CLEN CHIM
QUALITY MANAGEMENT
Quality Assurance (QA)
- includes maneuvers encountered in the pre-analytic, analytic, and post- analytic phases of laboratory testing
Pre-analytical phase includes
- test ordering
- specimen collection
- transport of the specimen in the laboratory
- processing of specimen
- entering patient information
- centrifuging
- separating aliquoting specimen
- delivery to proper laboratory location
Analytical phase includes
- specimen analysis (manual or automated)
- use of commercial controls
- record keeping
Post-analytical phase includes
- reporting out results of analysis (manual or computerized)
- physician contact
- monitors quality performance starting from the ordering of a laboratory determination to its reporting, interpretation of results, and then application to patient care
- involves total quality control which requires constant attention of all involved in the laboratory testing
Quality Control (QC)
- is concerned with the analytic phase of QA
- monitors the over-all reliability of laboratory results in terms of accuracy and precision
- relies on the diligent and persistent execution of the following QC associated activities:
1) assay of control samples
2) instrument maintenance
3) statistical data analyses
4) proficiency testing survey
- has two major types
External QC (Interlaboratory QC) was established by Belk which monitors primarily the accuracy of laboratory tests; the use of Youden plots.
Internal QC (Intralaboratory QC) was established by Levey which primarily monitors the day-to-day performance of laboratory tests - precision assessment. Intralaboratory QC can be based either from the results of control specimens or on the results of patient specimens; the use of Levey- Jennings chart
Errors
Analytical errors are usually systematic errors or determinate errors that are caused by some factors in the analytical system such as erroneously calibrated pipettor, deteriorating reagent and fluctuating electrical current.
Personnel or operator errors are usually called random errors or indeterminate errors that usually affect several analyses. Examples are mislabeling the specimen, wrong number entry and instability of needle due to electronic component of instrument.
Trend suggests a systematic drift or error. Values move continuously away from the mean in just one direction. Among its causes include deteriorating reagents, changes in standard concentration or failing instrument.
Shift also suggests systematic error. Seen as an abrupt change from the established mean and continue in a linear fashion parallel to the mean.
Increased dispersion suggests random error. High or low outliers are frequently observed. Causes of this are variations in operation of instrument, interfering substances, electronic fluctuations, and clerical errors.
Westgard Multi-Rule Technic
Rule Meaning
1:2s = one control observation exceeds the control limit set at M±2SD; warning rule
1:3s = reject when one observation exceeds M±3SD; suggests random error
2:2s = reject when two consecutive observations exceed the same M+2SD or the same M-2SD; suggests systematic error
R:4s = reject when one control observation in the run exceeds its M+2SD and another exceeds M-2SD; suggests random errors
4:1s = reject when four consecutive control observations exceed the same M+1SD limit or the same M-1SD limit; suggests systematic error
10:Mean= reject when 10 consecutive control observations fall on one side of the mean; suggests systematic error
Accuracy, Precision & Reliability
Accuracy is the extent to which the mean measurement is close to the true value. The accuracy of the method is reflected by its ability to obtain the same values of the reference samples of known concentration. Expressed as absolute error or relative error.
absolute error = true value – actual value true value
relative error = true value – actual value x 100%
true value
Precision is the reproducibility of a laboratory determination when it is run repeatedly under identical conditions. Precision is commonly expressed in terms of standard deviation (SD), variance or coefficient of variation (CV).
Standard deviation(SD) = Σ (M – x)2
n-1
Variance = (SD)2
Coefficient of variation (CV) = SD x 100%
M
Reliability refers to the ability of a test to maintain its accuracy and precision for an extended period of time.
SD is a measure of dispersion of the values around the mean and in normal or Gaussian distribution,
68% of the values fall within +/-1 SD around the mean
95% of the values fall within +/-2 SD around the mean
99.7% of the values fall within +/-3 SD around the mean
- most laboratories choose the 95% confidence limit in expressing precision
- the median is the value that is middlemost in an array of numbers while the mode is the value that occurs most frequently.
- in a Gaussian distribution, the mean, median and mode are very close in value as shown by its bell-shaped curve.
Sensitivity & Specificity
Sensitivity is the ability of the test to detect the smallest amount of the analyte in a solution or sample. It expresses the ability of the test to be positive in the presence of the analyte or the disease. A highly sensitive test is characterized by a decreased probability of obtaining false negative results.
Specificity refers to the ability of the test to detect analyte without detecting other analytes that are also present in the sample. It expresses the ability of the test to be negative in the absence of the analyte or the disease. A highly specific test leads to a decreased probability of obtaining false positive results.
Control & Standard
Control is a solution (usually pooled serum samples) whose constituents are diverse but are known (a range of values per analyte). This can be run simultaneously with the Test to check, verify or validate the accuracy of the results.
