CC1 - Mtap

Clinical Chemistry 1
-Laarni Hannah C. Lacorte, RMT, MSMLS
BASIC PRINCIPLES AND PRACTICES

Role of CC Laboratory
▪ Measure chemical changes in the body for diagnosis, therapy and prognosis of disease
▪ Measure the concentration of a particular constituent (the analyte) in body fluids
ROLE OF THE MEDICAL TECHNOLOGIST

▪ Must understand the tools:
▪ Equipment
▪ Reagents
▪ Principle of the testing methods
▪ Knowledge of the medical uses of the determinations
UNITS OF MEASURE
 Quantitative measurement is expressed in defined units
Components of quantitative laboratory results
1. Number
2. Unit (based on the SI system)
 Quantitative laboratory results
1. Substance concentration
▪ e.g: moles
2. Derived units
▪ e.g: mg/dL, g/dL, g/L, mEq/L, IU
REAGENTS
1. Chemicals
a. Analytical Grade (AR)
1. Suitable for most analytic procedures
2. Carry designations as AR or ACS and For Laboratory Use or ACS standard-Grade
Reference materials
b. Ultrapure Reagent
1. Suitable for techniques that require extremely pure chemicals (e.g. AAS, EIA,
MDx)
2. Carry designations of HPLC or chromatographic
c. Chemically Pure (CP)
1. Impurity limitations are not stated
2. Preparation is not uniform
3. Not recommended for clinical laboratories.
d. United States Pharmacopeia (USP) and National Formulary (NF) Grade
1. Used to manufacture drugs
2. Based on the criterion of not being injurious to man.
e. Technical or commercial grade
1. Used for manufacturing
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2. Reference Materials
a. Primary standard
▪ Substance of exact known concentration and purity.
b. Secondary standard
▪ Substance of lower purity with concentration determined by comparison with a
1° standard.
3. Water Specifications
a. Distilled water
▪ Purified by distillation
b. Deionized water
▪ Water purified by ion exchange
▪ Remove dissolved ionized solids and gases.
c. Reverse Osmosis (OS) water
▪ Uses pressure to force water through a semi permeable membrane.
d. Ultrafiltration and nanofiltered water, UV oxidation, sterilization or ozone treatment
e. Reagent grade water
▪Obtained by initial filter, followed by RO, deionization and a 0.2 mm filter.
Reagent grade water
▪ Type I water
• Trace metal analysis by FES and AAS
• Gas, pH, enzyme and electrolyte analysis
▪ Type II water
• For analytical preparations
• reagent, QC and standard preparation
▪ Type III/autoclave wash water
• Glassware washing
SOLUTION PROPERTIES
Solute + Solvent = Solution
a. Concentration
 Molarity
 Molality
 Normality
b. Collegative properties
c. Redox Potential
 Reducing agents = donate electrons
 Oxidizing agents = accept electrons
d. Conductivity
e. pH and Buffers
 Buffers – weak acids or bases
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CLINICAL LABORATORY SUPPLIES

1. Thermometers
 Analytical reactions occurs at an optimal temp. (circulating water or
heating/cooling metal blocks)
Liquid-in-glass
▪ Total Immersion
▪ Partial Immersion
▪ Surface thermometer
Electronic thermometer/Thermistor probe
Digital thermometer
2. Glassware and Plasticware

▪ Glass ware
i. Borosilicate (Kimax®/Pyrex®)
ii. Aluminosilicate (Corex®)
iii. High silica
iv. Acid /alkali resistant (Vycor®)
v. Amber colored (Low actinic)
vi. Lime soda (Flint glass)
▪ Plastic ware
i. Polystyrene
ii. Polyethylene
iii. Polypropylene
iv. Tygon®
v. Telon®
a. Laboratory vessels
i. Class A volumetric flask
1. Calibrated to hold one exact of liquid (TC)
ii. Erlenmeyer flasks and Griffin beaker
1. Hold different volume
2. Used in reagent preparation
iii. Graduated cylinder
1. Used to measure volumes of liquid
b. Pipets
▪ Glass or plastic utensils used to transfer liquids
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Design
To contain (TC)
o Used for viscous samples
o Uses mercury as calibrating medium
o Proper use requires rinsing technique
To Deliver (TD)
o Used for non-viscous samples
o Uses distilled water as calibrating medium
CALIBRATION MARKS OR DRAINING CHARACTERISTICS

To Deliver (TD)
Blowout pipet
o Serologic, (Ostwald Folin)
o With etched ring / two small continuous rings
Self-draining
o (Volumetric / Mohr)
o Without marking. Drains completely.
MEASURING OR GRADUATED PIPETS

 Graduated uniformly along its length
 Designed to deliver any amount within its capacity
Transfer pipet
Delivers an exact volume
Ostwald-Folin pipet
For viscous fluids (blow out pipet)
Volumetric pipet
For aqueous solutions (self-draining)
Mechanical or Automatic pipets
• Micropipet – deliver amount <1ml
• Macropipet – deliver amount >1ml
Dessicators and Desiccants

▪ Uses hygroscopic substance that take up water/moisture on exposure to air.
Balances
• Must be level and vibration-free
• Avoid air currents
• Kept clean
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▪ Electronic Top-loading balance

1. Top-loading balance
2. For knowing the mass of substances (greater quantity)
3. Used for preparative experiments.
▪ Analytical balance
1. for preparation of primary standards
2. With sliding transparent doors
3. Measure exact mass but with lower capacities (operating ranges 0.01 mg to
160g)
BASIC SEPARATION TECHNIQUES

1. Centrifugation
a. Consist of head/rotor (attached to the shaft of the motor), carrier and shields.
b. The speed/centrifugal force is expresses by:
c. Revolution per minute (RPM)
d. Relative centrifugal force (RCF) or gravities (g)
e. Centrifuged must be properly balanced and free from excessive vibrations.
2. Filtration
a. Paper, cellulose, polyester fibers and column materials
3. Dialysis
a. Separating macromolecules from a solvent or smaller substances
b. Semi-permeable membrane
c. Slow process
LABORATORY SAFETY AND REGULATIONS

Occupational Safety and Health Act (OSHA)
• Federal regulation enacted by Congress in 1970
• Goal: provide all employees with safe work environment
• On-site inspections to ensure compliance
• Administered by state agencies or federal administration
• Includes standards that regulate safety in many aspects of clinical laboratory
• Bloodborne Pathogens Standard
o Mandates “Universal Precautions” to prevent, evaluate, and manage exposure to
bloodborne pathogens
o Presumes all blood, tissue, fluids are infected
• Hazard Communication Standard
o Defines hazardous chemicals and ensures employers/employees are made aware of
hazards
• OSHA Lab Standard
o Requires Chemical Hygiene Officer and Chemical Hygiene Plan
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Safety Awareness
• Employer’s responsibilities
o Establish written laboratory work methods and safety policies
o Provide safety information, supervision, guidance, training, protective equipment,
medical surveillance to employees
o Provide and maintain adequate equipment and facilities
• Employee’s responsibilities
o Know and comply with work safety methods
o Notify supervisor of unsafe conditions/practices
• Use personal protective equipment
Signage and Labeling

o National Fire Protection Association’s standard hazards-identification system:
diamondshaped, color-coded symbol
o Manufacturers’ precautionary labels
▪ Statement of hazard
▪ Precautionary measures
▪ Specific hazard class ▪ First aid instructions
▪ Storage code, safety code, protective equipment needed
SAFETY EQUIPMENT
• Fume Hoods
o Expel noxious and hazardous fumes from chemical reagents
o Should be inspected for blockages, ventilation velocity
• Biosafety Cabinets
o Remove potentially harmful particles of infective biologic specimens
o Offer various levels of protection, depending on biosafety level of specific laboratory
• Chemical Storage Equipment o Safety carriers for transport of acids, alkalies, other solvents
o Safety cabinets, safety cans, and explosion-proof refrigerators for flammable liquids
o Gas-cylinder supports or clamps, use of handcarts for large tanks
• Personal Protective Equipment and Hygiene o Safety glasses, goggles, visors, work shields to
protect eyes
o Gloves, rubberized sleeves, full-length lab coat, proper footwear
o Proper respirator (i.e., HEPA)
o Hygiene—hand washing
BIOLOGIC SAFETY
• Use of gloves, gowns, face protection
• Consistent and thorough hand washing
• Capping of specimens during centrifugation and use of centrifuge with sealed cups
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• Cleanup of spills o Alert others; wear appropriate protective equipment o Use mechanical
devices to pick up broken glass/sharp objects.
o Absorb spill with paper towels, gauze pads, tissue.
o Clean spill site with detergent & disinfect with 10% bleach.
o Rinse spill site with water & dispose of all materials appropriately.
• Bloodborne Pathogens
o Written exposure control plan must be available to employees.
o Universal precautions policy should be followed.
• Airborne Pathogens
o Tuberculosis (TB) exposure plan must be developed.
o TB isolation areas with ventilation controls must be established.
o Workers in high-risk areas must wear respirators.
o Airborne pathogens other than TB
• Shipping: “infectious substances” and “diagnostic specimens” must be so labeled and packaged
as required
Chemical Safety
• Safety Data Sheet (SDS)
o Major source of safety information regarding a specific hazardous material, provided by
manufacturer or developed by employer
o Must, by law, include information on health hazard, handling, storage, protective
equipment needed, first aid procedures, etc.
• Toxic Effects from Hazardous Substances
o Toxic vapors from chemical solvents, mercury must be avoided.
o Air sampling, routine monitoring, spill kits are required.
o Storage and Handling of Chemicals
o Flammable/combustible chemicals
o Corrosive chemicals o Reactive chemicals
o Carcinogenic chemicals
o Chemical spills
Radiation Safety
• Caution signs and restricted access to areas with radioactive material
• Regular monitoring and decontamination of equipment
• Maintenance of records and appropriate licenses
• Proper training for work with radioisotopes
• Monitoring of employee exposure
• Engineered shielding and personal protective equipment
Fire Safety
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• Fire: chemical reaction involving rapid oxidation of combustible material or fuel, liberating heat
and light
• Four factors: fuel, ignition source, oxygen, reaction chain
• CLASSIFICATION OF FIRES
 Class A: ordinary combustible solid materials (paper, wood, etc.)
 Class B: flammable liquids/gases and petroleum products
 Class C: energized electrical equipment
 Class D: combustive/reactive metals (magnesium, sodium, etc.)
• TYPES/APPLICATIONS OF FIRE EXTINGUISHERS
 Class A: pressurized water, foam, multipurpose dry-chemical
 Classes B and C: multipurpose dry-chemical, carbon dioxide, halogenated hydrocarbon
(for computers)
 Class D: dry-chemical extinguishers (trained firefighters only)
• Fire drills conducted regularly
• Evacuation of all personnel in event of fire
CONTROL OF OTHER HAZARDS

• Electrical Hazards
o Use only explosion-proof and properly grounded equipment.
o Check for frayed electrical cords.
o Promptly report malfunctions; know location of electrical panel.
o Never operate on live electrical equipment or with wet hands.
• Compressed Gases Hazards o Store tanks vertically and always keep cylinders secured.
o Never store flammable liquids and compressed gases together.
o Do not force “frozen” cylinder valve.
• Cryogenics Materials Hazards
o Use only containers designed to withstand ultralow temperatures.
o Use eye/face protection and impermeable, loose-fitting gloves.
o Store cryogenic fluids in well-insulated but loosely stoppered containers.
• Mechanical Hazards
o Balance centrifuges; take special care with sharps and glassware.
• Ergonomic Hazards
o Avoid prolonged repetitive motions, and lift with the legs.
DISPOSAL OF HAZARDOUS MATERIALS

