LABORATORY INSTRUMENTATION

ANALYTIC TECHNIQUES

Automation

1. Optical Techniques
2. Electrochemistry
3. Electrophoresis
4. Chromatography

OPTICAL TECHNIQUES FLAME EMISSION SPECTROPHOTOMETRY

● Has something to do with light/radiant energy ● Measures light emitted by excited atoms.
● 1. Spectrophotometry
● 2. Reflectance photometry In your previous Chemistry, you have learned that atoms
● 3. Flame Emission Spectrophotometry can be excited or they can be placed in their un-excited or
● 4. Atomic Absorption Spectroscopy ground state. From ground state to excited state and from
● 5. Chemiluminescence excited state to ground state.
● 6. Fluorescence
● 7. Nephelometry
● 8. Turbidimetry

REFLECTANCE PHOTOMETRY

● Reflector – A filter photometer that measures the

quantity of light reflected by a liquid sample that
has been dispensed into a grainy or fibrous solid
support. ● That characteristic of atoms is explored in this
technique.
Remember, in your Clinical Microscopy, you made use of ● In Flame Emission, the atoms are placed in their
urine dipsticks. Those dipsticks use this principle excited state by subjecting them in hot flame, and
(Reflectance photometry). Urine - Liquid sample ; then each metallic element emits light as their
Dipstick pad/Urine pad - Fibrous solid support. characteristic.
● The electro-components used are almost the
same as those in absorbance photometry.
● The clinical application of this technique is seen in
your Dry-Film Chemistry Systems
○ Example of Dry-Film Chemistry system is
the Vitros Machine:

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 ● Sodium produces a yellow flame
● Potassium produces a violet flame
● In this technique, the light intensity of the
characteristic wavelength produced by each of
the atoms is directly proportional to the
concentration of the substance interest in the
sample.
Flame emission has been widely used to determine the
concentrations of Sodium, Potassium, and Lithium. But
with the development of ion selective electrode, today it is
no longer routinely used.
For example, in this setup, our target analyte is Sodium.
We will measure the concentration of Sodium from this
sample. So the sample will be exposed to flame in order
to excite the atoms. So, once the atoms are excited, they
will release light. So for Sodium, it’ll produce a yellow
light. But you have to remember that it is a mixture and is
not only composed of Sodium. It is possible that it also
contains Potassium or other elements. So when the
sample is subjected to the flame, the other elements will
also be excited and would also emit light. So it will emit a
polychromatic light. Since we’re only after the yellow
light/Sodium, we have to use a monochromator which
will isolate our target light. Once the yellow light has been
isolated, it will pass through the exit slit. After passing
through the exit slit, it will be detected in the detector, and
the amount of light that reaches the detector is
proportional to the amount of Sodium from the
sample.
ATOMIC ABSORPTION SPECTROSCOPY
(AAS)
● Widely used to measure elements such as
Aluminum, Calcium, Copper, Lead, Lithium,
Magnesium and Zinc.
● Used to measure concentration by detecting the
absorption of electromagnetic radiation by atoms
rather than by molecules.
● Said to be 100 times more sensitive than
Flame Emission because it uses
Hollow-cathode lamp.
● Hollow-cathode lamp - light source for AAS.
Such lamps are made of the metal of the
substance to be analyzed (they are different in
each analysis depending on your analyte).
○ Example if our target analyte is Sodium,
the cathode we will use is made of
Sodium, and then it will emit Sodium light
at a particular wavelength of 589
nanometer.
In AAS, the element is exposed to flame in order to
dissociate it and then place it in its ground state. So once
the element is in its ground state, it is capable of
absorbing radiation corresponding to its own wavelength.
The amount of light absorbed is proportional to the
concentration of the analyte we are after.
As the atom absorbs light, it also becomes excited, and it
emits light. In order to isolate the absorbed light, the
hollow-cathode lamp is modulated by inserting a
mechanical chopper or by pulsing the electric supply to
the lamp.
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● Light beam being absorbed is in pulses – the
transmitted light beam will also be in pulses.
● Two light signals from the flame
● Alternating signal – comes from the hollow
cathode
● Direct signal – comes from the excited atoms in
the flame
● Flameless AAS Technique
○ The strips of tantalum or platinum metal
are used as sample cups, and then the
sample cups with the sample are placed
in a depression or carbon rod in an
enclosed chamber.
○ Instead of flame, it uses an electric
furnace in order to break the chemical
bonds or to dissociate the atoms.
○ It is said to be more sensitive than the
conventional flame method.
● AAS
○ ADVANTAGES
○ More sensitive than flame emission
○ Sensitive and precise
○ Used to measure concentration of trace
elements not are not easily excited
○ DISADVANTAGES
○ Inability of the flame to dissociate ions
into free atoms
■ For example, Phosphate which
may interfere when you’re
measuring Calcium, which would
lead to the formation of Calcium
Phosphate. In order to remedy
that, you have to add
Lanthanum or Strontium to
compete with Calcium in binding
with Phosphate.
○ Ionization of atoms following dissociation
by flame
■ In order to remedy this, you have
to decrease the flame
temperature.
FLUOROMETRY
Many elements can absorb light and can undergo
electronic transition, from low to high energy levels. When
an atom or molecule absorbs light at a particular
wavelength, that molecule can pass from low energy to
high energy; from ground state to excited state. But that
molecule can only stay at its excited state for a particular
time, it will eventually return to its ground state. And as it
returns to its ground state, it re-emits light. And the
re-emitted light is of longer wavelength than the
absorbed light.
● Fluorescence – occurs when a molecule absorbs
light at one wavelength and re-emits light at a
longer wavelength.
● Fluorophore - an atom or molecule that
fluoresces.
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 ● Fluorometry – measurement of the emitted ○ Incandescent tungsten lamp - seldom
fluorescence light. used because they release little energy
● Stokes shift - the difference between the in the UV region
● MONOCHROMATOR - used for the isolation
excitation and emitted fluorescence
of incident light
○ Grating, Prisms, Filters
■ For spectrofluorometry, the
filters are replaced with
gratings and prisms
● DETECTOR
○ Photomultiplier tube (PM Tube) - has a
higher sensitivity to low light intensities

