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Clinical Biochemistry CSMLS Review

HANDOUT

For additional information and resources visit: www.uoitclinicalbiochemistry.weebly.com (password: westgard)

1. MISC Quality Control Information 42-53 questions (includes QM)

Control: Material that is chemically and physically similar (has similar MATRIX) to unknown specimens and is
tested in exactly the same manner. Can be liquid, ready to use or lyophilized (drawback is reconstitution errors)
External QC (EQA – External Quality Assurance, Proficiency Testing): Control material received from a proficiency
testing program that is to be analyzed in the same manner as patient samples. Results are reported to agency
(IQMH in ON), which compares them to results from other labs to determine acceptable lab performance.
Checking for ACCURACY of lab results (and on occasion checking for precision/reproducibility of reported results).
Mean: Sum of all observations divided by the number of observations (average)
Standard Deviation: Statistical expression of dispersion of values around mean.

Coefficient of variation (%CV): Expresses standard deviation as a percentage of the mean. Allows for comparison
of imprecision.
 CV % = (SD ÷ mean) × 100. The lower the CV, the better the precision (the lower the imprecision).
Normal Distribution: produces a bell-shaped (Gaussian) curve. Apparent when mean=median=mode.
 68% of values fall within ±1 SD of mean
 95% of values fall within ±2 SD of mean (95% confidence interval)
 99.7% of values fall within ±3 SD of mean
Levey-Jennings Chart: Normal distribution curve lying on its side, marked with mean, ±1, ±2, ±3 SD. Considered
BEST chart to detect quality control errors.
Control Limits: range of values in which control values must fall within in order for the assay to be considered
acceptable (valid). Most commonly used is mean +/- 2SD (95% Confidence interval) in which 1 determination in 20
will fall outside ±2 SD. This is an expected part of normal variation.
Accuracy: closeness to true value. Troubleshoot (assess) by running material with known values (ex. EQA vials).
Can reset accuracy by recalibrating.
Precision/Imprecision: reproducibility. Troubleshoot (assess) by running 10-20 replicates of a patient sample.
Recalibration does NOT correct for imprecision in an assay.
Random Error: Error that is not reproducible (doesn’t recur in a regular pattern). Related to imprecision. [12s, 13s,
R4s Westgard Rules]. Usually a 1-time error, controls can be rerun with success.
Systematic Error: Reproducible results due to an error inherent in the test procedure. [22s, 41s, 10x Westgard
Rules]. Requires investigation to determine cause (rerunning QC does not correct the problem). Includes SHIFTS
(consecutive control values on one side of the mean) and TRENDS (consecutive increasing or decreasing control
value).
False rejection: Rejection of run because QC results indicate problem when none is present. (use of multi-rules
reduces false rejections).
Calibration: process used to set accuracy. Uses standards (calibrators) with known concentrations to establish
correlation of measured signal (usually absorbance) to concentration of unknown samples.
Calibration Errors: standards can be prepared incorrectly (over or under diluted) or standard concentration can be
incorrect (too low or too high). Calibration errors lead to ACCURACY errors (most commonly a shift in QC) and
flagged QC for accuracy related multi-rules (most commonly 22s)
 Dilution errors
o Too much diluent (OVERdilution): unknown samples will be falsely HIGH
o Too little diluent (UNDERdilution): unknown samples will be falsely LOW
 Concentration errors
o STD value used is too HIGH (higher than actual stated value): unknown samples falsely HIGH
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o STD value used is too LOW (lower than actual stated value): unknown samples falsely LOW
Analytical Sensitivity: same as detection limit; lowest concentration of analyte that can be detected. High
sensitivity leads to reduced false NEGATIVES
Analytical Specificity: ability of a method to only detect the analyte that it’s supposed to be measuring. Related to
interferences. High specificity reduces false POSITIVES.
Diagnostic Sensitivity: percentage of population with the disease that tests positive for that assay

where: TP = True Positives and FN = False Negatives

Diagnostic Specificity: percentage of population without the disease that tests negative for that assay
where: TN = True Negatives and FP = False Positives

2. Pre Analytical Factors: 10-15 questions

Patient Variables that Affect Chemistry Values:
Factor Examples of Analytes Affected
Age Albumin, ALP, Phosphorus, Cholesterol, Bilirubin (NBIL)
Gender ALB, ALP, CREAT, URIC, CK, iron/ferritin
Diurnal Variation ↑ in AM: ACTH, cortisol, iron
↑ in PM: GH, PTH, TSH, ACP
Day to day variation >20% ALT, bilirubin, iron, TSH, trig
Recent food ingestion ↑ GLU, insulin, trig, gastrin, Iron
↓ Cl-, PHOS, K+
Foods/Beverages False POS:
-5HIAA (bananas, avocados, eggplant, plums, walnuts)
-VMA (bananas, vanilla)

Caffeine increases cortisol

Alcohol increases TRIG, glucose, cortisol (GGT elevated if liver damage)

Tests Requiring a Fasting Specimen: FBS, GTT, Lipid panel (Trig/calculated

HDL), Gastrin, Insulin
Posture ↑ when standing: TP/albumin, IGs, CHOL, TRIG, ALT, AST, ALP, T4
Activity ↑ in ambulatory patients: CK
↑ w/exercise: lactic acid, CREAT, TP, CK, Myoglobin, ALT/AST, LD
↓ w/exercise: CHOL, TRIG, Iron, K+, pCO2
Stress ↑ ACTH, cortisol, catecholamines
Pregnancy plasma volume increases 3X more than rbc mass
Smoking ↑CoHB above 10%, glu, HGH, cortisol, cholesterol, triglycerides, urea
Drugs Varying affects on varying assays. Always check for potential drug interference
when unexpected results are obtained!

Effect of Anticoagulant Contamination:

Anticoagulant Analytes Affected
EDTA (lavender, chelates calcium) ↓calcium, magnesium
Inhibits ALP, CK, AMY
↑ K+ (K2EDTA), Na+ (Na2EDTA)
Sodium Fluoride/potassium oxalate ↓ calcium, magnesium
(grey top tube: oxalate binds Ca++, fluoride inhibits Fluoride inhibits urea (by urease) and uric acid (by uricase)
glycolysis)
Heparin (green, neutralizes thrombin) Inhibits ALP
-can be lithium, ammonium or sodium heparin ↑ AMM (w/ammonium hep), LI (w/lithium hep), Na+ (w/ Na hep if
tube not filled)
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(must check when collecting)

Lithium Iodoacetate (grey, iodoacetate inhibits Inhibits CK
glycolysis) ↑ lithium
Sodium Citrate (light blue, binds Ca++ -requires 9:1 ↓ calcium
ratio of blood to anticoagulant) ↑ Na+

Specimen Collection/Handling Effects:

Factor Example of Analytes Affected
Squeezing site of capillary puncture ↑ K+
Pumping fist during venipuncture ↑ K+, Lactic acid, calcium, phosphorus
↓ pH
Tourniquet > 1 minute ↓ K+, TP, CHOL, iron
Drawn above IV Fluid Dependent upon IV fluid
↑ glucose (if glucose drip), Na+/Cl- (if saline drip), drugs
↓ other analytes due to dilutional effects -ex. TP!!
Hemolysis ↑ K+, Mg, phos, LD, ACP, AST, TP, ammonia, iron
Exposure to light (keep wrapped in foil) ↓ bilirubin, b-carotene, vit A, vit B12, erythrocyte
protoporphyrin
Delay in serum/plasma separation ↑ ammonia, lactic acid, K+, Mg, LD
↓ glucose (unless collected in NaFl tube)
Temp of storage ↓ at RT: glucose (unless collected in NaFl tube), ACP
↑ at RT: lactic acid, ammonia
↓ at 4oC: LD ↑at 4oC: ALP

K+ storage at cold temperature

ATPase pump in cell membrane actively transports Na+ out of
the cell and K+ into the cell against the electrochemical
gradient –requires energy!
0-4oC storage inhibits pump function, K+ leaks out from cell
into plasma
K+ will increase by 0.1 mmol/L the 1st hour and 0.4 mmol/L
each following hour!