• Chemical Waste
o Neutralize strong acids or bases before disposal.
o Never dispose of foul-smelling or potentially reactive chemicals down drain.
o Collect designated liquid wastes (i.e., flammable solvents) in approved containers and
segregate into compatible classes.
o Filter and redistill solvents such as xylene and acetone for reuse.
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o Burn flammable material in specially designed incinerators.

o Transform explosive substances into less hazardous forms.
o Bury wastes unsuitable for incineration in designated landfills.
• Radioactive Waste
o Consult radiation safety officer about policies dealing with radioactive waste disposal.
o Transfer radioactive materials to licensed receiver for disposal.
• Biohazardous Waste
o Establish and implement an infectious waste program.
o Place into bag marked with biohazard symbol and then into a leakproof, punctureresistant
container with tight-fitting lid.
o Place sharps into special puncture-resistant container.
o Never transport, recap, bend, or break by hand needles.
Accident Documentation and Investigation
• Report any accidents involving personal injuries to supervisor immediately.
• Maintain records of occupational injuries/illnesses for length of employment plus 30 years.
• Complete and forward log and summary of occupational injuries/illnesses to U.S. Department of
Labor.
• Investigate causes of all accidents.
QUALITY ASSURANCE & QUALITY CONTROL

Quality Assurance
• Sum of all activities which ensure correct results.
Quality Control
• Is a system of ensuring accuracy and precision in the laboratory by including quality control
reagents in every series of measurements.
PARAMETERS OF QC
• Sensitivity - measure the smallest concentration of the analyte of interest.
• Specificity - measure only the analyte of interest.
• Accuracy - Is the nearness or closeness of the assayed value to the true or target value.
• Precision or Reproducibility - to give repeated results on the same sample that agree with one
another.
• Analytical measurement Range – range of an analyte concentration that can be directly
measured without dilution or concentration
• Clinically reportable range - Range of analyte that a method can quantitatively report, allowing
for dilution, concentration used to extend AMR
• Limit of detection - Lowest amount of analyte accurately detected by a method
• Practicability - Is the degree by which a method is easily repeated
• Reliability - ability of an analytical method to maintain accuracy and precision over an extended
period of time during which equipment, reagents and personnel may change.
INTRALAB QC (Internal QC)
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• It involves the analyses of control samples together with the patient specimens.
• It detects changes in performance between present operation and the “stable” operation.
• It is important for the daily monitoring of accuracy and precision
• It detects both random and systematic errors within a one-week cycle
INTERLAB QC (External QC)
• It involves proficiency testing programs that periodically provide samples of unknown
concentrations to participating clinical laboratories
• It is important in maintaining long-term accuracy of the analytical methods
• The CAP proficiency programs are the gold standard of clinical laboratory external QC testing.
Standard
• Most specific known sample
• Contain one component/analyte only
• colorless
Control
• Resemble patient’s sample
• Contain several analytes
• Yellow just like serum
Objectives of QC:
• to check the stability of the machine
• to check the quality of reagents
• to check technical (operator errors )
CONTROL SOLUTIONS (QC MATERIALS)

• The accuracy of a solution depends on the control solutions, how they are originally constituted
and how they remain stable overtime.
• To establish statistical quality control on a new instrument or on new lot number of control
materials, the different levels of control material must be analysed for 20 consecutive days in
duplicates.
• For highly précised assays (with <1% CV) such as blood gases, analysis for 5 days is adequate.
CONTROL LIMITS (CONTROL VALUES)

• Control limits are calculated from the mean and SD.
• The ideal control/ reference limit is between +/-2SD
• When changing a new lot #, laboratorians use the newly calculated mean value as the target
mean but retain the previous SD value, but when more data are obtained, all values should be
averaged to get best estimates of the mean and SD
CHARACTERISTICS OF AN IDEAL QC MATERIALS:

1. Resemble human sample (human ctrl preferred)
2. Non-infectious
3. Inexpensive and stable for long periods
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4. No matrix effects/known matrix effects

5. With known analyte concentrations (assayed control)
6. Convenient packaging for easy dispensing and storage
Variations
- Are errors encountered in the collection, preparation and measurement of samples, including
transcription and releasing of laboratory results.
TYPES OF ERRORS:
1. Random error/ indeterminate/ unpredictable/imprecision
• Is present in all measurements; it is due to chance; variations in technique
• Best measured by: SD & CoV
2. Systematic Error/ Determinate/ Predictable/ Inaccuracy
• Is an error that influences observations consistently in one direction
(constant difference)
• Statistical tool to confirm: MEAN or AVERAGE Constant error
• Difference between the target value and the assayed value.
• Independent of sample concentration
3. Proportional/slope/percent error
• greater deviation from the target value due to higher sample concentrations
• exists when the difference between the test method and the comparative method
values is proportional to the analyte concentration
Statistics
• Mean - is a measure of central tendency. it is associated with symmetrical or normal distribution
• Standard deviation - is a measure of the dispersion of values from the mean. it helps describe
the curve. a measure of distribution range.
o it is the most frequently used measure of variation
o SD is inversely proportional to precision
• Coefficient of Variation (CV) - is the percentile expression of the mean; an index of precision.
o Ideal result = 2-4%
o CV = SD/ mean x 100
o Aka Total % error
• Variance - Is called the SD squared; a measure of variability. It represents the difference
between each value and the average of the data.
o V= (SD)2
QC CHART
• Is used to observe values of control materials over time to determine reliability of the analytical
method.
• Is utilized to detect and observe analytic errors such as inaccuracy and imprecision
1. Gaussian curve
• AKA Bell-Shaped Curve, Normal Distribution Curve
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•It occurs when the data set can be accurately described by the SD and the mean.
•It occurs when data elements are centered around the mean with most elements close
to the mean.
• It focuses on the distribution errors from the analytical method rather than the values
from a healthy or patient population.
• Can identify both random and systematic error.
2. Cumulative sum graph (cuSUM)
• It calculates the difference between QC results and the target means.
• Common method: V mask
• It identifies consistent bias problems; it requires computer implementation
• This plot will give the earliest indication of systematic errors (trend) and can be used
with the 13s rule.
• Results are out of control when the slope exceeds 45o or a decision (+ 2.7 SD) is
exceeded.
3. Youden/ twin plot
• Will identify systematic error
• It is used to compare results obtained on a high and low control serum from different
laboratories
It displays the results of the analyses by plotting the mean values for one specimen on
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the ordinate (y axis) and three other specimens on the abscissa (x-axis)
4. Shewhart Levy-Jenning’s chart
• Aka Dot Chart
• It is a graphic representation of the acceptable limits of variation in the results of an
analytical method
• It easily identifies random and systematic errors
ERRORS WHICH CAN BE OBSERVED ON LJ CHART:

1. Trend - Is formed by control values that either has gradual increase of decrease for 6 consecutive
days;
 Main cause: deterioration of reagents
2. Shift: 6 consecutive control values on the same side of the mean
 Main cause: improper calibration of the instrument
WESTGARD CONTROL CHART

13S
• Random error determination
• One ctrl result exceeds the mean + 3SD
22S
• Respond most often to systematic error
• The last two ctrl results exceed either the mean + 2SD
13S
• Random error determination
• One ctrl result exceeds the mean + 3SD
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41S
• Respond most often to systematic error
• The last four ctrl results exceed either the mean + 1SD
R4S
• Respond most often to random error
• The range or difference between the highest and lowest ctrl result exceeds 4SD
10x
• Systematic error
• Ten consecutive results are on the same side of the target mean; either higher or lower mean
AUTOMATION
History
• First Automated Analyzer: “AutoAnalyzer”
– Introduced by Technicon in 1957
– A continuous-flow, single-channel, sequential batch analyzer
– Capable of providing a single test result on about 40 samples/hr
• Second Generation: Simultaneous Multiple Analyzer
– Multiple channels working synchronously
– Produced 6–12 test results simultaneously at rate of 360–720 tests/hr
• First Commercial Centrifugal Analyzer Introduced in 1970
– Spin-off technology from NASA space research
– Developed by Dr. Norman Anderson at Oak Ridge National Laboratory
– An alternative to continuous-flow technology
• Automatic Clinical Analyzer (1970) introduced by DuPont
– First non-continuous flow, discrete analyzer
– First instrument to have random access capabilities
– Unique features: plastic test packs, positive patient identification, infrequent calibration
• Thin Film Analysis Technology Introduced in 1976
• Kodak Ektachem Analyzer Produced in 1978
– First instrument to use microsample volumes and reagents on slides for dry chemistry
analysis
– First instrument to incorporate computer technology extensively into its design and use
• Discrete Analyzers Since 1980
– Ion-selective electrodes, fiberoptics, polychromatic analysis
– Sophisticated computer hardware and software for data handling – Larger test
menus
RECENT ADVANCES
• Point-of-Care Benchtop Analyzers
– Small, portable, easy to operate
– Used in physician office laboratories, surgical & critical care units
• Immunochemistry Analyzers
– Assaying drugs, specific proteins, tumor markers, hormones
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– Instruments using fluorescence-polarization immunoassay, nephelometry, &

immunoassay with chemiluminescent detection
• Modular Analyzers
– Combination of chemistry and immunoassay
BASIC APPROACHES
• Continuous Flow
– Liquids are pumped through system of continuous tubing. – Samples are introduced
in a sequential manner.
• Centrifugal Analysis
– Force of centrifugation transfers & contains liquids .
– Capable of batch analysis
• Discrete Analysis
– Separation of each sample & reagent in a separate container
– Most popular type; can run multiple tests on one sample at a time or multiple samples
one test at a time
ANALYTICAL METHODS
• Energy – transmitted via electromagnetic waves that are characterized by their frequency
and wavelength.
• Wavelength – is the distance between two successive peaks and it is expressed in terms of
nanometer (nm).
o 400-700nm – Visible spectrum.
o <400nm – Ultraviolet region (UV)
o >700nm – Infrared region (IR)
 Light - is a form of electromagnetic energy

o Transmitted via electromagnetic waves
o Waves is measured in nanometer (wavelength).
 Frequency – is the number of vibrations of wave motion per second.
o The LOWER the wave frequency, the LONGER the wavelength.
o Wavelength is INVERSELY RELATED to frequency and energy.
 Optical Instruments
o Instruments that measure light energy
o Based on the property of colored solutions to absorb light of specific wavelength.
Colorimetry
A. Spectrophotometric Measurement
▪ Measurement of LIGHT INTENSITY in a NARROWER wavelength.
B. Photometric Measurement
▪ Measurement of light intensity without consideration of wavelength.
Spectrophotometry
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 It involves measurement of the light transmitted by a solution to determine the concentration of

the light-absorbing substances in the solution.
2 TYPES OF SPECTROPHOTOMETERS:
1. Single beam spectrophotometer
o It is the simplest type of absorption spectro.
o It is designed to make one measurement at a time at one specified
wavelength.
o The maximum absorption of the analyte must be known in advance when a
singlebeam instrument is used.
2. Double-beam spectrophotometer
o Is an instrument that splits the monochromatic light into two components:
 One beam passes through the SAMPLE, and the other through a
REFERENCE SOLUTION or BLANK.
o The additional beam corrects for variation in light source intensity.
o Absorbance of the sample can be recorded directly as the electrical output of the
sample beam.
BEER’S LAW (A = 2-Log%T)

