CLINICAL CHEMISTRY NOTES

Quality Control
 A system to monitor the analytical process and to detect errors that can affect accuracy and
precision

PARAMETERS OF QUALITY CONTROL

1. Accuracy
 The closeness of the assayed value to the true value
2. Precision / Reproducibility
 The closeness of the assayed value to a repeated value
3. Sensitivity / Detection Limit
 The ability of an analytical method to measure the smallest concentration of the analyte
of interest
4. Specificity
 The ability of an analytical method to measure only the analyte of interest
5. Diagnostic sensitivity
 The ability of an analytical method to detect the proportion of individuals with the
disease
 The ability of the test o generate more true-positives results and few false-negative
6. Diagnostic specificity
 The ability of an analytical method to detect the proportion of individuals without the
disease
 The ability of the test to detect true-negative results and few false-positives

KINDS OF QUALITY CONTROL

1. Intralab Quality Control (Internal QC)

 Involves the analyses of control samples together with the patient specimens
 Important for the daily monitoring of accuracy and precision of analytical methods
 Detects both random and systematic errors in a daily basis
2. Interlab Quality Control (External QC)
 Involves proficiency testing programs that periodically provide samples of unknown
concentrations to participating clinical laboratories
 Important for maintaining long-term accuracy of the analytical method

SOLUTIONS FOR CHECKING ACCURACY AND PRECISION

1. Standard
 A material of known composition available in highly purified form
 Universal color: Colorless
2. Control
 Material with physical and chemical properties closely resembling the test specimen and
containing preanalyzed concentrations of the substances being measured
 Universal color: Yellow

SOURCES OF CONTROL

1. Human-based control
  Non-infectious, non-icteric and nonhemolyzed
2. Bovine-based control

ANALYTICAL ERRORS

1. Systematic Error
 Error that occurs predictably once a pattern of recognition has been established

Causes:
a. Deterioration of reagents and control materials
b. Aging calibrators
c. Poorly written procedures
d. Wear and tear of instrument
e. Improperly made standard solutions

2. Random Error / Indeterminate

 Error that occurs unpredictably
 Basis of imprecision which results in varying differences between repeated measurements

Causes:
a. Variation in handling techniques: pipetting, mixing and timing
b. Mislabeling of samples
c. Temperature fluctuations
d. Calibrator reconstitution
e. Sample evaporation

QUALITY CONTROL CHARTS

1. Gaussian Curve (Bell-Shaped Curve)

 It occurs when data elements are centered around the mean with most elements close to
the mean
 Normal distribution curve: mean = median = mode
+1SD – 68.27%
+2SD – 95.45%
+3SD – 99.73%
2. Cummulative Sum Graph
 It calculates the difference between QC results and the target means
 Common method: V mask
 It gives the earliest indication of systematic errors and can be used with the 1 3s rule
3. Youden/Twin Plot
 It is used to compare results obtained on a high and low control serum from different
laboratories
 The points falling from a center but on the 45O line suggest a proportional error, and
points falling from the center but not on the 45O line suggest a constant error.
4. Shewhart Levey-Jennings Chart / Dot Chart
 Most commonly used chart for QC recording
 Analyte concentrations are in the Y-axis; day and time are plotted in the X-axis

Evaluation of LJ Chart
a. Trend
 Gradual deviation formed by control values that either increase or decrease for six
consecutive days
 Main cause: Deterioration of reagents
b. Shift
 Sudden or abrupt change formed by control values that distribute themselves on one
side or either side of the mean for six consecutive days
 Main cause: Improper calibration of the instrument
c. Outliers
 Highly deviating values that are far from the main set of values
5. Westgard Control Chart
 Use of upper and lower control limits is not enough to identify analytical problems
 Sensitivity of the multirole procedure can be increased to detect smaller systematic
errors by increasing the number of observations considered.

Westgard QC Rules
12s Used as screening or warning rule when a single QC result falls outside +2SD
13S Single QC result falls outside +3SD
R4S Two consecutive values differ by more than 4SD
22S Two consecutive QC results fall more than 2SD on the same side of the mean
41S Four consecutive QC results are more than 1SD away from the mean in the same
direction
10x Ten consecutive QC results are on the same side of the target mean

STATISTICAL TOOL FOR QA

1. Measure of Central Tendency

a. Mean
 Can be obtained by adding all the numbers in the set and dividing by the number of
values in that set
 Ultimate measure of inaccuracy or systematic error
b. Median
 Middle value in a set of numbers arranged from highest to lowest
c. Mode
 Most frequently obtained value in a set of numbers
2. Measure of Dispersion
a. Standard Deviation
 Dispersion of values around the mean
 Best measure of imprecision/inaccuracy
b. Coefficient of Variation
 Percentage comparison of the standard deviation divided by the mean
 Relative indicator of precision
c. Variance
 Measure of variability
 Also called standard deviation squared
3. Tests for Statistical Significance
a. T-test
 Used to determine whether there is a statistically significant difference between the
means of two groups of data
b. F-test
 Used to determine whether there is a statistically significant difference between the
standard deviations of two groups of data
4. Diagnostic Efficiency
a. Diagnostic Sensitivity
Diagnostic sensitivity = True positive X 100 / True positive + False negative
b. Diagnostic Specificity
Diagnostic specificity = True negative X 100 / True negative + False positive

Analytical Techniques and Instrumentation

A. Photometry/Spectrophotometry
1. Single-beam spectrophotometer
 Simplest type of absorption spectrophotometer
 Used to make one measurement at a time at one specified wavelength
2. Double-beam spectrophotometer
 Designed to compensate for possible variations in intensity of the light source by
splitting the light beam from the lamp and directing one portion to a reference cuvet
and the other to the sample cuvet
 2 types:
a. Double-beam-in-space – uses 2 photodetectors
b. Double-beam-in-time – uses one photodetector and a chopper

Parts of the Spectrophotometer:

1. Light
 Provides incident light for the system
 Spectrum of Light – measured in wavelength
 Wavelength – refers to the distance between the peaks of a light wave expressed
in nanometers
a. <400nm – IR region
b. 400 – 700nm – visible spectrum
c. >700nm – UV region
 2 types:
a. Continuum source
 Emits radiation that changes in intensity
b. Line source
 Emits limited radiation and wavelength

Sources:
a. Xenon discharge lamp and mercury arc lamp – UV and visible region
b. Deuterium and hydrogen lamp – UV region
c. Tungsten light bulb and tungsten iodide – Infrared and visible region
d. Merst glower and globar (silicone carbide) – Infrared region

 Beer’s Law: It states that the concentration of the unknown substance is directly
proportional to the absorbed light and inversely proportional to the amount of
transmitted light.
Formula:
(1) A = abc
(2) A = 2 – log% T
(3) Au/As X Cs
where A= absorbance
a = molar absorptivity
b = length of light thru the solution
c = concentration of absorbing molecules

2. Entrance slit
 Minimizes stray light and prevents the entrance of scattered light into the
monochromator system
 Stray light refers to any wavelength outside the band transmitted by the
monochromator, which is the most common cause of loss linearity at high analyte
concentration
 Can be detected through the use of glass-cut filters or solutions such as sodium
nitrite, sodium bromide, acetone and methylene blue.

3. Monochromator
 Isolates a specific wavelength of interest from the light source
a. Interference filter
 Simple, least expensive, not precise but useful
 Based on constructive interference of waves
 Sharp cut of filter – isolates stray light
b. Prisms
 Wedge-shaped made up of either aluminum, glass, sodium chloride or
quartz
 Separates white (visible) light into a continuous spectrum
c. Diffraction gratings
 Most commonly used
  Bends light and form wave fronts. Lights that are in phase reinforce one
another, if not it cancels out.