● FLUOROMETRY
○ ADVANTAGES
○ Specificity - increased specificity by
selecting the optimal wavelength for
both absorption and fluorescence
rather than just the absorption as seen
in spectrophotometry
○ Sensitivity - 1000 times more sensitive
than the conventional
spectrophotometry

○ DISADVANTAGES
○ Very sensitive to environmental
This demonstrates the components of a basic filter change - change in pH can affect the
fluorometer. The light source emits short wavelength high availability of the electrons and then
energy excitation light. Then a mechanical attenuator the UV light used for excitation can
controls the intensity of light. The first filter/primary filter is cause photo-chemical changes
found between the sample and the source. Then as the ■ Any decrease in fluorescence
resulting from the things
light reaches the sample, the molecules in the sample
mentioned above is called
re-emit light. So the sample fluoresces and emits QUENCHING.
radiant energy in all directions. Between the cuvette
and the photodetector is the secondary filter which A lot of factors can affect fluorometry that’s why
isolates the longer wavelengths of fluorescent light, extreme care is required.
and it prevents the incident light from striking the
detector. The electrical output of the detector is
directly proportional to the intensity of the
fluorescent light.
CHEMILUMINESCENCE
Components of Fluorometer:
● Is the emission of light when an electron
● LIGHT SOURCE returns from an excited or higher energy level
○ Mercury - used for filter fluorometry to a lower energy level.
○ Xenon Arc - used for ● Somehow similar to Fluorescence except that
spectrofluorometry in this one, the excitation is caused by a

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 Chemical reaction (Recall: in fluorescence,
the excitation event is caused by a radiant
energy in the light source)
● The chemical reaction involves the oxidation of an
organic compound such as: Luminol,
Isoluminol, Acridinium esters, or Luciferin
● The oxidation of an organic compound by an
oxidant such as Hydrogen peroxide, Hypochlorite,
or Oxygen
● Since we’re talking about chemical reactions, they
require the presence of a catalyst
● Chemiluminescence assays are ultrasensitive
● Automated immunoassay and DNA probe assay
systems
Basic components of Chemiluminescence
● Sample cell - housed in a light tight chamber
● Injection system - adds reagent to the sample
cell
● Detector - usually a photomultiplier tube,
however, charge-coupled detector such as x-ray
film and photographic film has also been used
● LIMITATIONS
○ Light leaks, light piping, and high
background luminescence from assay
reagents and reaction vessels are
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common factors that degrade the
analytical performance of luminescence
measurements
○ Requires stringent controls on the purity
of reagents and the solvents.
NEPHELOMETRY AND TURBIDIMETRY
● Used to measure the concentration of large
particles which cannot be measured using
absorption spectroscopy.
● Based on the scattering of radiation by particles in
suspension.
● Instrument operation is the same as for any
spectrophotometer
NEPHELOMETRY
● Is the measurement of light scattered by a
particulate in a solution.
● The light scattered by the particles is usually
measured at an angle, typically 15 to 90
degrees.
● The light scattering in Nephelometry depends on
the wavelength and particle size
○ For macromolecules with a size close to
or larger than the wavelength of the
incident light, sensitivity is increased by
measuring the forward light scatter
SETUP FOR NEPHELOMETRY
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 The light scattering depends on the wavelength of the These techniques make use of electrochemical cells
incident light and particle size. Photodetector is placed in
15-90 degrees in order to catch the scattered light. Two types of Electrochemical Cells:

TURBIDIMETRY
● The measurement in the reduction in light
transmission caused by particle formation.
● The amount of light absorbed by a suspension of
particles depends on the specimen concentration
and on the particle size.
● Protein quantification in biological fluids (such as
urine and CSF)
● Galvanic
● Electrolytic
GALVANIC ELECTROCHEMICAL CELL
● The basis for potentiometric methods
● Potentiometric analytical techniques
● It operates spontaneously and produces a
potential difference by the conversion of chemical
into electrical energy.

ELECTROLYTIC ELECTROCHEMICAL CELL

SETUP FOR TURBIDIMETRY
Form the basis for:

● Amperometry
● Conductometry
● Coulometry
● Voltammetry

- Chemical reaction occur by the application of an external

potential difference

The main difference from galvanic electrochemical cell is

The photodetector is placed in parallel with the light that it requires a external potential difference for a
source and cuvette. chemical reaction to occur

ANALYTIC TE#C Electrochemical Cell

HNIQUES
ELECTROCHEMISTRY

● Involves the measurement of current or voltage

generated by the activity of specific ions.