Transportation temperature Hot vehicles can falsely decrease K+ as Na+/K+ pump activity
is increased causing K+ to be pumped into the cell.

Specimens Requiring Special Handling:

Requirement Tests Misc Info

Chilling ABGs, ACTH, ammonia, gastrin, Place in crushed ice or ice w/water (ice
glucagon, lactate, PTH, iCAL, cubes alone lyse rbcs and do not
angiotensin converting enzyme (ACE), provide even temperature
acetone, catecholamines, free fatty distribution)
acids, pyruvate and renin.

Warming Cold agglutinins, cryoglobulins Place in 37oC heat block or hold in

hands
Chain of Custody Tests used in legal proceedings: dependent upon local regulations
alcohol, drug screens, DNA analysis

Differences in Analyte Concentrations:

Higher in Higher in Serum Higher in Higher in Higher in Higher in Higher in
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Plasma than than in Plasma plasma than Capillary than Venous than in RBCs than Plasma
in Serum in whole Venous blood Capillary Blood in Plasma than in
blood RBCs
TP K+ Glucose Glucose (in Calcium K+, PHOS, Na+, Cl-
LD Phosphorus postprandial TP Mg, LD, AST,
calcium Glucose specimen) iron,
CK K+ ammonia
Bicarbonate
ALP
Albumin
AST
triglycerides

Effects of Incorrect Order of Draw:

3. Analytical Considerations: (CSMLS 5.12 Demonstrates information management skills, e.g.

computer, laboratory information systems and related technology)

Automation/IS Terminology:
RAM (Random access memory): Working memory used for temporary storage of programs & data. Content is lost
each time computer is turned off (if power goes off you would lose RAM data).
ROM (Read-only memory): memory that is permanently protected from being modified, erased, or written over.
Not affected by power loss (data is NOT lost). Used for boot-level and other system instructions.
Unidirectional interface: Interface that transmits electronic information in 1 direction (ex. Vitros sending results
to LIS)
Bidirectional interface: Interface that transmits electronic information in 2 directions (ex. the LIS downloads orders
from the HIS to the analyzer and the analyzer uploads results to the HIS).
Discrete Analysis: Each sample reaction occurs in a separate compartment (ie. sample and reagent are added
together but each “mixture” is added into a separate cuvette, reaction vessel).
Random Access: ability to order/analyzer tests in any combination
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Batch Analysis: samples are all processed for the SAME test or combination of tests
Stand-alone analyzer: instrument from a single discipline with automated capability
Modular workcell: at least two instruments from a single discipline with one controller
Multiple Platform: instrument capable of performing tests from two disciplines
Throughput: maximum number of tests generated per hour
Turnaround: amount of time to generate a result
Dead Volume: amount of serum that cannot be aspirated (required test volume is added to the dead volume in
order to aspirate the sample)
Carryover: the contamination of one sample by a previously aspirated sample
Level-sensing: the ability of a sample or reagent probe to detect the amount of sample in a tube or container
Reflex Testing: Use of preliminary test results to determine if additional tests should be ordered (or cancelled!) on
a specimen. (ex. TSH as screening test in the Thyroid Pathway)
Kinetic Assays: determination of sample concentration based upon change in absorbance over time
Endpoint (Colorimetric) Assays: single absorbance reading taken after a predetermined incubation period
Pending (Outstanding) List: LIS generated or manual worklist that identifies which samples have NOT yet been
completed (have pending/outstanding tests). Used to check if patient testing is finished.

Instrumentation Highlights:

LIGHT MEASURING SYSTEMS 3 questions

General Information:
 Electromagnetic radiation (radiant energy) has both wave-like and particle-like properties. =Photons
(particles of light) of energy traveling in waves
 Wavelength: the distance traveled by one complete wave cycle (distance between two successive
“crests”) measured in nm
o The shorter the wavelength, the greater the energy in the light (the greater the number of
photons)
 Radiant energy that passes through an object will be partially reflected, absorbed, and transmitted
o Light scatter occurs when radiant energy collides with molecules in the solution. Scatter is in
different directions but majority is in forward direction (intensity changes with direction of
scatter)
 For radiant energy to be absorbed it must have the same frequency as a rotational or vibrational
frequency in the atom or molecule it strikes. Therefore, a particular molecule or atom will absorb only
certain energies (not all energies)
 When energy is absorbed as a photon, an electron moves to a higher energy level where it is unstable and
then returns to its original energy level (ground state). Upon return to ground state light of a
characteristic wavelength is emitted which can identify the molecule.
o Types of Emission Spectra:
 Continuous Spectrum: light of equal intensity is emitted over entire spectrum (ex.
Tungsten bulb)
 Band Spectrum: emission of light from an atom over a small range of wavelengths (ex.
Mg emits light between 370-380nm)
 Line Spectrum: characteristic atomic light emission at a specific wavelength (ex. Na+ at
589nm, K+ at 404 and 767nm)

Method Principle Component Use Miscellaneous info

Parts
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Absorbance Chemical reaction Light source, Many routine A = 2-log%T

produces color in which monochromator colorimetric/enzymatic
Spectrophotometry
absorbance is (glass filters, tests. %T is inversely proportional
proportional to interference filters in Many enzyme tests to the log of the
Follows Beer’s Law concentration of the photometers; measure increase or concentration
analyte diffraction grating or decrease in absorbance of
A=abc NADH or NADPH at 340nm
prisms in
spectrophotometers),
Bougher-Lambert Law: cuvette, detector
The longer the cuvette (converts light energy
path in cm (the LONGER into electrical signal,
the cuvette) the less light ex PMT), readout
transmitted.

