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Lesson 1 - Basic Principles & Practices I. Units of Measure ● International System of Units (SI) was devised to standardize scientific measurements worldwide. ● based on the metric system & on 7 fundamental/base units: a. meter (m) - length b. kilogram (kg) - mass c. second (s) - time d. mole (mol) - quantity of substance e. ampere (A) - electric current f. Kelvin (K) - thermodynamic temperature g. candela (cd) - luminous intensity ● important to learn conversion factors, conventional to SI, vice versa Units used in reporting lab results: ● Used in lab (SI)
- Moles ● Used in lab (Conventional Units)
- mg/dL
- g/dL
- mmol/L
- IU ● Glucose
- 120 mg/dL = 6.6 mmol/L ● Hemoglobin
- 10 g/dL = 100 g/L II. Reagents ● are substances or solution used in laboratory analyses ● due to automation, most reagents are in “kit” form, readily available ● Lyophilized
- Powder and tablet form
- (add distilled water and mix) ● Swirling mixing
- Name of mixing III. Chemicals ● The chemicals used to prepare reagents for chemical testing exist in varying degrees of purity. 1. Analytic reagent (AR) ● the specifications for AR grade chemicals have been set & chemical manufacturers will either meet or exceed these requirements. ● Labels on reagents state the actual impurities for each chemical lot or list the maximum allowable impurities. ● Chemicals of this grade are suitable for most analytic lab. Procedures ● Analytic Reagent grade for body fluids 2. Ultrapure ● chemicals that have been put through additional purification steps for use in specific procedures such as chromatography, atomic absorption, immunoassays, molecular diagnostics, standardization or other techniques that require extremely pure chemicals ● For sensitive tests ● Purity: 99.9 % 3. United States Pharmacopeia (USP) & National Formulary (NF) ● are used to manufacture drugs ● the limitations established for this group of chemicals are based only on the criterion of not being injurious to individuals ● Chemicals in this group maybe pure enough for use in most chemical procedures; however it should be recognized that the purity standards are not based on the needs of the laboratory & therefore may or may not meet all assay requirements. ● Ex. caffeine 4. Chemically pure (CP) or pure grade ● impurity limitations are not stated & that the preparation of these chemicals is not uniform ● It is not recommended for clinical lab use. ● Not used in lab ● Ex. bleach and alcohol 5. Technical/Commercial ● used primarily in manufacturing & should never be used in the clinical lab ● Not used in lab ● Ex. color, tela **Organic solvents:
- practical grade**
- with some impurities 2. chemically pure
- approaches the purity level of reagent grade chemicals 3. spectroscopic & chromatographic grade
- minimum purity of 99% to ensure minimal spectral
- interference & minimal residual contamination **International Union of Pure & Applied Chemistry (IUPAC):
- Grade A**
- atomic weight standard 2. Grade B
- ultimate standard 3. Grade C
- primary standard 4. Grade D
- working standard 5. Grade E
- secondary substances
IV. Water ● used to prepare reagents, as diluent for controls & calibrators, to flush & clean internal components of analyzers, to serve as heating bath for cuvets & to wash & rinse lab glassware ● Highest purity of water is needed for most lab uses while for rinsing glassware may be of lesser purity 3 grades of water purity according to CLSI & CAP: ● Type I
- highest purity & used for procedures that require maximum purity like preparation of controls, standard solutions, buffers, toxicology tests, electrophoresis, HPLC
- Normal pH: 7
- ultrapure ● Type II
- for most laboratory analysis
- For chemistry and hematology ● Type III
- wash water
- For urinalysis, parasitology and histology Clinical Laboratory Standards Institute (CLSI) categories of water:
- reagent grade water
- used for most lab analysis
- Type II
- special reagent water
- instrument feed water
- water supplied by method manufacturer
- autoclave water
- wash water Water parameters to be tested: ● microbiologic count (less than 10CFU/mL) ● pH: 7 ● resistivity (lack of conductivity of electricity) ● silicate (less than 0.05mg/L) ● particulate matter (contaminants) & organics **Purification process:
