Chemistry Unit #1

Reference Range

represents expected values for a healthy individual based on specific population served by the lab

Diagnostic Sensitivity

probability of detecting ALL patients with target disease, negative rules out. no false negatives

Analytical Sensitvity

how sensitive a test is in the analyzer

Diagnostic Specificity

probability of detecting ONLY target disease, positive rules in. No false positives

Analytical Specificity

how specific the analyzer is when running a test

Negative Prediction Value

probability that a negative result means a patient does not have the disease

Positive Prediction Value

probability that a positive result means the patient has the disease

PPV/NPV effect with rare conditions

PPV is lower, NPV is higher

PPV/NPV effect with common conditions

PPV is higher, NPV is lower

Specificity Formula

S = TN/TN+FP

PPV Formula

PV = TP/TP+FP

NPV Formula

PV = TN/TN+FP

Sensitivity Formula

S = TP/TP+FN

Quality Assurance Purpose

assess effectiveness of labs policies and procedures to assure accurate, reliable, and prompt reporting of results

Calibration

standard is used to establish analyzer detection of known value

When is calibration done?

after instrument installation, new lot of reagent, regular intervals, recalibrate after QC trend/shift

Analytical Measurement Range (AMR)

range of results in which instrument response and specimen value is linear, limitation of analyzer/reagent

Clinically Reportable Range (CRR)

may be different from AMR if linear range is extended with dilution

Quality Control

monitor and evaluation of quality of each analysis method, measures accuracy and precision

QC Material

matrix similar to actual specimen, ideally stable with little vial to vial variation

Lyophilized material

freeze dried, reconstitution is required and can be source of error

Assayed

already been tested, very specific target and range, expensive

Unassayed

target and range must be established, broader range requiring techs to run 30x over 30 days, less expensive

Standard Deviation

how widespread values are around the mean, higher is less precise

Coefficient of Variation Formula

SD/mean x 100

Coefficient of Variation

relative standard deviation expressed as %, compares methods

Trend

gradual increase or decrease, can be caused by reagent deterioration or light deterioration

Shift

abrupt increase or decrease, can be caused by change in calibration, reagent change, or analyzer issue, analyzer issue

Random Error

operator technique, pipette problems, incorrect reconstitution

Systematic Error

change of lot, deterioration, improper alignment of sample or reagnet pipette

Westgard Multirules

6 rules for evaluating QC data

1-2s

1 point outside 2SD. serves as warning, may indicate random error

1-3s

1 point outside 3SD. resolve error before releasing patient results, may indicate random error

R-4s

1 point outside +2SD and -2SD within same run. Resolve before releasing patient results, may indicate random error

2-2s

2 points outside 2SD. resolve error before releasing patient results, may indicate systematic error

4-1s

4 points outside 1SD. Resolve error before releasing patient results, may indicate systematic error

10X

10 points on one side of mean. Resolve error before releasing patient results, may indicate systematic error

Random Error troubleshooting

re-assay fresh aliquot, open/reconstitute new vial of control

Systematic Error troubleshooting

replace reagent, instrument/alarm issues

Delta Flag

LIS compares patients current results to previous and finds change greater than threshold

Proficiency Testing

surveys or external quality controls, required by CLIA, tested by CAP and API. Uses simulated patient samples to confirm analyzers are working in universal manner

Wavelength of visible light

400nm to 700nm

Spectrophotometry and Turbidimetry measures:

light absorbed by analyte

Fluorometry and Chemiluminescence measure:

light emitted by analyte

Nephelometry measures:

light scattered by analyte

Light Source

provides light of appropriate wavelength

Monochromator

selects a narrow wavelength bandpass to improve specificity of target analyte

Diffraction Grating

reflective surface with fine grooves that seperates light

Sample Holder

flow cell or cuvette

Photodetector

converts light into electrical signal

Phototube

photodetector that is largely obsolete

Photomultiplier Tube (PMT)

uses dynodes to amplify small signal

Photodiode Array (PDA)

measures multiple wavelength at once

Signal processing

converts electrical signal to result

Reflection Photometry (Light source, monochromator, sample holder, detector, measurement)