Reflectance Measures change in light Same as abs spect Routine chem. (Vitros Use SPECULAR reflectance
intensity from a chemical slides) and Urine (test from interaction of incident
Spectrophotometry
reaction that has -except NO cuvette! strip pad/Clinitek) light w/reaction layer
reflected from a matte molecules (uses absorbance
surface. and light scattering)

Intensity of reflected light Uses underlying reflective

is NON-linear (requires layer to reflect diffuse
algorithm to linearize reflectance to detector.
data) and INVERSELY
proportional to
concentration of analyte
Fluorometry Atoms absorb light of a UV light most often Hormones, amino acids, Detector is at 90o to light
particular λ and emit light (Mercury or xenon arc vitamins, drugs, source –allows only light
of a LONGER λ (LOWER lamp), 1o porphyrins emitted from sample to be
energy) monochromator, measured (NOT incident
quartz cuvettes, 2o light)
monochromator, (more sensitive than
detector, readout colorimetry)
QUENCHING: anything that
causes a false decrease in
fluorescence.
Nephelometry Measures light scattered Light source, cuvette, IGs, complement, specific Detector is at an angle
by particles (usually Ag- detector, readout proteins (usually 30-90o) to capture
Ab complexes) scattered light.
Changing angle changes
sensitivity
Turbidimetry Measures light Same as absorbance Drugs, specific proteins Detector is in line (180o
transmitted through a spectrophotometry (also in microbiology angle) w/light source to
solution containing (Vitek) and coag measure light that was not
particles (usually Ag-Ab analyzers) scattered by particles. Can
complexes) be used on routine analyzers
Misc Info:
Sample Blank: used to reduce effects of sample color on final color reaction (add volume of sample to volume of water in place of reagent)
Reagent Blank: used to reduce effects of reagent color on final color reaction (add volume of water in place of sample to volume of reagent)

Bichromatic Readings: measures reaction solution at 2 different wavelengths

λ1 = peak abs of compound of interest
λ2 = absorbance away from peak but in area of interfering substance (ex. hemolysis peak) = BLANKING

Spectrophotometer Quality Assurance:

Wavelength Accuracy: ensures that selected λ on spec corresponds to the actual λ of light being transmitted through the sample. Use
didymium or holmium oxide filters (have maximum abs at known wavelength)
Photometric Accuracy: ensures that the Abs displayed on the spect is the true absorbance
Stray Light: any light that reaches the detector that did not pass through the sample/ radiant energy reaching the detector through the
monochromator that is not of the selected wavelength Causes of stray light: scratches on optical surfaces, dust particles in the light path, aging
diffraction gratings. Use “cutoff” filters in visible range, NiSO4 NaNO2 or acetone in UV range.
 Increase in stray light results in loss of linearity
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Spectral Isolation:
Bandpass: the number (or range) of λs passing through the exit slit of the monochromator
Bandwidth: the width of the opening in the exit slit (measured in nm) of the monochromator
NOTE: the larger the bandwidth or bandpass, the poorer the resolution of the spectrophotometer
 In a spect with a bandwidth of 20nm set at 450nm, expect the wavelength range passing through to be 440-460nm
General rule: for peak absorbance readings to be w/in 99.5% of the true values, the spectral bandwidth should not exceed 10% of the
natural bandwidth of the solution being measured

Enzyme Reactions:
Factor Effect Info
Substrate First-order kinetics: [enz] > [sub] Assays measuring an enzyme use zero-
-reaction rate proportional to [sub] order kinetics
Concentration [sub] Zero-order kinetics: [sub] > [enz]  excess substrate present so that
-reaction rate proportional to [enz] reaction is proportional to amount of
enzyme in sample
Assays using an enzyme as the reagent
use 1st-order kinetics
 excess enzyme present as the reagent
so that reaction is proportional to the
amount of analyte [aka “substrate] in the
sample
Enzyme Concentration Velocity of reaction is proportional to [enz] as long as the Report enzymes to U/L (amt of enzyme that
[sub] > [enz] will catalyze the reaction of 1 umol of
[enz] substrate per minute under specified
(If the enz concentration is high the substrate is depleted) conditions)
pH Extremes of pH can denature enzymes Most assays at pH 7-8 and use buffers to
pH can drive a reaction (forward vs. reverse reactions ex. maintain pH
LD)
Temperature Increase of 10oC doubles the rate of reaction (up to 40- Most assays at 37oC, next common at 25oC
50oC where enzyme denaturation may occur)
Cofactors Activators: inorganic cofactors (metallic or nonmetallic The higher the [cofactors], the greater the
ions) rate of reaction.
Coenzymes: organic cofactors (nucleotide phosphates Must be present in excess in assays
and vitamins) Commonly used: NAD/NADH (note: it is
NADH/NADPH that absorb at 340nm which
is why most enzyme assays are read at
340nm)

Summary of Enzyme Elevations:

Cardiac Hepatic Skeletal Muscle Bone Pancreas
CK, LD, AST Hepatocellular: AST, LD, ALT CK, AST, LD ALP AMY, LIP
Biliary Obstruction: ALP, GGT
Chronic alcoholism: GGT

Cardiac Markers Summary:

CKMB LD1>LD2 Myoglobin Troponins
Elevation after chest ~4-8h ~12-24h ~3-4h ~4-12h
pain
Duration of elevation ~2-3 days ~5 days ~24h ~7-10d
Sensitivity/Specificity Not completely specific Insensitive Sensitive but NOT Better sens/spec than
for AMI Nonspecific specific (ex. elevated in CKMB
muscle disorders)
Use Classic “gold standard” No longer used Negative predictor used Replaces LDi
but being replaced by (previously used to to RULE-OUT AMI (no Elevated cTnI used to
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newer markers detect late presenters) increase w/in hours after help Rule-in AMI (now
chest pain) acting as the “gold
standard”

Creatinine Summary (Renal Function):

Types of renal Pre-Renal (loss of Renal (loss of functional kidney tissue) Post-Renal
disorders appropriate blood ACUTE: (blockage of urinary tract
supply) -Acute Glomerulonephritis preventing urine outflow)
-Tubular Necrosis
-decrease in plasma -Nephrotoxicity (ex. Heavy metals) -Renal stones
volume -septic shock -Prostate cancer
-reduced cardiac output -infections -Enlarged prostate
-occlusion of renal artery CHRONIC:
-glomerulonephritis
-Polycystic kidney disease
-Diabetic nephropathy
-Chronic pyelonephritis

Creatinine Clinical Significance: evaluate kidney function Factors Affecting Creatinine Concentration:
Plasma levels are the function of: Female: DECREASE: -Decreased generation
 Muscle mass* due to reduced muscle mass
 Rate of creatine turnover
 Kidney filtration rate African American: INCREASE: -Increased
In “normal”, healthy individuals creatinine levels in generation due to higher muscle mass
the blood are low and constant!! Vegetarian Diet: DECREASE: -less N intake
High Meat Diet: INCREASE: -more N intake
Malnutrition: DECREASE: -less N intake
Muscular: INCREASE: -increased muscle
mass
Obesity: NO EFFECT excess mass is fat, not muscle
Creatinine Method 1. The Jaffe Reaction:
Creatinine is oxidized by saturated picric acid in an alkaline solution to form creatinine picrate, a red-
orange chromogen read at 510-520nm
 Reminder: dried picric acid is explosive (must be removed by waste management services)

NOT specific due to positive interference from “non-creatinine chromogens”/other reducing agents that also
react with picric acid:
 Acetoacetate, acetone, ascorbate, glucose, pyruvate, uric acid, protein, cephalosporin
antibiotics
Negative interference from bilirubin and hemoglobin

2. Creatinase Method: enzymatic reaction that is specific for creatinine

Creatinine Clearance Requires collection of Proper collection of a
both a TIMED urine 24h urine sample: CALCULATION:
and plasma samples 1. Urine is voided and
Translation: the ratio from the patient DISCARDED. Time is
noted on the collection
of urine creatinine to container as the “start”
plasma creatinine time Where:
over a 24h period Need a substance that 2. Subsequent voidings U = creatinine in umol/L of 24h urine sample
is: are collected and put P = creatinine in umol/L of plasma or serum sample
1. freely filtered into the container for 24 V= volume of 24h urine in mL/d
through the hours. 86400 = number of seconds/day
glomerulus and not 3. Urine in bladder must A= patient body surface area in square meters
bound to protein be voided and placed 1.73 = average body surface area in square meters
2. not reabsorbed by into the container at 24h
the tubules from start time (end of
3. Is physiologically collection). Typical Corrected Creatinine Clearance RI:
inert 1.25 – 2.10 mL/s
The urine must be CrCl <1.00 mL/s indicates loss of about 50% of
Inulin is BEST substance refrigerated throughout functional nephron capacity and is classified as
but requires injection. the 24h collection period moderate kidney disease
Creatinine is next best.
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eGFR Calculation using serum creatinine (NO URINE REQUIRED) to estimate the glomerular filtration rate.

