygen passes through, mixes in a phosphate buffer solution and reacts with polarized **platinum** cathode - Reduction of oxygen produces flow of electrons and magnitude of current is directly proportional to amount of oxygen in sample AAJICWP0\_alt Osmometry --------- - Measurement of the osmolality of an aqueous solution such as serum, plasma, or urine - When a solute is added to a solvent, vapor pressure is lowered below that of pure solvent - As a result of change in vapor pressure, boiling point is raised above that of pure solvent - Freezing point is depressed below that of pure solvent - Colligative properties are directly related to the number of solute particles per mass of solvent - **Number of particles by mass of solvent** - Osmotic pressure represents hydrostatic pressure that develops and is maintained when two solutions of different concentrations exist on opposite sides of semipermeable barrier - Measures concentration of solute particles in a solution - Freezing-point osmometer - Sample in a small tube is lowered into a chamber with cold refrigerant circulating from cooling unit. - Thermistor is immersed in sample - Wire is used to stir sample until it is cooled to several degrees below its freezing point - Thermistor is a material that has less resistance when the temperature increases - Readout uses a Wheatstone bridge circuit that detects temperature change as proportional to change in thermistor resistance - Freezing point depression is proportional to the number of solute particles ### Osmolality - Osmolarity expresses concentration per volume of solution osmol/L - Osmolality as mOsmol/kg H2O and identifies number of moles of a particle per kilogram of water, **not the kind of particle** - **Osmolality is better since not dependant on temperature** - Measured value: - Freezing-point depression osmometry - Vapor pressure osmometry - **Calculated osmolality = 2x Na + (urea+glucose)** - Osmolality gap #### Osmolality gap - Measured osmolality-calculated osmolality = gap - Normal gap is close to zero - Abnormal gap may indicate presence of significant amounts of unmeasured substances in blood (alcohols, acetone (ketone) and other organic solvents) ### Techniques - Freezing-Point Depression - Cryptoscopic method - Convenient - Rapid - Small volumes - Most often used method - Osmotic Pressure - Slow to equilibrate - Need large sample volume - Membrane behaviour is not always reproducible - Vapor-Pressure Depression - Most accurate - Slow and requires very precise temp - Boiling Point Elevation - Not favoured for biological samples (structural degradation) #### Freezing Point Depression or Vapor Pressure Vapor Pressure - Vapor Pressure is better method BUT DOES NOT measure volatiles such as alcohols making it unsuitable for emergency room patients with possible alcohol ingestion Freezing Point Osmometer - Consists of sample chamber containing a stirrer and a thermistor (temperature sensing device) connected to readout device - Sample is rapidly super cooled to several degrees below freezing point - Sample is agitated to initiate freezing - As ice crystals form, heat of fusion is released. - Equilibrium temperature stays constant (freezing point) stays constant once reached. - Detected by thermistor and converted to milliosmoles per kg of water Quality COntrol =============== Lean vs. Six Sigma ------------------ - Lean principles work to eliminate waste (lean down the process) - Six Sigma seeks to improve performance of a process by eliminating causes of defects (developing the process) - Lean Six Sigma uses a problem cause solution method to improve processes - **DMAIC**: Define, measure, analyze, improve, control - One difference is that six sigma analyzes what taking away *or* adding to a process would be beneficial Sigma Metrics ------------- - Quantitatively measures error or variation in a system - Process sigma represents a capability of process to meet or exceed defined criteria for acceptability - Represents number of defects pers million opportunities - Also refers to number of SD away from mean a process can move before outside acceptable limits - **IQMH** is the governing body for determining what is and isn't acceptable in terms of QC values. They set minimum acceptable ranges but we try to exceed that - Sodium test has six sigma then the mean could shift by 6SD and still meet requirements for accuracy and precision but we want to do better - **6 Sigma** is very precise - 3 errors in every million tests - **3 Sigma** is not as precise - 26, 674 errors per million tests - 12% of lab errors impact pt health and 0.0375% happen in analytical stage so sigma can be used for more than that ### Sigma and QC - If a test has excellent precision/accuracy, then fewer errors occur - Takes a large shift in mean to fail - Fewer QC rules would be needed to identify errors if you're precise and accurate already - Rules maximise chance of detecting a problem but if it's built into your assay then you don't need to worry about it as much - Sigma=[\$\\frac{Total\\ allowable\\ error\\ - bias}{\\text{coefficient\~of\~variation}}\$]{.math.inline} - Total allowable error (IQMH dictated) - Bias (how close we are to a result relevant to peer groups) - Coefficient (IQMH tells us how precise we must be) ![A diagram of a function Description automatically generated](media/image22.png) A diagram of a function Description automatically generated ![](media/image24.png)*NOTE: You lose a bit of precision and accuracy (curve is flatter) and now it's down to 3 Sigma.