Standard is a solution of a particular analyte of known characteristics and known value (exact concentration). It is used as reference for the calculation of the value of the Unknown.
Figures of Merit of Analytical Methods
Major
1. Accuracy
2. Precision
3. Limit of detection
4. Applicable concentration range or linear range
5. Sensitivity
6. Selectivity or specificity
Minor
1. Speed
2. Ease and convenience
3. Skill required of the operator
4. Cost of analysis and availability of equipment
5. Per sample cost
MEASUREMENT
1. Direct measurement
A + B AB
e.g., Condensation methods
One-step methods
2. Indirect measurement
A + B C + D (either C or D is measured)
e.g., Enzymatic methods (Degradative methods)
Measurement of coenzymes
Oxygen consumption tests
3. Substitution or subtraction
A + B = C, thus, having known A & C, B can be determined.
e.g., Determination of indirect bilirubin
Determination of VLDL and LDL in lipid profiling
Mass measurement with Mettler analytical balance
Chemical Measurement
1. Absolute methods
a. Gravimetric
b. Volumetric
2. Relative methods (Instrumental methods)
INSTRUMENTATION AND ANALYTICAL PRINCIPLES
Colorimetry
This involves Beer’s law which states that the concentration of an analyte is directly proportional to the amount of light it absorbs. There are two types:
1) visual colorimetry which solely on the human eye (Duboscq colorimeter)
2) photoelectric colorimetry which employs a photoelectric device (Leitz photrometer).
Photometric methods are based on the Beer’s law, which is applicable only for monochromatic light. A monochromator is a device for selecting a narrow band of wavelengths from a continuous spectrum such as filters, prism and diffraction grating.
Spectrophotometry
In spectrophotometry, molecules in solution will cause incident light to be absorbed while the remaining light energy will be transmitted.
Absorbance (OD) is the term used to describe the monochromatic light that is absorbed by the sample and transmittance describes the light and passes through the sample.
A = 2 - log%T
In turn, when the absorbance increases exponentially with an increase in the light path, the Lambert law is followed. Incorporation of these two laws, together with the contribution of Bouguer, may be stated as
A = abc
where A = absorbance,
a = absorptivity of the substance being measured,
b = light in cm, and
c = concentration of the measured substance.
When the Beer-Lambert law is applied to spectrophotometric analyses of standards and unknown samples that are being measured, the following equation is derived:
Au x Cs = Cu
As
Where Au = absorbance of unknown
Cs = concentration of standard
As = absorbance of standard
Cu = concentration of the unknown
This formula is applied to assays that exhibit linear relationships between changes in absorbance with changes in concentration to calculate the concentration of the unknown sample.
The general parts of the spectrophotometer are
1. Light source. This provides the radiant energy.
A tungsten filament lamp is the most common light source for photometry in the visible region.
- continuous spectrum (360 - 800 nm) from the near-infrared (NIR) through the visible to the ultraviolet (UV) region.
- most of the radiant energy is in the NIR.
- only about 15% is in the visible region - the region usually used. Because of the large emission in the near IR, tungsten lamps generate a significant amount of heat.
Hydrogen and deuterium lamps are used for work in the UV region with 200 - 375 nm range. NADH consumption tests are tests employing the UV region of the emitted light.
The mercury vapor lamp does not provide a continuous spectrum, emitting radiation at a specific wavelength.
2. Entrance slit. It minimizes stray light emitted by the lamp and prevents scattered light from entering the monochromator.
3. Monochromator. It sharply isolates specific wavelengths of light which will made to pass the cuvets. The three kinds of monochromators are filters, prisms and diffraction gratings. It replaced the filters in photometers to increase sensitivity of measurements.
4. Exit slit. It controls the amount of emergent light that passes through the cuvet.
5. Cuvet holder. It holds the cuvet holding a solution containing the analytes to be measured. A cuvet is also known as an analytical cell or absorption cell.
- for strongly acidic solutions, aluminosilicate glass cuvets should be used.
- for strongly alkaline solutions, silicate glass cuvets should be used.
- Plastic or quartz cuvets should be used for assays using wavelength range of the UV region (200 - 380 nm).
6. Detector. It converts light signal into an electrical signal which can be detected by a galvanometer.
A photomultiplier tube (PMT) responds to the radiant energy it absorbs by emitting electrons in a proportional amount to the initial light absorbed. - These electrons then go through a series of stages where amplification occurs.
- The cascade effect, as the electrons go through 10 - 15 stages, results in a final current that may be one million times the initial current.
- The PMT exhibits rapid response time and sensitivity.
- PMT has two functions
1) as a transducer that converts light to electricity
2) as an amplifier of the signal within the tube
An iron plate and a layer of selenium are partial descriptions of the composition of a photocell or barrier layer cells.