The MDRD equation is most commonly used in laboratories:

 The equation requires 4 variables:
 Serum, or plasma creatinine (sCr) in umol/L
 Age in years (18 years or older)
 Sex
 Race (African American or not)

eGFR-MDRD: <15 mL/min/1.73m2 - Consistent with kidney failure.

Summary of Lipoproteins:
HDL LDL VLDL Chylomicrons
Density (g/mL) 1.063 – 1.2 1.006 – 1.06 1.0006 <1.0006
(most dense) (float to the top upon standing)
Particle size (A) 70-100 100-300 2000 >2000
(largest)
% PROTEIN 50 32 2-13 0.5
(highest in protein)
%TRIG 77 7 64-80 90
%CHOL 17 35 8-13 6
%Phospholipids 27 25 6-15 4
Mobility (EP) Alpha Beta Pre-beta Origin
Lipid:protein 50:50 80:20 90:10 99:1
ADDITIONAL INFORMATION ON LIPIDS:
 12h fast required for trigs/LDL (not req for chol/HDL)
• Lipemic samples are the result of triglycerides (CHYLOMICRONS) in the sample (insoluble, high levels
visible to the eye)
• TC = HDL + LDL + VLDL (VLDL = TG/2.2)
• Friedewald Formula: (in mmol/L):
• [LDLchol] = [Total chol] - [HDLchol] - ([TG]/2.2) -formula is used to calculate LDL but can be used to
calculate whichever test result is missing.
• The Friedewald equation should NOT be used:
• when chylomicrons are present
• when plasma TG concentration exceeds 4.52 mmol/L

Summary of Proteins/ Protein Electrophoresis: 1-3 questions

Total Protein: measured by Biuret reaction which requires 2 or more peptide bonds
Albumin: measured by dye-binding techniques (BCG/BCP) which causes a spectral shift.
Globulin: calculation: TP-ALB
A/G Ratio: = ALB/GLOB
Ampholytes/zwitterions: Ability to be negatively or positively charged (charges change relative to pH)
Isoelectric Point: pH at which protein has net zero charge (least stable, least soluble=precipitation)
Electrophoresis: method of separating charged particles across a support medium by their rates of migration in an
electric field (voltage most often applied)
Resolution: degree of separation obtained or the ability to identify two species as separate bands
 ↑TIME: improves resolution (bands further apart), excessive time causes diffusion and reduced resolution
Increased ionic strength improves resolution (sharper bands)
Rate of Migration Depends on size, shape, charge of molecule. Migration is the result of driving forces and
resisting forces on the molecules. Main determining factor is the Charge-to-Mass ratio.
 The greater the net charge, the greater the mobility (faster migration).
 The larger/more asymmetrical the molecule the slower the migration.
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 The less mass of a molecule, the faster the migration

The medium is the largest resisting force.
Support medium Agarose, cellulose acetate
Stains Ponceau S (cellulose acetate), Amido Blue, Bromphenol blue, Coomassie brilliant blue
(agarose)
Charge Buffer imparts pH of soln. At pH 8.6 (barbital or tris-boric-EDTA buffer): proteins negatively charged
and move to anode (which is positively charged)
Order of migration Albumin (homogenous), alpha-1(ἀ1antitrypsin, AFP, ἀ1acid glycoprotein), alpha-2 (ἀ2
(fastest to slowest macroglobulin, ceruloplasmin, haptoglobin), beta (transferrin, complement, fibrinogen, LDL),
gamma (Igs)
toward the anode)
Electroendosmosis Buffer flow toward cathode. Causes gamma region to be cathodic to point of application (ie.
behind the point of application)
Effects on Migration ↑ Rate of Migration:
-increased net charge, increased voltage/current, increased pH, increased
temperature (until proteins denature), increased pore size of medium, decreased
viscosity of medium
↓ Rate of Migration:
-increased molecular size, increased ionic strength of buffer, increased viscosity of
medium, decreased temperature (increased temp if proteins denaturing), decreased
pH, decreased voltage
Hemolyzed serum Band between alpha-2 and beta (or increased beta)
sample
Plasma sample Extra band between beta and gamma (presence of fibrinogen)
Urine sample Must be concentrated. Bence-Jones protein in gamma region
SF sample Must be concentrated. Has pre-albumin (transthyretin) band. See oligoclonal banding in
multiple sclerosis
Monoclonal Sharp increase in one IG (called paraprotein, M-spike). Often see decrease in other IGs
gammopathy (hypogammaglobulinemia may suggest BJ protein –request a urine PE)
Polyclonal gammopathy Diffuse increase of many immunoglobulins in gamma region (ex. infection)
Cirrhosis pattern Polyclonal increase in gamma with beta-gamma bridging (bridging due to increased
production of IgA which migrates to the beta side of the gamma region)
Nephrotic syndrome ↓ alb ↑alpha-2, beta
pattern
Inflammation pattern ↑alpha-1, alpha-2, beta
(acute phase response)
Alpha-1-antitrypsin ↓ alpha-1
deficiency
Summary of Chromatography: 1-3 questions
Chromatography: the physical separation of solutes (analytes) from a mixture based upon their distribution in two
different phases
Stationary Phase (SP): does not move: solid, gel, liquid (distributed on a solid)
Mobile Phase (MP): gas or liquid that flows through or across the stationary phase
Modes of separation: Partition, Adsorption, Steric Exclusion, Ion Exchange, Affinity
HPLC Normal Phase: Stationary phase is polar
HPLC Reverse Phase: Mobile phase is polar (elution is faster), most common
Resolution: the ability of a column to discriminate (“resolve”) chromatographic peaks between two solutes
Retention time: time interval between injection of component into a column and detection of the component
(time it elutes out of the column). Used to IDENTIFY a compound (requires reproducibility).
Peak Height or Peak Area: used to QUANTITATE a compound (height/area = concentration)
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Summary of Mass Spectrometry: 1-3 questions