4. Exit slit
 Controls the width of bandpass – total range of wavelengths transmitted

5. Sample Cell
 Also known as cuvet/analytical cell, absorption cell
 Holds the solution of which the absorption is to be measured
 Should be free from scratches – it would scatter the light causing absorbance to
decrease
 High alkali solution must not stay in cuvet longer than 30 minutes – alkali
solution can dissolve cuvet components leading to light scattering
a. Glass cuvet
 Visible range
b. Quartz
 UV and visible range
c. Borosilicate glass
 Visible and near infrared region

6. Photodetector
 Converts transmitted radiant energy into an equivalent amount of electrical energy
a. Photocell/Barrier layer cell/Photovoltaic cell/Selenide cell
 Simpliest detector and least expensive
 Composed of selenium ion in silver
 Generates electromotive force by electrons (no external voltage)
 Output is not amplified (sufficient illumination)
b. Phototube
 Contains cathode and anode enclosed in a glass case
 Similar to photocell but requires external voltage
c. Photomultiplier tube
 Most commonly used detector which detects and amplifies radiant energy
(200x sensitive)
 Can detect smallest amount of light energy transmitted to the cuvet
 Most sensitive and most rapid response to light energy transmission
d. Phototransistors
 Uses a photosensitive negative-positive junction diode to produce
photocurrent

7. Meter or Read-out device

 A moving needle on a dial or a digital display which indicates the amount
of light passing through a sample

Calibration of Spectrophotometers
A. Setting transmittance and Absorbance Limits
1. Zeroing – set transmittance to 0%
2. Blanking – set transmittance to 100%
 Means the blank contains serum but without the reagent to complete the assay
 Sample blank measures the absorbance of sample and reagent in the absence
of end-product
 Reagent blank corrects absorbance caused by the color of reagents – the
absorbance of reagents is automatically substracted from each of unknown
reading
B. Linearity Checks/Proportionality Check
 Determine the lowest and highest values that can be accurately measured by a
particular method
 Use of neutral density filters
C. Wavelength Calibration
1. Holmium – 360 nm
2. Didymium – 600 nm
 3. Atomic Emission Spectrophotometry
 Principle: Measurement of the amount of light emitted from the excitation of
electrons by heat energy
 Components:
a. Nebulizer (Atomizer)
 Delivers a fine spray of sample containing the metallic ion to the burner
b. Burner
 A fuel gas (propane) with an oxidizing agent (compressed air) burned to
produce the flame
c. Monochromator system (filter)
 Allows only emitted light spectrum of specific element to strike the
photodetector
d. Photodetector: Photocell
 NO CUVET because the flame is the sample holder
 Employs an internal standard – used to correct interferences brought about by
atomizer and flame variations
a. Lithium (IS) – red
b. If lithium is measured, the IS is Cesium
c. Sodium – yellow
d. Potassium – violet g. Copper II chloride, Arsenic, Antimony, Lead - blue
e. Calcium – orange h. Barium, Manganese (Manganese chloride) – light green
f. Strontium –red i. Copper, thalium, boron (boric acid) – green

4. Atomic Absorption Spectrophotometry

 Principle: The atom under consideration is dissociated from its chemical bonds, then
it absorbs light of a specific wavelength in its ground state
 Components:
a. Light source – hallow cathode lamp
b. Beam chopper – modulates the light source
c. Nebulizer/atomizer – used to convert ion to atoms
d. Burner/cuvet
e. Monochromator: Diffraction gratings
f. Photomultiplier tube
g. Read-out device
 Advantages: More sensitive than FEP, accurate, precise and very specific; does not
require an internal standard; reference method for calcium and magnesium – trace
metals that are not easily excited
 Disadvantages: Inability of the flame to dissociate samples into free atoms
 Zeeman Effect/Background correction technique – presence of an intense magnetic
field will cause a shift in wavelength

B. Luminescence
1. Fluorometry
 Principle: Measurement of the amount of light emitted by a molecule after excitation
by electromagnetic radiation
 Components:
a. Light source – emits short wavelength high-energy excitation light
1. Mercury-vapor lamps – filter fluorometer
2. Xenon-arc lamps - spectrofluorometer
b. Attenuator – controls the light intensity
c. Primary filter – selects the wavelength that is best absorbed by the sample
d. Sample holder
e. Secondary filter – passes longer wavelengths of fluorescent light, preventing
incident light from striking the photodetector
f. Photodetector: photomultiplier tube – measures fluorescence light and incidence
light
 Advantages: 1000X more sensitive than spectrophotometry
 Disadvantages: Quenching interference – pH and temperature changes, chemical
contaminants, UV light changes which reduces intensity of fluorescence
 Stokes Effect – difference between the maximum wavelength of the excitation and
emitted fluorescence
 2. Chemiluminescence
 Principle: Process of exciting molecules by chemical or electrochemical means and
measuring the light emitted as molecules return to their ground state
 Requires no excitation radiation and no monochromator needed
a. Involves oxidation reactions of luminol, acridinium ester and dioxetane
 Advantages: subpicomolar detection limits, speed, ease of use, simple
instrumentation
 Application: Immunoassays

3. Turbidity and Nephelometry

Theories of Light Scatters:
 Rayleigh Theory – If the wavelength of light is much larger than the diameter of
the particle, the light scatter is symmetric around the particle
 Mie Theory – If the wavelength of light is much smaller than the diameter of the
particle, the light scatters forward owing to destructive out-of-phase back scatter
 Rayleigh-Debye Theory – If the wavelength of light is approximately the same as
the particle size, more light scatters in the forward direction than in other
directions

a. Turbidimetry
 Principle: Measurement of the reduction in light transmission caused by particle
formation or of the amount of light blocked by a suspension of particles
 Application: Measurement of the concentration of antigen-antibody complexes,
prealbumin and other serum proteins, detect bacterial growth in broth cultures,
antibiotic sensitivity from cultures and detection of clot formation

b. Nephelometry
 Principle: Measurement of the amount of light scattered b a particulate solution
 Application: Measurement of antigen-antibody reactions
 Light sources: mercury-arc lamp, tungsten-filament lamp, light emitting diode,
laser

4. LASER Applications (Light Amplification by stimulated emission of radiation)

 Principle: The interaction of radiant energy and suitably excited atoms or molecules
leads to stimulated emission of radiation
 Application: Differential analysis of WBC

C. Electroanalytical Techniques
1. Potentiometry
 Involves direct measurement of electrical potential (voltage) due to the activity of free
ions
 Follows the Nernst equation
a. Ion Selective Electrode (ISE)
 Concentration of ions in a solution is calculated from the measured potential
difference between the reference and indicator electrodes
 Application: pH and pCO2 tests (Severinghaus electrode)

a. Reference electrode
 Consists of a metal and its salt in contact with a solution containing the same
anion
 Calomel (Hg/HgCl2) electrode – mercurous chloride in direct contact with
metallic mercury in an electrolyte solution of KCl
 Ag/AgCl electrode – consists of a silver electrode immersed in a solution of
potassium chloride that has been saturated with silver chloride

b. ISE Membranes
 Sodium – glass aluminum silicate
 Potassium – Valinomycin gel
 Calcium and lithium - Organic liquid membrane ion exchanger
2. Amperometry
 Measurement of the current flow produced by an oxidation reaction
 Application: Used in the determination of pO2 (Clarke electrode) glucose and
peroxidase

3. Cuolometry
 Measurement of the amount of electricity in coulombs at a fixed potential
 Electrochemical titration in which the titrant is electrochemically generated and the
endpoint is detected by amperometry
 Follows Faraday’s Law
 Application: Serum and sweat chloride analysis

4. Voltammetry
 Measurement of current after which a potential is applied to an electrochemical cell
 Allows sample to be preconcentrated, thus utilizing minimal analyte
 Anodic stripping voltammetry – for lead and iron testing

5. Electrophoresis
 Migration of charged solutes or particles in an electrical field
 Iontophoresis – migration of small charged particles
 Zone electrophoresis – migration of charged macromolecules
 The rate of migration is directly proportional to the net charge of the particle and
inversely proportional to its size and the viscosity of the buffer