- Prefiltration** ● filtration cartridges are made up of glass, cotton, activated charcoal & submicron filters (≤0.2mm) 2. Distillation ● is the process by which a liquid is vaporized & condensed & is used to purify or concentrate a substance or separate a volatile substance from a less volatile volatile substances ● effectively removes bacteria, pyrogens, particulate matter, dissolved ionized solids & to lesser extent dissolved organic contaminants ● does not remove volatiles such as carbon dioxide, ammonia & chlorine ● Used in clinical lab 3. Deionization ● water is passed through a bed of mixed cation & anion exchange resins (deionizer) ● excellent for removal of dissolved ions but is ineffective for other contaminants ● Removes ion 4. Reverse Osmosis ● water is forced under pressure through a semi-permeable membrane & remnants of dissolved organic, ionic & suspended impurities including bacterial & viral contaminants are left behind ● does not remove dissolved gases effectively ● used frequently to pre-treat water before deionization process ● To obtain ultrapure water ● Destroy bacteria 5. Ultraviolet Oxidation & Sterilization ● are used after other purification process are performed to remove trace quantities of organic contaminants (oxidation) & bacteria (sterilization) V. Solutions & Concentration ● Solute
- substance that is dissolved in a liquid ● Solvent
- liquid in which the solute is dissolved ● Dilute solution
- little amount of solute ● Concentrated solution
- large quantity of solute ● Saturated solution
- excess amounts of undissolved solute particles ● Supersaturated solution
- more solute in solution than saturated solution of the same substance; thermodynamically unstable ● Solutions
- expressed in percent solution, molarity, molality & normality VI. Reference Materials ● a material or substance, one or more physical or chemical properties of which are sufficiently well established to be used for the calibration of an apparatus, the verification of measurement method, or for assigning values to materials
3. Pipettes ● are used for reconstitution of controls & calibrators, preparing serum or plasma dilutions & aliquoting specimens ● high degree of accuracy & precision is required **Classification of Pipettes:
- By design: a. To Contain (TC)** ● holds a particular volume but does not transfer the exact amount ● Blow out or Rinse Out ● Calibrated by mercury for viscous solutions b. To Deliver ● transfers the exact amount ● Self draining, by gravity, do not blow the last drop ● Calibrated by distilled water for non-viscous solutions 2. By drainage characteristics: a. To Blow out
- indicated by double etched rings b. Self-draining
- no markings **Classified according to: A. Type
- Measuring or graduated** ● delivers particular amount of liquid contained between 2 graduation marks on a cylindrical tube a. Mohr
- calibrated between 2 marks on a stem
- self-draining b. Serologic
- larger orifice; graduation marks down to the tip
- Blow out 2. Volumetric or transfer ● calibrated to deliver a fixed volume of liquid by drainage ● have cylindrical bulb joined at both ends to narrow glass tubing a. Volumetric
- calibrated to deliver accurately a fixed volume of dilute aqueous solution b. Ostwald-Folin
- similar to volumetric but bulb is closer to the delivery tip & are used for accurate measurement of viscous fluids such as blood or serum 3. Automatic pipettes ● most routinely used ● Greater accuracy = microvolume
- 10 ul and 20 ul ● comes in fixed volume, variable volume & multi-channel ● automatic meaning the mechanism that draws up & dispenses the liquid is an integral part of the pipette ● maybe fully automated or self-operating, semi-automatic or manually operated ● single channel pipette
- 1 sample at a time ● multi-channel pipette
- multiple samples at a time ● Mechanism of operation: a. air-displacement pipette ● Naas taas ang piston ● relies on a piston for creating suction to draw the sample into the tip ● the piston does not come in contact with the sample ● tip must be replaced each use b. positive-displacement pipette ● Had a contact with the sample ● operates by moving the piston in the pipette tip or barrel ● does not require different tip for each use but requires rinsing & blotting between samples to prevent carry-over c. dispensers and dilutor ● Can be adjusted to a fixed volume ● obtain liquid from a common reservoir & dispense it repeatedly ● often combines sampling & dispensing functions
Lesson 2 - Pre Analysis Pre-analysis
- 32 to 75 %
- All testing errors
- Patient-related variables