Mass Spectrometer: Instrument (detector) that uses principle of charged particles moving through a magnetic or
electric field, with ions being separated from other charged particles according to their mass-to-charge ratio (m/z).
Provides positive molecular identification of compound.
 Hard Ionization: ionization process that produces extensive Matrix-Assisted Laser
fragmentation Ex. Electron Ionization (EI)
Desorption/Ionization (MALDI): Soft
 Soft Ionization: ionization process that produces minimal
fragmentation Ex. Chemical Ionization (CI) ionization technique (does not need
 Base Peak: ion in the mass spectrum with the highest coupling to HLPC or GC). Analyte
abundance. Assigned relative abundance of 100%. (sample) dissolved in the matrix (low
 Mass Spectrum: plot of the relative abundances of each
separated ion as a function of its mass-to-charge ratio.
molecular weight absorbing
ICP-MS (Inductively Coupled Plasma Mass Spectrometry): used for compound). Solution of co-crystallized
metal/trace metal analysis (arsenic, lead, copper, mercury, zinc). Uses matrix and analyte molecules placed
inductively coupled hot plasma (ICP) to fully decompose a sample into onto target plate in the analyzer. UV
its constituent elements and transform the elements, which laser then used to irradiate the
traditionally did not easily form ions, into ions. It then uses MS to mixture causing vaporization of small
separate and quantitate the ions. amounts of matrix and analyte into a
plume of ions. Plume of ions then
Summary of Electrochemistry: 3 questions directed into the mass analyzer. This
technique is used in the microbiology
ELECTROCHEMISTRY: The measurement of electrical signals
laboratory to identify bacteria.
associated with chemical systems that are incorporated into an
electrochemical cell (electrodes and the solution in which they
are submersed)
 Electrons flow from an electrode of higher electron affinity to an electrode of lower electron affinity if the
electrodes are connected via a salt bridge.
o characterized by a half-cell reaction and a half-cell potential (voltage)
o ANODE: electrode from which electrons flow
o CATHODE: electrode that accepts electrons
 Page 12 of 21

Electricity: flow of electrons

Conductor: a substance whose high-energy electrons can be easily moved as free electrons
Insulator: high energy electrons are held tightly in the orbits and cannot be easily moved as free
electrons
Semiconductors: electronic conductivity between conductors and insulators
Electromotive Force: the force produced in a substance when there is an excess of electrons on one
end and an electron deficit on the other end (voltage)
Volt: unit of measure of electromotive force
Direct Current: produced when chemical energy is converted to electrical energy; a steady current in
one direction in a circuit
Alternating Current: produced when a rotating mechanism produces mechanical energy and converts it
to electrical energy by induction. A current that reverses direction in regular cycles.
Electron theory: electrons leave the negative pole of an electrical source, travel through the circuit,
then travel back to the positive pole
Circuit: closed loop of a conductor
Coulomb (Q): unit for measuring charge (1 coulomb = charge of 6.24 x 1018 electrons)
Current: rate at which a charge moves through a conductor. Measures in amperes (A). 1 ampere is
when 1 coulomb of charge passes one point in a circuit every second
Voltage (E): force that moves electrons through a conductor (electromotive force). EMF is measured in
volts
Potential: whenever there are 2 poles of different charges, a potential for producing current exists
Resistance: resistance to current flow in a circuit. Measured in ohms. 1 ohm = resistance when 1 volt
maintains a current of 1 ampere.

 Electrochemical cell (2 half-cells) composed of reference electrode, salt bridge, indicator

electrode, liquid junction, and detector.
 Reference Electrode: has known, constant potential (Standard Hydrogen Electrode/SHE
arbitrarily assigned potential =0.0 volt)
o Calomel reference electrode: mercury covered by layer of mercurous chloride in
contact with saturated KCl
o Silver/Silver chloride reference electrode: most common
 Indicator Electrode: changes potential according to concentration of analyte to be measured.
o Compares voltage between reference and indicator electrodes to determine analyte
concentration (activity)
Description Measures Info
POTENTIOMETRY Measures change in electric Potential develops when there is an Includes pH, pCO2 and ISE electrodes.
potential (VOLTAGE) interface between a metal and the ions of
between 2 electrodes that metal in a solution Ecell = log of ion activity
(indicator and reference OR
electrode) immersed in a When different concentrations of an The Nernst Equation relates the
solution in which current is ion are separated by a membrane that electrochemical cell potential to
kept at zero. is semi-permeable to that ion concentration
Reference Electrodes:  for every 0.059V, there is a
SHE, calomel, Ag/AgCl Requires voltmeter for detection change of 1 pH unit)
Na+/K+/Cl- As above As above Na/K+ are ISE electrodes with liquid-
polymer membranes that contain an
 Page 13 of 21

Ion Exchange ionophore (VALINOMYCIN for K+) that

gives specificity to the membrane
Electrodes (ISE)
Na+ polymer: false POS from lithium

Cl- Subject to interference by other

halides: Br-, I-, F-, CN-, OH-, S2-

Direct ISE: measures ion activity in

undiluted sample

Indirect ISE: uses diluted samples to

measure ion concentration in total
serum volume –affected by
abnormal HIGH levels of lipids and
proteins which leads to
pseudohyponatremia (aka The
Electrolyte Exclusion Effect)!!
pH electrode Glass membrane sensitive [H+] Potentiometric Calibrate w/2 phosphate buffers w/
to H+ -exchange of Na+ and H+ across the known pH
Calomel reference membrane develops potential difference
electrode + Ag/AgCl wire in between inside and outside of electrode Loses sensitivity above pH 10 (in the
saturated electrolyte presence of Na+)
pCO2 electrode pH electrode covered with Dissolved CO2, Potentiometric Calibrate w/2 gases of known pCO2
CO2 gas permeable
(Severinghaus) membrane. Bicarbonate CO2 from sample crosses the membrane,
buffer between membrane enters electrolyte soln layer and forms Reminder: pCO2 refers to the partial
and electrode carbonic acid. Carbonic acid dissociates, pressure of CO2
releasing H+ and shifting the pH. -this is the part of the total gas
CO2 + H2O ↔ H2CO3 ↔ H+ + CO3- The pressure contributed by CO2.
inner pH electrode senses pH change

AMPEROMETRY Electrochemical reaction By keeping voltage at a constant level, pO2 electrode

driven by a constant voltage any change in electron flow (CURRENT)
which results in a is indicative of the ion activity
measurable current (concentration)
(electron flow)
Requires ammeter/amperometer for
detection
pO2 electrode Inner pH electrode Dissolved O2 Amperometric Calibrate w/2 gases of known pO2
(Platinum cathode and
(Clark) Ag/AgCl anode) covered Ag/AgCl anode is oxidized and releases Oxygen is CONSUMED in this
with semipermeable gas e. O2 at the cathode is reduced by the e, electrode (measurement taken at
membrane. plateau of current just before readings
generating a flow of electrons (current) fall due to consumption)
The flow of current between anode and
cathode is directly proportional to pO2 in pO2 also referred to as polarographic
the sample solution (more O2 available = (The measurement of the gain or loss
more e- taken up at cathode = greater of electrons in a chemical reaction by
current flow) electrical means)
CARE AND MAINTENANCE OF ELECTRODES:
Electrode must be clean *Note: electrode function can usually be restored by cleaning the membrane surface.
 Protein build-up: requires scheduled cleaning with pepsin, bleach, or 0.1 M HCL
 Inorganic deposits: can be removed with EDTA or acid cleaning solutions.
 After cleaning must wash thoroughly with dH2O
CALIBRATION PRINCIPLES
Electrode Calibration: A process of normalizing electrode output by measuring a series of two or more known concentration
solutions (Calibrators). The analyzer then calculates the offset (intercept) and slope (the slope and intercept describe the line in
the calibration curve) of the electrode and uses them to compute the concentration of unknown samples. The slope and intercept
values must fall within designated ranges in order for the calibration to “pass”.
Calibration Curve: A plot of electrode potential (or current for pO2) versus activity from two or more standard
 Page 14 of 21

solutions. Unknown sample activity is determined by converting electrode potential (or current) to activity using the curve.
• 2-point cal: Low and high solns required at a minimum of once every 4 hours
• 1-point cal: One soln performed between analyses, minimum once every 2 hours
pH: uses 2-point calibration, on-board calibrator solutions at pH 6.840 and pH 7.384
pCO2, pO2: use 2 gas tanks (2 levels) with precise concentrations of CO2 and O2 (ex: 5% and 10% mixture of CO2 in an inert
gas)
Troubleshooting: If repeated calibration failures check condition of buffers, gas tanks, electrode membrane, and electrode