TECHNIQUES

1. Potentiometry
2. Amperometry
3. Coulometry This is a schematic diagram of an electrochemical cell
4. Polarography that consists of two (2) half cells or electrodes, cathode

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 and anode and is connected by a salt bridge. The salt
bridge is saturated with electrolytes.
POTENTIOMETRY
● The measurement of an electrical potential
difference between two electrodes in an
electrochemical cell
● The electrochemical cell used for this technique is
the galvanic electrochemical cell
● The pH meter is a special type of potentiometer
The galvanic cell and potentiometry consists of two
electrodes, the reference electrode and the indicator
electrode.
Reference Electrode
● The electrode with constant voltage
● Most commonly used reference electrodes in the
clinical setting are calomel and silver/ silver
chloride electrodes.
● The potential in this electrode is known, constant
and completely insensitive to the composition of
the solution understudy
Indicator Electrode
● Considered to be the measuring electrode
● The most commonly used indicator electrodes are
the ion selective electrode and the glass
electrode.
A schematic diagram of an ion selective membrane
electrode based potentiometric cell. The reference
electrode is a silver/silver chloride and the indicator
electrode is an ion selective membrane.
pH Meter
● is a potentiometer that measures the hydrogen
ion concentration in a sample
● Acidic = ↑ Hydrogen ions
● Basic = ↓ Hydrogen ions
A pH meter consists of an indicator electrode and a
reference electrode. The indicator electrode consists of
a silver wire covered with silver chloride. It is immersed in
an internal solution of hydrogen chloride and the tip is
sensitive to hydrogen ions. This is a special membrane
glass tip. On the other hand, the reference electrode can
either be a silver chloride or calomel and is immersed in
an internal solution of potassium chloride. There is a filling
hole used to add KCl or potassium chloride solution.
There is also a tiny opening at the bottom which is
required for the completion of electric contact between the
reference and indicator electrode.
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The leftmost is the indicator electrode with a silver wire
filled with internal HCl solution. This is the sensitive glass
membrane tip that is sensitive to hydrogen ions. The one
next to it is the reference electrode. The right image
shows an indicator and reference electrode connected to
a voltmeter.
As the pH electrode is placed in a solution like hydrogen
chloride, the hydrogen from the HCL solution does not
necessarily penetrate the glass membrane but ion
exchange is involved. The hydrogen ions from the test
solution will move near the tip of the glass membrane as
well as the hydrogen ions from the internal solution.
Remember that there is a hydrogen chloride internal
solution in the indicator electrode. So, the hydrogen
from the test solution will move near the membrane as
well the hydrogen from the internal solution of the
indicator electrode. Therefore, creating a potential
difference between the internal and test solution. This
potential difference is measured as the pH and read
by the voltmeter.
If the hydrogen ion concentration is greater on the outside
of the solution, the solution is said to be acidic. If the
hydrogen ion concentration is greater on the inside
solution, the solution is said to be basic. Overall, the
general electromotive force generated by the hydrogen
ions in the glass tip is described by the nernst equation.
If the hydrogen ion concentration on the inside and
outside is equal, then no potential difference is created
thus, the solution/pH is neutral or 7.
VOLTAMMETRY/AMPEROMETRY
● This is another electroanalytical technique and is
the most sensitive
● Widely applicable
● This method is based on electrolytic
electrochemical cell which an external voltage is
applied to polarize working electrode
● The application of this method is used in the
determination of partial oxygen in the blood
COULOMETRY
● This measures the electrical charge passing
between two electrodes in an electrochemical cell
● it is used in the determination of chloride
ELECTROPHORESIS
● an analytical technique that is capable of
separating and analyzing a diverse range of
ionized analytes.
● is the migration of charged solutes or particles in
an electric field.
Iontophoresis
● migration of small ions
Zone Electrophoresis
● migration of charged macromolecules
○ Macromolecules that are of interest in
clinical laboratories are proteins in serum,
urine, CSF, and other body fluids.
● most commonly used technique in clinical
applications
To completely understand electrophoresis, it is important
to know that chemical species, once ionized, they take on
an electric charge.
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 1. Cations - positively (+) charged ions
2. Anions - negatively (-) charged ions
As in the law of physics, opposite attracts. So, positively
charged ions are attracted to a negative electrode called
Cathode while negatively charged ions are attracted to a
positive electrode called Anode.
3. Ampholyte is a molecule that is either positively
or negatively charged. If a solution is acidic than
its isoelectric point, ampholyte takes on a positive
charge and in a more alkaline solution and
ampholyte takes on a negative charge
This is the point of origin (rectangular-shaped boxes)
where the samples have been introduced, and the
particles (black lines). Each particle traveled differently.
The particle (lowest line) traveled the farthest from the
point of origin, meaning that this particle has the lowest or
smallest molecular mass. These two particles (two
uppermost lines) almost stayed within the point of origin.
To conclude, they have the highest or largest molecular
mass compared to the other particles.
WHY IS ELECTROPHORESIS VALUABLE?