• The concentration of the substance is:
• DIRECTLY PROPORTIONAL to the amount of light absorbed. (absorbance/OD)
• INVERSELY PROPORTIONAL to the amount of transmitted light (%transmittance)
COMPONENTS OF SPECTROPHOTOMETRY
1. Light/Radiant Source
o It provides polychromatic light and must generate sufficient radiant energy or power
to measure the analyte of interest.
2 types:
o Continuum source – emits radiation that changes in intensity; widely used in
laboratory.
Examples: Tungsten, Deuterium and xenon lamps
▪ Tungsten light bulb – is the commonly used light source in the visible
and near infrared region.
▪ Deuterium lamp – routinely used to provide UV radiation in AS.
▪ Xenon discharge lamp – produces a continuous source of radiation,
which covers both the UV and visible range.
o Line Source – emits limited radiation and wavelength.
▪ Line sources that emit a few discrete lines find wide use in atomic
absorption, molecular, and fluorescent spectroscopy.
2. Entrance Slit
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 It minimizes unwanted or stray light and prevents the entrance of scattered light
into the monochromator sys.
 Stray Light
▪ refers to any wavelengths OUTSIDE the band transmitted by the monochromator
▪ It does not originate from polychromatic light.
▪ Causes ABSORBANCE ERROR.
▪ limits the max absorbance that a spectro can achieve.
▪ MOST COMMON cause of LOSS OF LINEARITY at high-analyte concentration.
3. Monochromator - It ISOLATES specific or individual wavelength of light.

KINDS OF MONOCHROMATORS:
o Prism – are wedge-shaped piece of glass, quartz or NaCl.
▪ Can be rotated, allowing only the desired wavelength to pass through an exit
slit.
▪ A narrow light focused on a prism is refracted as it enters the denser glass.
o Diffraction gratings – MOST COMMONLY USED.
▪ Better resolution than prism
▪ Made by cutting parallel grooves or slits into an aluminized surface of a flat
piece of crown glass – wavelengths are bent as they pass a sharp corner.
o Filter – simple and least expensive, not precise but useful.
▪ Made by placing semi-transparent silver films on both sides of a dielectric
such as magnesium fluoride.
▪ It produces monochromatic light based on the principle of constructive
interference of waves.
▪ Isolates stray light.
o Holographic gratings - type of diffraction grating formed by an interference-fringe field of
two laser beams whose standing-wave pattern is exposed to a polished substrate coated
with photoresist.
4. Exit Slit
• It controls the width of light beam (bandpass) – allows only a narrow fraction of the
spectrum to reach the sample cuvette.
• BANDPASS – total range of wavelengths transmitted.
• The NARROWER the bandpass, the GREATER resolution.
5. Sample Cell
o Also called Absorption cell/ Sample Cell/ Analytical cell.
o It holds the solution whose concentration is to be measured.
o Kinds of Cuvettes:
▪ Alumina Silica Glass – most commonly used
▪ Quartz/ Plastic – used for measurement of solution requiring UV spectrum
▪ Borosilicate Glass – for high alkali solution
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▪ Soft Glass
6. Photodetector - Detects and converts transmitted light into photoelectric energy.
KINDS OF PHOTODETECTORS:
o Photocell (Barrier layer cell/ Selenide cell/ Photovoltaic cell)
▪ Simplest detector and least expensive but temperature-sensitive.
▪ It requires NO external voltage source but utilized internal electron transfer for current
production.
▪ It is used in filter photometers with a wide bandpass.
▪ It is composed of selenium on a plate of iron covered with transparent layer of silver. o
Phototube
▪ Similar with photocell but requires external voltage.
▪ It contains cathode and anode enclosed in a glass case.
▪ It has a photosensitive material that gives off electron when light energy strikes it.
o Photomultiplier tube (PMT)
▪ Most commonly used detector – measures visible and UV regions.
▪ It has excellent sensitivity and has rapid response.
▪ Detects and amplifies radiant energy (200x sensitive)
▪ It should never be exposed to room light because it will burn out.
o Photodiode
▪ It is not as sensitive as PMT but with excellent linearity.
▪ It measures light at a multitude of wavelengths – detects less amount of light.
▪ It has a lower dynamic range and higher noise compared to PMT.
▪ It is most useful as a simultaneous multichannel detector.
7. Meter or Read-out Device - It displays output of the detection system.

Ex: Galvanometer / Ammeter/ Light-Emitting
▪ diode (LED)
Flame Emission Photometry (FEP)

• It measures the light emitted by a single atom burned in a flame.
• It is used for the measurement of excited ions (Na & K)
Principle: Excitation of electrons from lower to higher energy state.
Light Source: FLAME (also serves as the cuvette)
Method: Indirect Internal Standard Method
Internal Standard: Lithium/Cesium- corrects variations in flame and atomizer characteristics.
COMPONENT OF FEP/FES
• Nebulizer (atomizer) – deliver a fine spray of sample containing the metallic ion in the burner.
• Burner – a fuel gas (propane) with an oxidizing agent burned to produce the flame.
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• Monochromator System – allow only emitted light spectrum of specific element to strike the
PMT.
• Photosensitive Detector (Photocell/PMT)
Atomic Absorption Spectrophotometry (AAS) - It measures the LIGHT ABSORBED by atoms

dissociated by heat.
Principle: Element is not excited by merely dissociated from its chemical bond and place in an
unionized, unexcited, ground state
Light source: HOLLOW-CATHODE LAMP
Interference: Chemical, matrix and ionization.
 It is more sensitive than FEP/FES; accurate, precise and specific.
 It is used for measurement of UNEXCITED trace metals. (Ca & Mg)
 REFERENCE METHOD in measurement of CALCIUM & Mg.
 Uses PMT (photomultiplier tube)
 Lanthanum/Strontium Chloride – added to samples to form stable complexes with
phosphate.
VOLUMETRIC AND DENSITOMETRY
Volumetric- A.k.a Titrimetric

 Principle: The UNKNOWN sample is made to react with a KNOWN solution in the presence of
an INDICATOR.
 Examples: Schales and Schales Method and EDTA Titration Method
Densitometry
 It measures the absorbance of stain – concentration of the dye and protein fraction.
 It scans and quantitates electrophoretic pattern.
 It is the quantitative measurement of Optical Density in light-sensitive materials, such as
photographic paper or photographic film, due to exposure to light.
Fluorometry (A.k.a. Molecular Luminescence Spectrophotometry )

 Principle: Light emitted by a molecule after excitation by electromagnetic radiation.
 Light source: mercury arc or xenon lamp.
 Light detector: PMT or phototube.
Uses 2 monochromators (either filters, prisms or gratings)

 Primary Filter – wavelength that is best absorbed by the solution to be measured is selected.
 Secondary Filter – incident light is prevented.
 Advantage: 1000x more sensitive than spectro
 Disadv: affected by quenching and changes in pH,temp.,light
COMPONENTS OF FILTER FLOUROMETERS

1. Gas discharge lamps
 Emits excitation light
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 Mercury-vapor lamps
 Xenon-arc lamp
2. Attenuator
 Controls the light intensity
3. Primary Filter
 Selects the wavelength that is best absorbed by the sample
4. Sample holder
 The fluorescing sample emits fluorescent light in all directions
5. Secondary filter
 Passes longer wavelengths of fluorescent light
 Prevents incident light from striking the photodetector
6. Photodetector
Chemiluminescence
• It differs from fluorescence and phosphorescence in that the emission of light is created from a
chemical or electrochemical reaction.
• Principle: Process of exciting molecules by chemical means.
• Light emitted is measured as the molecules return to their unexcited state.
• More sensitive that fluorescence.
• Simple and Fast instrumentation.
• Requires no excitation radiation and monochromator.
Turbidimetry
• For measuring abundant large particles (proteins) and bacterial suspension.
• Principle: It determines the amount of LIGHT BLOCKED by a particulate matter in a turbid
solution.
• Dependent on specimen concentration and particle size.
• The measurement of light reduction is due to particle formation.
• Solutions requiring quantitation by turbidimetry are measured using visible Photometers or
visible spectrophotometers.
Nephelometry
• For measuring the amount of Antigen-Antibody Complexes.
• Principle: It determines the amount of SCATTERED LIGHT by a particulate matter suspended in a
turbid solution.
• Light scattering depends on wavelengths and particle size.
• Principle: Light scattered by the small particles is measured at an angle (forward or 90°) to the
incident light.
Components:
1. Light source (Mercury-Arc Lamp, Tungsten, Light emitting diode and a Laser)
2. Collimator (device that narrows a beam of particles or waves)
3. Monochromator
4. Sample cuvette
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5. Photodetector and Stray light Trap
Electrophoresis
▪ The process of separating the charged constituents of a sample by means of an electrical
current.
▪ The migration of charged particles in an electric field.
▪ Components: electrical power, support medium, buffer, sample and detecting system.
▪ Buffer: Barbital (pH 8.6)
Terms:
➢ Amphoteric – has a net charge that can be either positive or negative. (pH)
➢ Electroendosmosis – movement of buffer ions and solvent relative to the fixed support.
➢ Iontophoresis – is the migration of small charged ions.
➢ Zone Electrophoresis – is the migration of charged macromolecules.
Supporting Media:
➢ Cellulose Acetate – separates by molecular size
➢ Agarose Gel – separates by electrical charge
➢ Polyacrylamide Gel – separates on the basis of charge and molecular size. (SDS-PAGE)
➢ Electrophoretogram
➢ Result of electrophoresis consisting of separated strands of a macromolecule
▪ Factors that affects migration:

