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UOIT Chemistry 1

Laboratories working with infectious agents not known to cause disease in healthy humans. BSL1
Labs handling infectious agents of moderate potential hazard to personnel or the environment, and required for laboratories working with specimens of blood or body fluids. BSL2
Required when working with agents that cause serious or potentially lethal disease via inhalation. Stringent engineering and safety precautions are required. BSL3
moderate individual and low community risk Risk Group 2
1) Product Name 2) Safe handling instructions 3) MSDS Workplace Label
Blue: health Red: Fire Yellow: Reactivity White: Specific Hazard NFPA (National Fire and Prevention Agency) communication label
patient is inconvenienced or harmed as a result of an improperly processed sample Critical Incidence
the state or job of being in charge of someone or something and of making sure that what they do or what happens to them is right or satisfactory Responsibility
Having the necessary knowledge, skills and judgment to perform safely, effectively and ethically and applying that knowledge, skill and judgment to ensure safe, effective and ethical outcomes for the patient. Competence
Being responsible for one’s decisions and actions, including those undertaken independently or collectively as a member of a team. Accountability
the measure by which the accuracy or quality of performance of an individual is judged Standard of Practice
-Graduate from an accredited program in medical laboratory science -Write the CSMLS exam and pass -Register with the CMLTO What do you need to do to obtain a job in Ontario?
1) Laboratory and Specimen Collection Centre Licensing Act R.R.O. 1990, Regulation 682 2) Regulated Health Professions Act 1991 3) Medical Laboratory Technology Act 1991 3 major acts
Cannot operate a laboratory in Ontario without obtaining a license from MOHLTC Laboratories Branch Laboratory and Specimen Collection Centre Licensing Act
Lab Requires a Laboratory Director (medical practitioner certified by Royal College of Physicians and Surgeons) in order to obtain a license Specimen Collection Act
person who is responsible for the administration of the scientific and technical operation of a laboratory including the supervision of the test and the reporting of the results of the tests laboratory director
person who under the supervision of a laboratory director supervises laboratory personnel and who may perform tests requiring special scientific skills Supervisor/technical specialist
person who under general supervision performs tests which require the exercise of independent judgment laboratory technologist
person who under direct supervision performs laboratory tests which require limited technical skill and responsibilities laboratory technician
acts that only qualified persons of RHP are authorized to perform as these activities could cause harm to the public controlled acts
is a controlled act procedure, or a portion of a controlled act procedure, that is authorized to a specific profession to perform under the appropriate health profession Act (MLT Act). Authorized Act
a controlled act procedure, that is performed under the authority of delegation from someone who is authorized to perform the act Delegated Act
The college is established under the name College of Medical Laboratory Technologists of Ontario (CMLTO) Established College for MLTs
7-11 persons who are members elected in accordance to the bylaws. 7-10 persons appointed by the Lieutenant Governor who are NOT members (or members of other colleges). 1 person selected from members who are faculty members of an educational institution. college Council
lawsuit involves private individuals suing someone for a negligent act that violates their private rights CIVIL Negligence
more serious and involves committing a crime against society and intending to commit it. CRIMINAL Negligence
Consent must be informed and may be expressed or implied Principles of Consent in the Health Care Consent Act, 1996
“Incapable” patients may include minors, hearing impaired, legally not competent, physically unable to write, doesn’t speak language Consent
7 Committees Executive Registration Quality Assurance Complaints Discipline Fitness to Practice Patient Relations COLLEGE STRUCTURE Committees CMLTO
Components of this program include: Professional Portfolio Practice Review Competence Evaluation Quality Assurance
A minimum of 30 hours of professional enhancement/continuing education each year (previously 60 hours over two years): document what you’ve done. Reading articles etc. Professional Portfolio
Selected members must answer 25 questions, online, related to the CMLTO standards of practice and the application of professional knowledge, skill and judgment. Practice Review
Takes place if the QAC is of the opinion that the member's knowledge, skill, and judgment are unsatisfactory.Decision based on review of member’s professional portfolio, Practice Review assessment, or any other written information provided to the QAC. Competence Evaluation
Manual tests with every batch Following the introduction of a new reagent lot When tests results (in particular controls) are suspect Following certain maintenance procedures Following changing or adjusting a lamp At a minimum of 6 mos When do we need to calibrate?
a measure of both accuracy and precision of a method Reliability
Random error signals a change in IMPRECISION!!! Random Error: An error which varies in an unpredictable manner, in magnitude and sign
Systematic error signals a change in ACCURACY!!! Systematic Error: An error which remains constant when measurements are made under the same conditions, or varies according to a definite law when conditions change.