* ***For Data QC Images (right and below):*** - *1~3S~ rule is all you need because assay is good enough* - *N = number of control levels needed* - *R = number of runs needed* - ![A diagram of a diagram Description automatically generated](media/image26.png) Method EVALUATION ----------------- - Methods and lab equipment must be evaluated for a number of criteria before they are suitable for patient testing - New method may be needed if current no longer meets needs or a new analyzer is required to replace an aging one - Most methods today are commercially developed - *TEST QUESTION: should know linearity. We can be confident in a method up to the limit of its linearity. If it hits the point of plateau we can declare we're confident in the assay up to a certain point or we can dilute to within an acceptable known range of our linearity* ### Method Selection - Collect technical information from: - Colleagues - Scientific presentations and literature - Manufacturer claims - Consider what type and volume of samples are required - Things you must consider with a new method: - Analytic sensitivity - Analytic specificity - Linear range - Interfering substances - Estimate of precision and accuracy - Series of standards nee to be analyzed to ensure linear range - QC should be done in replicates (8) to ensure short term precision - Those two things must pass before further evaluation can be done - NIST traceable ### Estimation of Inaccuracy Three types of studies are needed: - Recovery - Shows whether method can accurately measure analyte - "Spiked" sample used to determine how much of the analyte is detected - Original patient sample should not be diluted more than 10% (then you can lower the dilutions needed to find the lowest amount detectable) - Done to determine if specific compounds affect the accuracy of the lab tests - Interference - Common interferants include: bilirubin, hemolysis, and lipemia. - Interferants affect measurements by absorbing or scattering light, reacting with reagents, or affect reaction rate - If interferant is present and has and effect, the result may not be reported out or may be reported with a comment - Patient sample comparison - Examine pt samples by the methods being evaluated and by the reference method - 40-100 specimens ea ch day over 8-20 days, preferably tested within 4 hours - Samples should span clinical range expected in population - 25% should be below, 50% within range, and 25% above - Samples should be analyzed in duplicate - Daily pt samples should be analyzed(2-5 patients if 40 specimens used, 20 days if 100 specimens used) - Methods should produce straight line - Correlation Coefficient (r) = defines strength of relationship between two variables. Ranges from -1 to +1. A values of zero indicates no correlation or a random relationship between variables. - Coefficient of determination (r^2^)= proportion of variation explained by one variable to predict another. It indicates how well the line represents the data. #### Allowable Analytical Error - Limits that specify max error allowed - Standards used to determine acceptability of clinical chemistry analyzer performance - If a test does not meet allowable error criteria, it must be modified or rejected ### Carryover - Can be a problem when a high result is carried over into the next sample and falsely elevates it - Tests with high known results are deliberately followed with tests of low known results to determine the carry over. If carryover is detected, corrective action must be taken ### Reference Intervals - Test are used to make medical diagnoses and thus a reference interval is designed so that essential 95% of patients (healthy) would typically fall within the upper and lower limits - Reference ranges often referred to as "normal ranges" and that is not correct. Reference ranges are often normal ranges but **can differ for patients on drugs (therapeutic or toxic)** - **Reference Range Interval: a pair of medical decision points that span the limits of results expected for a defined healthy population** - Developing reference ranges: - If no existing range, test a minimum of 120 samples up to 700 - If existing range is available but you're switching methods, you have to test whether or not you can still sue that interval (often done by linear regression) and if you can, then you have to provide proof that you can. This is done by