1. Net electric charge of the molecule
2. Size and shape of the molecule
3. Electric field strength
4. Nature of supporting medium
5. Temperature of operation
▪ Stains for Visualization of Fraction/Bands:
1. Amido black
2. Ponceau S
3. Oil Red O
4. Sudan Black
5. Fat Red 7B
6. Coomassie Blue
7. Gold/Silver Stain
Chromatography
 Group of techniques used to separate complex mixtures on the basis of different
physical interactions between the individual compounds and the stationary
phase of the system.
MODES OF SEPARATION:
1. Adsorption Chromatography (Liquid-Solid Chromatography)
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▪ Based on the competition between the sample and the mobile phase for
adsorptive sites on the stationary phase.
▪ The molecules that are most soluble in the mobile phase, move fastest; the least
soluble, move slowest.
2. Partition (Liquid-Liquid Chromatography)
 Separation of solute is based on relative solubility in an organic (nonpolar)
solvent and an aqueous (polar) solvent.
3. Steric Exclusion
 A.k.a Gel filtration/Gel permeation/Size Exclusion/Molecular Sieve
 Used to separate solute molecules on the basis of size and shape.
4. Affinity Chromatography
 It uses immobilized biochemical ligands as the stationary phase to separate a
few solutes from other unretained solutes.
 Used for separation of LPP, Carbohydrates and HbA1c; Antibodies.
5. Ion-exchange Chromatography
 Solute mixtures are separated by virtue of the magnitude and charge of ionic
species.
 Used for separation of amino acids, proteins and nucleic acids.
CHROMATOGRAPHIC PROCEDURES:
1. Planar Chromatography
o Paper Chromatography
▪ Used for fractionation of sugar and amino acids.
▪ Sorbent (stationary phase) – Whatman Paper.
▪ The solvent move up through through the paper and the fractions move up at
different rates.
o Thin Layer Chromatography (TLC)
▪ It is used for semi quantitative drug screening test.
▪ Each drug has a characteristic Rf value and it must match the Rf value of the
drug standard.
▪ Extraction of drug is pH dependent – the pH must be adjusted to reduce the
solubility of the drug in the aqueous phase.
2. Columnar Chromatography - packed into a tube or coated onto the inner surface of
tube/column.
o Liquid Chromatography
▪ It is based on the distribution of solutes between a liquid mobile phase and a
stationary phase.
 High-performance liquid chromatography
▪ HPLC is the MOST WIDELY USED liquid chromatography.
▪ used in fractionation of drugs, hormones, lipids, carbs and proteins; separation
and quantitation of various Hgb associated with specific disease and RAPID
HbA1c Tests.
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 Liquid Chromatography-Mass Spectroscopy (LC-MS) – used for detecting

nonvolatile substances in body fluids; also used in therapeutic drug monitoring,
toxicology and drug metabolites study.
o Column Chromatography
 Liquid chromatography - Based on the distribution of solutes between a liquid
mobile phase and stationary phase.
 High-performance liquid chromatography - Uses pressure for fast separation of
thermolabile substance
o Gas Chromatography
▪ Separate mixture of compounds that are volatile or can be easily made into a
volatile form.
▪ It is used for separation of steroids, barbiturates, blood, alcohol and lipids.
▪ if the molecule of interest is NOT VOLATILE enough for direct injection, it is
necessary to derivatize it into a more volatile form.
Samples: URINE or BLOOD are introduced into the GC column using a hypodermic
syringe or an automated sampler.
Osmometry - Is the measurement of the osmolality of an aqueous solution such as serum, plasma or
urine.
 Principle: It is based on measuring changes in the colligative properties of solutions that occur
owing to variations in particle concentration.
 Colligative properties of the solution: osmotic pressure, boiling point, freezing point, and vapor
pressure.
 Osmotic particles: Glucose, Urea nitrogen and sodium.
 When active osmotic particles are added to a solution, OSMOLALITY INCREASES.
 As the osmolality of a solution increases:
o Osmotic Pressure INCREASES
o Boiling point is ELEVATED
o Freezing point is DEPRESSED
o Vapor Pressure is DEPRESSED
Freezing point depression osmometry

o MOST COMMONLY USED method for measuring the changes in colligative properties of
solution.
o It is based on the principle that addition of solute molecules lowers the temperature at which
solution freezes.
Electrochemistry
o Is the measurement of current or voltage generated by the activity of a specific ion.
o Measures: Blood pH, Blood Gas, Electrolytes, Glucose, Urea, Ionized calcium, lead and chloride.
o Reference electrode: (half-cell) calomel and silver-silver chloride
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 Electrode with a constant voltage

 Reference potential against unknown voltage.
Mass Spectrometry (MS)

o Based on fragmentation and ionization of molecules using a suitable source of energy.
o Before a compound can be detected and quantifies by MS, it must be separated by GC.
o It can also detect structural information and determination of molecular weight.
o Definitive identification and quantitation of samples or compounds eluting from GC or HPLC
columns.
o Applications
 Measuring drugs of abuse in urine.
 Measuring low-level and mixed-polarity analytes.
▪ Vitamin D, testosterone, immunosuppressant drugs
Refractometry Principle:
o Measures the extent to which light is bent (refracted) when it moves from air into a
sample
o Is typically used to determine the index of refraction, (refractive index, (RI) or n) of a
liquid sample.
o Measurement of refractive index of liquids and solid are quick and easy using
refractometers.
Refractive index (n)
 The ratio of the speed of light in a vacuum to the speed of light in another substance is defined
as the for the substance.
 The density of a liquid usually decreases with temperature, it is not surprising that the speed of
light in a liquid will normally increase as the temperature increases.
 The index of refraction normally decreases as the temperature increases for a liquid.
 For many organic liquids the index of refraction decreases by approximately 0.0005 for every 1
°C increase in temperature
Phlebotomy
 The process of collecting blood “to cut a vein”
 Two main phlebotomy procedures:
I. Venipuncture
II. Capillary puncture
Public Relations & Client Interaction

1. Professionalism
 Appearance, Attitude, Communication skills, Bedside Manner
2. Patient Consent (implied consent)
3. Infection Control (PPE, hand hygiene, isolation)
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The Vascular System - Network of arteries, veins and capillaries.
ARTERIES AND VEINS ARE COMPRISED OF THREE LAYERS OF TISSUE:

1. Tunica intima
 Innermost, smooth layer
2. Tunica media
 Middle, thickest layer
3. Tunica adventitia
 Outer covering
“Capillaries comprise only one layer of tissue. “
Arterial Blood
• Has a larger concentration of oxygen than carbon dioxide
• Pumped by the heart to the body cells
Venous Blood
• Has a larger concentration of carbon dioxide
• Pumped by the heart to the lungs
Arteries
 Elastic, muscular and thick walled
 Arterial blood is bright red (oxygenated blood)
Veins
 Thinner walls
 Venous blood is dark red (deoxygenated blood)
Capillaries
 Smallest blood vessels.
 One cell thick to allow for gas and nutrient exchange.
PHLEBOTOMY SITES - The most commonly used veins for venipuncture are located in the antecubital
fossa.
Vein of choice:
• Median cubital vein
• Cephalic vein
• Basilic vein Note:
• Hand veins are smaller and less anchored.
• This can be very painful for the patient.
TYPES OF BLOOD SPECIMENS

1. Whole blood
Contains the plasma and the cellular components
1. Arterial
2. Venous
2. Plasma
The liquid portion of an anticoagulated blood
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Contains fibrinogen
3. Serum
The liquid portion of clotted blood
Plasma minus the fibrinogen
VENIPUNCTURE EQUIPMENT
i. Tourniquet
 made of pliable rubber or a strip with Velcro
 Used to locate the patient’s veins
 applied to a patient’s arm during venipuncture.
 must not be left on longer than 1 minute ii.
ii. Needle
 Size – gauge and bore are inversely related
 21 gauge – standard for routine venipuncture.
Three (3) Basic Methods
 Evacuated Tube System
i. Multisample needle
ii. Tube holder (barrel/adapter)
iii. Evacuated tubes
 Needle and Syringe
i. Syringe needles
ii. Syringe
iii. Transfer device
 Winged Infusion Set (Butterfly)
i. Luer fitting for syringe
ii. Luer adapter for ETS
o Used to collect blood from elderly and children
o Provides greater control with non-stable patients
ORDER OF DRAW
Reduce the risk of specimen contamination by microorganisms and additive carry-over.
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CAPILLARY SPECIMEN COLLECTION

 Blood is collected from a skin puncture (lancet)
1. Collection site
2. Materials
3. Capillary Order of Draw
4. Indications for Capillary Puncture
Collection site
 Outer area of the bottom of the foot (heel stick)
 Fleshy part of the last phalanx of the 3rd/4th finger
 Fleshy portion of the earlobe
Capillary Order of Draw

i. EDTA specimens
ii. Other additive specimen
iii. Serum
Indications of Capillary Puncture
i. No accessible veins
ii. Veins are fragile
iii. Thrombotic tendencies
iv. POCT (glucose monitoring)
v. Small blood volume
vi. Prevent Injury by restraining
vii. New born screening
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SPECIMEN PROCESSING
1. Routine handling
a. Mixing Tubes
 Inverti 3-8x
b. Transporting specimens
 Plastic bag with a biohazard logo, closure and slip pocket
c. Delivery time limits
 45 minutes of collection
 Centrifuged within 1 hour
2. Special handling
 Maintained at 37ºC (heat block)
 Chilled (using crushed ice)
 Wrapped on foil
3. Specimen Suitability
a. Hemolyzed
b. Collection in the wrong tube
c. Failure to follow timing and handling requirements
d. Quantity not sufficient (QNS)
e. Clotting in WB or plasma specimen
4. Centrifugation
a. Specimens must be completely clotted (30-60 minutes at room temp.)
b. Visually check
1. Lipemic
2. Hemolyzed
3. Icteric
UNACCEPTABLE BLOOD SPECIMENS
1. Lipemic
▪ A cloudy turbid appearance, presence of lipid, indicates a non-fasting specimen.
▪ Interferes with colorimetric analysis
2. Hemolyzed
▪ Destruction of RBC results in plasma/serum appearing red to pink due
▪ Affects potassium and enzyme testing
3. Icteric
▪ Specimen with a yellowish appearance due to increased bilirubin content
5. Stopper Removal
 Stopper removal devices
 Face Shield
 Splash Shield
6. Aliquot Preparation
 For multiple tests in a single specimen.
 Stored at 4ºC – 20ºC for 8 hours
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CARBOHYDRATES
Importance of Carbohydrates
 Major food source & energy supply of the body
 Source of energy of brain, erythrocytes & retinal cells
 Formation of the structural framework of RNA and DNA
 Structural elements in the cell walls of bacteria, plants) and animals.
 They are linked to many proteins and lipids.
 As "food" for energy supply and production of fats.
 Stored at liver as glycogen
General Characteristics
 It contains CARBON, HYDROGEN & OXYGEN
 Contains ketone and aldehyde group
Can be classified according to
 Size of the base carbon chain
 Location of the CO function group
 Stereochemistry of the compound
 Number of the sugar units
o Monosaccharide
o Disaccharide
o Oligosaccharide
o Polysaccharide
Glycoside bonds: bridges of oxygen atoms
GLUCOSE METABOLISM
Glycolysis
• Metabolism of glucose to lactate or pyruvate for production of energy
Gluconeogenesis
• Formation of glucose-6-phosphate from non-carbohydrate source
• Fats & Proteins
Glycogenolysis
• Breakdown of glycogen to glucose for use as energy
Glycogenesis
• Conversion of glucose to glycogen for storage
Lipogenesis
• Conversion of carbohydrates to fatty acids
Lipolysis
• Decomposition of fat
BIOCHEMICAL PATHWAYS IN CARBOHYDRATE BREAKDOWN

Embden-Meyerhoff pathway
 Converts glucose to pyruvate/lactate
 Primary energy source for humans
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Hexose monophosphate shunt

 Oxidizes glucose to ribose and CO2
 Produces NADPH as an energy source
Glycogenesis
 Converts glucose to glycogen
REGULATION OF GLUCOSE METABOLISM