Five Components:
a. Driving force (electrical power)
b. Support Medium
 Paper
 Cellulose acetate – separates by molecular size
 Agarose gel
 Polyacrylamide gel
 Involves separation of protein on the basis of charge and molecular size
 Separates serum proteins into 20 or more fractions rather than the usual 5
fractions separated by cellulose acetate or agarose
 Widely used to study isoenzymes
 Starch gel
 Separates proteins on the basis of surface charge and molecular size
c. Buffer
 Two buffer properties that affect the charge of ampholytes: pH and ionic strength
 Ampholytes – a molecule, such as protein, whose net charge can either be positive
or negative
 Isoelectric point – is the point at which the number of positively charged groups
equals the number of negatively charged groups in protein
 If the buffer is more acidic than the isoelectric point (pI) of the ampholyte, it
binds H+ ions, becomes positively charged, and migrates toward the cathode
 If the buffer is more basic than the pI, the ampholyte loses H+ ions, becomes
negatively charged, and migrates toward the anode
 Charged particles migrate toward the opposite charged electrode
d. Sample
e. Detecting system
 Densitometer – a specialized colorimeter designed to measure the absorbance of
the stain on a support medium

Factors that affect the rate of migration:

1. Net charge of the particle
2. Size and shape of the particle
3. Strength of the electric field
4. Chemical and physical properties of the supporting medium
5. Electrophoretic temperature
 Procedure:
1. The sample is soaked in hydrated support for approximately 5 minutes
2. The support is put into the electrophoresis chamber, which was previously filled with
the buffer
3. Electrophoresis is carried by applying a constant voltage or constant current for a
specific time
4. The support is then removed and placed in a fixative or rapidly dried to prevent
diffusion of the sample
5. This is followed by staining the zones with appropriate dye
6. The uptake of dye b the sample is proportional to sample concentration

6. Isoelectric Focusing
 Charged proteins migrate through a support medium that has a continuous gradient
 pH gradient is created by adding acid to the anodic area of the electrolyte cell and
adding base on the cathode area
 Molecules move towards the medium at its optimum pH. When pH is reached, the
molecule loses its charge and so movement stops
 Application: measurement of isoenzymes of ACP, CK and ALP and oligoclonal
bands in CSF

7. Capillary Electrophoresis
 Separation is performed in narrow-bore fuse silica capillaries
 Sample molecules are separated by electro-osmotic flow
 Cations migrate fastest because both EOF and electrophoretic attraction are toward
the cathode
 Neutral molecules are all carried by the EOF but are not separated from each other
 Anions move slowest because, although they are carried to the cathode by the EOF,
they are attracted to the anode and repelled by the cathode

D. Chromatography
 Techniques used to separate complex mixtures on the basis of different physical
interactions between the individual compounds and the stationary phase of the system

Composition:
a. Mobile phase (gas/liquid) – carries the complex mixture
b. Stationary phase (solid/liquid) – through which the mobile phase flows
c. Column holding the stationary phase
d. Eluates (separated components)

1. Gas Chromatography
 Used to separate mixture of compounds that are naturally volatile or can easily be
converted into a volatile form

Components:
a. Gas cylinder (mobile phase)
 Must be chemically inert (helium, hydrogen, nitrogen)
b. Sample injector
 Hypodermic syringe or automated sample
 Each injection part is heated to very high temp
c. Column
 Capillary columns – generally have higher efficiency and better detection
 Packed columns – larger specimen or sample capacity, useful in purification work
d. Detectors
 Flame ionization detector – widely used and capable of detecting almost all
organic compounds
 Thermal conductivity
e. Recorders

2. Liquid Chromatography
 Uses lower temperatures for separation, thereby achieving better separation of
thermolabile compounds
 Forms:
a. Paper
 Solvent move up through the paper by capillary action and the fractions move up
at different rates
 Sorbent (stationary phase) – Whatman paper
b. Thin-layer Chromatography
 The stationary phase is a thin layer sorbent (silica gel) coated on a glass plate or a
plastic sheet
 The mobile phase (solvent) is place in one edge of the plate which migrates up the
thin layer
 Retention factor = distance travelled by a specific component / total distance
travelled by the mobile phase
c. High-Performance Liquid Chromatography (HPLC)
 Uses pressure for fast separation of thermolabile substances like drugs and
hormones
 Analysis times are usually shorter
 Reproducibility is greatly improved
 Application: Separation of amino acids, proteins and nucleic acids

Separation Techniques in Liquid Chromatography:

a. Adsorption
 Also known as liquid-solid chromatography
 Based on the competition between the sample and the mobile phase for adsorptive
sites on the solid stationary phase (silica or alumina)
b. Partition
 Also known as liquid-liquid chromatography
 Separation of solute is based on relative solubility between a liquid mobile phase
and a liquid stationary phase coated on a solid support
 Application: Separation of therapeutic drugs and metabolites
c. Steric/Size Exclusion
 Separation of molecules based on difference in size and shape
 As solutes travel through, the small molecules can enter the pores, whereas the
largest ones cannot and will elute first from the column
d. Affinity Chromatography
 Uses immobilized biochemical ligands as the stationary phase
 Uses the so-called lock-and-key binding that is widely present in biological
systems

E. Mass Spectrometry
 Based on the fragmentation and ionization of molecules using a suitable source of energy
 Definitive identification and quantitation of samples or compounds eluting from GC or
HPLC columns
 Application: Measurement of drugs of abuse in urine toxicology confirmations; analysis
of low levels and mixed polarity analytes such as Vitamin D, testosterone, and
immunosuppressant drugs
 GC-MS – gold standard for drug testing

F. Osmometry
 Measure of solute particles in a solution in terms of thei colligative properties
 As the osmolality of the solution increases:
a. Osmotic pressure increases
b. Boiling point is elevated
c. Freezing point is depressed
d. Vapor pressure is depressed
 Freezing point osmometry – most commonly used method for measuring the changes in
colligative properties of a solution; based on the principle that addition of solute
molecules lowers the temperature at which a solution freezes
Laboratory Equipment and Reagent Preparation
A. Centrifuge
 Used to separate substances of different mass or density
1. Horizontal head
 Swinging bucket type; the centrifuge tubes are held in a vertical position when not
moving, but are horizontal when the centrifuge is fully in motion
 Recommended for serum separator tubes
2. Angle Head
 Has a fixed 25 -52 angle at which the tubes are held during centrifugation
3. Ultracentrifuge
 Generates the highest speeds
 In order to reduce the heat produced by the friction generated by high centrifugal
speeds, ultracentrifuges are refrigerated
 Recommended for lipoprotein analysis since refrigeration enhances separation

Centrifuge Maintenance Procedure:

 Weekly – clean interior components with soap and water followed by freshly made 10%
v/v bleach solution, including sample buckets
 Monthly – check for unusual vibrations, braking mechanisms to ensure a smooth, gradual
stop, the timer of the centrifuge using a stopwatch
 Quarterly – check the revolutions per minute at several commonly used speeds while
centrifuging a balanced load using a tachometer or strobe light

B. Pipet
According to Design:
1. To contain – holds the particular volume but does not dispense the exact volume
2. To deliver – will dispense the exact volume indicated

According to Drainage Characteristics:

1. Blow-out – has a continuous etched ring located near the top of the pipet; exact volume is
obtained when the last drop is blown out
2. Self-draining – no etched rings; liquid is allowed to drain by gravity

According to Purpose:
1. Transfer pipet
 Volumetric – designed to transfer nonviscous or aqueous solutions and is self-
draining; has the greatest degree of accuracy and precision
 Ostwald-Folin – viscous fluid; blow-out pipet
 Pasteur pipet
2. Graduated/Measuring pipet
 Mohr pipet – without graduations to the tip; self-draining
 Serological pipet – with graduations to the tip; blow-out
 Bacteriological pipet

C. Chemicals Used for Reagent Preparation

1. Analytical Reagent Grade
 For quantitative and qualitative analyses
2. Ultrapure Reagent
 Type of reagent that has been put through additional purification steps
3. Chemically Pure or Pure Grade
 Limitations of this type are not usually stated and is not recommended for research
and analytical chemistry
4. Technical or Commercial Grade
 Used primarily in manufacturing and should never be used in clinical laboratory
setting
5. United States Pharmacopoeia (USP) and National Formulary (NF)
 Approved for human consumption but not applicable for laboratory analysis
D. Grades of Reagent Water
1. Type I Reagent Water
 Used for test methods requiring minimum interference
 Used for procedures that require maximum water purity for accuracy and precision
2. Type II Reagent Water
 Acceptable for preparation of reagents and quality control materials
3. Type III Reagent Water
 Used for washing glasswares