running as few as 40 samples ### Diagnostic EFFICIENCY - Ideal would be a test where if you are healthy it is normal and if you are not, it is abnormal but we often have overlap - To determine how good a test is at detecting and predicting disease states there are a number of parameters that are used. These are termed **diagnostic efficiency** which are broken down into: - Sensitivity - Specificity - Predictive values #### Definitions - **Diagnostic Sensitivity -** ability of a test to detect a given disease or condition - **Diagnostic Specificity -** ability of a test to correctly identify the absence of a given disease or condition - **Positive predictive value -** chance of an individual having a given disease or condition if the test is abnormal - **Negative predictive value -** chance of an individual not having a given disease or condition if the test is within the reference interval. - **True positives-** patients that have the disease that test positive - **False positives**-patients that don't have the disease but test positive - **True negatives-** patients that do not have the disease and test negative - **False negatives-** people that do have the disease but test negative - **Analytical sensitivity -** refers to the limit of detection (how low can you go and still pick it up?) - **Clinical sensitivity** is proportion that have the disease that test positive for the test #### Calculating Diagnostic Efficiency - Diagnostic Sensitivity =[\$\\frac{\\text{TP}}{TP + FN}\$]{.math.inline} - Specificity= [\$\\frac{\\text{TN}}{TN + FP}\$]{.math.inline} - If you have 100% specificity and 100% sensitivity it means the test detects everyone with the disease and everyone without will test negative. - Positive Predictive Values refers to the probability of an individual having the disease if the test is positive and negative predictive value means that the patient does not have disease if the result is within the reference interval. - PPV= [\$\\frac{\\text{TP}}{TP + FP}\$]{.math.inline} - NPV= [\$\\frac{\\text{TN}}{TN + FN}\$]{.math.inline} Chemistry QC ============ QC Statistics ------------- - ![](media/image29.png)Reference range for a particular measurement in most cases is related to a normal bell-shaped curve **Mean:** - Often used in lab measurements - Essential for estimating control values **Data Collection:** - Obtain a min of 20 data points from separate testing runs - If 20 runs are not feasible, use at least 7 runs (with 3 replicate per run - Provisional ranges can be set using mean and SD **Updating Mean and Limits:** - Replace mean and limits derived from abbreviated data when 20 separate runs' data become available - Regularly reassess to ensure accuracy and reliability Standard Deviation ------------------ Definition: - SD measures the spread or variability in a dataset - Calculated as the square root of the variance of values - SD is our **confidence limit** QC Tasks: - Establish QC targets and ranges (SD) for assay controls - Determine mean and SD for new QC sera lots - Revised standards allow for calculating target mean using 10 control specimens over 10 days Statistical Information: - In a normal population: - 68% of values fall within 1SD - 95% of values fall within 2 SD - 99.7% of values fall within 3SD Reference Values: - Reference interval includes 95% of test results for a healthy population - Replaces terms like "normal values" Confidence Intervals -------------------- - CI is represented by 2SDs around the mean - It encompasses 95% of values and considers day-to-day shifts in analytical procedures - The purpose of 95% CI is that it accounts for **variability** and **method imprecision** - Laboratory reference ranges - Manufacturer's ranges and QC limits on patient population - Control material analysis - New control lots should be analyzed alongside existing material - This ensures accurate mean and limits of quality control Coefficient of Variation ------------------------ - **Definition:** The CV, expressed as a percentage, is **the ratio of the** **standard deviation (SD) to the mean.** It quantifies the variability of a data set - **Purpose:** to normalize variability and allow for the comparison of SDs across different means. It's useful for assessing precision differences in assays and methods - **Comparison:** when comparing SDs, consider the mean. Directly comparing SDs without considering the mean can be misleading - Control limits: in QC, setting control limits at +/- 2SD is common, but it may lead to high false rejection rates Determination of Control Range ------------------------------ ### Control Solution Assessment After purchasing an unassayed control solution, labs must determine an acceptable control range for a specific analysis ### Method for Establishing Range One approach is to assay an aliquot of the control serum alongside regular batches of assays over 15 to 25 days. Treat the control sample like an unknown specimen ### Calculating ACCEPTABLE