- Specimen collection / labelling technique
- Specimen preservatives / anticoagulant
- Specimen transport
- Specimen processing
- Specimen storage LABORATORY TESTING PROCESS I. PRE-ANALYTICAL PHASE ● refers to all steps that must take place before a sample can be analyzed. ● This stage is a major source of residual ‘error’ & or variables that can affect the results. ● The laboratory results are only as good as the specimen tested. ● Pre-analytic factors include: A. Pre-collection variables
- also known as patient-related variables 1. Age ● a number of lab tests have different ranges for different ages. ● newborns especially have many different normal ranges than adults or older children for substances in blood & other body fluids ● Hgb F is for newborns ● Hgb A1 is for adults
- If adult have high Hgb F, they have anemia ● coagulation factors do not reach adult levels for many months after birth ● cholesterol level rises with age. 2. Gender ● ex. testosterone & estradiol ● Men have higher ALP, AST, CK & aldolase levels than women due to larger muscle mass. ● Women have lower levels of Mg++, Ca++, Fe++, Hgb 3. Diurnal variation ● hormones such as cortisol, ACTH, aldosterone & iron have higher levels in AM. ● Prolactin have higher levels between 4am - 8am & 8pm - 10pm. ● GH, PTH & ACP have higher levels in PM. 4. Diet ● an individual’s food intake can greatly affect lab results ● The effect maybe transient & easily controlled ● Glucose & TAG absorbed from food increase after eating ● Eating a meal depending on the fat content may elevate K+, TAG, ALP, etc. 5. Patient preparation ● for certain lab tests there are a number of certain preparations of the px that are necessary to provide the most clinically useful, accurate & precise results. ● 8 - 12 hours ● FBS: 6 - 8 hours 6. Exercise ● physical activity elevates creatinine, protein, CK, AST & LDH. ● It also activates coagulation, fibrinolysis & platelets. ● 5 minutes to calm 7. Stress ● mental & physical stress induce the production of ACTH, cortisol & catecholamines. ● Hyperventilation affects the acid-base balance, elevates leukocyte counts & serum lactate. 8. Smoking ● smokers have high carboxyhemoglobin levels, plasma catecholamines, serum cortisol & WBC count. ● It also affects the body’s immune response; lower Ig levels. ● Decreased sperm counts & abnormal morphology have been reported in male smokers when compared to non-smokers. 9. Posture ● changing from supine (lying) position to sitting or standing results in a shift of body water from inside the blood vessels to the interstitial spaces. ● An upright position increases hydrostatic pressure, causing a reduction of plasma volume & increased concentration of proteins. ● Ex. plasma renin activity: higher standing than supine ● Out patient
EDTA ALP, CK,
Leucineamino peptidase inhibits calcium, iron decrease PT, PTT, sodium, potassium increase platelet aggregation prevents Oxalate ACP, ALP, Amylase, LDH inhibits calcium decrease sodium, potassium increase cell morphology distorts Citrate ALT, AST, ALP, inhibits ACP stimulates Amylase, calcium decrease sodium, potassium increase labile coagulation factors preserves Heparin Triiodothyronin e, thyroxine, PT, PTT increase Wright’s stain causes blue background lithium (LiHep), sodium (NaHep) increase Fluorides ACP, ALP, Amylase, CK, ALT, AST decrease cell morphology distorts Table 5. Order of Draw EVACUATED TUBE/SYRINGE METHOD
CAPILLARY METHOD
- Blood culture tube 1. EDTA
- Citrate 2. Other additives
- Plain 3. Serum
- Heparin
- EDTA
- Fluoride D. Interferences in Laboratory Tests 1. Analytical interferences - Hemolysis - elevated bilirubin - lipemia 2. Impact of drugs on lab test results ● drugs can affect lab tests in 2 ways: - as an interfering substance in the lab test - by producing an effect in the body that alters a lab test result Urine & Other Body Fluids Collection 1. Urine ● testing generally falls under 3 categories: chemical, bacteriologic & microscopic ● proper specimen collection must be observed ● ‘timed specimes’ (usually for chemistry) are obtained at designated intervals, starting from ‘time zero.’ Collection time is noted on each subsequent container. ● Urine specimens for 24-hr total volume collection must be placed in a clean 4L, plastic container & the patient must be given instructions carefully. ● Urine must also be stored (refrigeration) & preserved (NaF, boric acid) properly to avoid falsely decreased or elevated results. Table 6. Changes in Urine with Delayed Testing RESULT REASON change in color breakdown/alteration of chromogen changes in odor bacterial growth, decomposition increased turbidity increased bacteria, crystal formation, precipitation of amorphous materials falsely low pH glucose converted to acids & alcohol by bacteria producing ammonia, CO 2 lost