Automation
Principles:
1. Single channel – able to perform only one test with a dedicated portion of the instrument
2. Multi-channel – able to perform a variety of tests at the same time with separate dedicated
instrument components
3. Random access – test can be performed in a variety of sequence
4. Batch analysis – group of samples are analyzed at the same time for the same test
5. Sequential analysis – performing a set of tests in a particular order one after another on
given specimen
6. Open reagent system – reagents can be purchased from a variety of sources
7. Closed reagent system – reagents are purchased only from the manufacturer of the instrument
because of unique container or format

A. Continuous Flow Analyzer

 Sample is aspirated through the sample probe into a continuous reagent stream
 Liquids are pumped through a system of continuous tubing
 Samples are introduced in a sequential manner following each other on the same network.
Air bubbles serve as separating and cleaning media
 Shows significant carryover problems

B. Centrifugal Analyzer
 Uses the force generated by centrifugation to transfer and then contain liquids in separate
cuvets for measurement at the perimeter of a spinning rotor
 Most capable of running multiple samples, one test at a time, in a batch

C. Discrete Analyzer
 Capability of running multiple tests one sample at a time or multiple samples one test at a
time.
 Most popular and versatile analyzer

Blood Collection and Specimen Considerations

Methods of Collection
A. Capillary Puncture
 Preferred way to obtain blood from pediatric population
 Length of lancet: 1.75mm (to avoid penetrating the bone)
 Depth of incision should be less than 2.0 mm for infants and children and less than 2.5
mm for adults

1. Sites of Puncture
 Lateral or medial plantar heel surface
 Palmar surface of the last phalanx of the third or fourth finger
 Plantar surface of the big toe
 Lateral side of the finger adjacent to the nail
 Earlobe

2. General Precautions
 Site of puncture must not be edematous or a previous puncture site
 Warm the puncture site with a warm, moist towel to increase blood flow
 Do not milk the puncture site
 3. Order of Draw
 Blood gases
 Slides/smears
 EDTA specimens
 Other additive specimens
 Serum specimens

4. Indications
 Patients with severe burns, thrombotic disorders
 Obesity
 Microspecimens are needed
 Geriatric patients

5. Advantages
 Capillary sites are accessible
 Only a small quantity of blood is needed

6. Disadvantages
 Tissue fluid could mix with blood flow resulting to dilution effect
 Squeezing can cause hemolysis of the blood
 Small quantity so cannot provide enough blood when multiple tests are to be done
 Painful especially to the fingertips

B. Venipuncture

1. Sites of Collection
 Veins of the antecubital fossa

Two Basic Vein Patterns

a. H Pattern – displayed by approximately 70% of the population
b. M Pattern

 Median cubital vein is the best site because it is the largest and the best anchored vein
 Cephalic vein is the second choice
 Basilic vein should not be chosen due to its proximity to the brachial artery

2. Basic Venipuncture Methods

a. Evacuated Tube System
 Most common blood collection system which uses a vacuum to pull blood into a
container
 Preferred method because blood is collected directly from the vein into a tube,
minimizing the risks of specimen contamination and exposure to the blood
b. Needle and Syringe
 Used on small, fragile, damaged veins

Needle Specifications:
 The gauge of the needle is inversely related to the size of the needle, the larger the
gauge number, the smaller the bore and length
 Standard: 21-gauge needle
 23-gauge needle: used for children and for small and difficult veins
 Needle length: 1 inch or 1.5 inches – 21-23 gauge
½ to ¾ inch – butterfly needle

c. Butterfly/Winged Infusion Set

 Can be used with an ETS or syringe
 Gauge-23 needle is most commonly used
 Indications: infants and children, hand veins, and difficult-draw situations

3. General Precautions
a. If a fasting specimen is required, confirm that the fasting order has been followed
  8 – 12 hours: glucose, lipid and lipoproteins
 48 hours: Increase serum bilirubin
 72 hours of fasting: Increase of plasma triglyceride while glucose is decreased to
45mg/dL
 Fasting specimen: FBS, GTT, triglycerides, lipids, lipoproteins, insulin and
gastrin
b. Do not extract from patients while they are receiving IV medications because these
solutions my influence the chemical analysis
 In cases when both arms are involved in therapy and IV could not be
discontinued, a site below the IV line is selected. Initial sample (5mL) is drawn
and discarded.
 As little as 10% contamination with 5% dextrose will increase glucose in a blood
sample by 500 mg/dL or more.
c. Never draw out the syringe without removing first the tourniquet to avoid hematoma
 Recommended application of tourniquet is 1-minute only
 Avoid prolonged application of tourniquet to avoid hemoconcentration
 May increase potassium, lactate, proteins, cholesterol and ammonia
d. Blood specimens obtained must be placed in appropriate containers for each specific
test
 Yellow – blood culture tubes
 Light blue – trisodium citrate
 Serum/red/SST – with or without clot activator or gel separator
1. Plain red-stoppered tubes with no additives take about 60 minutes to clot
completely
2. Clotting times for tubes with gel separators is approximately 30 minutes
3. Clotting times for tubes with clot activators such as thrombin take 5 minutes
 Green – Heparin (sodium, lithium or ammonium)
 Lavender – EDTA
 Gray – sodium fluoride and potassium oxalate or iodoacetate and heparin

4. Complications of Venipuncture
a. Vascular complication
 Bleeding from the site of puncture, hematoma formation, vein damage
b. Infections
 Second most common complication of venipuncture
c. Anemia

C. Arterial Puncture
1. Site of Puncture: radial artery, brachial artery, femoral artery
2. General Precautions
 Before blood is collected from the radial artery, modified Allen test should be done t
determine whether the ulnar artery can provide collateral circulation to the hand after
the radial artery puncture

D. Central Venous Access (CVA) Collection Technique

 Drawing blood from indwelling catheters that are surgically inserted in the cephalic vein,
or internal jugular or femoral veins
1. Order of Draw
 Draw 3 – 5 mL in a syringe and discard
 Blood for blood culture
 Blood for anticoagulated tubes
 Blood for clot tubes

2. Advantages
 Provides ready access to patient’s circulation
 Eliminates multiplr phlebotomies
 Useful in critical care and surgical situations
Carbohydrates
Pancreas
1. Exocrine – secretes amylase for breakdown of ingested complex carbohydrates
2. Endocrine
a. Insulin – beta cells
b. Glucagon – alpha cells
c. Somatostatin – delta cells
d. Pancreatic polypeptide – PP or F cells

 Ratio of insulin-to-glucagon is influenced by:

o Somatostatin
o Neural input
o Intestinal peptides
o Concentrations of glucose
 Ratio of C-peptide and insulin levels: 5:1 to 15:1
 Glycol aldehyde is the simplest form of carbohydrate
 2/3 of glucose utilization in resting adults occurs in the CNS

Insulin
 Responsible for glucose uptake
 HYPOGLYCEMIC AGENT
o ↑ glycogenesis, lipogenesis
o ↓ glycogenolysis
 Falsely decreased in the presence of hemolysis
 Proinsulin is a precursor of insulin. Increased proinsulin levels is associated with
insulinomas; glucose level (<50mg/dL)
 Incretins also regulates insulin secretion
o Glucagon-like peptide 1 (GLP-1)
o Glucose-dependent insulinotropic peptide (GIP)/Gastric inhibitory polypeptide

Glucagon
 Primary HYPERGLYCEMIC AGENT
 Fasting plasma glucagon: 25 – 50 pg/mL
 ↑ glycogenolysis

Hyperglycemic Agents:
1. Glucocorticoids
 Decreased intestinal entry of glucose into the cell
 ↑ gluconeogenesis and lipolysis
2. Catecholamines
 Inhibits insulin secretion
 ↑ glycogenolysis and lipolysis
3. Growth hormone
 Decrease entry of glucose into the cell
 ↑ glycogenolysis and glycolysis
4. Thyroid hormone
 Promotes intestinal absorption of gluose
 ↑ glycogenolysis and gluconeogenesis
5. ACTH
6. Somatostatin
 Inhibits action of insulin, glucagon and growth hormone