No. of gram equivalent weights per 1L of soln Gram equivalent weight=gmw of a substance divided by its valence Valence: how many “parts” a compound will dissociate into HCl valence = 1 H2SO4 valence = 2 Normality
Absorption spectrophotometry Emission spectrophotometry Reflectometry Turbidimetry light measuring systems
Wavelength calibration Photometric Accuracy Photometric Linearity Stray Light Spectrophotometer Quality Assurance
-Absorption spectrophotometry -Reflectometry -Emission Spectrometry(Flame Photometry, Atomic Absorption Spectroscopy, Fluorescence and Fluorescent Polarization, and Chemiluminesence) -Light scattering (Turbidimetry and nephelometry) LMS
A = 2-log%T Beer’s Law
A = abc where A = absorbance a = absorptivity constant b = light path in cm c = concentration of analyte Beer-Lambert Law
The wavelength selected for analysis is usually at the peak of maximal absorbance to achieve maximum sensitivity!!! Spectrophotometry
-Related to the chemical nature of the solution -L/mol cm -Relationship:compound with higher molar absorptivity has a higher absorbance for a given molar concentration than one with a lower molar absorptivity. Absorptivity constant - Assays select chromogens that have higher molar absorptivity (increased sensitivity)
the degree of isolation is the function of the type of monochromator used and the width of entrance and exit slits used Spectral Isolation
the number (or range) of λs passing through the exit slit Bandpass
the width of the opening in the exit slit (measured in nm) Bandwidth
Band Width is inversely related to peak resolution =the larger the bandwidth, the poorer the resolution Effect of Bandwidth size
often 5-15nm bandwidths =narrow-bandpass filters Interference filters
Double-beam has a single light source which is reflected off a mirror to 2 sets of components (two detectors) Double Beam Spectrophotometer in SPACE
a single light source in which a “CHOPPER” alternately directs the light beam from the sample to reference pathways and mirrors return the 2 light paths to a single detector Double Beam Spectrophotometer in TIME
The light changes direction (similar to reflectance) but is scattered in different directions dependent upon the size of the particle that it collides with Light scatter occurs when radiant energy collides with the molecules in a solution
-Rayleigh Scatter:Light is scattered in all directions around the particle -Rayleigh-Debye Scatter:light scattered in forward direction, some perpendicular direction, no backward direction -Mie Scatter: light scatters in the forward direction Light scatter
measurement of decrease in light intensity (ABS) caused by scattering, reflectance, and absorbance of incident light (due to turbidity from particles in solution). Used to determine amount of particulate matter in a solution Turbidimetry
Amount of light transmitted to detector is inversely proportional to concentration Turbidimetry
Clinical Use: Most often used to detect formation of IMMUNE COMPLEXES (Ag-Ab complexes) in immunoassays TDM assays, specific proteins (IGs, Complement) Light Scattering Techniques
Measurement of light SCATTERED in forward direction by particulate solution.Amount scatter directly proportional to number and size of particles in solution.Detector not in direct path of transmitted light read at an angle(30–90o) to the incident light Nephelometry
When photons strike the surface of certain materials, they can emit electrons or light.