Limits Repeated determinations yield a normal bell-shaped curve, Calculate the mean (X) and SD. Most labs used 2SD from the mean, but others use it as a warning limit ### CONTROl Implementation Once the acceptable range is established, include control specimens from each batch. If a control falls outside of limits, repeat the procedure before reporting patient results. Accreditation bodies mandate written procedures for monitoring and resolving out-of-control situations. ### Steps - Step 1: Review procedures used - Step 2: Search for recent events that could cause change, such as a new reagent kit or lot, component replacement, or environmental condition (e.g. temp and humidity) - Step 3: Prepare new control materials - Step 4: Follow manufacturer's troubleshooting guide - Step 5: Contact manufacturers of instruments, reagent materials and controls ### Sources of Variance and Error Obtaining identical results for a specific specimen is generally impossible due to inherent variability. The variance, or error, arises from limitations in the procedure itself and sampling mechanisms - **Sampling factors:** reliable results depend on proper sampling. Variance related to the sample includes factors like collection time, patient position, physical activity, fasting duration and storage conditions - **Procedural factors**: aging chemicals, personal bias, and variations in standards, reagents, environment, etc. contribute to variance. Experimental errors may also arise from method changes, instrument variations or personnel shifts - Instruments should be run by about three techs Levey-Jennings Charts --------------------- - **Daily Control Specimen Values:** - Labs must plot the values on a **QC chart**. Many modern instruments automatically generate Qc charts daily, flagging out-of-control results - **Levey-Jennings Charts:** - Traditionally used, these charts identify unacceptable runs and evaluate deviations. Software automates control value plotting, aiding stability of the analytical measuring - **Purpose:** - Control charting ensures reliable and stable lab processes ![](media/image31.png)*Correction was made but new "mean" is too low. You would ask if you changed the lot or did something to bring down the mean. The imprecision at the end of the chart needs further investigation (could be sample error like a faulty pipettor)* - **Control Samples:** - QC involves using at least two different control samples for a specific analyte. Includes **normal** and **abnormal** controls. - **Control Limits**: - The mean value and acceptable error limits are indicated on the chart. Control limits are typically set at ±2 SD or ±3 SD from the mean. The 2-SD value serves as a **warning limit**, and the 3-SD value acts as an **action limit**. - **Daily Monitoring**: - Each day, the control value is plotted on the chart. Any value falling "out of control" is easily identified. Control charts provide visual documentation and help detect trends or drift over time, especially during procedural changes - **Validation of New Procedures**: - Laboratories validate new procedures before routine use. - Reproducibility and confidence limits are determined. - Acceptable variation limits for control specimens are established. - **Quality Control (QC) Program**: - Calculates mean and standard deviation (SD) for each procedure. - Generates control charts for monitoring performance. - Regular assessment routines detect issues promptly. - **Timely Corrections**: - If problems arise, corrections are made promptly. - Patient results are reported only after resolving issues [**Shifts, Trends, and Dispersion**:] - **Shifts**: - **Definition**: A sudden and sustained change in one direction in control sample values. - **Indication**: May occur due to sudden instrument malfunction. - Visual inspection of control charts helps detect these changes - **Trends or Systematic Drift**: - **Definition**: Gradual change in control sample results. - **Indication**: Control value direction consistently shifts from the mean for **at least 3 days**, signaling potential issues like reagent deterioration or control sample problems. - **Dispersion**: - **Definition**: Dispersion refers to an increase in **random error** or **lack of precision**. It signifies variability in measurement results. - **Significance**: Dispersion may indicate **instability problems**. When measurements scatter widely, it can affect the reliability of data *Actual range stays the same in photo, but if it flattens out it's a precision problem.* Quality Control Multi-Rules --------------------------- - Shall be applied before reporting patient data - Designed to detect random and systematic errors - Multiple quality control rules are used to decrease probability of false rejection while at the same time maximize the ability to detect true error Rates of False Rejection - With 1 control: 5% - With 2 controls: 10% - With 3 controls: 15% ![tab\_04\_10](media/image33.jpeg) *For full descriptions and images of the Westgard rules, see slide 37-end of Chemistry QC PowerPoint.