Hyperglycemia
 FBS level = ≥ 126 mg/dL
 Causes: stress, dehydration, pancreatectomy, insulin deficiency, hemochromatosis, abnormal
insulin receptor

LAB FINDINGS:
1. ↑ glucose in plasma and urine
 2. ↑ urine specific gravity
3. ↑ ketones
4. Acidosis
5. ↑ K, ↓ Na, ↓ HCO3, - Kussmaul-Kien respiration (deep respiration)

 Period of plateau – urine glucose matches plasma glucose at 300 mg/dL to 500 mg/dL

Hypoglycemia
 Grading
o 50 – 65 mg/dL – observable symptoms appear
o <60 mg/dL – strongly suggest hypoglycemia
o 65 – 75 mg/dL – glucagon and hyperglycemic hormones are released into the
circulation
 Diagnoses by Whipple’s triad
o ↓ blood glucose concentration
o Typical symptoms appear
o Symptoms alleviated by glucose administration
 Associated with:
1. Alcohol intake
2. Renal, hepatic ad cardiac failure
3. Hormonal deficiency
4. Leukemia
5. GSD, Reye’s syndrome
6. Reactive – 4 hours after a meal

Diabetes mellitus
Type 1 DM (IDDM) Type 2 DM (NIDDM)
B-cells destruction PATHOGENESIS Insulin resistance
Childhood (5 – 10%) ONSET Aged (90 – 95%)
(+) autoantibodies PREDIABETES (-) autoantibodies
a. Glutamic acid a. Sedentary lifestyle
decarboxylase 65 b. Obesity
b. Insulin autoantibodies – c. PCOS
associated with HLA DR3
and DR4 (chrom 6)
↓/undetectable C-PEPTIDE Detectable
Abrupt SYMPTOMS Gradual
Insulin absolute MEDICATION Oral agents
Juvenile Onset AKA Maturity Onset
Brittle diabetes Stable diabetes
Ketosis prone Ketosis-resistant
Receptor-deficient

Criteria for DM:

1. Impaired fasting glucose
2. BP > 30/80 mmHg
3. TG > 150 mg/dL
4. HDL > 40 mg/dL for men; < 50 mg/dL for women

 Untreated DM Type 2: Nonketotic hyperosmolar coma; glucose (>300mg/dL) accompanied

by dehydration, electrolyte imbalance, and ↑ BUN and creatinine
 Adult ages 45 and older – screened for diabetes every 3 years

Glucose Methodologies
 Glucose in whole blood is 10 – 15% lower than in serum or plasma
 Venous glucose is 7 mg/dL ↓ than capillary blood
 Glucose is metabolized at room temperature at a rate of 7mg/dL; at 4C, the loss is
approximately 2mg/dL/hour.

1. Alkaline Copper Reduction Method

 Nelson-Somogyi Reduction of copper in hot alkaline solution to cuprous ion which in
turn reduces arsenomolybdic acid in a greenish blue complex
Folin-Wu Reduction of copper in hot alkaline solution to cuprous ion which in
turn reduces phosphomolybdic acid forming a blue complex of
molybdenum oxide
Neocuproine Reduction of cupric ions to cuprous ions (2,9-dimethyl-1,10-
phenanthroline hydrochloride) complexes with cuprous ions to form a
yellow color
Benedict’s Modification of Folin-Wu. Used citrate or tartrate as stabilizing agent

2. Alkaline Ferric Reduction Method (Hagedorn Jensen)

 Redox of yellow ferricyanide to a colorless ferrocyanide by glucose (inverse colorimetry)

3. Condensation Method
 O-toluidine (Dubowski method) – reaction of glucose with aromatic amines forming
glycosylamine and schiff’s base (OD 630) by the action of glacial acetic acid and heat

4. Glucose Oxidase Method

 Measures B-D-glucose
 False ↓: Ascorbic acid, uric acid, glutathione, creatinine, dopamine and methyldopa
a. Saifer Gernstenfield Method (same reactions with reagent strip test – Trinder
reaction)
 Incorporated with mutarotase t convert other forms of glucose to B-D-glucose
b. Polarographic Method
 Measures rate of oxygen consumption which is proportional to glucose
concentration

5. Hexokinase Method
 Reference method; most specific glucose method
 Affected by hemoglobin

Glucose + ATP – hexokinase  Glucose-6-phosphate + ADP

G6P + NADP – G6PD  6-phosphogluconolactone + NADPH

6. Dextrostics (cellular strip)

 Capillary blood glucose of > 140 mg/dL, should be rescreened with
a. Fasting plasma glucose
b. HbA1c
c. OGTT

Gestational Diabetes mellitus

 Screening performed between 24 – 48 weeks of gestation

Diagnostic Criteria:
1. FBS > 92 mg/dL
2. 1hr GTT > 180 mg/dL
3. 2hr GTT > 153 mg/dL
 Infants are at increased risk for Respiratory Distress Syndrome, hypocalcemia and
hyperbilirubinema
 Large % of patients develop DM within 5-10 years

Glucose Measurement
1. RBS – required during insulin shock and hyperglycemic ketonic coma
2. FBS – measure overall glucose homeostasis; requires NPO for 8 hours
3. 2hr-Post Prandial Blood Sugar – evaluate hyperglycemia and hypoglycemia
4. GTT – performed to diagnose gestational diabetes
a. Oral Glucose Tolerance Test (OGTT)
1. Single dose / Janey Isaacson – most common
 2. Double dose / Exton Rose
Added plasma glucose after intake of glucose load:
a. 30 mins 30 – 60 mg/dL above fasting
b. 1 hour 20 – 50 mg/dL above fasting
c. 2 hour 5 – 15 mg/dL above fasting
d. 3 hour fasting level or below
** O’ Sullivan Test (1hr GDM Test) – screening at 1st prenatal visit, then again at 24
– 48 weeks
Reference range: 130 – 140 mg/dL (1hr after 50g of glucose)

Requirements for OGTT:

1. Patient is ambulatory
2. Unrestricted diet of 150g carbohydrate for 3 days
3. No smoking or drinking alcohol
4. Fasting for 8 – 12 hours
5. Glucose load: 75g – standard – drink within 5 minutes
100g – pregnant
1.75g/kg body wt – children

b. Intravenous GTT
 For DM patients with GIT disorders
0.5g of glucose/kg of body weight (within 3 minutes)
 2nd blood collection is after 5 minutes of IV glucose

Fasting Plasma Glucose

1. Non-diabetic < 100 mg/dL
2. Impaired Plasma Glucose 100 – 125 mg/dL
3. DM > 126 mg/dL

Oral Glucose Tolerance Test

1. Normal < 140 mg/dL
2. Impaired GTT 140 – 199 mg/dL
3. DM > 200 mg/dL

Diabetes mellitus
1. RBS > 200 mg/dL
2. FBS > 126 mg/dL
3. 2hr post glucose blood > 200 mg/dL
4. HbA1c > 6.5%

Glycosylated Hemoglobin (HbA1c)

 HbA that is irreversibly glycosylated at one or both N-terminal valines of the B-chains of
tetrametric hemoglobin
 Monitoring of long-term glucose control (2 – 4 months) performed every 3 – 6 months
 Levels:
o > 6.5% on at least 2 occasions – indicative of DM
o 5.7 – 6.4% - prediabetes
 IDA and older red cells - ↑ HbA1c levels
 1% change in value – 35mg/dL is added to plasma glucose

↑ HbA1c in Non-diabetics ↓ HbA1c

1. IDA 1. Hemolytic Anemia
2. Splenectomy 2. Chronic blood loss
3. Alcohol toxicity 3. Pregnancy
4. Lead toxicity 4. Chronic renal failure

Fructosamine/Glycosylated Albumin/Plasma Protein Ketoamine

 Short-term glucose control (3 – 6 weeks)
 Monitoring diabetic individuals with chronic hemolytic anemia and hemoglobin variants –
decreased RBC lifespan
 Reference value: 205 – 285 umol/L
Inborn Errors of Carbohydrate Metabolism
1. Galactosemia – deficiency in galactokinase, galactose-1-phosphate uridyl transferase and
uridine diphosphate galactose-4-epimerase
2. Essential fructosuria – fructokinase deficiency
3. Hereditary fructose intolerance – defect of fructose-1,6-biphosphate aldolase B activity
4. Fructose-1,6-biphosphate deficiency – failure of hepatic glucose generation by
gluconeogenic precursors such as lactate and glycerol
5. Glycogen Storage Disease
a. Liver damage – I, III, IV, VI, IX and O
b. Muscle damage – V, VII