FACTORS AFFECTING THE RATE OF

MIGRATION

The rate of migration in the electrophoresis is dependent

on different factors.
1. Net electrical charge of the molecule
- rate of migration is directly proportional to the net
charge of the particle.
2. Size and shape of the molecule
The results obtained from electrophoresis enables us to
- rate of migration is inversely proportional to the
characterize proteins. From these results, we can
size of the molecule. The higher the molecular
characterize different proteins or if known proteins have
mass, the slower it will move into the electric field.
already been described from research, we can simply
3. Electrical field strength
detect their presence or absence in the sample, which
4. Properties of supporting medium
one is elevated or decreased using electrophoresis.
5. Temperature of operation
Electrophoretic mobility is directly proportional to net COMPONENTS OF ELECTROPHORESIS
charge and inversely proportional to molecular size. APPARATUS
In simple terms, the higher the charge, the greater the
mobility. The smaller the molecule, the greater the mobility
as well.

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 COMPONENTS OF ELECTROPHORESIS
● Electrodes
● Electrophoretic support
● Buffer
● Cover
● Power supply
The 2 buffer chambers containing buffer solution. The
support media is where the separation takes place. It
can either be in direct contact with the buffer or it can be
wick into the buffer. There are also the electrodes
(negative and positive) and power supply.
1. POWER SUPPLY
- the primary function of a power supply in an
electrophoretic process is to supply electric
power. The commercially available power
supplies allow operation under:
● Current (Constant Current) - Isoelectric
focusing Electrophoresis
● Voltage (Constant Voltage) - Capillary
electrophoresis
● Power (Constant Power)
Whatever you use, either current voltage or power, should
be constant in order to minimize heat evolution during
electrophoresis. This can affect both migration rate of
the protein and the rate of evaporation of water from the
stationary medium.
2. BUFFER
Functions:
● Carries the applied current
● Establishes the pH at which electrophoresis is
performed
● Determines the electrical charge of the solutes
● Most widely used buffers are made of monovalent
ions because their ionic strength and molality are
equal.
● IONIC STRENGTH: important property of a buffer
because it influences the ffg.
○ Conductance of the support
○ Thickness of the ionic clouds surrounding
a charged molecule
○ Rate of migration
○ Sharpness of the electrophoretic zones
● High ionic strength buffers yield sharper bond
separations
Thickness of the ionic cloud surrounding a charged
molecule
With the increasing buffer concentration, the ionic cloud
increases as well. The ionic cloud surrounds the particle
and inhibits or slows down the particle as it moves into the
electric field. Therefore, it influences or affects the rate of
migration.
3. SUPPORT MATERIALS
- provides the matrix in which the separation of the
molecules takes place. Various support medium
are used in electrophoresis such as:
- Cellulose acetate
- Agarose gel
- Polyacrylamide gel
- Starch gel

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● Cellulose acetate ● Polyacrylamide Gel
○ Dry, brittle film, 80% air space ○ Thermostable
○ When soaked in the buffer, the air spaces ○ Transparent
are filled with electrolyte and the film ○ Durable
becomes pliable. ○ Relatively chemically inert
○ Cellulose acetate is made by treating ○ Uncharged - eliminates endosmosis
cellulose with acetic anhydride, they ○ It separates proteins based on the
require clearing before scanning for charge to mass ratio and molecular
densitometry. size.
○ Cellulose acetate, once dried up, can be
stored for a long period of time.
○ These are also used for isoelectric
focusing.

Acrylamide is carcinogenic. Therefore, when you are

working with this type of material you have to exert extra
caution.
● Agarose Gel ● Starch Gel
○ Another widely used support medium - First material to be used as a support medium.
○ Agarose is purified essentially neutral - It separates macromolecules based on surface
fraction of agar. It does not produce charge and molecular size
electroendosmosis. - It is not widely used - because of technical
○ It does not bind protein, therefore, not difficulty in preparing the gel.
affecting the rate of migration.
○ Another advantage is that its native clarity PROCEDURE
after drying.
○ It requires only a small amount of sample
and can be stored indefinitely.
○ The separation of protein in agarose gel
is based on charge to mass ratio of the
protein

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 Generally, the preparation of the sample is done
according to the suggestion of the manufacturer of the
A. As you can see in the diagram, the casting tree is
supplies, so you have to read the inserts.
where you will place your support medium
(Agarose gel). STAINING
B. The gel is poured into the casting tray and then
● Once the electrophoretic separation is done, the
wait for it to solidify.
support medium is removed from the chamber
C. Once solidified, a comb is pushed down in order
then dried or placed into fixative. It is stained to
to create depressions or wells.
visualize the individual protein zones. The stains
D. The depressions will introduce or charge our
can be used to reveal their locations.
samples. Pipette the samples into the wells then
● Example of stains:
soak the samples for approximately 5 minutes in
○ Amido Black B
a hydrated support.
○ Coomassie Brilliant Blue
E. Once the casting tray is ready, it is placed into the
electrophoresis chamber. The electrophoresis
chamber is previously filled with a buffer solution
(transparent blue). Make sure that the support
medium is in contact with the buffer solution, if
not, you may add more buffer. After ensuring that
everything is good, turn on the power supply.
After a specific amount of time, the separation
takes place and then the medium is taken out of
the chamber and then it is fixed. if you do not
want to fix, make sure that it is completely dry in
order for the zones to not to diffuse. In order to DETECTION AND QUANTIFICATION
observe the different zones of separation, stain or ● UV Light - simplest way to accomplish detection
other methods can be used. ● Densitometry - most common and reliable way
for quantification of the individual zones.