Insulin
 Responsible for the entry of glucose in the cell
 Beta cells of islet of Langerhans
 ↑ glycogenesis, glycolysis, lipogenesis
 ↓ glycogenolysis.
 Stored in the liver, fat & muscle Hypoglycemic Agent
Glucagon
 responsible for increasing blood glucose
 Αlpha cells of the islets of Langerhan
 ↑ glycogenolysis and gluconeogenesis
 Release during stress & fasting
 Hyperglycemic Agent
Epinephrine
• Adrenal medulla
• Inhibit insulin secretion
• ↑ glycogenolysis and lipolysis
Cortisol
• Adrenal cortex
• ↓ intestinal entry of glucose
Growth hormone
• Anterior pituitary gland
• ↓ entry of glucose into the cell
Thyroid hormone
• Thyroid gland
• ↑ glycogenolysis, gluconeogenesis & intestinal absorption
Diabetes
- Metabolic disease characterized by hyperglycemia and glucose intolerance resulting from the
defect in insulin secretion, insulin action or both.
WHO CATEGORIES FOR DIABETES

o Type 1 diabetes (IDDM)
o Type 2 diabetes (NIDDM)
o Other specific types of diabetes
o Gestational diabetes mellitus (GDM)
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Type I (IDDM)
 AKA: Juvenile onset, insulin dependent & ketosis tendency.
 deficiency of insulin due to the destruction or degeneration of their pancreatic islet beta cells
 Insulin resistance with an insulin secretory defect
 Replacement therapy or pancreas implants
Type II (NIDDM)
 Adult-onset DM.
 Functional pancreatic beta cells
 insulin resistance & insulin secretory defect
 Controlled by reducing weight or by oral therapy
 Mostly of the patient is obese
 Hyperosmolar coma
Gestational DM
 Diagnosed during the latter half of pregnancy
 Increase secretion of placental hormone, placental lactogen inhibits the action of insulin
Other specific types of diabetes

May be associated with genetic defects of beta cell function or insulin action, pancreatic disease,
disease of endocrine origin, drug or chemical induced insulin receptor abnormalities
DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS

Categories of Fasting Plasma Glucose
 Normal fasting glucose: 70 – 99 mg/dL (3.9-5.5 mmol/L)
 Impaired: 100 – 125 mg/dL (5.6-6.9 mmol/L)
 Provisional Diabetes Diagnosis: ≥ 126 mg/dL (≥ 7.0 mmol/L)
Hypoglycemia
- It results from an imbalance between glucose utilization & production Warning
- signs: related to CNS
1. Neurogenic: tremors, palpitations, anxiety
2.Neuroglycopenic: dizziness, tingling. Blurred vision confusion
Diagnostic Test
 Fasting Blood Sugar
 Glycosylated Hemoglobin
 Oral Glucose Tolerance Test
 Random Blood Sugar
 2 Hour post prandial blood sugar
Glucose Determination
Enzymatic
• Glucose oxidase
• Hexokinase
• Clinitest
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Non-enzymatic/ Chemical
• Nelson Somogyi
• Hagedorn Jensen
• Ortho-toluidine
Lipids and Lipoproteins

Roles of Lipids
 Source of energy
 Integral part of cellular membranes that assist in cell structure
 Converted to hormones or hormone precursors
 Insulators for nerve conduction and heat retention
TYPES OF LIPIDS
 Fatty Acids
o Linear chain of C-H bonds
o Terminate with a carboxyl group
o Integral part of triglycerides/phospholipids
o Body makes most fatty acids
o Store large amounts of energy
o Essential fatty acids: linolenic and linoleic
acid
o Acquired by diet
Classification - Dependent on the number of C=C
 Saturated – no double bonds
 Monounsaturated – one double bond
 Polyunsaturated – two or more double bonds
 Triglyceride
o Composed of 3 fatty acid molecules, which includes glycerol
o Hydrophobic = Water insoluble
o Comprises 95% of fat stored in tissue
 Triglycerides with unsaturated fatty acids tend to turn oils at room temp
 Triglycerides with saturated fatty acids tend to be solid at room temp
▪ Exogenous
• Come from the diet
• Plant or animal sources
▪ Endogenous
• Synthesized by the body
 Phospholipids
o Composed of 2 fatty acid molecules
o Amphipathic
o Has both hydrophilic and hydrophobic parts
o Found on surfaces of lipid layers.
o Synthesized in the liver
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 Cholesterol
o An unsaturated steroid alcohol containing 4 rings & single side chain tail
o Synthesized in animals (liver)
Functions
 Promotes fat absorption in intestine via bile acids
 Produces some hormones
 Transforms Vitamin D in the skin
 Component of cell membranes
o Amphipathic
• Exogenous
 Originates in animal products
 Also absorbed via biliary secretions, intestinal secretions, and turnover of
intestinal mucosal cells
• Endogenous
 Produced in the liver and intestine from acetyl-CoA
 Cholesteryl esters
o Hydrophobic
o Located in the center of lipoproteins
GENERAL STRUCTURE OF LIPOPROTEINS

 Size of the molecule correlates with lipid content
 Composed of both lipids and proteins
 Transports lipids
 Outer layer of proteins called the apolipoprotein
CLASSIFICATION OF LIPOPROTEINS
Chylomicron
 Largest and least dense of the lipoproteins
 Produced by intestine
 Lipid-rich transport vessel that carries triglyceride in circulatory system to cells
 Observed as a creamy layer in samples
VLDL: very low-density lipoproteins
 Carry endogenous triglycerides to cells for energy use and storage
 Transfer triglycerides from liver to peripheral tissue
 Liver-made
 Specimen appears turbid in fasting samples
HDL: High density lipoproteins
 Gather excess cholesterol and return them to liver
 Made in liver and intestine
 HDL
 Smallest and most dense
 Synthesized by liver and intestine
 Can exist either as disk-shaped or spherical particles
 Removes excess cholesterol from peripheral cell and transporting it back to the liver
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LDL: Low-density lipoproteins

 Deliver cholesterol to peripheral cells and liver following triglyceride removal
 Carries mostly cholesterol
 Forms as a result of lipolysis of VLDL
 Readily taken up by cells via LDL receptor
 High level promotes atherosclerosis
LDL METHODS
Friedwald calculation
(If <400 mg/dl triglycerides)
LDL=Chol – HDL – Trig/5
Beta-quantification: most common; combine ultracentrifugation & chemical precipitation

(If >400 mg/dl triglycerides)
FUNCTION OF APOLIPOPROTEINS
 Maintain structural integrity

 Binding site for cell receptors
 Activator/Inhibitor of various enzymes
TYPES OF APOLIPOPROTEINS
1. Apo A-I
 Major protein on HDL
2. Apo B
 Principal protein on LDL, VLDL and chylomicrons
Two forms:
 B-100 = LDL and VLDL, acts as an LDL receptor; critical in the uptake of LDL by cells
 B-48 = only in chylomicrons
3. Apo C
 Activates lipoprotein lipase (LPL) to break down triglycerides
4. Apo E
 Promotes binding of LDL, VLDL
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PHYSIOLOGY AND METABOLISM
Three pathways
1. Lipid absorption
o During digestion, pancreatic lipase cuts off fatty acids and converts dietary lipids to
compounds with amphipathic properties
o Triglycerides, phospholipids and cholesterol esters are also transformed to amphipathic
lipids
o These lipids form aggregates with bile acids in the intestine-called micelles
o Absorption occurs when micelles contact membranes of the intestinal mucosal cells
o Short chain fatty acids
o Enter circulation, picked up by albumin, taken to liver
o Long chain fatty acids, monoglycerides, diglycerides
o Re-esterified in intestinal cells to form triglycerides and cholestyl esters
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2. Exogenous
o Transport of dietary lipids
o Chylomicron remnants are taken up by the liver
o Once inside the liver, lysosomal enzymes break down the remnants to release fatty acids,
free cholesterol and amino acids
o Some cholesterol is converted to bile acids
o Bile acids and free cholesterol are directly excreted into the bile, but not all exit the body
o Half is reabsorbed by the intestine
o Remainder found in stool
3. Endogenous
o Transport of hepatic-derived lipids
o VLDL loses core lipids once secreted in the circulation
o Loss of core lipids leads to conversion of VLDL to remnants
o About half of the remnants are converted to LDL, and half are taken in by the liver
– Depend on apo-B lipoprotein particles

– Transport dietary lipids and hepatic-derived lipids to peripheral cells
– Critical transport mechanism of fatty acids to peripheral cells
The fourth pathway – Reverse cholesterol transport

• Maintains cholesterol equilibrium
• Mediated by HDL
• Excess cholesterol from peripheral cells is transported back to the liver
• HDL serves to taxi cholesteryl esters to chylomicrons/VLDL remnants to liver
• Conversion of cholesterol into bile acids for removal
Population Distribution of Lipids

 Concentration differs between men, women and children due to sex hormone
concentration and age
Women:
 Higher HDL
 Lower Cholesterol, triglyceride
Aging
 Men and women increase in total cholesterol, LDL cholesterol and triglyceride
LIPIDS AND LIPOPROTEIN DISORDERS
Dyslipidemias
• Disease associated with abnormal lipid concentrations
• Subdivided into two major categories
a. Hyperlipoproteinemias
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b. Hypolipoproteinemias
• Usually due to Congestive Heart Disease (CHD) or arteriosclerosis
Acquired
• Environmental/lifestyle imbalance
Congenital
• Genetic abnormalities
Secondary
• Due to other diseases
Arteriosclerosis
• Hypertension, elevated lipid and chronic endothelial damage contribute
• Effects both men and women; however, women present later in life
• Disease stems from the deposition of lipids, in form of esterified cholesterol, in artery walls.
High Cholesterol with High LDL-C

• Polygenic (Nonfamilial) Hypercholesterolemia
• Familial Hypercholesterolemia
• Familial Defective ApoB
• Sitosterolemia
• Autosomal Dominant Hypercholesterolemia
• Autosomal Recessive Hypercholesterolemia
High Triglycerides with Normal Cholesterol

• Diabetic Dyslipidemia
• Familial Hypertriglyceridemia
• Lipoprotein Lipase Deficiency (Hyperlipoproteinemia Type 1 or Hyperchylomicronemia)
• ApoC-II Deficiency
• ApoC-III Excess
• ApoA-V
High Cholesterol with High Triglycerides

• Familial Combined Hyperlipidemia (Type 2B)
• Acquired Combined Hyperlipidemia
• Dysbetalipoproteinemia (Type 3)
• Hepatic Lipase Deficiency
• Cholesterol 7-Alpha-Hydroxylase Deficiency
Low Total Cholesterol and Triglyceride
• Abetalipoproteinemia
• Hypobetalipoproteinemia
• Chylomicron Retention Disease
Isolated Low HDL-C
• Familial Hypoalphalipoproteinemia
• ApoA-I Defciency and ApoC-III Deficiency
• ApoA-I Variants
• Tangier Disease
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• Lecithin:Cholesterol Acyltransferase Deficiency

Isolated High HDL-C
• Cholesteryl Ester Transfer Protein Gene Defects
High Cholesterol with HIGH LDL-C

• Polygenic (Nonfamilial) Hypercholesterolemia
• Multifactorial
• 85% of hypercholesterolemia fall under this category
• term to describe patients who develop age-related increases in cholesterol that do not respond
to lifestyle modification.
Familial Hypercholesterolemia (FH)
• autosomal dominant disorder caused by one of several mutations in the LDL-receptor gene on
chromosome 19
• cannot bind or clear LDL from the circulation
• LDL receptor gene mutation affect:
o Synthesis
o Transport
o Function
• Homozygous FH presents in childhood with LDL level of >400 mg/dl
• Homozygotes are less affected by environmental factors in terms of LDL-C levels
• Large valvular and supravalvular cholesterol deposits can produce symptomatic aortic stenosis
• stigmata of the disease:
• corneal arcus
• tendinous xanthomata
• xanthelasma
Hypercholesterolemia
• Clinical signs and Symptoms
o Heart attacks occur at an early age (teenage years)
o Patient exhibit xanthomas, which are
cholesterol deposits under the skin
o Cholesterol can range from 300-1000
mg/dL
Familial Defective ApoB
• autosomal dominant disorder of the apoB gene on chromosome 2 that interferes with the
recognition of apoB-100 by the LDL receptor
• Missense mutation
• similar physical stigmata to those of FH:
o Tendinous xanthomata o xanthelasma
o premature coronary disease
• Statin drugs are effective
Sitosterolemia
• extremely rare autosomal recessive disorder
• phytosterols (plant sterols) are absorbed and accumulate in plasma and peripheral tissues.
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• mutations in the ABCG8 or ABCG5 gene at chromosome 2p21