Type of AKA Enzyme Deficient Clinical Features

GSD
Ia Von Gierke Glucose-6-phophatase Hepatomegaly, hyperlipidemia, seizure
Ib Glucose-6-phosphatase Same as Ia, recurrent bacterial infection
translocase
II Pompe 1,4-glucosidase Cardiomegaly, infantile death
IIIa Cori Forbes De brancher (L and M) Hepatomegaly, cardiomyopathy,
muscle weakness
IIIb De brancher (L) Same as IIIa except muscle weakness
IV Andersen/Amyl Brancher Cirrhosis, esophageal varices, ascites
opectinosis
V McArdle Muscle phosphorylase Myoglobinuria, muscle cramps
VI Hers Liver phosphorylase Hepatomegaly, hypoglycemia
VII Tarui Phosphofructokinase Pain and stiffness on exertion
VIII Adenyl kinase Urinary excretion of catecholamines
IXa Phosphorylase kinase Hepatomegaly, hypoglycemia
(L)
IXb Phosphorylase (L and Hepatomegaly, muscle hypotonia
M)
X Cyclic AMP-dependent Hepatomegaly
kinase
XI Fanconi-Beckel Glucose transporter-2 Hepatomegaly, rickets
O Glycogen synthase No hepatomegaly, hypoglycemic
symptoms in morning; growth delay

C-Peptide Test
 Formed during conversion of proinsulin to insulin
 To monitor responses to pancreatic surgery
 Requires fasting serum
 ↑ in insulinoma, type 2 DM, ingestion of hypoglycemic drugs

Lipids
A. Phospholipid (Conjugated Lipid)
 Produced from conjugation of two fatty acids and a phosphorylated glycerol
 Amphiphatic lipid: Polar hydrophilic head; nonpolar hydrophobic side chains
 Forms:
o Lecithin 70% aka phosphatidyl choline
o Sphingomyelin 20% only phospholipid membrane that is not derived from
glycerol but from an amino alcohol called sphingosine
o Cephalin 10%
 Estimation of serum lipid phosphorus: each mole of phosphorus contributes about 4%
to the total phospholipid mass; phospholipid mass = phospholipid phosphorus X 25

B. Cholesterol
 Unsaturated steroid alcohol with 4 rings
 Precursor of steroids
  When converted to 7-dehydrocholesterol, can also be transformed to Vitamin D3 in the
skin by irradiation from sunlight
 Reference values:
o Desirable < 200 mg/dL
o Desirable high 200 – 239 mg/dL
o High > 240 mg/dL

 Forms:
1. Cholesterol Ester (70%)
 Undergoes esterification by LCAT
 LCAT – enables HDL to accumulate cholesterol as cholesterol ester
Apo-A1 is the activator
2. Free cholesterol (30%)
 Found in plasma, serum, and RBCs – produced via lysosomal hydrolysis

Methods:
1. CDC Reference Method: Abell, Levy and Brodie Method
 Uses hexane extraction after hydrolysis with alcoholic KOH followed by reaction
with Liebermann-Burchardt reagent
2. Chemical Method
 Liebermann-Burchardt/Colorimetric Reaction
 End-product: Cholestadienyl Monosulfonic Acid (green)
 Salkowski
 End-product: Cholestadienyl Disulfonic Acid (red)

 Color developer
a. Glacial Acetic Acid
b. Acetic anhydride
c. Concentrated Sulfuric Acid

 Precautions
a. Avoid hemolyzed blood – falsely ↑
b. Avoid icteric specimen

 General Method
1. 1-step: Colorimetry (Pearson, Stern and Mac Gavack)
2. 2-step: Extraction + Colorimetry (Bloors)
3. 3-step:Saponification (alcoholic KOH) + Extraction (Phenol) + Colorimetry
(Abell-Kendall)
4. 4-step: Saponification + Extraction + Colorimetry + Precipitation (Schoenheimer,
Sperry, Parekh and Jung)

3. Cholesterol Oxidase
 Interference: ↑ ascorbic acid (↓ TC)
Bilirubin >5 mg/dL ↓ TC by 5 – 15%

Recommended Cutoff Points for Cholesterol

Age Moderate Risk High Risk
40 and over >240 >260
30 – 39 >220 >240
20 – 29 >200 >220
2 – 19 >170 >185

↑ Cholesterol ↓ Cholesterol
1. Hyperlipoproteinemia I, III, V 1. Severe hepatocellular disease
2. Biliary cirrhosis 2. Malnutrition
3. Nephrotic syndrome 3. Severe burns
4. Poorly controlled DM 4. Malabsorption syndrome
5. Alcoholism 5. Hyperthyroidism
6. Primary hypothyroidism
C. Triglyceride (Neutral Fat)
 3 molecules of fatty acid and 1 molecule of glycerol
 Main storage lipid in man
 Fasting requirement: 12 – 14 hours
 Reference Values:
Normal < 150 mg/dL
Borderline high 150 – 199 mg/dL
High TAG 200 – 499 mg/dL
Very high > 500 mg/dL (acute and recurrent pancreatitis)
 Fasting TAG > 200 mg/dL – risk for coronary artery disease. Postural changes ↓ TAG by
50%
 Lipemia occurs when TAG levels exceed 4.6 mmol/L (400mg/dL). Inhibition of assays
for amylase, urate, urea, CK, bilirubin, and total protein may be observed.

Methods:
1. CDC Reference Method: Modified Van Handel and Zilversmith
 Saponification (alcoholic KOH)  extraction (chloroform) treated with silicic acid
 pink end color
2. Colorimetric Method (Van Handel and Zilversmith)
 Triglycerides – alcoholic KOHglycerol + Fatty acid
Glycerol oxidized by PERIODIC ACID  formaldehyde
Formaldehyde + Chromotropic acid  (+) blue
3. Fluorometric Method (Hantzsch Condensation)
 Triglycerides – alcoholic KOHglycerol + Fatty acid
Glycerol oxidized by PERIODIC ACID  formaldehyde
Formaldehyde + diacetyl acetone + NH3  Diacetyl Lutidine Compound
4. Glycerol kinase Method
 Disappearance of NADH is measured at 340nm
 Interference: Glycerol – normally present in plasma in concentration ↓ 0.163mmol/L
is equal to TAG about 14 mg/dL

Lipoproteins
 Complexes of lipids with specialized proteins known as apolipoproteins
 Vitamin E depends upon CM for absorption and relies upon VLDL and LDL for delivery to
tissues

Protein % Cholesterol % Chole Ester % TAG % PPL %

CM 1–2 1–3 2–4 80 – 95 3–6
VLDL 6 – 10 4–8 16 – 22 45 – 65 15 – 20
IDL Intermediate between VLDL and LDL
LDL 18 – 22 6–8 45 – 50 4–8 20 – 24
HDL 45 – 55 3–5 15 – 20 2–7 26 – 32

1. Chylomicrons
 Largest and least dense; transports exogenous/dietary TAG to tissues
 Apolipoproteins: Apo-B48, Apo-A1, C and E
 Density: < 0.95 kg/L
2. VLDL/ Pre-B Lipoprotein
 Transports endogenous TAG from the liver to tissues
 Apolipoproteins: Apo B-100, C and E
 Density: 0.95 – 1.006 kg/L
3. LDL/ B-Lipoprotein
 50% of the total lipoproteins in plasma – major source of cholesterol for tissues
 Transports cholesterol to peripheral tissues
 Apolipoproteins: Apo B-100, Apo-E
 Research method: B-quantification (EDTA) – combines ultracentrifugation and chemical
precipitation
 Reference value:
Optimal < 100 mg/dL
 Near/above Normal 100 – 129 mg/dL
Borderline high 130 – 159 mg/dL
High 160 – 189 mg/dL
Very high > 190 mg/dL
 Density: 1.019 – 1.063 kg/L
4. HDL/A-Lipoprotein
 Smallest lipoprotein but the most dense; nascent disk-shaped particles in liver and
intestine
 Transports excess cholesterol to the liver
Common method: Homogenous Assay
Common error: Presence of Apo-B containing lipoproteins
Interference: ↑ TAG
 Apolipoproteins: Apo-A1, Apo-A-II, Apo-C