LED, filters, test strip/pad, photodiode, reflected light intensity

Spectrophotometry (Light source, monochromator, sample holder, detector, measurement)

Tungsten-Halogen/Deuterium, diffraction grating, cuvette, photodiode/PDA, decrease in transmitted light

Turbidmetry (Light source, monochromator, sample holder, detector, measurement)

Tungsten-Halogen/Deuterium/Xenon, diffraction grating, cuvette, photodiode, decrease in transmitted light

Nephelometry (Light source, monochromator, sample holder, detector, measurement)

laser, filters, cuvette, photodiode/photomultiplier tube, scattered light

Fluorometry (Light source, monochromator, sample holder, detector, measurement)

LED/laser, filters, cuvette, photomultiplier tube, emitted light

Chemiluminescence (Light source, monochromator, sample holder, detector, measurement)

none, filters, reaction cuvette (magnets), photomultiplier tube, light emitted during chemical reaction

Deuterium

Light source for UV

Tungsten/Tungsten-halogen

Light source for visible light

Xenon

Light source for UV and visible light

Ion-Selective Electrode (ISE)

measures for electrolytes using potential voltage between two electrodes

Electrolytes

sodium, potassium, and chloride

Peak position

id

Peak height/area

concentration

Electrophoresis

use of an electric field to separate charges based on charge and size

Anode

Positive

Cathode

Negative

Components of Electrophoresis

electric field, support medium, buffer, sample, detecting system

Immunofixation

antibodies used to identify protein present in bands

Isoelectric Focusing

pH gradient seperates proteins by their isoelectric point

Chromatography

consists of mobile phase and stationary phase. Stationary compounds retain longer, like dissolves like

Retention Time

Property of analyte, how long to elute off

Resolution

property of system, how well does it separate compounds in sample

Absorption

liquid mobile and solid surface stationary

Partition

polar (aqueous) liquid phase and nonpolar (organic) liquid phase

Steric Exclusion

liquid mobile phase and solid stationary phase, based on size and shape

Ion Exchange

opposite charge liquid mobile phase and solid stationary phase

Thin Layer Chromatography (TLC)

mostly obselete, uses Retention factor (Rf)

Retention Factor

Rf = distance of component/total distance of solvent

High-Performance liquid chromatography

therapeutic drug monitoring and toxicology

Gas Chromatography

therapeutic drug monitoring and toxicology

Mass Spectrometry

identify and quantify molecules in a sample based on mass to charge ratio

MALDI

ionized by source of ions for mass spectrometry

Immunoassay

the binding of antibody to antigen for the specific and sensitive detection of an analyte

Competitive Assay Binding

labeled reagent competes with patient analyte for antibody binding sites, signal is inversely proportional to analyte concetration

Non-competitive Assay Binding

labeled reagent used to bind to and directly detect patient analyte. signal is directly proportional to analyte concentration

Heterogenous

requires separation of immune complexes from free antibodies and antigen to measure labeled complex

Homogenous

does not require separation, unbound label will not interfere

Fluorescence Polarization Immunoassay (FPIA)

homogenous competitive. polarized light excites the fluorescent label. Obsolete, less sensitive than CLIA/EIA. Therapeutic drug monitoring and drug testing

ELISA

solid phase heterogenous assay. detection by spectrophotometry. tests infectious disease and autoimmune antibodies

Horseradish peroixdase and tetramethylbenzidine

turns blue then yellow after stop

alkaline phosphatase and p-nitrophenyl phosphate

turns yellow

Chemiluminescent Immunoassay (CLIA)

heterogenous asasy. luminescent labels emit a photon of light as result of chemical reaction

HAMA

human anti-mouse anitbodies in patient sample, exposure to mice

Heterophile antibodies

weak general antibodies that bind to assay

High Dose Hook Effect

false negative/decreased result due to high amounts of analyte

Prozone

antibody excess causing high dose hook effect