• Disrupts ability of liver to secrete absorbed phytosterols into bile
• high plasma LDL-C levels during childhood
• present with:
o tendinous xanthomata
o normal to high levels of LDL
o Premature CHD
o Cholesterol levels may be normal or
elevated.
Autosomal Dominant Hypercholesterolemia

• autosomal dominant disorder of the PCSK9 gene on chromosome 1 that is involved in
cholesterol homeostasis in the liver
• Two known mutations of the gene may lead to gain of function of PCSK9 :
o S127R
o F216L
• present clinically with increased plasma levels of LDL-C and exhibit higher risk of coronary heart
disease
Autosomal Recessive Hypercholesterolemia (ARH)

• ARH gene on chromosome 1
• ARH is also known as low density lipoprotein receptor adaptor protein 1 (LDLRAP1 )
• LDLR expression is normal, but LDL clearance rates are low 9888888888
HIGH TRIGLYCERIDES WITH NORMAL CHOLESTEROL
1. Diabetic Dyslipidemia
• consists of atherogenic dyslipidemia in type 2 DM patient
• treatment of LDL-C as the primary target in patients with this disorder
2. Familial Hypertriglyceridemia
• Isolated hypertriglyceridemia (or Type 4 hyperlipidemia)
• autosomal dominant disorder
• presents in adulthood with fasting triglyceride levels in the 200–500 mg/dL range
• Increased VLDL production with normal apoB resulting in fluffy triglyceride rich VLDL particles
• Premature CHD possibly from hypertriglyceridemia or coexisting obesity and insulin resistance
3. Lipoprotein Lipase Deficiency (Hyperlipoproteinemia Type 1 or Hyperchylomicronemia)
• rare, autosomal recessive disorder
• childhood with abdominal pain and pancreatitis
• Defective or absent LPL
• Fasting triglyceride levels may be >100 mg/dL and may rise to >10,000 mg/dL postprandially
• LPL deficiency do not develop premature CHD
• Treatment:
o Low fat diet
o Fat soluble vitamins
o Drug therapy
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ApoC-II Deficiency
• ApoC-II is an activating cofactor for LPL
• rare autosomal recessive form of familial hyperchylomicronemia
• presents in children and young adults
• abdominal pain and pancreatitis
• treated with plasma transfusions during severe hypertriglyceridemia providing apoC-II, which
will activate endogenous LPL
ApoC-III Excess
• interferes with the activity of lipoprotein lipase
• independent risk factor for CHD
ApoC-III levels can be increased:
o diabetes type 2
o hyperbilirubinemia
o kidney deficiency
o thyroid dysfunction
Affected by:
o Age
o Alcohol consumption in men and women
o Oral contraceptive use in women
ApoA-V
• highly hydrophobic protein that has a preference for binding to lipids and HDL particles
• hypothesized as being involved in VLDL assembly
• ApoA-V may be involved in activation of LPL-mediated triglyceride hydrolysis
• low levels of ApoA-V may promote hypertriglyceridemia, whereas high levels of ApoA-V would
have the opposite effect
HIGH CHOLESTEROL WITH HIGH TRIGLYCERIDES
Familial Combined Hyperlipidemia (Type 2B)

• relatively common disorder wherein affected individuals may have:
o simple hypercholesterolemia
o simple hypertriglyceridemia
o Mixed
Acquired Combined Hyperlipidemia
• common in patients who have metabolic syndrome
• the disease stems from a syndrome of conditions that include:
o type 2 diabetes mellitus
o Hypertension
o central obesity
o coronary heart disease
• liver increases the production of VLDL through high levels of free fatty acids in plasma.
• increased VLDL levels mature to LDL until this process is saturated
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Dysbetalipoproteinemia (Type 3)
• ApoE helps to clear these lipoproteins from circulation by binding to LDL receptor
Three common electrophoretic isoforms of apoE

1. apoE-3 most common
2. apoE-4
3. apoE-2 lower affinity to LDL
Hepatic Lipase Deficiency
• mutations of the HL gene,
• rare familial disorder is associated with combined hyperlipidemia
• Levels of HDL-C are normal or increased
• In contrast to Type 3 hyperlipidemia, although b-VLDL is increased, the TC/TG ratio is not
increased
Cholesterol 7-Alpha-Hydroxylase Deficiency
• recessive disorder of the CYP7A1 gene that encodes cholesterol 7-alpha-hydroxylase protein
• resistant to statin therapy
• lower plasma cholesterol with atorvastatin and niacin therapy
LOW TOTAL CHOLESTEROL AND TRIGLYCERIDE
Abetalipoproteinemia
• also known as Bassen-Kornzweig syndrome
• rare, autosomal recessive disorder
• Mutations in the Microsomal Triglyceride Transfer Protein (MTP) gene located on chromosome
4
• Absence of lipoproteins containing Apo-B
o Includes: LDL and VLDL
• Hallmarks
o Difficulty in weight gain and growth
o Fat absorption problems
o RBC membrane defects
o Usually effects infants
Hypobetalipoproteinemia
• autosomal dominant disorder explained by nonsense or missense mutations in the apoB gene,
leading to synthesis of various truncated forms of apoB.
• Homozygous individuals have TC levels <50 mg/dL and present at an early age with fat
malabsorption and low plasma cholesterol levels
• They develop:
o progressive neurologic degenerative disease
o retinitis pigmentosa
o acanthocytosis
40
•
Chylomicron Retention Disease
• Chylomicron retention disease (Anderson’s disease) presents in childhood with fat
malabsorption and low levels of plasma lipids
• only apoB-48 is affected
• characterized by:
o hypocholesterolemia
o chronic diarrhea
o failure to thrive
o deficiency of fat-soluble vitamins
ISOLATED LOW HDL-C
Familial Hypoalphalipoproteinemia
• common autosomal dominant disorder that occurs in 1 in 400 people
• Affected men have HDL-C levels <30 mg/dL
• women have HDL-C levels <40 mg/dL
• criteria for familial hypoalphalipoproteinemia:
o low HDL cholesterol in the presence of
normal VLDL cholesterol and LDL
cholesterol levels
o absence of diseases or factors that lead
to secondary effects of
hypoalphalipoproteinemia
o the presence of a similar lipoprotein
pattern in a frst-degree relative
• Premature CHD is typically present
ApoA-I DefIciency and ApoC-III Deficiency
• rare autosomal recessive condition characterized by a reduction in the formation of HDL
• linked to point mutations in the apoA-I gene and to deletions/gene rearrangements at the
apoA-I/C-III/A-IV gene locus on the long arm of chromosome 11
• HDL-C levels are <5 mg/dL
Signs:
o corneal opacification
ApoA-I Variants
• rare specific amino acid substitutions in the apoA-I gene
• increase catabolism of HDL and apoA-I
• Homozygous patients generally present with autosomal recessive inheritance of low HDL-C
levels (approximately 10 mg/dL)
• Signs:
o corneal opacifcations
o xanthomata
41
•
Tangier Disease
• rare autosomal recessive disorder characterized by complete absence of HDL due to a
mutation in the ATP Binding Cassette A1 (ABCA1) gene on chromosome 9
• In the homozygous state, patients present with:
o low or undetectable HDL in plasma
o Hepatosplenomegaly
o peripheral neuropathy
o orange tonsils
Lecithin:Cholesterol Acyltransferase Deficiency

• very rare autosomal recessive disorder that occurs in two forms:
o a classic (or complete) familial LCAT
deficiency
▪ anemia
▪ increased proteinuria
▪ renal failure
o milder partial deficiency phenotype known as fish-eye disease

▪ progressive corneal opacifcation
▪ low plasma HDL levels (<10 mg/dL)
▪ variable hypertriglyceridemia
• In complete deficiency, HDL-C total cholesterol levels are normal or high.
• Without LCAT, most cholesterol remains unesterifed and HDL synthesis is impeded
ISOLATED HIGH HDL-C
Cholesteryl Ester Transfer Protein(CETP) Gene Defects

• CETP is involved in reverse transport of cholesterol by facilitating the transfer of cholesterol
esters from HDL
• CETP deficiency is an autosomal recessive disorder in which the transfer of cholesterol esters is
inhibited
• HDL particles are large and laden with cholesterol ester, and apoA-I is increased, as is HDL-C
(typically >100 mg/dL)
Lipids And Lipoproteins Assessment

Specimen Collection for Lipid Analysis
• Serum
• Fasting
o 12 hour fast preferred
o Avoid lipemia
▪ Presence of fat droplets suspended in a solution.
▪ Lipemia affects assays by affecting absorbance due to refraction of light
42
Ultracentrifuge
• High plasma lipid concentrations can cause excessive plasma turbidity and interfere with
spectrophotometric methods.
• Lipoproteins can be spun down in this special centrifuge.
• Plasma is placed inside the “donut.” Lipids spin to the outside of the donut.
HISTORICAL TOTAL CHOLESTEROL METHODOLOGIES

 Colorimetric measurement procedures are less costly but are subject to interfering substances
and may require extraction steps and strong acids
 Classical method = Liebermann-Burchard
 Involved extraction & hydrolysis. Uses sulfuric & acetic acids
 Results in formation of a green color, proportional to the cholesterol concentration
HDL Methodology
Methods
• Precipitation Reaction
 Dextran sulfate or phosphotungsate acid with magnesium chloride precipitates LDL and
VLDL lipoproteins
 HDL left in the supernatant is tested using cholesterol assay. The answer represents
the amount of HDL in the sample
Precipitation Method
 Drawbacks
 Elevated triglyceride levels
 Results in overestimation of HDL
 Many labs will not perform HDL testing when triglyceride concentrations exceed 400
mg/dL
 Homogeneous Reaction
 Detergents or enzymes binds sites of VLDL and LDL particles. HDL is then left to react
with colored products and can be measured
 Disadvantages- lacks specificity for HDL
 Desirable range ( > 60 mg/dL )
 Gray area (40-59 mg/dL )
 High risk (< 40 mg/dL )
TRIGLYCERIDE MEASUREMENT
 Methods
 Enzymatic
 Colorimetric
43
 Involve the liberation of glycerol by lipase

o Recommended ( <150 mg/dL )
o Borderline high ( 150-199 mg/dL )
o High ( 200-499 mg/dL )
o Very high ( > 500 mg/dL )
LDL Measurement
 Direct measurement of LDL is uncommon because of technical difficulities
 Friedewald estimation ( calculation )
 Test: Total Cholesterol, Total Triglycerides and HDL with routine procedure
 Estimate the LDL with the following :
• LDL= Chol- (HDL + VLDL)
• VLDL= Triglycerides/5
AMINO ACIDS
Peptide Bonds
• A chemical bond formed bet the carboxyl group of one amino acid and the amino group of
another
• Oligopeptides- 5 chains
• Polypeptides-6-30 chains
• PROTEINS – greater than 40 chains
• At least 1 of both amino &carboxylic acid functional group
• Building blocks of proteins
• Chemical properties determine biological activity
• Origin
o Majority made in the human
▪ Generated from amino acid pool
▪ Generated from breakdown of proteins
o Metabolism
▪ 10 amino acids needed by human can be synthesized by body
▪ 10 amino acids (essential) must be supplied in proteins in food
o Essential Amino acids
▪ Ingested in diet
▪ “MILL PATH TV” Essential Amino Acids
• The liver and other tissues draw from this pool for synthesis of plasma and intracellular protein
• The liver and kidneys also play a role in excretion
• Transamination and deamination
• Deaminaion produces ammonium ions w/c are used in the synthesis of urea
• Urea is then excreted by the kidneys
44
Amino Acids Concentration