TREATMENT AND APPLICATION OF SAMPLE

If Cellulose acetate and agarose gel are used as a
support medium, they require:
● 2 to 5 mL of sample
● Serum specimens - DILUTED
○ serum specimens contain high
concentrations of protein,
especially albumin.
● Urine and CSF - CONCENTRATED
● 5 minutes - Once the specimens are
prepared, it is allowed into the gel for
approximately 5 minutes. To remove
excess specimen you have to blot the
template. examples of DRIED, FIXED, STAINED SUPPORT
MEDIUM

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 ● Each individual zone is separated sharply.
● It can now be kept as a part of record-keeping as
to how long it depends on what type of medium is
used.
CHROMATOGRAPHY
● Analytical technique used in clinical laboratories
to separate and quantify different clinically
relevant analytes
● Refers to the 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.
Chromatographic procedures
● Thin Layer Chromatography
● High-Performance Liquid Chromatography
(HPLC)
● Gas Chromatography
Basic components in chromatographic techniques:
● Mobile phase (gas or liquid)
○ Carries the sample mixture
● Stationary phase (solid or liquid)
○ Where the mobile phase flows
● Column
○ Holds the stationary phase
● Eluate
○ Separated components
Setup for HPLC
As the mobile phase carrying the solution enters the
column, the components of the solution has three phases.
1. The solutes may reside only on the stationary
phase.
2. The solutes may reside only on the mobile phase.
3. Distributed between the two phases - depending
on the interaction of the solutes with the
stationary and mobile phase
Basic Concepts
● Solutes with higher affinity for the stationary
phase reside in the stationary phase.
● Less affinity with the stationary phase reside
mostly in the mobile phase and migrate faster.
● Solutes with lower affinity to the stationary phase
separates from the solutes with higher affinity to
the stationary phase.
THIN LAYER CHROMATOGRAPHY (TLC)
● Column chromatography
● Mobile phase – closed container
● Stationary phase – glass or plastic plate
○ Alumina
○ Silica gel
○ Cellulose
○ Cross-linked dextran
But if the thin layer consists of particles with smaller
diameters, the technique is called High-Performance Thin
Layer Chromatography (HPTLC). HPTLC is said to be
more efficient and more reproducible because particles of
small diameters are used.
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 SETUP OF TLC

As the mobile phase migrates upward via capillary action,

it carries with it your sample.

● TLC
○ Advantages
○ Simplicity
○ Rapidity
○ Versatility
○ Ability to process large amount of
samples in minimal time
○ Low cost of reagents and equipment
Another illustration. TLC is commonly used as a
semi-quantitative screening test
● The sample components in TLC are identified by GAS CHROMATOGRAPHY
comparison with the standards on the same plate.
● Retention factor (Rf) - the distance a component ● To separate mixtures of compounds that are
migrates compared with the distance the solvent volatile or can be made volatile.
front moves ● Gas-solid chromatography (GSC) - solid state
● Then further information is obtained after the ● Gas-liquid chromatography (GLC) - liquid state
plate is dried. After the plate is dried, the ● Gas – mobile phase - gaseous state
separated component can be located and Mobile phase is gas. The common carrier gases used in
identified using: this type of chromatography are:
○ UV illumination
○ Fluorescence ● Nitrogen
○ Color-generating reagents ● Helium
○ Autoradiography ● Argon
The selection of carrier gas is primarily determined by the
detector used in the setup.

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 Example of setup in Gas Chromatography
LIQUID CHROMATOGRAPHY (LC)
● The separation of the solutes in LC is based on
the distribution of the solutes between the liquid
mobile phase and a stationary phase.
● Liquid – mobile phase
● Stationary phase (solid or liquid)
● High-performance or High-pressure liquid
chromatography is the most widely used form of
LC
Example of setup in HPLC
The sample is injected, it can either be injected as a gas,
or if it’s not yet in a gaseous state, the temperature of the
injection port should be made higher than the boiling point
of the sample so that upon injection, sample will vaporize.
The sample is injected then it will be swept through the
column by the mobile phase, and then as it reaches the
column, the solutes or the components will be partially
dissolved in the liquid stationary phase and then they will
exit, and reach the detector then the recorder.
● Volatile compounds that are present mainly in the
It is almost similar to that of Gas Chromatography, there is
gas phase will have low partition coefficient.
just the absence of the injection port.
● Compounds with higher boiling points will slowly
move though the column There is a pump/motor. The mobile phase will be injected
through the column at a higher velocity because of this
A more volatile solute will leave the column faster
motor. Then, the column is packed with silica gel.
than a less volatile one.
The column effluent carries separated solutes to the
detector in order of their elusion and then as the effluent
passes through the detector, they will create an electrical
signal proportional to their concentration and this electrical
signal will be recorded in the recorder.
● Similar retention time – identified qualitatively
The sample mixture is injected and is carried by the
● Peak size – proportional to the amount of solute
mobile phase, eventually entering the column which
detected
contains the stationary phase.