Light and heat provide energy sources to allow electrons to jump to higher energy levels
atomic absorption (from ground state) atomic emission (thermal excitation) atomic fluorescence (light excitation)
Flame photometry involves the excitation of ATOMS to a higher energy level, followed by spontaneous emission of a CHARACTERISTIC λ of light when returning to the ground state The heat of a flame excites the atoms. Flame Photometry
When a controlled volume of sample is introduced into the flame: the intensity of the characteristic wavelength of light emitted is indicative of the number of excited electrons and therefore is DIRECTLY proportional to the concentration. Flame Photometry
Aspirator and atomizer (makes fine mist droplets of the sample) to allow the sample to be vaporized in the flame Utilizes the Venturi effect: pulls sample up into the flame by negative force Flame Photometry
Flame: acts as light source and sample cuvette Needs constant temp, must be steady Uses propane-air at 1900oC: mixture determines temperature of flame Flame Photometry
-Not normally found in humans! -Emitted λ must be different than analyte λ -Should have similar solubility and excitation characteristics Requirements for the internal standard
Atomic absorption involves absorbance of characteristic λ of light by ground state ATOMS *Flame (heat) dissociates chemical bonds to generate free atoms in ground state.Energy level of heat not high enough for electrons to jump to higher energy levels Atomic Absorption
Hollow Cathode Lamp: hollow quartz cylinder with a metal cathode lined with element being measured and filled with an inert gas (helium, argon) Components of AA Spectrophotometry
Principle: current causes ionization of gas which moves to cathode and dislodges metal ions which collide with gases and get excited! When the metal ions of the cathode return to the ground state, characteristic energy of that metal is emitted Hollow Cathode Lamp -Need to change the lamp for each analyte to be analyzed!
1) Nebulizer: converts sample into a fine spray (nebulization) 2) Flame: heats spray mixture , evaporates solvent, leaves particles that disintegrate from heat to atoms Components of AA Spectrophotometry
Uses Graphite Furnace to replace flame! Sample dried onto a carbon support platform Electric current passes through support creating a temperature high enough to vaporize the analyte. Required to vaporize heavier metals More sensitive than flame Flameless AAS
Monochromator located AFTER the sample. WHY?? Removes background energy (light) produced by the flame itself! Components of AA Spectrophotometry
Why is there a chopper? Excited atoms in flame emit light at same wavelength as lamp. When chopper blocks lamp emission; detector picks up flame emission form excited atoms. Concentration is difference between 2 readings = similar to blanking. Components of AA Spectrophotometry
Add competing metal Lanthanum or Strontium which forms a tighter bond with phosphorus and removes it from Ca++ or Mg, allowing for the analysis of those metals Atomic Absorption Spectrophotometry
-Spectral Interference: absorbance from otherclosely related absorbing species.Reduced by using very narrow bandwidths --Chemical:cations of interest form complexes with anions that cannot be dissociated by the flame.+ metal Lanthanum or Strontium Atomic Absorption Spectrophotometry -Interferences: potential sources of error
-Ionization: lower flame -Matrix: sample and standards have dissimilar properties. Dilute sample. Atomic Absorption Spectrophotometry -Interferences: potential sources of error
Luminescence: the emission of radiant energy when an electron returns from an excited state (higher energy level) to a lower energy level Usually at a molecular level not atomic Excitation NOT from heat but from light Emission Spectroscopy –Luminescence
-Fluorescence -Phosphorescence -Bioluminescence -Chemiluminescence -Electrochemiluminescence Types of Luminescence
Fluorescence is the immediate emission of light from a MOLECULE following absorption of light *Uses a light, NO Flame Fluorescence
Occurs when a molecule absorbs light at one wavelength and re-emits light at a longer wavelength Fluorescence
Stokes Shift: difference between max λ of excitation light and max λ of emitted light Fluorescence
There is a time delay of 10-8 to 10-7 seconds between absorption and emission Fluorescence (emission) has a rapid (10-8 sec) decay time Therefore, reading MUST take place during quick burst of light (emission!) Fluorescence