 Reference value:
Cutoff 40 mg/dL
High risk for CHD < 35 mg/dL
High (protective) > 60 mg/dL
 Density: 1.063 – 1.21 kg/L

 Standing Plasma Test – to differentiate VLDL from chylomicrons; store at 4C , do not

agitate
a. Creamy layer – floating creamy layer
b. VLDL – homogenous turbidity

Minor Lipoproteins
1. Intermediate Density Lipoproteins
 Product of VLDL – “VLDL remnant” which is converted to LDL
 Migrates on Pre-beta or beta-region (electrophoresis)
 Defective clearance in Type 3 HLPP – deficiency in Apo E-100
 Major Apolipoprotein: Apo B-100
 Density: 1.006 – 1.019 kg/L causing it to float on the 1.063 density potassium bromide
solution
2. Lipoprotein (a) / Lp (a)
 Density is similar to LDL but migrates similarly to VLDL on electrophoresis
 SINKING PRE-B-LIPOPROTEIN

Abnormal Lipoproteins
1. Lipoprotein X – only LPP that moves toward the cathode
 Found in obstructive jaundice, LCAT deficiency and sensitive and specific indicator of
cholestasis
 Mostly phospholipid and free cholesterol (90%) lipid content
2. B-VLDL/Floating B-Lipoprotein
 Found in Type 3 hyperlipoproteinemia
 Known as “VLDL rich in cholesterol” due to defective catabolism of VLDL
 Found in VLDL density range but migrates electrophoretically with or near LDL

Patient Prepartion:
1. Fasting: 12 – 14 hours
 Fasting state: Plasma TAG is present in VLDL
 Nonfasting state: Plasma TAG is present in Chylomicrons
 Fasting samples: Test for TAG and LDL-C
 Nonfasting samples: Test for TC and HDL-C

Methods:
 Lipoprotein measurements be made no sooner than 8 weeks after any form of trauma or
acute bacterial or viral infection, and 3 – 4 months after childbirth
1. Ultracentrifugation (density gradient) – reference method
 Lipids have density of 1.0 g/mL; proteins have 1.4 mg/L
 Expressed in Svedverg units
  Frozen samples are not appropriate
 Reagent: Potassium bromide solution with 1.063 density
2. Electrophoresis (HDL, VLDL, LDL, CM)
 Preferred supporting medium: Agarose gel

Formula for LDL-C:

LDL-C = TC – HDL – Plasma TAG / 2.175

Friedewald Method De Long Method

VLDL = TAG / 2.175 (mmol/L) VLDL = TAG / 2.825 (mmol/L)
VLDL = TAG / 5 (mg/dL) VLDL = TAG / 6.5 (mg/dL)

Hyperlipoproteinemia
Type I – Hyperchylomicronemia Extremely elevated TAG due to presence of
Familial LPL deficiency CM
Type IIa – Familial Hypercholesterolemia Elevated LDL, cholesterol
Type IIb – Mixed Defect Elevated LDL, VLDL, TAG and cholesterol
Familial Combined Hyperlipidemia
Type III – Familial Dysbetalipoproteinemia Elevated Cholesterol, presence of B-VLDL;
TAG
Type IV – Familial Triglyceridemia Elevated VLDL, TAG
Type V Elevated VLDL, presence of CM, TAG and
cholesterol

NCEP Guidelines for Acceptable Measurement Error

Total Error Bias CV*
Cholesterol < 9% < 3% < 3%
TAG < 15% < 5% < 5%
HDL < 13% < 5% < 4%
LDL < 12% < 4% < 4%

Apolipoproteins
Apo- A1 Major structural protein in HDL Liver and intestine
Activates LCAT
Ligand for HDL binding
Apo- A2 Stuctural protein in HDL Liver
Activate LCAT
Enhances hepatic triglyceride lipase
activity
Apo- A-IV Component of intestinal LPP Intestine
Apo B-100 Major structural protein in VLDL and Liver
LDL
Ligand for LDL receptor
Apo B-48 Primary structural protein in CM Intestine
Apo C-I Activates lipoprotein lipase/LPF Liver
Apo C-II Activates lipoprotein lipase; LCAT Liver
Apo C-III Inhibits lipoprotein lipase and recognition Liver
of Apo E
Apo E Binds to LDL-receptor and remnant Liver
receptor; E (4) higher risk of CHD and
Alzheimer’s disease
Apo (a) May inhibit plasminogen binding Liver

Proteins

 Amphoteric because of their acid and basic amino acid compositions

 Provides 12 – 20% of the total daily body requirement
 50 – 70% of the cell’s dry weight

Structures
1. Primary structure
 Linear sequence of amino acid
 Determines identity of protein, molecular structure, binding capacity and recognition
ability

2. Secondary structure
 Winding of polypeptide chain
3. Tertiary structure
 Actual 3-dimensional configuration; folding pattern of the protein
4. Quaternary structure
 Association of 2 or more polypeptide chains to form a functional protein molecule

Specific Proteins
Prealbumin / Transthyretin Indicator of malnutrition; binds T3 and T4 and retinol-binding
protein
Albumin Sensitive and highly prognostic marker of cystic fibrosis
Alpha-1 globulin Alpha-1 acid glycoprotein – greatest affinity for progesterone
Alpha-1 antichymotrypsin – binds and inactivates PSA; associated
With pathogenesis of Alzheimer’s
Alpha-1 antitrypsin – protease inhibitor
Alpha-2 globulin Ceruloplasmin, haptoglobin, alpha-2 macroglobulin, lipoproteins
Beta-globulin Transferrin, hemopexin, complement system, lipoproteins
Gamma-globulin Immunoglobulins

Kidney Function Tests

SYNTHESIS EPO, renin and prostaglandins

METABOLISM Inactivation of aldosterone, glucagon and insulin
Activation of Vitamin D, formation of creatine
EXCRETION Ammonia, urea, uric acid, several minerals, toxic substances

 Plasma contains 20 – 35 mg/dL of NPN

Urea 45%
Amino acid 20%
Uric acid 20%
Creatinine 5%
Creatine 1 – 2%
Ammonia 0.2%

Blood Urea Nitrogen

 Major product of protein and amino acid catabolism
 1st metabolite to elevate in kidney disease
 To obtain concentration of urea from BUN
a. 2.14 X BUN (mmol/L)
b. 0.357 X BUN (mg/dL)
 Reference value: 8 – 23mg/dL
 BUN: Creatinine ratio: 10:1 – 20:1

Methods:

Methods Comments
Colorimetric: diacetyl Inexpesive, lacks specificity
Enzymatic: Ammonia formation Greater specificity, more expensive
Reference method: IDMS

A. Direct Method (Chemical Method)

 Diacetyl monoxime + Urea –Fearon’s reaction  Yellow Diazine Derivative

B. Indirect Method (Enzymatic Method)

1. Urease (production of ammonia and carbon dioxide
2. Coupled Urease / Glutamate dehydrogenase method
 Most common enzymatic method; best as kinetic measurement
 Reduced, NAD is measured at 340nm
PATHOPHYSIOLOGY
 Azotemia – elevated urea concentration in the blood
 Uremia – very high plasma urea concentration accompanied by renal failure

A. INCREASED UREA CONCENTRATION

1. Prerenal – caused by reduced renal blood flow
a. Congestive heart failure
b. Shock
c. Hemorrhage
d. Dehydration
e. High protein diet
f. Increased protein catabolism (stress, fever)
2. Renal
a. Acute/chronic renal failure, glomerular nephritis, and tubular necrosis
3. Postrenal
a. Urinary tract obstruction, renal calculi, tumors of urinary tract, severe infection