Photodiode array detectors are designed with 256 - 2048 photodiodes that are arranged in a linear fashion. This arrangement allows each photodiode to respond to a specific wavelength that results in a continuous UV/visible spectrum. The resolution is generally 1 - 2 nm.
7. Read-out device. It provides the actual value or numerical data needed in the calculation of the value of analyte present in the sample. Examples of these are
- printer
- monitor or digital window
- galvanometer scale
Spectrophotometric Methods involving Suspensions
1) Turbidimetry is the measurement of the amount of light blocked by particulate matter in passing through a turbid solution.
The amount of light blocked depends on the number and the size of the particles. Hence the particle size in the standards and samples must be comparable.
Consistent timing of sample preparation and assay helps to avoid errors resulting from aggregation or settling of particles.
The procedure is usually carried out at RT. Slight variations in temperature are not critical.
2) Nephelometry is the measurement of the amount of light scattered by particles in suspension.
The amount of light scattered depends on the size and shape of the particles and on the wavelength of the incident light.
UV light should not be used because it might produce some fluorescence, which would lead to erroneously high results.
It is more sensitive than turbidimetry because of the 90 degree instrument configuration.
Sensitive Types of Spectrophotometric Methods
1) In the dry reagent slide technique, Reflectance spectrophotometry, as light from a radiant energy source passes through an interference filter, it is projected to the slide at a 45-degree angle.
The light then follows a path through the clear support material and reagent layer and hits a white spreading layer; the unabsorbed light is then reflected back through the reagent and support layers.
This reflected light impinges on the photodetector, which is positioned at a 90-degree angle to the slide.
Since reflectance values are neither linearly proportional to transmission values nor consequently to dye concentration, the microcomputer utilizes an algorithm as a linearizing transformation of reflectance values to transmission values so that concentration may be calculated.
2) Fluorescence occurs when a molecule absorbs light of a particular short wavelength and is thereby stimulated to emit light of a longer wavelength.
The emitted light has a characteristic spectrum, the emission spectrum, that is unique for each flurorescing molecule. Hence, fluorometric methods are extremely sensitive and highly specific.
Because of this extreme sensitivity, reagents used must be of higher degree of purity than is required for spectroscopy, since even slight traces of impurities may fluoresce.
In a fluorimeter (Fluorescence spectrophotometry), light from the excitation lamp travels in a straight line, whereas the fluorescent light is radiated in all directions.
If the detector for the emitted fluorescent light is placed at a right angle to the path of the excitation light, the excitation light will not fall on the detector. In addition, baffles can be placed around a cuvet to avoid reflection of exciting light from the surface of the cuvet to the detector.
The right-angle configuration does not prevent loss of the exciting or the emitted light.
3) Instrumentation employing fluorescence polarization is used for measuring therapeutic drug levels and for fetal lung maturity testing.
In these immunologic assays, plane-polarized light excites fluorophors in the sample cuvet.
The free-fluorophore-labeled ligands rotate freely because of their small size and primarily emit depolarized light.
The labeled ligand-antibody complexes rotate slower because of their large size and emit polarized fluorescent light.
Because of the differences in emitted light, it is not necessary to separate the free from bound fluorophore-labeled ligands, allowing for use of the homogeneous assay technique.
The emitted fluorescence intensity is measured by a polarization analyzer in the vertical plane, followed by its 90-degree movement for measurement in the horizontal plane.
The amount of polarized light detected is inversely proportional to the concentration of ligand in the serum sample.
4) Bioluminescence is a type of chemiluminescence in which the excitation energy is supplied by a chemical reaction rather than by radiant energy, as in fluorescence and phosphorescence.
Bioluminescence assays may employ either an NADH:FMN oxidoreductase- bacterial luciferase or an ATP-firefly luciferase system.
Bioluminescence assays are nonradioactive, having sensitivity levels in the attomole (10-18) to zeptomole (10-21) ranges, which make them more sensitive than direct fluorescent tests.
Bioluminescence has been applied in the development of immunoassays.
5) Atomic absorption spectrophotometry (AAS) is based on the principle that atoms in a basic ground state are capable of absorbing energy in the form of light at a specific wavelength.
The analyte must first be converted to nonionized atoms by heating in a flame. About 99% of the atoms of the analyte in the flame are in the ground state and therefore, are capable of absorbing energy at the appropriate wavelength.
Hence, light absorbed is essentially proportional to the concentration of the analyte.
The light source in AAS is a hollow cathode lamp in which the cathode contains the element that is to be measured. The hollow cathode lamp supplies the emission line of light required for the analysis.
The basis of AAS is the measurement of light, at a specific wavelength, that is absorbed by the element whose atoms are in the ground state.
The flame in AAS serves two functions, to accept the sample, thus serving as a cuvet, and to supply heat for converting the element, which is usually present in the sample in molecular form, into its atomic form at ground state energy level.