* Chromatography ============== Chromatography Overview ----------------------- - Group of techniques used to separate complex mixtures on the basis of different physical interactions - Basic components: - Mobile phase (the part that moves) - Gas or liquid - Stationary phase (doesn't move) - Solid or liquid - Column holding stationary phase - Holds stationary phase in specific types of gas chromatography - Separated Components (eluate) - Attached to something then eluted from it ### Chromatograms - Instruments provide a response that is related to the amount of compound eluted from column as function of elution time or volume passed through system - Resulting plot of **response vs. time** is known as a chromatogram - Average time for particular chemicals to pass through a column is known as **retention time** - Values increase with strength and degree that chemical interacts with stationary phase - Most separations are carried out by injecting small volume or amount of sample into chromatographic system - Retention time helps **identify** compound - Area or height of peak **quantitates** that is present - Width of peak represents separating performance of efficiency of system - Sharp peaks are best and means the system is functioning better (system is able to separate peaks with similar interactions...resolutions) - Broad peaks introduce lower limits of performance - A graph of a graph Description automatically generated ![A black line with red text Description automatically generated](media/image35.jpeg) A diagram of a curve Description automatically generated - **Peaks are only relevant to the column used as different substances will react with different stationary phases so you must use references for the column you have** - There are factors that affect the chromatographic efficiency: - Column length - Particle size of support - Uniformity in size, shape and packing of support - Flow rate - Mobile phase viscosity - Initial injection volume Methods of Separation --------------------- - **Adsorption** -- "sticking to something." Liquid-solid. Based on competition between the sample and mobile phase for adsorptive sites on the solid stationary phase - **Partition** -- Liquid-liquid. Separation based on relative solubility in an organic (non-polar) solvent and aqueous (polar) solvent - **Ion-exchange** -- solute mixtures are separated by magnitude and charge of ionic species - Good for removing interfering substances from solution - Concentrating dilute ion solutions - Separating charged molecules like amino acids ![A diagram of separation mechanism Description automatically generated](media/image37.jpeg) ### ion Exchange Chromatography - Based on an exchange of ions between a charged stationary phase and ions of the opposite charge in the mobile phase - Retention of components by stationary phase is adjusted by changing the ionic strength or pH of the mobile phase - Amino acids, glycated hemoglobin, hemoglobin variants, oligonucleotides ### Partition Chromatography - Differential distribution of solutes between two immiscible liquids is the basis for separation - Classified as **gas-liquid** chromatography or as **liquid-liquid** chromatography - Used for separation of biological, organic, and inorganic molecules by differences in polarity ### ADSORPTION Chromatography - Differential adsorption of solutes on the surface of the stationary phase - Retention depends on the surface area of the stationary phase and the affinity of the solutes for the stationary phase - In GC this mode is used to separate low molecular weight compounds that are normally gases at RT ### Size-Exclusion Chromatography - AKA molecular-exclusion, gel filtration, molecular-sieve chromatography - Separates solutes based on their molecular size in solution. - Commonly used to separate large molecules such as proteins and nucleic acids from small molecules such as salts or oligonucleotides. ### HPLC -- High Pressure Liquid Chromatography - Uses pressure for fast separations, controlled temperature, (usually ambient/RT) in-line detectors, and gradient elution techniques - Components: pumps, columns, sample injectors, detectors, recorders - Silica gel often used in column - Eluate monitored as it leaves column which produces an electronic signal proportional to concentration. - Photodiode array often used for **drug analysis** in urine - Separates and **measures hemoglobin A1c** and **beta thalassemia** - Mechanical pump provides precise and accurate flow Gas CHROMATOGRAPHY ------------------ Separation technique that uses "carrier gas" to move compounds through a stationary phase located within a column. - Carrier gas is **inert** -- usually helium or nitrogen ### COmponents - Basic design of GC consists of: - Carrier gas supply - Sample injection device and GC inlet - Column - Detector - Data system #### Column - Packed - Open tubular or capillary - **Capillary often used due to high efficiency** - Stainless steel - Fused silica -- would take out water - Teflon -- doesn't react - Housed in ovens that provide high temperatures for required separation - Longer columns are more efficient but require increased carrier gas pressures and analysis times - Narrow columns are more efficient but wider columns have increased sample capacities ### Process - Sample must be injected through septum as gas or temperature port must be above the boiling point of components, so they vaporize upon injection - Injection port usually 30-50 degrees higher than column to vaporize what you're looking for, so they travel through column - Samples with high boiling points move slow - Chromatogram used to identify compounds by retention time and by determining the area under the peak. - Detector usually thermal conductivity or flame ionization detectors (FID) since most stable - Retention determined by vapour pressure and volatility which depends on interaction with stationary phase #### Injection Problems - Septum leaks - Adsorption of sample components onto septum -- septum should be inert so it doesn't act like a second column - Thermal decomposition (spurious peaks or "ghost peaks") - Teflon septum or low bleed septum can illuminate this ### Analyte Identification - Internal standard may be added to specimens to check on consistency of retention times - Irregular gas flow - Leaks in the system (because we know the amount we're putting in) - Changes in temperature control (if oven not maintained, you'll get strange results) - Degradation of the stationary phase may lead to problems with stable retention times. - Acts as quality control measure and calibration - Seems weird to add something to the sample but it's important because the internal standard is something that doesn't usually appear there. **Internal Calibration** - Internal calibration is the internal standard. -- added to each reference solution and to each sample - A calibration curve is then established for the ratio of detector response of the analyte versus the internal standard and this curve is applied to ratios of signal from analyte versus internal standard in patient specimens **Analyte Quantification** - Electronic signals generated by the detector are also used to produce quantitative information - External and internal calibrating techniques have been used. **External Calibration** - Reference solutions containing known quantities of analytes are analyzed to establish detector responses per amount of analyte - A calibration curve can be constructed - Peak height - Peak area #### Limit of Detection - Analytical limit of detection can be improved by: - Increasing chromatographic efficiency (taller and sharper peaks) - Increasing amount of specimen loaded - Higher analyte concentration - Proper settings of detectors #### Detectors - Must provide: - High sensitivity - Good stability and reproducibility - Linear response over wide concentrations - Expand temp range 300-400 Celsius - Short response times (so things don't build up in the column - Non-destructivity of samples (so you don't burn up your sample) - Types of detectors: - FID -- Flame Ionization - Quantitative analysis can be accomplished by assaying series of standards - Integrating area of peaks or calculating peak height ratios ### Liquid Chromatography Advantages - Liquid chromatography is better than gas in that: - Less extensive extraction procedures saving time and expense. - Polar and heat labile compounds fare better in LC (ambient temp) - L. Chromatography is often less robust than GC resulting in wider peaks and more variable retention times and potentially more maintenance. - Less reproducible mass spectra for LC and long run times. For high volume testing several pumps may be installed. - LC great for vitamin D, testosterone, immunosuppressant drugs. Can detect multiple analytes in one run Mass Spectrometry ----------------- Technique to identify unknown compounds and determine concentrations of known substances. Can also study molecular structure and chemical composition of organic and inorganic material. - Determine how drugs are used in body - You can get the metabolites of drugs in the body -- can be helpful for testing new drugs because MS can help you figure out what is/can be produced by the drug - Identify illegitimate steroids in athletes - Determine damage to human genes due to environmental causes - Identify and characterize proteins - Detect presence of metabolic disorders in infants -- PKU - Used as a detector to identify samples eluting from gas chromatographic or HPLC columns - When coupled with other techniques, has powerful analytical capabilities with widespread clinical applications ### Clinical Applications - One of the most common applications of GC-MS consists of **drug testing for clinical or forensic purposes** - Many drugs have relatively small molecular weights and are