B. DECREASED UREA CONCENTRATION

 Low protein intake, severe vomiting and diarrhea, liver disease and pregnancy

Low Ratio (BUN:Creatinine) Low protein diet

<10:1 Acute tubular necrosis
Repeated dialysis
Hepatic disease
High Ratio >20:1 Postrenal azotemia
With increased creatinine Prerenal azotemia with renal disease
Renal failure
High Ratio >20:1 Prerenal azotemia
With normal creatinine Dehydration
Catabolic states
GI hemorrhage
High protein diet

Cretininine
 End product of muscle metabolism derived from creatine and also produced from 3 amino
acids such as methionine, arginine and lysine

Methods Comments
Colorimetric: endpoint Simple, nonspecific
Colorimetric: kinetic Rapid, increased specificity
Enzymatic Measure ammonia colorimetrically or
with ISE

A. Chemical Method – Direct Jaffe Method

 Red-orange chromogen of creatinine picrate is formed when creatinine is mixed with
alkaline picrate reagent
a. Saturated picric acid b. 10% sodium hydroxide
 Interferences: False ↑: ascorbate, glucose, uric acid, and alpha-ketoacids
False ↓: bilirubin and hemoglobin
a. Foln Wu – sensitive but nonspecific
b. Lloyd/Fuller’s Earth Method – sensitive and specific
 Adsorbent (removes interferences):
1. Lloyd’s - sodium aluminum silicate
 2. Fuller’s – aluminum magnesium silicate

B. Kinetic Jaffe Method

 Requires automated equipment for precision
 Serum is mixed with alkaline picrate solution and the rate of change in absorbance is
measured between 2 points

C. Enzymatic Method
a. Creatinine aminohydrolase Method / CK Method
 Requires large volume of preincubated sample; NAD is measured
b. Creatinase-Hydrogen Peroxide Method
 Has a potential to replace Jaffe Method
 Product is benzoquinonemine dye (red)

D. IDMS – reference method

Uric Acid
 Major product of purine (adenine, guanine) catabolism and breakdown of nucleic acids
 formed from xanthine by the action of xanthine oxidase in liver and intestine
 TOPHI – uric acid in tissues

Methods Comments
Colorimetric Problems with turbidity, several common
drugs interfere
Enzymatic : UV Needs special instrument and optical cells
Enzymatic : Hydrogen peroxide Interference by reducing substances

A. Chemical Method
 Principle: Reduction-oxidation reaction

Uric acid + Phosphotungstic acid – NaCN/NaCO3  Tungsten blue + Allantoin + CO2

 NaCN – Folin Newton Benedict (Brown)

 NaCO3 – Archibald Caraway Henry – lacks specificity

B. Enzymatic Method
 Uricase Method: Specific method which measures UV absorbance of uric acid at 293 nm.
EDTA and fluoride should not be used

Ammonia
 Severe liver disease is the most common cause of disturbed metabolism
 Elevated levels are seen in Reye’s syndrome, occurring most commonly in children preceded
by a viral infection and administration of aspirin
 Cigarette smoking is a significant source of ammonia concentration

A. Enzymatic (Glutamate dehydrogenase) – most common

Measurement of Ammonia:
1. Nesslerization Reaction
 Yellow end color – nitrogen low to moderate
 Orange-brown – nitrogen is high
2. Berthelot Reaction

NH3 + Phenol + hypochlorite – Sodium Nitroprusside  Indophenol blue

Specimen of Choice: Arterial blood (Heparin/EDTA)

Plasma/serum kept in ice water immediately
Liver Function Tests
Function Examples
SYNTHESIS Proteins – albumin, cholinesterase,
coagulation proteins, cholesterol, bile salts
and glycogen
METABOLISM Glucose to acetyl-CoA, gluconeogenesis,
amino acid conversions, fatty acid
DETOXIFICATION Bilirubin, drugs, ammonia
EXCRETION Bile acids

Test Measuring Hepatic Synthetic Function:

 Serum albumin and vitamin K dependent coagulation factors provide the most useful indices
for assessing severity of liver disease

A. Total Protein
 Plama levels of total protein is 0.2 – 0.4 g/dL higher than serum due to fibrinogen
 Reference value: 6.5 – 8.3 g/dL

1. Kjeldahl Method
 Reference method but not routinely used
 Based on measurement of nitrogen; nitrogen content is 15.1 – 16.8%
 1g of Nitrogen = 6.54g of proteins
 Kjeldahlization – conversion of nitrogen to ammonia using sulfuric acid as digesting
agent

2. Biuret Method
 Most widely used method; requires at least 2 peptide bonds and an alkaline medium
 Reagent: Alkaline Copper Sulfate
Tartrate salt/Rochelle salt/NaK tartrate – to complex cupric ions to prevent
precipitation in alkaline solution
 (+) Reddish-violet complex
 Measured at absorbance at 540nm

3. Folin-Ciocalteu (Lowry) Method

 Highest analytical sensitivity
 Principle: oxidation of phenolic compounds such as tyrosine, tryptophan and histidine
to give a deep blue color
 Main reagent: Phosphotungstic acid-molybdic acid or phenol reagent
 Color enhance: Biuret reagent

4. Dye-binding
 Albumin binds to dye which causes shift in absorption maximum
a. Bromcresol green – most commonly used (630nm)
b. Bromcresol purple – most specific
c. Methyl orange - nonspecific
d. Hydorxyazobenzene benzoic acid – many interferences (salicylates, bilirubin)

5. Ultraviolet Adsorption Method

 Protein absorbs light at 280nm (tryptophan, tyrosine, phenylalanine) and at 210nm

6. Salt Fractionation
 Globulins can be separated from albumin by salting-out procedures using sodium
salts

7. Serum Protein Electrophoresis

Abnormal Serum Protein Electrophoretic Patterns

Gamma spike Multiple myeloma – caused by IgG
 (monoclonal gammpathy)
Beta-gamma bridging Hepatic cirrhosis – caused by IgA
(polyclonal gmmopathy)
Alpha-2 globulin spike Nephrotic syndrome
Alpha- globulin flat curve Juvenile cirrhosis (AAT deficiency)

Albumin/Globulin Ratio
 Determined to validate if globulin is higher than albumin

Total Albumin Globulin

Protein
N, ↓ ↓ ↓ Salt retention syndrome
↓ ↓ N Nephrosis, malabsorption
N, ↓ ↓ ↑ Cirrhosis, hepatitis,
obstructive jaundice
↑ ↑ ↑ Dehydration
↑ N ↑ Multiple myeloma

Biliubin
 Reference range: 0.2 – 1.0mg/dL; 250 – 35 mg/day – amount of bilirubin production

Metabolism

Hemoglobin  Heme  bilirubin bound by albumin (unconjugated bilirubin)  flows to

sinusoidal space and is released from albumin to be picked up by ligandin conjugation with
UDPGT  conjugated bilirubin secreted to canaliculi  uribilinogen (colorless) 
urobilin/stercobilin  excreted

 Remaining 20% urobilinogen will enter extrahepatic circulation and be recycled

in the liver

Bilirubin Fractions
1. Unconjugated Bilirubin / B1
 Noncovalently attached to albumin
 Does not react with the color reagent until the bilirubin is first dissociated from the
protein
 Indirect bilirubin
2. Conjugated Bilirubin / B2
 Contains 2 or more attached glucuronic acid molecules
 Reacts directly with the color reagent
 Direct biirubin
3. Delta Bilirubin
 Bilirubin fraction that is covalently attaché to protein
 React directly with the color reagent and contributes to the direct bilirubin value

Types of Hyperbilirubinemia
1. Prehepatic
 Problem causing jaundice occurs prior to liver metabolism
 Cases of acute and chronic hemolytic anemia
2. Posthepatic
 Impaired bilirubin excretion
3. Hepatic
 Intrinsic liver defect or disease due to disorders of bilirubin metabolism and transport
defects or due to diseases resulting in hepatocellular injury

Derangements of Bilirubin Metabolism

1. Gilbert’s Syndrome – Bilirubin Transport Deficit
2. Crigler-Najjar Syndrome – Conjugation Deficit
3. Dubin-Johnson Syndrome and Rotor Syndrome – Bilirubin Excretion Deficit
4. Lucey-Driscoll Syndrome