Summary of Electrolytes:

Analyte Reference Clinical Significance MISC Info

Interval
Na+ 135-145 mmol/L ↑ (hypernatremia): Due to ↑ intake or Major extracellular cation. Greatest
IV administration, hyperaldosteronism, contributor to plasma osmolality.
excessive sweating, burns, diabetes insipidus. Maintains normal distribution of
Causes tremors, irritability, confusion, coma. water & osmotic pressure. Levels
↓ (hyponatremia): Due to renal or extrarenal regulated by aldosterone. Ion-
loss (vomiting, diarrhea, sweating, burns) or selective electrode (ISE) is most
↑ extracellular fluid volume. Causes weakness, common method.
nausea, altered mental status.
K+ 3.5-5.0 mmol/L ↑ (hyperkalemia): Due to ↑ intake, Major intracellular cation. False ↑
↓ excretion, crush injuries, metabolic acidosis. due to squeezing site of capillary
Can cause muscle weakness, confusion, puncture, prolonged tourniquet,
cardiac arrhythmia, cardiac arrest. pumping fist during venipuncture,
>6.0 mmol/L =CRITICAL VALUE that must be phoned contamination with K+ IV fluid,
IMMEDIATELY hemolysis, prolonged contact with
↓ (hypokalemia): Due to ↑ GI or urinary loss, RBCs, leukocytosis,
use of diuretics, metabolic alkalosis. Can thrombocytosis.
cause muscle weakness, paralysis, breathing Serum 0.1–0.2 mmol/L higher than
problems, cardiac arrhythmia, death. plasma due to release from
platelets during clotting. Most
common method is ISE with
valinomycin membrane.
Cl- 98-106 mmol/L ↑ (hyperchloremia): Due to same conditions as ↑ Major extracellular ion. Helps
Na+ & excess loss of HCO3–. maintain osmolality, blood volume,
↓ (hypochloremia): From prolonged vomiting, electrical neutrality.
diabetic ketoacidosis, aldosterone deficiency, Passively follows Na+. Most
salt-losing renal diseases, metabolic alkalosis, common method is ISE. Sweat
compensated respiratory acidosis chloride test for cystic fibrosis.
TCO2 22-28 mmol/L ↑ in metabolic alkalosis, compensated >90% is bicarbonate/HCO3–;
respiratory acidosis remainder is carbonic acid (H2CO3)
↓ in metabolic acidosis, compensated & dissolved CO2.
respiratory alkalosis Important in maintaining acid-base
balance.
Keep sample capped to prevent
loss of CO2. Measured by ISE or
enzymatic method.
Anion Gap 8-16 mmol/L Calculated by: Na+ - (Cl- + TCO2) Useful QC check. Can’t be negative
=Difference between unmeasured anions and number (=lab error) If all gaps
unmeasured cations. are high or low, possible
↑ in renal failure; diabetic acidosis; lactic acidosis; instrument error in 1 of the
methanol,ethanol, ethylene glycol, or 4 electrolytes. Requires
salicylate poisoning. troubleshooting.

Calcium 2.20-2.60 ↑ with primary hyperparathyroidism, Most abundant mineral in body.

cancer, multiple myeloma. 99% in bones. Regulated by
mmol/L Can cause weakness, coma, GI symptoms, renal parathyroid hormone (PTH), vitamin
calculi. D, calcitonin. Anticoagulants
↓ with hypoparathyroidism, malabsorption, vitamin other than heparin bind Ca++.
D deficiency, renal tubular acidosis. TCAL increases with increased
 Page 15 of 21

Leads to tetany (muscle spasms), seizures, cardiac albumin.

arrhythmias. Ionized Ca is biologically active
form, better indicator of CAL status.
Measured by ISE. ICAL increases
with decreased pH
Phosphorous 0.85-1.45 ↑ hypoparathyroidism, renal disease Major intracellular anion. Mostly in
↓ with hyperparathyroidism, vitamin D bones.
mmol/L deficiency, renal tubular acidosis. Component of nucleic acids,
coenzymes. Important reservoir of
energy (ATP). Limited value alone.
Should be correlated with CAL
(normally reciprocal
relationship). Higher in children.
Citrate, oxalate, EDTA interfere.
More in RBCs than plasma. Avoid
hemolysis. Separate promptly.
Magnesium 0.65-1.00 ↑ due to renal failure, ↑ intake (ex. antacids), Essential cofactor for many
dehydration, bone cancer, endocrine disorders. Can enzymes.
mmol/L cause cardiac abnormalities, paralysis, respiratory 10× more concentrated in RBCs.
arrest, coma. Avoid hemolysis. EDTA, citrate,
↓ due to severe illness, GI disorders, endocrine oxalate bind Mg2+.
disorders, renal loss. Can lead to Colorimetric methods are most
cardiac arrhythmias, tremors, tetany, paralysis, common
psychosis, coma. Rare in nonhospitalized patients.

Summary of Acid-Base Info:

Henderson-Hasselbalch pH = 6.1 + log [HCO3-] base or pH = 6.1 + log [HCO3-]
Equation [H2CO3] acid [pCO2 x 0.03]
pH = 7.4 when there is a 20:1 ratio of base to acid. (change in either base
or acid concentration alters pH)
Relationship:
pH is directly proportional to bicarbonate and indirectly proportional to acid
(carbonic acid/ pCO2)
Major buffering system: Bicarbonate-Carbonic Acid system: MOST important, makes up ¾ of body’s
buffering capacity

TCO2 All forms of CO2 (HCO3- + H2CO3)

pCO2 Amount of dissolved CO2. -regulated by the lungs
Represents respiratory component (THINK: pCO2 inversely proportional to respiration
rate)
Bicarbonate HCO3- = (TCO2 – 1) -regulated by kidneys.
Represents metabolic component
Hypoxemia Low O2 content in blood
Hypoxia Lack of O2 at cellular level
pO2 pressure exerted by oxygen in a mixture of gases. Represents dissolved O2 (in plasma)
=DIRECT measurement (amperometric electrode). Assesses pulmonary function
O2SAT Amount of O2 combined w/ hemoglobin out of all hemoglobin.capable of binding O2
O2Hb 1g of Hb can combine w/ 1.34 mL of O2
O2Hb measured by co-oximetry (differential absorbance): Specific absorption spectra for
each species of Hb. Able to identify hemoglobin variants by taking readings at numerous
wavelengths simultaneously.