Attenuator controls light intensity Fluorescence
Why at right angle? Blocks light from excitation source Fluorescence
Fluorescence intensity is DIRECTLY proportional to the concentration of the fluorophor (analyte). Fluorescence
Immunoassays use fluorescence by using fluorophors as labels attached to an Ab or Ag Measure hormones, porphyrins, amino acids, vitamins, drugs, etc Fluorescence
-Concentration effects:High concentration of fluorophor results in non-uniform distribution of fluorescence -no longer linear Light Scattering Effects: Loss of fluorescent signal due to background light scatter Limitations of Fluorometry
-Solvent Effects:interaction between fluorophor and solvent or another solute in the solvent.Loss of signal due to energy transfer -Cuvet Effects:Plastic cuvets contain UV absorbers that fluoresce and contribute to background fluorescence.QUARTZ cuvets! Limitations of Fluorometry
-Temperature Effects:fluorescence intensity decreases by 1-5% for every increase of 1 degree in temperature.Must regulate temp to within ± 0.1 oC Samples may contain compounds that fluoresce (bilirubin, protein) or scatter light (lipemia) Limitations of Fluorometry
pH Effects: fluorescence is pH dependent because many molecules must be in either the ionized or un-ionized form to fluoresce Sample Matrix effects: Limitations of Fluorometry
Emission of light as a product of a chemical reaction. NOT photolumination = no excitation light! NOT heat = no flame!!! Oxidation of certain organic compounds by an oxidant results in light emitted from the excited object MOST sensitive of all LMS Chemiluminescence
A maximum limit of TWO HOURS from the time of collection is recommended. Exceptions:-K+, ACTH, cortisol, catecholamines, lactate, homocysteine must be separated in <1 hour Serum or plasma (S/P) to be separated from red cells asap. Note:studies show that by 2 hours: -glucose is decreased -K+ is increased -LD is increased
Separated Serum or Plasma (S/P) should remain at RT for NO longer than 8h (less for some analytes) If >8h, then S/P should be refrigerated (2-8oC) If >48h, S/P should be frozen at or below -20oC Specimen Handling in Biochemistry
S/P samples are NOT to be repeatedly frozen and thawed! Some proteins denatured. To be thawed only ONCE Frost-Free freezers are NOT to be used in the clinical lab WHY?? Accreditation thing. Specimen Handling in Biochemistry
LD, AST, K, Plasma Hb, iCal decreased Hemolysis seriously affecting results (Increased)
Iron, ALT, decreased T4 Hemolysis Noticeably affecting results (Increased)
Phosphorus, total protein, albumin, magnesium, calcium, ACP Hemolysis slightly affecting results (Increased)
Bilirubin, Vit A, Vit B6, beta-carotene, porphyrins Sensitive to light
Chilled Specimens: 2-8oC slows metabolism of blood cells and stabilizes thermolabile constituents Examples: Ammonia, catecholamines, lactate, pyruvate, gastrin, PTH, arterial blood gases Specimen Handling in Biochemistry
do not store sample for electrolytes on ice!!! Cold inhibits glycolysis which is required for pumping K+ into the cells. W/O glycolysis, K+ leaks out into the plasma (false increase) Specimen Handling in Biochemistry
Mostly synthesized in the liver and secreted into the circulation Exception is immunoglobulins which are synthesized in lymphocytes/plasma cells Proteins
POSITIVE Nitrogen Balance: intake exceeds loss of Nitrogen Children Pregnancy Adults recovering from major illness Proteins
NEGATIVE Nitrogen Balance: loss of Nitrogen exceeds intake Burns (excessive tissue destruction) Wasting diseases High fever Starvation Proteins
Nonpolar: hydrophobic side chains saturated with hydrogen Uncharged polar: hydrophilic uncharged side chains Acidic: hydrophilic acidic (COO-) side chains Basic: hydrophilic basic side (NH3+) chains. Amino Acids
Can be classified by Function: Enzymes Hormones Transport proteins Defensive proteins Structural proteins Motor proteins Receptors Signaling proteins Storage proteins Proteins
Simple Those proteins which upon hydrolysis yield only amino acids Conjugated Upon hydrolysis yield both amino acids and a non-protein prosthetic group Derived Group of proteins that are derived from other proteins Ex.Pepton Classification by Structure of proteins
Plasma pH = 7.4 Plasma Proteins have net negative charge and remain in solution!!