falsely elevated pH breakdown of urea by bacteria forming ammonia falsely negative glucose utilization by bacteria (glycolysis) falsely negative ketone volatilization of acetone, breakdown of acetoacetate by bacteria falsely negative bilirubin destroyed by light, oxidation to biliverdin falsely negative urobilinogen destroyed by light falsely positive nitrite nitrite produced by bacteria after specimen is voided falsely negative nitrite nitrite converts to nitrogen & evaporates increased bacteriuria bacteria multiply in specimen before analysis disintegration of cell/casts unstable environment, especially in alkaline urine, hypotonic urine or both
2. Cerebrospinal fluid (CSF) ● lumbar punctures (LPs) or spinal taps are performed to collect CSF (bet. 3rd or 4th^ lumbar vertebrae or bet. the 4th or 5th) for lab evaluation to establish diagnosis of infection, malignancy, subarachnoid hemorrhage, multiple sclerosis or demyelinating diseases ● before collection, pressure should be maintained at 90-180 mmHg, on closing the pressure should be between 10-30 mmHg 3 Tubes are collected/order: ● Tube 1
- chemistry/serology ● Tube 2
- microbiology ● Tube 3
- hematology for cell counts 3. Synovial fluid ● an ultrafiltrate of plasma that is passed through fenestrations of the subsynovial capillary endothelium into the synovial cavity. ● Once in the cavity it is combined with hyaluronic acid secreted by the synovial lining cells. ● collected by arthrocentesis ● anticoagulant used is 25units NaHeparin per mL of synovial fluid ● oxalate, powdered EDTA, LiHeparin should not be used bec they produce crystalline artifacts similar to monosodium urate crystals ● transferred to 3 tubes : sterile, EDTA or heparin, red top 4. Pleural, pericardial, peritoneal fluids a. Pleural ● ultrafiltrate of plasma formed in the pleural cavity ● Thoracentesis
- is a surgical procedure to drain fluids (effusions) from the thoracic cavity & is helpful in diagnosing inflammation on neoplastic disease ● Surrounds lungs, blood vessels, nerves, bronchi b. Pericardial
- collected by Pericardiocentesis
- Surrounds hearts c. Peritoneal
- collected by paracentesis Specimen Transport ● all specimens must be transported in a safe, & convenient manner to prevent biohazard exposure or contamination of the specimen ● avoid hemolysis, exposure to light, kept at right temperature, proper container ● proper labeling is required on all containers used to store, transport, ship or dispose of blood or other potentially infectious materials Chain of Custody ● When laboratory tests are evidence of crime or accident they become forensic in nature. In these cases, documented specimen identification is required at each phase of the process. Each facility has its own forms & protocols; however the patient & usually a witness must identify the sample. It should have a tamper proof seal. Any individual in contact with the sample must document receipt of the sample, the condition of the sample at the time of receipt & the date & time it was received. **Specimen Processing
- Precentrifugation Phase** ● ideally all specimens should be tested within 45mins to 1 hour after collection ● if it is not possible, appropriate steps must be done for storage or preservation to avoid alteration of analytes prior testing
Phlebotomy Clinical Chemistry
- Biochemical process associated with health and idease 3 main biomolecules in Clinical Chemistry ● Carbohydrates ● Lipids / Fats ● Protein Analytic procedures for biochemical tests ● Principles and method of the tests ● Instrumentation ● Reference ranges Serum
- Normal color: Yellow
- Hemolyzed color: Pink or reddish
- Golden yellow: icteric Phlebotomy
- 70% of sisoprophyl or ethanol was used for disinfection
- Alcohol was applied to skin for 30 seconds to 1 minute before extracting
- Alcohol should be applied first, then iodine. Do it for two times, to ensure na nawala ng skin flora
- If the patient is allergic to alcohol or iodine, we can use benzakium chloride
- To prevent hemoconcentration, tourniquet must be placed for 1 minute only Syringe
- Single use or draw