sufficiently volatile for analysis by GC. - Limitation is that GC-MS is that compounds must be sufficiently volatile to allow transfer from solid phase to the mobile carrier gas and thus elution from the column to the detector - Structural and molecular weight determinations, identification and quantitation - GC/MS- drugs of abuse - LC/MS- low level Vitamin D, testosterone, immunosuppressant drugs. - Superior sensitivity to immunoassays - Free from antibody interferences like in immunoassays - Can detect multiple analytes in one run #### New-Born Screening - Because of technological advances -- Mass Spectrometry often included in newborn screening programs. - Newborn screening is a public health activity aimed at: - Early identification of conditions for which timely intervention is expected to result in elimination or reduction of morbidity, mortality and disabilities. - **Guthrie Test** - Originally instituted in the 1960's by Robert **Guthrie** and colleagues who developed a screening assay known as the **Guthrie test** which measures the phenylalanine content of a dried blood spot on filter paper collected from blood of newborn babies. - **PKU** -- Phenylketone Uria - PKU is a disorder of phenylalanine metabolism that results from the absence of phenylalanine hydroxylase activity leading to the accumulation of phenylalanine and production of phenylketones that are excreted in urine. - Patients are clinically asymptomatic at birth with developmental delays and neurological manifestations becoming evident at several months of life when brain damage has already occurred ### Sample Introduction and Ionization - Sample volatilized, ionized to form charged molecules then **separated based on mass charge ratio** - Electron ionization- most common form of ionization- GC/MS - Atmospheric pressure ionization-LC/MS - Electrospray ionization-"soft" ionization techniques that leave molecular ion largely intact. Most common ionization source. - Atmospheric pressure chemical ionization - Electron ionization -- hard or destructive - Atmospheric pressure ionization -- soft - Diff between soft and hard is soft leaves molecule intact but hard can break up the particles ### Atomic Mass Spectrophotometry Steps - Atomization - **Atomization** -- separates into individual atoms - Conversion of a substantial fraction of the atoms to stream of ions (usually single charged and positive) - Separation of ions based on mass to charge ratio (m/z) - Counting the number of ions by measuring current produced when ions strike transducer - **Transducer** -- changes electrical current into something we can detect - Obtained by **dividing the atomic or molecular mass by the number of charges that the ion bears** ### Components of Mass Spec **3 Basic Components:** - Ion source - Some way to atomize into ions - Mass analyzer - Gives us the first value (mass of what they are) - Ion detector - Gives us the charge - Mass spectroscopy determines the mass-to-charge ratio (m/z) of a molecule that has been ionized. - Mass spectrum is a 2-D plot of m/z versus ion abundance #### Ion Sources - Electrospray ionization (ESI) - soft - Matrix-Assisted Laser Desorption Ionization (MALDI) - Common in micro because you can identify proteins in microbes - Sum of all ions produced is displayed as a function of time to yield a total ion chromatogram (TIC) - When only a few analytes are of interest for quantitative analysis and their mass spectrum is known, mass spectrometer is programmed to monitor only those ions of interest - selected ion monitoring - All MS techniques require an ionization step in which an ion is produced from neutral atom or molecule. - Electron ionization and chemical ionization are ionization techniques that are used when gas phase molecules are introduced directly into the analyzer from GC. - In EI, gas phase molecules are bombarded by electrons emitted from a heated filament and attracted to a collector electrode. - Process must occur in a vacuum **[Electrospray ionization]** - Sample is ionized at atmospheric pressure before introduced to mass analyzer. - soft ionization technique meaning that relatively little fragmentation is produced during the ionization process. - Doesn't break protein, just adjusts charge - Whole thing *isn't* done in a vacuum ### Liquid Chromatography-Mass Spectrometry - **More difficult** since analytes are dissolved in liquid rather than gas. Causes difficulties for vacuum pumping system of mass spec. - HPLC-MS is used clinically in screening and confirmation of genetic disorders and inborn errors of metabolism. Anti-retrovirals, steroid hormones ### MALDI-TOF and SELDI-TOF - TOF = time of flight - Pathogen Identification- resulting in protein fingerprint of species which can be compared to library - MALDI TOF MS requires inexpensive reagents and only takes 5-10 min per run making it faster and cheaper than traditional automated techniques

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