In solution more acidic than the pI (pH<pI), the COO- in the amino acid accepts a proton. pH and ionization
In solutions more basic than the pI (pH>pI), the —NH3+ in the amino acid donates a proton. pH and ionization
Measurement of Total Protein (TP) provides general information reflecting disease states in many organ systems and is not diagnostic on its own
In alkaline medium and presence > 2 peptide bonds,Cu2+ form complexes with peptide bonds to produce a violet colored chelate Sodium potassium tartrate:prevent precipitation of copper in the alkaline medium. Potassium iodide:prevent oxidation Biuret alkaline solution of copper II sulfate
Biuret can NOT be used for: Urine analysis: ammonium ions in urine interfere with the method Spinal Fluid analysis: the method is not SENSITIVE enough to detect low levels of protein found in sf Use dye binding methods instead Measurement of Total Protein
Disease caused by inherited enzyme defect that blocks normal metabolism of amino acid Aminoaciduria categorized as Overflow: arise from increased plasma concentrations Renal: defects in renal tubule reabsorption of aa
Once albumin binds to dye, dye has different absorbance maximum (different λ) than free dye. Amount of albumin quantitated by measurement of absorbance at λ of albumin-dye complex. BCG –bromcresyl green (most common) BCP –bromcresyl purple Measurement of Albumin
Thin Layer Chromatography with Ninhydrin detection Reaction with Ninhydrin to produce a purple color (except proline and hydroxyproline produce characteristic yellow color) AA Methods of analysis
Phenylketonuria (PKU) Absence of PAH PHE accumulates in blood, urine and CSF (toxic in high concentrations) PAH converts PHE to tyrosine, without it you get toxic byproduct instead: phenylpyruvic acid and metabolites Excreted in the urine
Uses strain of Bacillus subtilis which only grow in culture medium in presence of PHE. Small disk of filter paper is punched out and placed on an agar gel plate containing Bacillus subtilis and B-2-thienylalanine which inhibits bacterial growth Guthrie Test for PKU (Semiquantitative) .If there is normal concentration of PHE on the disk, the bacteria will not grow!
Infant must be 24 hrs old. Sampling should be done before administration of antibiotics or blood transfusion. False positives seen in premature infants: Immaturity of the liver’s enzyme systems Guthrie Test for PKU (Semiquantitative)
albumin: 35-50g/L TP: 60-80g/L FBS:4.0-6.0mmol/L Normal albumin in urine is <20mg/L RI
Responsible for ~80% of intravascular oncotic pressure.Binds to other analytes for transport in blood (promotes solubility). Functions of Albumin: Plays a major role in nutritional status =Source or reserve for amino acids Acts as plasma buffer.Provides antioxidant activity (binds FFAs, copper ions, bilirubin)
Tests for the presence of starch in the form of Amylose.Amylose forms long, tight, unbranched helices that are ideal for its storage function -allows iodine to insert itself into the helices. Iodine Test for Starch The immediate formation of a vivid blue color indicates amylose.
Reducing monosaccharides are oxidized by copper ion to form carboxylic acid and a reddish precipitate of copper (I) oxide within 3-5 minutes. Reducing disaccharides undergo the same reaction, but do so at a slower rate. Barfoed’s Test for Monosaccharides
Dehydrates ketohexoses to form 5-hydroxymethylfurfural which further reacts with resorcinol to produce an orange-red product within 2-5 minutes. Aldohexoses react to form the same product, but do so more slowly. Seliwanoff’s (Resorcinol) Test for Ketoses Note: Sucrose is positive as HCL hydrolyzes it to glucose and fructose
Detects the presence of reducing sugars:in hot alkaline solution, copper sulfate reacts with electrons from aldehyde functional group of reducing sugar to form cuprous oxide (Cu2O).POSITIVE =reducing sugars is the appearance of green to brick red color Benedict’s Test for Reducing Sugars
The appearance of a reducing substance other than glucose may be indicative of an inborn error of metabolism Galactose Lactose Fructose
Deficiency of galactose-1-phosphate uridyltransferase (prevents the conversion of galactose to glucose) =Build up of galactilol and galactonate which have direct toxic effects IEMs: Galactosemia
Hereditary Fructose Intolerance: a metabolic disease caused by the absence of the enzyme 1-phosphofructalsolase IEMs: Fructose Intolerance
Urine is normally NEGATIVE for glucose GLU filtered by the glomerulus and completely reabsorbed at the proximal renal tubule Blood glucose must rise above the renal threshold of 10.0 mmol/L before it spills into the urine Glycosuria: presence of glucose in urine Indicative of a pathological event
Strip contains glucose oxidase and organic dye.Glucose oxidase oxidizes glucose to gluconic acid with formation of hydrogen peroxide.Hydrogen peroxide reacts with o-toluidine to produce color. Color of strip related to the amount of glucose present. glucose oxidase method This test is specific for glucose since the glucose oxidase enzyme does not react with other reducing sugars that are readily oxidized and give positive Benedict's tests.