- Shaft: length of 1 to 1.5 inches Evacuated Tube System (ETS) or Vacutainer
- Multiple draw ETS or Multisample Needle ● 16 gauge
- Blood donation ● 21 gauge
- Green
- Biggest needle
- Recommended for extraction ● 22 gauge
- black ● 23 gauge
- Blue
- Small
- fragile
- Used for babies ● 25 gauge
- Orange
- Insulin
- Sharp
- Medication Major classes of tubes ● Plain tubes
- Red
- Composition of glass ● Additives
- Chemicals added Additives
- Clot activator
- Anticoagulant
- All of the tubes have 8 time of inversion except for Citrate it has 4 times of inversion Clot Activator
- Promotes blood coagulation, in less than 5 minutes the blood was clotted na
- Red top but plastic composition
- Gold: Serum Separator Gel - 5 times invert
- Clot Activator & SSG - 15 minutes to clog Anticoagulant
- Prevents coagulation **Order of Draw: ETS or Syringe
- Blood Culture Tube**
- Gold or yellow 2. Citrate
- blue 3. Plain
- red 4. Heparin
- green 5. EDTA
- lavender 6. Oxalate
- Grey Blood Culture Tube
- Gold or yellow
- SSG or Serum Separator Gel
- Clot activator and serum separator
- Serum is clearly separated from clotted red cells
- Contains: Sodium Polyanethol Sulfonate (SPS)
- Inhibits: Phagocytosis Citrate
● Blue
- Concentration: 3.2% - 3.8%
- Electrolytes, its partner is Na Citrate
- Sodium citrate: very particular sa volume at proper ratio (blood to anticoagulant)
- Ratio: 9 is to 1
- 9 parts of blood to 1 part of sodium citrate
- Very senstive and clots easily
- Binds calcium to prevent coagulation
- Coagulation Test a. Prothrombin Time b. Partial Thromboplastin Time ● Black
- Ratio: 4 is to 1
- Can used in Hematology but the specific used was for Erythrocyte Sedimentation Rate (ESR) Plain
- Red Glass Plastic
- plain - With clot activator Heparin
- Green
- Anti-thrombin or anti-thromboplastin
- Natural anticoagulant a. Sodium b. Lithium c. Ammonium
- Can used in every section
- Mostly special test like glucose EDTA
- Lavender
- Ethylenediaminetetraacetic acid ● K2 EDTA
- Powdered form
- Hematology: preserves all blood cells, rbc, wbc, platelets ● K3 EDTA
- Liquid form
- Hematology: preserves all blood cells, rbc, wbc, platelets Oxalate
- Gray
- Used in CC, plasma and glucose specimen
- Lactate acid
- by product of glycolysis
- no need to use torniquet
- Paired with sodium, added additive is fluoride
- Fluoride
- anti glycolysis
- preserves glucose for 3 days 3 types of blood specimens used: ● Whole blood
- Extracted blood was preserved ● Serum
- Liquid portion of clotted or coagulated blood
- Has no more fibrinogen sinced it was consumed for blood clotting ● Plasma
- Liquid portion of anticoagulant blood
- Fibrinogen
- coagulation factor
- likes blood clot
- is prerserved, it is still present or not consumed
● It is important for the daily monitoring of accuracy & precision of analytical methods. ● It detects both random & systematic errors in a daily basis. ● It allows identification of analytic errors within a one-week cycle. ● Control material
- commercially manufactured lyophillized that have the same matrix as patient specimens & are used to evaluate test process.
- handled exactly like patient specimens
- specifically selected so that values will be at medically significant levels
- normal control & abnormal control materials 2. Interlab QC (external QC) ● is performed by lab. personnel when analyzing specimens sent to the lab. by an external agency & the results generated are submitted to the agency for assessment. ● This type of assessment is known as proficiency testing. It is required by federal regulations for all labs. providing results for human diagnosis & or treatment. ● It is important in maintaining long-term accuracy of the analytical methods. ● Is important to ensure the laboratory is performing to an external standard
- External Quality Assurance
- Philippine Council for Quality assurance in Clinical Laboratories
- Send unknown specimens to labs Reference Laboratory ● Chemistry
- lung center
- Lung center of the philippines ● Hematology & Blood Banking
- National / Kidney Transplant Institute ● Infectious disease
- Non-donor
- sacel ● Donor
- Research Institute for tropical medicine ● Toxicology
- EAST AB **Parameters of QC:
- Sensitivity**
- is the ability of an analytical method to measure the smallest concentration of the analyte of interest. 2. Specificity
- the ability of an analytical method to measure only the analyte of interest (no interfering substances).