Carboxyhemoglobin formed through reversible interaction of carbon monoxide (CO) bound to hemoglobin usually
(CoHb) from inhalation of CO through exposure to products of incomplete combustion
Whole blood appears CHERRY RED. Higher in smokers
Methemoglobin (MetHb) Hb in which ferrous iron (normal) is oxidized to ferric iron. Provides a measure of CYANOSIS
 Page 16 of 21

as hemoglobin unable to bind O2. Whole blood is CHOCOLATE BROWN color

NORMAL ABG exposed ↑pH, ↓pCO2, ↑pO2 Room air: “0” mmHg pCO2, 150 mmHg pO2
to air (bubbles in Note: pts on high oxygen treatment (pO2>150 mmHg) will have falsely lowered pO2
sample)
NORMAL ABG at RT (or ↓ pH, ↑pCO2, ↓pO2 effects of glycolysis
↑ temp)
Clots in sample Clots result in lowered Hb, all parameters determined by THb are affected. Heparin is
anticoagulant of choice
Plastic syringe plastic syringes stored on ice cause ↑O2 permeability through plastic, resulting in pO2 errors.
Inadequate Mixing Inhomogenous sample error, uneven distribution of rbcs in sample, inaccurate results
Time Not required to cool ABG sample if analyzed within 30 minutes
When analysis is expected to be delayed for more than 30 minutes, the use of glass syringes
and storage in ice slurry is recommended

Summary of Acid-base disorders:

Disorder pH pCO2 HCO3- Compensation INTERPRETATION TIPS
1. Check pH to determine if acidosis or alkalosis
Respiratory acidosis ↓ ↑ N Kidneys retain HCO3-

↓ N ↓ Hyperventilation 2. Check pCO2 and HCO- to see if they are

Metabolic acidosis
within the normal range. If only one is
(blow off CO2) abnormal it is an uncompensated disorder. If
-↑AG indicates metabolic
acidosis due to both are abnormal compensation is suggested.
ketoacidosis, lactate
acidosis, ingestion of EG, 3. Check to see if the abnormal HCO3- is in the
Salicylate SAME or DIFFERENT direction as the pH (ie: are
-NORMAL AG in presence both the pH and HCO3- elevated/decreased?).
of metabolic acidosis due
to renal tubular acidosis,
diarrhea (Cl- shift) -If the SAME, the disorder is primary
↑ ↓ N Kidneys excrete HCO3- MEtabolic.
Respiratory alkalosis
-If DIFFERENT, the disorder is primary
Metabolic alkalosis ↑ N ↑ Hypoventilation REspiratory
Hypokalemia and (retain CO2)
hypochloremia

Summary of OSMOMETRY (measure of osmotic pressure): 3 questions

Osmometry: Determines osmolality which is the measurement of # of dissolved particles in solution,
irrespective of molecular weight, size, density, or type.
 note: S.G.by refractometry measures density of a solution and IS affected by molecular weight/size/type.
You will see higher results for refractometer SG in samples containing these types of particles
 note: S.G. by dipstick only detects ions and like osmolality is NOT affected by molecular weight, size of
particles
OSMOLALITY: the measure of the number of moles of dissolved osmotically active particles per kilogram of
water. Serum expressed as mosm/kg water. Normal: 280-300mosm/kg
 Clinical indicator of electrolyte balance, kidney function (concentrating ability), presence of unmeasured
anions in a sample
 INCREASED Osmolality (dehydration) stimulates ADH, THIRST
 DECREASED Osmolality (overhydration) suppressed ADH
 In diabetes insipidus: decreased ADH, normal serum glucose, elevated serum osmolality, low urine
osmolality
 Page 17 of 21

 In diabetes mellitus: decreased (or ineffective) insulin, elevated glucose, elevated serum osmolality,
elevated urine osmolality
 NOTE: Particulate samples (ex. Urine) should be centrifuged before analysis
Colligative properties: properties of a solution that are influenced by the number of molecules (solute
particles) in the solution but NOT their individual composition
As more solute is dissolved in a solvent the following properties of the solvent change:
 Osmotic pressure INCREASES
 Boiling point INCREASES
 Vapor pressure DECREASES
 Freezing point DECREASES
Freezing Point Depression Osmometry: MOST common (Cooling bath, thermistor, probe, stirring wire). Uses
principles of supercooling and Heat of Fusion. FP temperature is depressed 1.86oC for each mole of particles
dissolved per kg of water (FPD water = -1.86oC). FPD Osmometry detects VOLATILES (Vapor Pressure osmometry
does NOT detect volatiles).
Calculated Osmolality: used when osmometer not available (or to determine osmolal gap).
 = (2 x Na+) + GLU + UREA
Osmolal Gap: difference between measured osmolality and calculated osmolality. Normal: within +/-2 mosm/kg
 Most often used to screen for VOLATILES (alcohols)
 Also screens for presence of KETONES (ketoacidosis) and Mannitol (substance used as an osmotic diuretic
in cerebral edema, Gap determines therapeutic level required to reduce edema)

Summary of Immunoassays: 3 questions

IA: analytical methods that use an antibody (most commonly) as a reagent to detect an analyte (acts
as an antigen) in a patient sample
 Detection Limit: smallest amount of analyte (analyte sometimes referred to as ligand) that can be reliably
distinguished from zero. IAs have increased SENSITIVITY
 Ab Specificity: ability to bind to only the Ag (analyte) of interest
o %Cross-reactivity: degree to which an Ab reacts with Ag analogues (similar antigenic structure to
analyte). Increased cross-reactivity leads to poor specificity
o NSB (non-specific binding): binding of Ag to substances other than the intended reagent Ab. Leads to
poor specificity. Causes of NSB: heterophile Abs (HAMA), genetic variants of analyte (have different
epitopes), contaminants. If a method is binding generally (NSB) then SPECIFICITY needs to be
improved.
 Polyclonal Antiserum: Abs produced in animal from many cell clones (heterogeneous mixture of Abs)
 Monoclonal Antiserum: Abs produced from a single clone or plasma cell line (homogeneous Abs)
 Competitive-binding: based on competition between unlabeled Ag (sample analyte) and labeled Ag (reagent)
for limited reagent Ab sites
 Heterogenous Assay: requires physical separation of free and bound Ag (often uses a wash step)
 Homogeneous Assay: does NOT require physical separation of free and bound Ag
 Precipitin Curve: principle is that a lattice forms when there is an optimal proportion of Ag and Ab which
causes precipitation of the Ab-Ag complex. Consists of 3 zones:
o Prozone: Ab excess. All Ag sites are bound w/Ab so lattice doesn’t form (no precipitate)
o Zone of Equivalency: Optimal Ab-Ag proportion (2-3Ab molecules/Ag) produces lattice formation and
precipitate
o Postzone: Ag excess. All Ab sites are bound with Ag. Lattice doesn’t form (“breaks apart”).
 Related to High Dose Hook Effect: Ag (analyte) in very high concentration causes false low
result due to inadequate lattice formation. Sample requires dilution to obtain true analyte
concentration (diluted result>undil result)
 Page 18 of 21