Glucosuria Glucose in the urine Polyuria Excessive urination Polydipsia Excessive thirst Polyphagia Excessive eating (hunger) Unexplained weight loss Type I Diabetes complication: ketoacidosis
Insulin Resistance: exhibit a decreased biological response to normal levels of insulin (either by reduced receptor numbers or post-receptor abnormalities). Type 2 Diabetes complications:Hyperosmolar nonketotic hyperglycemia
2008 Practice Guidelines: CDA diagnosis of diabetes can be made if: FBS > 7.0 mmol/L (alone) OR Casual PG (RBS) > 11.1 + symptoms of diabetes (polyuria, polydypsia, unexplained weight loss) OR 2hPG in a 75g OGTT > 11.1 mmol/L
In separated (centrifuged and removed from cells), non-hemolyzed, bacteria-free serum, glucose concentration is stable for up to 8h at 25oC (room temperature) and up to 72h at 4oC (refrigerated). Stability of Glucose
Fasting (8-12h but not >16h): Venous:lowest glucose Capillary:0.1 – 0.3 mmol/L (7%) higher than venous Arterial:0.4 - 1.2 mmol/L higher than venous and capillary FBS is diurnal:higher in the morning than evening draw blood in am to avoid false “neg” Measurement of Glucose
-Specific for B-D-Glucose.Enzyme mutarotase is added to rgt=convert α-to β- form -Peroxidase step non-specific:Interferences from uric acid,ascorbic acid,bilirubin,hemoglobin inhibit reaction by competing with chromogen for H2O2.Falsely LOW glucose. Limitations of the Glucose Oxidase reaction Interference from Bleach (strong oxidizing reagent) results in falsely ELEVATED glucose results.
Hexokinase The amount of NADPH produced is directly proportional to the amount of glucose in the sample and measured at 340nm Accepted as the REFERENCE METHOD for Glucose NO interference from ascorbic acid or uric acid or bleach
Always a STAT test!! Normal glucose levels in Sf are approximately 60% of the blood glucose. Spinal Fluid Glucose
Ketone bodies are produced as by-products in the liver through the metabolism of fatty acids (Lipolysis) ketoacidosis occurs in Type I diabetes
3 ketone bodies are produced always in the following ratio Acetoacetate: 20% Acetone: 2% B-hydroxybutyrate: 78%
Nitroprusside reaction: Sodium nitroprusside reacts w/ acetoacetic acid in an alkaline medium to form a purple color If glycerin is added to the reagent then acetone is also detected Used in urine dipsticks and the Acetest Tablet tests Measurement of Ketones
Enzymatic reaction: Measures b-hydroxybutyrate or acetoacetate depending upon the pH of the solution Measurement of Ketones
Hyperglycemia POSITIVE urine glucose Ketonemia POSITIVE urine ketones Elevated urine specific gravity Low pH (metabolic acidosis) Decreased CO2 Typical Lab results in DKA Normal to elevated K+ (electrolyte imbalance) Elevated anion gap Elevated Urea Elevated Osmolality Elevated lipids
Characterized by HIGH glucose above 35.0 mmol/L with NO ketoacidosis Type 2 acute crisis situation Hyperosmotic Nonketotic (HONK) Hyperglycemia
Normal albumin in urine is <20mg/L 20-200mg/L is positive for microalbumin Normal urine creatinine is 1-26 mmol/L The normal ratio is 3.4mg ALB per mmol of creatinine Microalbumin Test “POS” results are indicative of early renal disease
Glycosylated Hb levels reflect the average blood glucose level over the past 2-3 months! Provides physician with a picture of patient’s glucose control over past 2-3 months. More reliable than random plasma glucose level *in Canada GHb is reported as a ratio. Target value: <0.070 =adequate diabetic control
New recommendations >6.5% is diagnostic for diabetes 5.7-6.4% is an indication of prediabetes Glycosylated Hemogobin
Values represent SHORT-TERM (1-3 wks) glycemic control Fructosamine Glycoprotein formed between glucose and primarily albumin
Created by: mastergogo