• Vary during the day by 30%
• Highest during the first 5 days of life, esp premature neonates
• Maternal values are low in the first half of pregnancy
• Urine excretion varies with age
Aminoacidopathies
• Rare inherited disorders
• Enzyme defect that inhibits the body’s ability to metabolize certain amino acids
• Abnormality due to problem with enzyme activity or the membrane transport system for amino
acids
• Cause severe medical problems due to the buildup of toxic amino acids and/or byproducts of
amino acid metabolism in blood
Phenylketonuria (PKU)
• Absence of phenylalanine hydroxylase (PAH)
• Musty odor of urine
• Mental retardation and microencephaly
Tyrosinemia I
• Absence of Fumarylacetoacetate hydrolase
• Failure to thrive, jaundice, cabbage like odor
Tyrosinemia II
• Absence of Tyrosine aminotranferase
• Symptoms include excessive tearing,
abnormal sensitivity to light (photophobia),
eye pain and redness, and painful skin lesions
on the palms and soles.
• half of individuals with type II tyrosinemia are
also mentally challenged.
Tyrosinemia III
• Absence of 4-Hydroxyphenylpyruvate oxidase
• Symptoms inclue mild mental retardation, seizures and intermittent ataxia
Alkaptonuria
• Lack of homogentisate oxidase
• ↑ homogentesic acid in urine
• Blackening of urine/brownish-black urine
• Clinical feature:
▪ Ochronosis (tissue pigmentation)
45
Maple syrup urine disease

▪ Absence or reduction of alpha-ketoacid decarboxylase blocking normal metabolism of 3
essential amino acids (leucine, isoleucine, valine)
▪ Hallmark feature is the odor of maple syrup or burnt sugar in the urine, breath and skin
▪ Clinical features:
• Failure to thrive, muscular rigidity, mental retardation, hypoglycemia
Isovaleric acidemia
▪ Lack of Isovaleryl-CoA dehydrogenase
▪ Has a characteristic Sweaty feet odor
▪ Can damage the brain
Citrullinemia
▪ Arginosuccinic acid synthetase
▪ ammonia builds up in the body, they develop a lack of energy (lethargy), poor feeding,
vomiting, seizures, and loss of consciousness.
Arginosuccinic aciduria
▪ Argininosuccinic acid lyase
▪ unwilling to eat and mental retardation
Homocystinuria
▪ Absence of cystathionine-beta-synthetase
▪ High level of Cysteine to methionine in urine and plasma
▪ Clinical features:
• Osteoporosis, dislocated eye lenses, mental retardation
Cystinuria
▪ Defect in amino acid transport system not metabolic enzyme deficiency
▪ Results in the formation of stones
• Inadequate reabsorption of cystine in the kidneys
• Hematuria, UTI, pain in the side (kidney pain)
AMINO ACID ANALYSIS

• Blood samples should be drawn after at least a 6- to 8-hour fast.
• Sample is collected in a heparin tube & plasma removed promptly from cells.
• Take care not to aspirate platelet & white cell layer, no hemolysis.
• Deproteinization within 30 minutes of sample collection
• Analysis performed immediately or sample frozen at –20°C to –40°C
 Urinary analysis performed on random specimen for screening
 Method of choice for screening is thin-layer chromatography.
PROTEINS
• Comes from the greek word “proteis” meaning “First rank of importance”
• Composed of one or more unbranched chains of amino acids.
• Most plasma proteins are synthesized in the liver and secreted by hepatocyte into circulation
▪ Except for immunoglobulins
▪ Synthesized in the plasma cells
• Built from one or more chains of amino acids
• Thousands of different proteins in the body
46
• Responsible for 50-70% cell’s dry weight

• Structural elements of cells and tissues
• Soluble in ICF and ECF
• Contain carbon, hydrogen, oxygen, sulfur and nitrogen
• Nucleotide sequence in the genes dictates the amino acid sequence of the protein
• Nitrogen Content: 16% in serum protein
• Charge & Isoelectric Point
 Isoelectric point (pI): pH at which protein has no net charge
 If pH > pI, charge is negative; if pH < pI, charge is positive.
• Solubility: > charge = > solubility in water
Synthesis
o Plasma proteins made in liver then secreted by hepatocyte into circulation
o Immunoglobulins made in plasma cells
o Insufficient dietary quantities of amino acids will limit synthesis and lower body levels of
proteins
Catabolism (Breakdown)
o Goal is to remove nitrogen from the system
o Occurs in the digestive tract, kidneys, and liver
o Produces ammonia, then urea
 Urea excreted via urine
o Produces ketoacids
 Ketoacids converted to glucose or fat
Classification
o By function: enzyme, hormone, transport, immunoglobulin, structural, storage, energy source,
osmotic force
o By structure: database (manual & automated), simple, conjugated
Simple proteins
 Proteins made of only amino acids
 Further classified as globular or fibrous
Conjugated proteins
 Proteins with nonprotein groups attached
 Metalloproteins
 Lipoproteins
 Glycoproteins
 Mucoproteins
 Nucleoproteins
 Phosphoproteins
Fibrous
 Mainly structural
 Fibrinogen, troponin, collagen, myosin
Globular
47
 Hemoglobin, enzymes, peptide hormones, plasma proteins

 Compact
 Narrow pH window
 Denaturation
Denaturation
• Disruption of structure
• Results in loss of function and protein chemical properties
• Caused by:
 Heat
 pH changes
 Mechanical forces
 Exposure to UV light
 Exposure to chemicals
Plasma Proteins
• Hundreds of them present in blood
• Synthesized in the liver (mostly)
o Acute-phase reaction proteins
o carrier proteins
o Fibrinogen/other coagulation proteins
o Complement proteins
o Immunolglobulins
o Enzyme inhibitors
o Precursors
TWO GROUPS
1. Albumin
• Most abundant in the plasma
• Synthesized in the liver by the hepatic parenchymal cells
• Functions
o Maintenance of colloid osmotic pressure
o Buffers pH
o Negative acute phase reactant Binds substances in the blood
• Analbuminemia
o rare genetic deficiency
o Related to abnormal lipid transport
• Inflammation
o Most common cause of hypoalbuminemia
2. Globulins
• Group consisting of α1, β, α2, and gamma fractions
• Focus will be on globulins most often encountered in the lab
Hypoproteinemia
48
 Total protein level <6.4 g/dL

 Due to a negative nitrogen balance
 Causes
◦ Excessive loss
 renal disease, blood loss, burns
◦ Decreased intake
 Malnutrition, intestinal malabsorption
◦ Decreased synthesis
 Liver disease, inherited immunodeficiency
◦ Acceleration of catabolism of proteins
 Burns, trauma
Hyperproteinemia
• Total protein level > 8.3 g/dL
• Causes
 Dehydration
o Excess water loss leads to the increased concentration of proteins
o Examples: vomiting, diarrhea, diabetic acidosis, hypoaldosteronism
 Excessive Production of gamma globulins

o Examples: multiple myeloma, Waldenstrom’s macroglobulinemia
ASSESSMENT OF PROTEINSTOTAL
Nitrogen
 Measures all chemically bound nitrogen in a sample using chemiluminescence
 Useful in assessing nitrogen balance
Total Protein
 Specimen Collection
 Serum
 Avoid hemolysis and lipemia
 Reference Range (6.4-8.3 g/dL )
 Methods : Kjeldahl, refractometry, biuret, dye binding
A/G Ratio
 Globulin concentration can be calculated by subtracting the albumin from total protein. Globulin
= Total Protein (g/dL) – Albumin (g/dL)
 The A/G ratio can then be determined by dividing the albumin concentration by the calculated
globulin.
Urinary Protein
 Sources
 Blood
 Kidney, urinary tract, vagina and prostate
 When proteins appear in the urine, they have not been reabsorbed by the renal tubules
49
 Screen: urine dipstick

 Quantitative: 12- or 24-hour urine
CSF proteins
 Reference range (15-45 mg/dL )
 Increased total CSF proteins
 Bacterial, viral, fungal meningitis
 Traumatic tap
 Multiple sclerosis
 neoplasm
Non - Protein Nitrogen

• NPN (Non - Protein Nitrogen) is a “funky” term that can be used for a bunch of different
substances that have the element nitrogen in them, but are NOT proteins.
• Term originated when analytic methodology required removal of protein from sample before
analysis
• Concentration of nitrogen-containing compounds was quantified spectrophotometrically by
converting nitrogen to ammonia
• This is a little unusual, because most of the body’s nitrogen is associated with proteins.
• There are many different unrelated NPNs, but we are only interested in 4 of them:
• Creatinine, Blood Urea Nitrogen (BUN), Uric Acid and Ammonia
• In general, plasma NPNs are increased in renal failure and are commonly ordered as blood
tests to check renal function
Most important NPNs

 BUN (Blood Urea Nitrogen)
 Creatinine
 Uric acid
 Ammonia
Urea (Blood Urea Nitrogen)

o Blood Urea Nitrogen = BUN = Urea
o 50% of the NPNs
o Product of protein catabolism which produces ammonia o Ammonia is very toxic –
converted to urea by the liver
o Liver converts ammonia and CO2
o Filtered by the glomerulus but also reabsorbed by renal tubules (40%)
o Some is lost through the skin and the GI tract (< 10%)
o Plasma BUN is affected by :
▪ Renal function
▪ Dietary protein
▪ Protein catabolism
Physiology
50
• Nitrogen is released as a result of protein and amino acid catabolism, converted to urea and
excreted as a waste product
• After synthesis in liver, urea is carries in blood to kidney and filtered out
• Most urea in glomerular filtrate is excreted in urine, but some is reabsorbed in renal tubules
▪ Azotemia = Elevated plasma BUN
▪ Uremia = very high plasma urea concentration with renal failure
▪ Prerenal  BUN (Not related to renal function )
o Low Blood Pressure (CHF, Shock, hemorrhage, dehydration)
o Decreased blood flow to kidney = No filtration
o Increased dietary protein or protein catabolism
▪ Prerenal  BUN (Not related to renal function)
o Decreased dietary protein
o Increased protein synthesis ( Pregnant women , children )
Renal causes of  BUN

Renal disease with decreased glomerular filtration
o Glomerular nephritis
o Renal failure form Diabetes Mellitus
Post renal causes of  BUN (not related to renal function)

Obstruction of urine flow
o Kidney stones
o Bladder or prostate tumors
o UTIs
Methods
 Enzymatic (urease), electrode reference method using isotope-dilution mass spectrometry
 Enzymatic methods (indirect)
o Berthelot method
o Glutamate dehydrogenase method
 Chemical methods (direct)
o Fearon method
o Fearon Diazine method
Specimen: Plasma, serum, urine