- Accuracy
- is the nearness or closeness of the assayed value to the true or target value.
- statistical measures of accuracy are the mean (average), median (middle value) & mode (most common value). 4. Precision or reproducibility
- is the ability of an analytical method to give repeated results on the same sample that agree with one another.
- statistical measures of precision are the standard deviation (SD) & coefficient of variation (CV). 5. Practicability
- is the degree by which a method is easily repeated. 6. Reliability
- is the ability of an analytical method to maintain accuracy & precision oven an extended period of time during which equipment, reagents & personnel may change. 7. Diagnostic sensitivity
- is the ability of an analytical method to detect the proportion of individuals with the disease.
- it indicates the ability of the test to generate more true-positive results & few false-negative.
- screening tests require high sensitivity so that no case is missed. Diagnostic sensitivity (%)= TP x 100 (TP + FN)
- Mataas ang TF, kulang ang FN
- TP divided by to TP + FN multiply to 100
- Mataas dapat ang Diagnostic Sensitivity Screening Tests ● Pregnancy Test
- Diagnostic specificity
- is the ability of the analytical method to detect the proportion of individuals without the disease.
- it reflects the ability of the method to detect true-negatives with very few-false positives.
- confirmatory tests require high specificity to be certain of the diagnosis.
Diagnostic specificity (%)= TN x 100 (TN + FP)
- Kapag sinabing walang sakit, wala kasing sakit Confirmatory Tests ● HIV 9. Predictive value
- utilizes the parameters of test sensitivity & specificity as well as disease prevalence (i.e. incidence of a disease or condition) a. positive predictive value
- is the % of people with positive test results who have the disease b. negative predictive value
- is the % of people with negative test results who do not have the disease Standard & Control reagents:
- Standard
- is a material of known concentration that is used to calibrate an instrument or develop a standard curve.
- Control
- is a material of known value that is analyzed with patient samples to determine acceptability of results.
- substances having a known or determined “range” of values
- it should simulate real patient specimens
- it should span the clinically important analytical range a. assayed control
- values are assigned by the manufacturer b. unassayed control
- values are determined by each individual laboratory for their methods or instruments ● the accuracy of any assay depends on the control solutions, how they are originally constituted & how they remain stable overtime ● general chemistry assays use 2 levels of control solutions, while immunoassays use 3 levels ● levels of control solutions: normal & abnormal; positive & negative; low, mid & high ● To establish statistical quality control on a new instrument or on new lot numbers (batch) of control materials, the different levels must be analyzed for 20 days. ● the ideal control/reference limit is between ± 2 SD ● controls are plotted on Levy-Jenninings chart QC Normal
- 70 to 110 QC Abnormal
- 150
- 60 Characteristics of ideal QC material:
- resembles human sample
- inexpensive & stable for long periods
- non-infectious
- does not came from humans
- no matrix effects/known matrix effects
- convenient packaging for easy dispensing & storage **Statistical methods in QC:
- Mean**
- is the mathematical average 2. Median
- is the middle value 3. Mode
- is the most frequent observation 4. Standard deviation
- it reflects the variation of data values around the mean. It helps to describe the normal curve.
- a measure of the distribution range; most frequently used measure of variation 5. Coefficient of variation
- reflects random variation of analytical methods in units that are independent of methodology, because it is a percentage comparison of the standard deviation divided by the mean.
- percentile expression of the mean
- it is an index of precision
Westgard Multirule Technique
- it is used to establish criteria for deciding whether an analytic process is out of control (^1) 2S
- one control value is outside the ± 2SD;
- this is a warning sign of a possible error in instrument or method malfunction
- Accept
- Random error (^1) 3S
- one control value is outside ± 3SD
- detects random error
- Reject (^2) 2S
- two consecutive control values are outside the ± 2SD limit
- detects systematic error
- Reject R4S
- one control value exceeds the + 2SD & a second control value exceeds the - 2SD, creating a 4SD spread
- detects random error
- Reject (^4) 1S
- 4 consecutive values are recorded on the same side of the mean & exceeds the ± 1SD
- Detects systematic error (^10) X
- 10 consecutive control values are plotted one 1 side of the mean (either above or below)
- detects systematic error Rule
- First In First Out
- Unang dumating, unang gamitin.