● Columns – made of glass or stainless

● Detectors – Thermal conductivity or flame
ionization detector

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 Example of a Chromatogram that records the results
in Chromatography

The column is covered with particles (non-polar particles -

gray colored in the image). As the sample mixture is
brought inside the stationary phase, the non-polar solutes
(red) will interact or will have a greater attraction with the
non-polar stationary phase, resulting in a slow movement.
Whereas the polar solutes (yellow), will have a low
attraction to the non-polar stationary phase, allowing them
to migrate faster. In this case, the stationary phase is
non-polar so we call it Reverse phase HPLC.

Analyte Identification
● In Gas and Liquid Chromatography, they use a
recorder and then this recorder records the
detector signal starting from the time of sample
injection until the time the mobile phase passes
through the instrument
● Retention time - used to identify compounds
when compared with standard retention times run
under identical conditions
● The appearance of a solute peak, band, or spot
at the same time as that of a reference compound
is consistent with the two compounds being the
same
Analyte Quantification
● Peak area - proportional to concentration of the
compounds that produced the peak.

Kong, Ponce, and D. 16

Use with caution. Char. Goodluck, future RMT <3
 INTRODUCTION
LABORATORY QUALITY (acc. to WHO)
1 Accuracy of result
• The laboratory results must be as
accurate as possible.
2 Reliability of result
• All aspects of the laboratory operations
must be reliable.
3 Timeliness of result
• Reporting must be timely in order to be
useful in a clinical or public health setting.
SUMMARY: Laboratories produce test results that
are widely used and health outcomes depend on the
accuracy of the testing and reporting.
CONSEQUENCES OF INNACURATE RESULTS
1 Unnecessary treatment
2 Treatment complications
3 Failure to provide proper treatment
4 Delay in correct diagnosis
5 Additional and unnecessary diagnostic testing
• Consequences of inaccurate results increase
cost in time and personal effort and often, poor
patient outcomes.
QUALITY MANAGEMENT SYSTEM
• The laboratory is a complex system involving
many steps of activities and people. The
complexity of the system requires that many
processes and procedures be performed
properly. Therefore, the quality management
system model which is built at the entire system
is important for achieving good laboratory
practice.
• Can be defined as Coordinated activities to
direct and control an organization with regard
to quality.
This meaning is used by:
▪International Organization for
Standardization (ISO)
▪ Clinical and Laboratory Standards
Institute (CLSI)
Additional note: Both groups (ISO and CLSI) are
internationally recognized laboratory standards
organization
12 QUALITY SYSTEM ESSENTIALS
Additional note: These are a set of coordinated
activities that serve as building blocks for quality
management. Each must be addressed if overall
quality improvement is to be achieved.
1 Organization
2 Personnel
3 Equipment
4 Purchasing and inventory
5 Process control
6 Information management
7 Documents and recording
8 Occurrence management
9 Assessment
10 Process improvement
11 Customer service
12 Facilities and Safety
• Many of the 12 quality system essentials
overlap and all 12 must be addressed to ensure
accurate, reliable, and timely lab results and to
have quality throughout the lab operations.
QUALITY ASSURANCE
• A part or component of quality
management. Focused on providing
confidence that quality requirements will be
fulfilled.
ISO standards group lab processes or quality
assurance into:
1 Pre-examination or pre-analytic or pre-test.
2 Examination or analytic or test.
3 Post-examination or post-analytic or post-test.
PATH OF WORKFLOW
Is The entire sets of operations that occur in testing
• Begins with patient sample collection and ends
in reporting of results and interpretation.
• The organizational structure of the lab must
support an optimal path of workflow by
allowing processes that yield efficient sample
handling while minimizing error. In short,
quality assurance monitors quality lab
performance from the ordering of lab requests
up to its reporting.
PRE-ANALYTICAL PHASE
Includes factors involving:
a. Acquiring,
b. Handling,
c. Transporting, and;
d. Specimen processing prior to actual analysis
It also includes activities such as:
a. Patient preparation fasting - before the
sample reaches the section of laboratory.
ANALYTICAL PHASE
• Involves all factors related to the test platform
and to the testing process itself done in the
laboratory.
• Actual measurement performed on the sample.
POST-ANALTICAL TEST
• Involves all steps after testing and consists of
recording and reporting of patient data.
ERRORS
• An error in any part of the cycle can produce a TOTAL QUALITY MANAGEMENT
poor lab result. For example, a sample that is
INTRODUCTION TO QUALITY
damaged or altered as a result of improper
ACCURACY
collection or transport can’t provide a reliable
•
What do we mean when we say quality in the
result. A medical report that is delayed, lost, or
laboratory?
poorly written will negate all the effort of
Laboratory Quality can be defined with three
performing the test well.
concepts/terms;
- First one is Accuracy.
QUALITY CONTROL Accuracy- meaning the laboratory results must
• Is a Component of quality assurance. be accurate as possible. Accurate, meaning that
• It is used to monitor the examination or analytic they are the true values for the patient, or they
phase of testing. are the correct results for the patients and that
• Essential element of the quality management there is no error.
system. RELIABILITY
• The goal of Quality control is to provide • Second concept is Reliability
laboratory with confidence. • Reliability- is that laboratory operations must
PROVISE LABORATORY WITH CONFIDENCE BY: be reliable, so that the end result is that we
(All are done before patient results are reported)
have an accurate and reliable result.
1 Detecting
TIMELINESS OF RESULTS
2 Evaluating
• Third and Last, is the Timeliness of Results
3 Correcting Errors
• Timeliness of Results- it is important that the
DUE TO:
1 Test system failure reporting of these results be given in a timely
2
manner, so they can be useful for the patient
Environmental conditions
3
and the requesting physician.
Operator performance
• E.g. If the result is accurate, and the
• QC is done by examining control materials of
processes were reliable, but the result was
known substances along with patient samples to
not given on time, then it is too late for the
monitor the accuracy and precision of the
patient and for the physician to use the
complete analytic process.
results. Which defies the purpose of having a
laboratory result to aid in the recovery or in
ACHIEVING, MAINTAINING AND IMPROVING the diagnosis of the physician for the patient.
ACCURACY, TIMELINESS AND RELIABILITY
• Another example: The result was given on
• Achieving, maintaining, and improving time, but the result was not accurate, or that
accuracy, timeliness, and reliability are major the result was not processed properly. This
challenges for health laboratories. In order to scenario also defies the purpose of a
achieve the highest level of accuracy and laboratory result. Again, this means that the
reliability, it is essential to perform all processes results should be Accurate, Reliable, and
and procedures in the best possible way. should be given on the correct time for us to
say that there is quality in the laboratory.
• Laboratories not implementing a good lab
management system are guaranteed with
many errors and problems occurring that may
go undetected.