 In plasma, avoid ammonium ions and high citrate and fluoride
 Susceptible to bacterial decomposition; use quickly or chill
 Reference intervals for adults
o In plasma or serum: 6-20 mg/dl (2.1-7.1 mmol urea/day)
o Urine, 24-hour: 12-20 g/day (0.43-0.71 mmol urea/day)
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CREATININE/CREATINE - Creatinine is formed from creatine and creatine phosphate in muscle

(oxidation of creatine) and is excreted into plasma at rate related to muscle mass
o Plasma creatinine is inversely related to glomerular filtration rate (GFR) and is commonly
used to assess renal filtration function
Physiology
o Creatine is synthesized mainly in liver from arginine, glycine and methionine
o It is transported to other tissues and converted to creatine phosphate, which serves a
highenergy source.
o Creatine phosphate and creatine form creatinine, which diffuses into plasma and is excreted
in urine
Clinical Application
o Sufficiency of kidney function and severity of kidney damage and to monitor progression of
kidney disease
o Creatinine clearance: measure amount of creatinine eliminated from blood by kidneys.
Clearance is defined as the volume of plasma from which a measured amount of substance
can be completely eliminated into the urine per unit time
CREATININE DISEASE CORRELATIONS
Creatinine
o Elevated concentration associated with abnormal renal function, esp as it relates to
glomerular function
o When plasma creatinine is elevated, GFR is decreased, indicating renal damage
Creatine
o Elevated concentration associated with muscle disease: muscular dystrophy, poliomyelitis,
hyperthyroidism, trauma
o Not elevated in renal disease
CREATININE DISEASE CORRELATIONS
o Increased plasma creatinine associated with decreased glomerular filtration (renal function)
o Glomerular filtration may be 50 % of normal before plasma creatinine is elevated
o Plasma creatinine is unaffected by diet
o Plasma creatinine is the most common test used to evaluate renal function
o Plasma creatinine concentrations are very stable from day to day - If there is a delta check ,
its very suspicious and must be investigated
METHODS
1. Jaffe reaction (time consuming, not readily automated)
o Creatinine reacts with picric acid in alkaline solution to form red-orange chromogen
2. Kinetic Jaffe method (rapid, inexpensive, easy to perform)
o Serum is mixed with alkaline picrate and rate of change in absorbance is measured
3. Coupled enzymatic methods (improved specificity)
4. Isotope dilution mass spectrometry (reference method)
Specimen: Plasma, serum, urine
 Hemolyzed and icteric samples should be avoided
52
 May be refrigerated for 4 days; frozen for longer storage

Sources of error
 Ascorbate, glucose, alpha-ketoacids, and uric acids may increase creatinine
concentration measured by Jaffe reaction (and some enzymatic methods)
 Bilirubin causes negative bias in both Jaffe and enzymatic methods
 Patient use of cephalosporin antibiotics, dopamine, lidocaine
CREATININE CLEARANCE
o Calculated measurement of the rate at which creatinine is removed from the plasma by the
kidneys
o Measurement of glomerular filtration (renal function)
o A good test of glomerular filtration because
▪ Creatinine is an endogenous substance (not affected by diet)
▪ Creatinine is filtered by the glomerulus, but not secreted or re-absorbed by the
renal tubules
Creatinine Clearance specimens

o 24-hour urine specimen
o Plasma / serum creatinine collected during the urine collection
o 24 Hour Creatinine Clearance Formula
CREATININE CLEARANCE =
U = Creatinine concentration of the 24-hour urine (mg / dl)

V = 24 hour urine volume (mls) per minute - V / 1440 = mls / minute
P = Plasma creatinine concentration ( mg / dl )
A = Correction factor accounts for differences in body surface area
obtained from a height – weight chart
Procedure for 24 Hour Urine Collection

 Have the patient empty his / her bladder (discard this urine).
 Note the time. For the next 24 hours, have the patient collect and save all urine in an
appropriate container.
 At the end of the 24-hour period have the patient void one last time into the urine
container. This completes the collection.
 If possible, keep the urine specimen refrigerated.
Uric Acid
• Breakdown product of purines (nucleic acid / DNA)
• Purines from cellular breakdown are converted to uric acid by the liver
• Uric acid is filtered by the glomerulus. Most is reabsorbed in proximal tubules and reused
• Relatively insoluble to plasma. Elevated plasma uric acid can promote formation of solid uric
acid crystals in joints and urine
53
Physiology
o Purines are converted to uric acid in liver
o Uric acid is transported in plasma from liver to kidney and filtered by glomerulus
o 70% is eliminated by renal excretion; remainder passes into GI tract and is degraded by
bacterial enzymes
o Assessment of inherited disorders of purine metabolism
o Confirmation of diagnosis and monitoring of treatment of gout
o Assistance in diagnosis of renal calculi
o Prevention of uric acid nephropathy in chemotherapy
o Detection of kidney dysfunction
URIC ACID DISEASES

▪ Hyperuricemia = elevated levels of uric acid found in:
• Inherited disorders of purine metabolism
• Gout
o Most in the form of Monosodium urate
o Insoluble at the pH of plasma
o Will precipitate out when >6.4 mg/dl in plasma
o Crystals will form in urine at a pH <5.75
o Increased plasma uric acid
o Painful uric acid crystals in joints
o Usually in older males (> 30 years-old)
o Associated with alcohol consumption
o Uric acid may also form kidney stones
▪ Other causes of increased uric acid

• Leukemias and lymphomas (  DNA catabolism)
• Megaloblastic anemias (  DNA catabolism)
• Renal disease (but not very specific)
▪ Hypouricemia = decreased levels of uric acid found in:

• Liver disease
• Defective tubular resorption
• chemotherapy
Methods
1. Caraway method
2. Uricase methods
3. Coupled enzyme method
4. Isotope dilution mass spectrometry
o Specimen: Heparinized Plasma, serum, urine
o Remove serum from cells quickly to prevent dilution by intracellular contents
o Diet may affect concentration overall, but fasting not necessary
54
Reference intervals for adults

o In plasma or serum: male, 3.5-7.2 mg/dl; female 2.6-6.0 mg/dl
o Child: 2.0-5.5 mg/dl
o Urine, 24-hour: 250-750 mg/dl
Ammonia
o Produced from the deamination of amino acids in the muscle and from bacteria in the GI
tract
o Ammonia is very toxic - The liver converts ammonia into urea
o Urea is less toxic and can be removed from the plasma by the kidneys
o In severe hepatic disease, the liver fails to convert ammonia into urea, resulting in increased
plasma ammonia levels
Increased plasma ammonia concentrations in:
o Liver failure
o Reye’s Disease
Physiology
 Produced in catabolism of amino acids and by bacterial metabolism in lumen of intestine
 Some results from anaerobic metabolic reactions in muscle during exercise
 Excreted as ammonium ion by kidney and acts to buffer urine
 Determination of prognosis for severe liver disease
 Determination of severity and prognosis of Reye’s syndrome
 Diagnosis of inherited deficiency of urea cycle enzymes
 Monitoring of hyperalimentation therapy
Elevated concentrations are seen in following conditions:

 Severe liver disease
 Encephalopathy
 Inherited deficiency of enzymes of urea cycle
Methods
o 2-step approach in which ammonia is isolated from samples and then assayed
o Direct measurement of ammonia by enzymatic method or ion-selective electrode
o Low concentration, volatile nature, instability, easy contamination – testing difficult
Historical Methods
o Conway 1935 – volatilize, absorbed then titrated
o Dowex 50 cation-exchange column + Berthelot reaction
Glutamate dehydrogenase
55
o Decrease in absorbance at 340 as NADPH is consumed (oxidized)
Direct ISE
 Change in pH of solution as ammonia diffuses through semi-permeable membrane
 Reference Interval: Adult Plasma 19 – 60 μg / dl
Specimen:
 Venous blood should be obtained without trauma and placed on ice immediately
 EDTA or Heparin are suitable anticoagulants
 Samples should be centrifuged at 0-4C within 20 minutes of collection and plasma or serum
removed
 Patient should not smoke for several hour before collection
 Delayed testing caused false increased values
Sources of error:
 Eliminate sources of contamination: tobacco smoke, urine ammonia in detergents, glassware,
reagents and water
56

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Clinical Chemistry 1

-Laarni Hannah C. Lacorte, RMT, MSMLS

BASIC PRINCIPLES AND PRACTICES

ROLE OF THE MEDICAL TECHNOLOGIST

CLINICAL LABORATORY SUPPLIES

2. Glassware and Plasticware

ii. Aluminosilicate (Corex®)

iii. High silica

iv. Acid /alkali resistant (Vycor®)

v. Amber colored (Low actinic)

vi. Lime soda (Flint glass)

CALIBRATION MARKS OR DRAINING CHARACTERISTICS

MEASURING OR GRADUATED PIPETS

Dessicators and Desiccants

▪ Electronic Top-loading balance

BASIC SEPARATION TECHNIQUES

LABORATORY SAFETY AND REGULATIONS

Signage and Labeling

CONTROL OF OTHER HAZARDS

DISPOSAL OF HAZARDOUS MATERIALS

o Burn flammable material in specially designed incinerators.

QUALITY ASSURANCE & QUALITY CONTROL

INTRALAB QC (Internal QC)

CONTROL SOLUTIONS (QC MATERIALS)

CONTROL LIMITS (CONTROL VALUES)

CHARACTERISTICS OF AN IDEAL QC MATERIALS:

4. No matrix effects/known matrix effects

ERRORS WHICH CAN BE OBSERVED ON LJ CHART:

WESTGARD CONTROL CHART

– Instruments using fluorescence-polarization immunoassay, nephelometry, &

 Light - is a form of electromagnetic energy

 It involves measurement of the light transmitted by a solution to determine the concentration of

BEER’S LAW (A = 2-Log%T)

3. Monochromator - It ISOLATES specific or individual wavelength of light.

6. Photodetector - Detects and converts transmitted light into photoelectric energy.

7. Meter or Read-out Device - It displays output of the detection system.

Flame Emission Photometry (FEP)

Atomic Absorption Spectrophotometry (AAS) - It measures the LIGHT ABSORBED by atoms

VOLUMETRIC AND DENSITOMETRY

Volumetric- A.k.a Titrimetric

Fluorometry (A.k.a. Molecular Luminescence Spectrophotometry )

Uses 2 monochromators (either filters, prisms or gratings)

COMPONENTS OF FILTER FLOUROMETERS

5. Photodetector and Stray light Trap

▪ Factors that affects migration:

 Liquid Chromatography-Mass Spectroscopy (LC-MS) – used for detecting

Freezing point depression osmometry

 Electrode with a constant voltage

Mass Spectrometry (MS)

Public Relations & Client Interaction

The Vascular System - Network of arteries, veins and capillaries.

ARTERIES AND VEINS ARE COMPRISED OF THREE LAYERS OF TISSUE:

TYPES OF BLOOD SPECIMENS

CAPILLARY SPECIMEN COLLECTION

Capillary Order of Draw

BIOCHEMICAL PATHWAYS IN CARBOHYDRATE BREAKDOWN

Hexose monophosphate shunt

REGULATION OF GLUCOSE METABOLISM

WHO CATEGORIES FOR DIABETES