- Kapag nabuksan reagent, mag label ng "Date open"
- MFG DATE
- EXP DATE Linearity check
- determines the lowest & highest values that can be accurately measured by a particular method.
- This is an example of a non-statistical QC parameter. Delta check
- assess the patient’s most recent results for a particular test as compared to the patient’s previous value
Lesson 4 - Analytical Techniques: Principles of Instrumentation ● The productivity of modern lab depends on instrumentation. ● The high output & accuracy of results obtained by modern analytical devices could not be achieved by the tedious manual separation & quantitation methods of old. ● Without a thorough understanding of the necessary principles, the operator will be ill-equipped to perform maintenance procedures, calibration & troubleshoot problems that may arise ● With an understanding of basic concepts, one can master the purpose & function of any analytical instrument, in spite of the flashing lights & complex computer-driven components Objective
- to learn about basic principles of instrumentation in the clinical laboratory **4 basic categories of measurement techniques:
- Spectrometry**
- spectrophotometry, atomic absorption, mass spectrophotometry
- Measures the light that being absorbed 2. Luminescence
- fluorescence, chemiluminescence, bioiluminescence & nephelometry
- Measures the light that the chemical provides 3. Electroanalytical methods
- electrophoresis, potentiometry & amperometry
- Utilise charges, negative and positive (plus and minus) 4. Chromatography
- gas, liquid & thin layer chromatography
- Uses separation techniques Most measurements in the clinical laboratory are based on radiant energy that are:
- Absorbed
- photometry, spectrophotometry, reflectance spectrophotometry, atomic absorption
- Emitted
- fluorometry, luminometry (chemiluminescent, bioiluminescent), flame emission spectrophotometry
- Scattered
- nephelometry, turbidimetry
- Polarized/transmitted
- fluorescence polarization spectroscopy, polarimetry I. Photometry & Spectrophotometry Basic concepts: ● Photometry
- is the measurement of luminous intensity of light or the amount of luminous light falling on a surface from such a source.
- You cannot select the wavelength of light ● Spectrophotometry
- is the measurement of the intensity of light at selected wavelengths.
- Selected ang wavelength ng light, can choose. ● In absorbance spectrophotometry – the absorption of light at a particular wavelength & comparing it with the absorption of light by a known standard solution measured at the same time & with the same wavelength. ● The intensity of the color
- is directly proportional to the concentration of the substance present.
- Mas mataas ang kulay, mas mataas ang glucose ng analyte
- Kapag mas may kulay, mas makakaabsorb ng light The Nature of Light
- uses EMR, in the form of light waves
- Electromagnetic radiation Visible Region / Visible Light
- Colors that we can see
- 700mm
- 600mm
- 500mm
- 400mm Infrared
e. Mercury Vapor lamp
- Monochromator ● a filter that isolates a specific region of the electromagnetic spectrum ● a system of isolating a desired wavelength & excluding others ● Light shines on a wavelength selector & is spread into a wide band of rays ● By moving the selector, we can direct the specific desired wavelength through the sample. ● The degree of wavelength isolation depends on the type of device used & the width of the entrance & exit slits. ● types of devices a. colored glass filters
- inexpensive, simple & useful;
- usually pass a relatively wide band of radiant energy & have low transmittance of the selected wavelength;
- used in photometers b. prism
- a narrow beam of light that is focused on a prism is refracted as it enters the more dense glass.
- Short wavelengths are refracted more than long wavelengths, resulting in dispersion of white light in continuous spectrum.
- It can be rotated allowing only the desired wavelength to pass through an exit slit c. diffraction gratings
- most commonly used monochromator.
- It consists of many parallel grooves (15,000-30,000/inch) etched onto polished surface.
- Diffraction is the separation of light into component wavelengths, is based on the principle that wavelengths bend as they pass a sharp corner.
- Sample cell holder/cuvette
- holds the specimen sample.