SUMMARY
QMS
• Quality management systems coordinate all 12
quality system essentials.
QA
• Quality assurance focuses on the path of
workflow that includes pre-analytic, analytic,
and post-analytic activities.
QC TOTAL QUALITY MANAGEMENT
• Quality control monitors the processes related 1 Structured approach to overall organizational
to the analytic or examination phase and of management.
testing, controls materials, and allows for - The keyword here is organizational,
detecting of errors in the testing system. because TQM involves the whole
• Implementing a quality management system organization, and it’s just not the
may not guarantee an error free lab but it does laboratory, so this would be the hospital,
yield a high-quality laboratory that detects where the laboratory is in or if the
errors and prevents them from recurring. laboratory is inside the company, then
TQM would involve the company itself.

e.g. The laboratory is part of a hospital, that
means the laboratory would have to
communicate with other departments inside the
hospital. That can be the ancillary department,
the nursing department, the admissions
department, etc.
Another example, is that if the laboratory is part
of a company or is inside a company, then that
means that the laboratory would have to
collaborate with the different branches inside of
that institution.
Again, it’s called organizational, because it
involves the whole organization.
2 Involvement of all the employees working at the
different levels to contribute to achieving
business objectives including customer
satisfaction.
- It does not matter what your work is if
you are working for that organization, company,
or hospital, then you are part of the TQM. These
are employees at their different levels, different
positions, different job descriptions, different
work. All of them contribute to a common
objective, or towards a common goal which is
the IMPROVEMENT OF QUALITY. End result is
that we would have customer satisfaction, in the
healthcare setting, our main customers are the
patients. Lastly, management means that there
is a management approach, so that there is a
long-term success. There is a managerial
process involved in the whole TQM.
“Again, TQM involves the whole organization
and everyone working in it focuses on improving
the quality of the whole organization
5 Q FRAMEWORK
• The principles and concepts of total quality
management can be summarized in this
framework.
• Used for managing quality in a healthcare
• All of these 5 components work together in a
feedback loop
1 Quality Planning (QP)
2 Quality Laboratory Processes (QLP)
3 Quality Control (QC)
4 Quality Assessment (QA)
5 Quality Improvement (QI)
QUALITY LAB PROCESSES (QLP)
• The laboratory processes are all implemented
through here
• This includes Analytical Processes:
Policies
Practices
Procedures
• -All of these define how work is done
QUALITY CONTROL (QC)
• All of the processes will be measured through
here (QC) and (QA)
• Emphasizes the Statistical procedure
• But it can also include non-statistical
procedures like Linearity check, Reagent &
Standard used for testing and check
Temperature monitoring of machines and so on
QUALITY ASSESSMENT (QA)
• All of the processes will be measured through
here (QA) and (QC)
• Concerned with the broader measures and
Monitors of Lab performances
• Ex: Turn Around Time
Specimen & Patient Identification
Test Utility
QUALITY IMPROVEMENT
• Any error seen in QC and QA will be improved
through here
• Provides the processes for problem solving
• First, we have to identify the root cause of the
problem and after that we have to identify the
remedy for that problem
QUALITY PLANNING
• Re-planning will occur to remove the error
through here
• The new processes done through this stage will
then be implemented in QLP and this will
restart the loop giving a Continuous Quality
Improvement
• This is necessary so that we can standardized
the remedy that was identified in QI
• We have to establish the measures for
monitoring the performances
• Ensure that the new performances, achieve
and satisfy the quality requirements
• Document the NEW QLP
• After that, we should have an improved quality
PDCA CYCLE
• May also be used like the TQM framework or
the 5Q framework
• PDCA stands for Plan Do Check Act
• It has 4 stages which is based on the scientific
method of problem solving.
• This is popularized by Dr. W. Edwards Deming
who is considered by many to be the Father of
Modern Quality Control
1 PLAN
Provides the planning step
Similar to QP
2 DO
Establish standard processes for doing things
Similar to QLP
3 Check
Provides the different measures for checking how
well things are done
Similar to QC&QA
4 Act
Provides a mechanism for acting on those
measures
Similar to QI
 CLINICAL CHEMISTRY (MTCC1) - LECTURE
MODULE 3: QUALITY ASSURANCE
Topic Outline:
● Introduction of Quality Assurance
● Path of workflow
● Pre-Analytical, Analytical, and Post-Analytical
QUALITY ASSURANCE
INTRODUCTION
● Clinical laboratories generate a large amount of