Types of Immunoassays:
Label Hetero- Competitive Dose-Response Misc Info
geneous Curve
ELISA Enzyme YES NO Direct Sandwich assay
(ALP, Prone to High Dose Hook Effect
Horseradish
peroxidase)
EMIT Enzyme NO YES Direct E-hapten bound to Ab loses enzyme activity
(binding to Ab changes enzyme-substrate
key) = BLOCKED enzyme
FPIA Fluorophor NO YES Inverse immune complex:
(fluorescein, large, rotates slowly in solution and emits
umbelliferone) polarized light which is detected
MEIA Enzyme Yes NO Direct Detects immune complex formation on the
surface of latex microparticles by
capturing the immune complex on a glass
fiber matrix
CLIA Chemilum Variable Variable Variable MOST sensitive
(isoluminol, Requires luminometer
acridinium (light source not required)
ester)
RIA Radionuclide YES YES Variable Reagent (radioactive Ag) referred to as
(I125 most “tracer”
common) Requires special SAFETY precautions:
(harm from exposure related to time of
exposure and dose/magnitude of exposure)
 Increase distance
 Increase shielding (ex. Gamma
rays require 2 inches of lead
shielding)
Require gamma or scintillation counters for
detection of radionuclides
IRMA radionuclide YES NO Direct Similar to ELISA
HDH effect

Summary of Iron status:

Disease Serum TIBC %Sat Serum Additional Info

Iron (Indirect Ferritin
measure of (storage form
transferrin of iron)
)
Iron deficiency anemia ↓ ↑ ↓ ↓ *if iron is low, ferritin should be
(ferritin* decreases before low EXCEPT in inflammation.
serum iron) Ferritin is a positive Acute Phase
Anemia of chronic ↓ ↓ ↓ N or ↑ Reactant and will be high in
infection inflammation/infection. This
could mask an iron deficiency if
Primary hemochromatosis ↑ ↓ ↑ ↑
ferritin alone is performed (iron is
not affected)
 Page 19 of 21

Summary of Bilirubin Status:

Disorder TBIL IBIL/Bu DBIL/Bc Urine Urine Feces
(unconjugated) (conjugated) bilirubin urobilinogen (urobilin)
NOT water Water soluble (must be conjugated
soluble and water soluble)

Prehepatic jaundice ↑ ↑ N NEG ↑ Dark

(hemolytic) brown
Hepatic jaundice ↑ ↑ ↑ POS ↓ Brown or
(Variable) (Variable) pale
Posthepatic jaundice ↑ N ↑ POS ↓ Clay/white
(obstructive) (acholic)
colored

MISC Endocrinology:
Estrogens:
 Estradiol: produced in the ovaries; major estrogen in non-pregnant women
 Estriol: produced in the placenta; major estrogen in pregnancy

Thyroid:
Hormone Regulation:

THYROID DISORDERS:

 Primary hypothyroidism: Hashimoto disease (autoimmune), Myxedema, Cretinism (congenital)

 Primary hyperthyroidism (Thyrotoxicosis) : Graves disease (autoimmune)

 Page 20 of 21

Parathyroid Hormone (PTH):

 Increases CAL through: increased CAL resorption from bone, increased CAL reabsorption in renal tubules,
increased intestinal absorption of CAL (stimulates production of VitD)
 Decreases PHOS through: decreased reabsorption of PHOS in renal tubules, increases PHOS through:
increased intestinal absorption (stimulate production of Vit D)
 Primary Hyperparathyroidism:
o SERUM: CAL increased, PHOS decreased (or normal), URINE: CAL and PHOS increased
 Primary Hypoparathyroidism:
o SERUM: CAL decreased, PHOS increased

MISC Tumor Markers (aka Cancer Markers):

Tumor Markers: substances synthesized and released by cancer cells or made by other tissues in response to
the presence of cancer cells. Can be present in blood and other body fluids, on cells, or within cells.

Terminology:
Hyperplasia: a general term referring to the proliferation of cells within an organ or tissue beyond that
which is ordinarily seen. May result in the gross enlargement of an organ
Neoplasia: abnormal mass of tissue as a result of abnormal proliferation of cells. Usually causes a lump
or tumor. Neoplasms may be benign, pre-malignant or malignant
Angiogenesis: formation of new blood vessels to supply oxygen and nutrients to cells
Apoptosis: process of programmed cell death.
Oncogene: gene encoding a protein when mutated promotes uncontrolled cell growth.
Tumor suppressor gene: gene encoding a protein that protects cells from unregulated growth.
 Page 21 of 21

Incidence: frequency of disease over time period in relation to the population in which the disease
occurs.
Prevalence: the number of cases of a disease in a specific population at a given time.

Types of Tumour Markers:

1. ONCOFETAL ANTIGENS (GLYCOPROTEINS): derived from fetal tissue (normal proteins in the embryo and
fetus), can be found in small amounts in normal tissue
2. CARBOHYDRATE-ASSOCIATED ANTIGENS: high-molecular weight mucin-like glycoproteins that become
overly expressed
3. PLACENTAL PROTEINS: synthesized by placental trophoblasts and certain tumors
4. HORMONES: elevations can occur due to excess production of endocrine (eutopic) or nonendocrine
(ectopic) tissues
5. PROTEINS: elevations of normally occurring proteins related to malignancies
6. ENZYMES: elevations of naturally occurring enzymes associated with malignancies

Tumour Marker Type Info

Alpha fetoprotein Oncofetal Synthesized in liver, yolk sac and GI tract of fetus
(AFP) antigen Used to determine presence of hepatic tumors (hepatomas). Considered the
most specific test currently available for liver carcinoma!
Carcinoembryonic Oncofetal Normally found in epithelial cells of the fetal GI tract
antigen (CEA) antigen Originally associated with assessment of tumors of the colon (colorectal
cancers).
Non-specific: CEA is elevated in many malignancies: Also used in
management of liver, pancreas, and lung tumors, found in inflammatory
bowel syndromes, ulcerative colitis, GI tract tumors, and in cigarette smokers
CA-125 Oncofetal Appears in serum of patients with ovarian cancer, especially useful in
antigen postmenopausal women.
CA 19-9 Oncofetal Blood group antigen, derivative of the Lewis blood group system
antigen Useful in assessment of pancreatic, colon, gastric, hepatobiliary cancers
CA 15-3 Oncofetal Most commonly used for assessment of breast cancer
antigen Also appears in ovarian, pancreatic, lung, stomach, and liver cancers
Prostatic-specific Oncofetal Produced by prostate epithelial cells, secreted into seminal plasma.
antigen (PSA) antigen Functions in the liquification of seminal coagulum
Currently used to screen for prostatic cancer and to confirm diagnosis
following a digital rectal exam
Consists in TWO forms: complexed to a protease inhibitor and free PSA (not
complexed)
TOTAL PSA: measures complexed and free PSA
FREE PSA: measures only non-complexed form
bHCG Placental Secreted by the trophoblastic cells of the placenta
protein Composed of alpha and beta subunits. The B-subunit is specific to HCG, the
alpha subunit is common in other hormones (can cause cross reactivity in
immunoassays)
Performed as a stat assay if ectopic pregnancy suspected
High levels associated with testicular (free-B form) and ovarian cancer
Very high levels seen in trophoblastic tumors such as in hydatiform moles
and choriocarcinomas.
Prone to High Dose Hook Effect
Don’t forget FOB!!!