- Sample holder / container ● It is a glass or plastic container (either round or square) which is designed to pass most of the incident light through without absorbing it. ● Square cuvettes minimize the light scatter than round cuvettes because it has plane-parallel optical surfaces & a constant light path. ● Quartz cuvettes are for below 330 nm.
- Photodetector ● is used to determine how much light passes through the sample in the cuvette. ● It responds to light striking it. ● When transmitted light hits the tube, an electrical signal is generated going through the detector system to a read-out device. ● Photomultiplier tube Types : a. photocell/barrier cell layer
- least expensive;
- composed of a film of light sensitive material, frequently selenium on a plate of iron b. phototube
- similar to barrier cell layer but requires outside voltage for operation c. photomultiplier tube
- detects & amplifies radiant energy. It is 200x more sensitive than phototube.
- It is used in instruments designed to be extremely sensitive to very low light levels & light flashes of very short duration
- Used in laboratory d. photodiode
- absorption of radiant energy by a reversed-biased positive-negative junction diode produces a photocurrent that is proportional to the incident radiant power.
- Readout device
- electrical energy from a detector is displayed on some type of digital display or readout system.
- It maybe a chart recorder or a computer printout. Quality Assurance in Spectrophotometry Quality control testing for spectrophotometry consists of checking:
- Wavelength accuracy check
- ensures that the wavelength on the dial is the wavelength of light passing through the monochromator.
- This is checked with standard absorbing solution or filters with maximum absorbance of known wavelength.
- Didymium or holmium oxide in glass is stable & frequently used.
- Stray light check
- detects any wavelength outside the band transmitted by the
monochromator (checks scratched cuvettes or higher order spectra produced by diffraction gratings).
- Stray light is detected by using cut-off filters which eliminate other wavelengths except for the wavelength of interest.
- Linearity check
- is demonstrated when a change in concentration results in a straight line calibration curve. Reagent na colored na, without adding a chemical or serum:
- Hemolyzed
- Lipemia (need mag fasting para maiwasan ang prob)
- Bilirubin **Blanking Solutions
- Reagent blank**
- used to compensate for any unwanted light absorption from materials in the reaction mixture
- Reagent lang laman ng cuvette
- Wala pang serum pero may color na, kapag nalagyan ng serum mas titingkad yung color
- SC-RB = Concentration 2. Sample blank
- used to compensate for interfering substances in the sample such as hemolysis, high bilirubin levels, lipemic
- Kapag hemolysis no need to run na, repeat collection dapat 3. Water blank
- used to calibrate the spectrophotometer, set to 0
- Distilled water II. Atomic Absorption Spectrophotometry ● Principle: measures concentration by detecting absorption of EMR by atoms rather than molecules ● The sample is atomized in flame where the atoms of the metal to be quantified are maintained at ground state. Then a beam of light from a hollow cathode lamp is passed through a chopper to the flame.
- The ground state atoms in the flame absorb the same wavelengths of light from the lamp as the atoms emit when excited.
- The light not absorbed by the atoms is measured as a decrease in light intensity by the detector.
- The detector will selectively read the pulsed light from the chopper that passes through the flame & will not detect any light emitted by the excited atoms when they return to ground state.
- The difference in the amount of light leaving the lamp & the amount of light measured by the detector is indirectly proportional to the concentration of the metal analyte in the sample.
- Components : 1. Light source
- Hollow cathode lamp 2. Monochromator
- diffraction gratings **3. Flame
- Sample holder
- Photomultiplier tube III. Flame Emission Photometry**
- Principle: excitation of an atom’s electrons by heat energy causes unstable electrons from a higher energy state to a lower energy state.
- Energy is put into a system, producing electron transitions within an ion. As the excited electrons drop back to the ground state, light energy of a specific wavelength is emitted by the ion. This light can be detected & measured using optical systems.
- Primarily used to determine the concentration of sodium, potassium & lithium
- The internal standard used when measuring sodium is lithium.
- The internal standard used when measuring lithium is cesium. - No longer routinely used because of ion selective electrode. IV. Fluorometry
- Principle: atoms absorb energy & the electrons are raised to a higher orbital & electrons release energy as they return to ground state by emitting light of longer wavelength
- Fluorescence measurement involves the interaction of light w/a chemical compound. In this case, the compound emits light (usu. of longer wavelength) in response to light striking it.