information everyday. These information are the values for
specific biochemical parameters that we release as
results. In chemistry, for example we have fasting blood
sugar, cholesterol, albumin, bilirubin and so on and so
forth
● How reliable are the results? Are they correct? Are they
accurate? Can we believe this? Do they signify the truth in
relation to the patient’s health
● If these results are not reliable, What does this value mean
in the physiologic health of the patient? Will it be
important that the patient receives accurate and precise
result, the answer is YES
● The main purpose of the laboratory is to be able to reflect
a condition of the patient so that we may be able to help
them
● It is important that we provide confidence in the
Laboratory data we furnish to patients
● For this reason, we now have QA
● QA
○ Make sure there is quality in the laboratory
○ There is quality in the results that we give
CONSEQUENCES OF POOR QUALITY IN THE
LABORATORY
INAPPROPRIATE ACTION
● Poor laboratory results = incorrect results = incorrect
treatment (leading the doctor to a diagnosis that is not
true)
○ Over treatment – causes extra effort and time from
everyone
○ Non treatment
○ Over investigation
○ Under investigation – patient may have a condition
but since poor results is given, it will not be
investigated causing the patient to progress in its
disease
Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.
“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 1 --
○ Delayed action – related to under investigation
● the patient will exert a lot of financial effort because of
these consequences
LOSS OF CREDIBILITY
● Does not
○ Support the provision of high quality health care
○ Enumerate credibility of the laboratory
○ Generate confidence in laboratory results
■ If these does happen, and if patients know,
doctors know that you are releasing an
inaccurate results, this can cause trouble, for
example if you released an abnormal results
yung mga doctors pwedeng sumugod dyan
kase alam nila na may possibility na mali yung
results mo
■ If nothing is coinciding with their diagnosis, they
can easily blame their laboratory for not giving
accurate results and we don’t want that
LEGAL ACTION
● Suspension of license
○ Laboratory - laboratory will not be able to operate
anymore and then all the employees will lose their job
○ Pathologist, Medical technologist - if you offer poor
quality and something happens, legal action happens
they can revoke your license, lahat ng pinagpaguran
mo mawawala and of course we have to prevent that.
How do we prevent it? By making sure that the work
we do has quality that the end result has quality, to
make sure that none of these, the examples of poor
quality in the laboratory, to make sure that they do
not happen
Importance of Quality Assurance: Make sure that we
provide good and quality results. It is not something to be
neglected but should be taken seriously to avoid
consequences
QUALITY ASSURANCE
● Refers to all planned and systematic activities to provide
confidence in the result that we generated are correct.
The purpose of this is to maintain the overall quality of the
patient result.
● A.K.A Quality Assurance System. We use the system
because it is vast and involves a lot of different factors.
● These factors are the pre-analytical, analytical, and
post-analytical.
● It concerns the total process from the start to finish; it
starts with the doctor’s requisition and finishes when the
doctor receives the result.
● Not only involves the laboratory but also includes the
COMMON ERRORS IN QUALITY ASSURANCE
doctors, patients, and anyone involved inside the
process.
○ Examples of the processes in the laboratory: TAT,
PRE-ANALYTICAL PHASE
patient identification, and test utility.
● Quality assurance makes sure that everything is done
properly and you perform with quality. Everything you do in the reception area and preparation area
● Quality assurance also involves documents. These are the
plans, policies, procedures that are involved in the ● Patient preparation
administrative structure in the laboratory. 1. Preparation before the extraction- correct fasting time
○ Other examples: Log books and results 2. Nutritional status of the patient- drunk alcohol, lack
● Monitors quality performance from request to reporting of sleep, intake of other medicine that can affect to
● Recognizes and minimizes errors and eliminate them in certain tests result
the future ● Test ordering
● Represent best practices recommended to be followed to 1. Lab request- hand written request may not be eligible
achieve desired quality goals and results enough to read by the medical technologist.
● Specimen Acquisition/Collection
PRE-ANALYTICAL 1. Wrong specimen- wrong specimen draw or may be a
wrong anticoagulant used.
● Factors before the sample reaches the laboratory and 2. Wrong identification of patient
tested 3. Quantity not sufficient (QNS)
● Starts with the doctor’s requisition until it reaches the 4. Prolonged tourniquet application- can cause
section of the laboratory where the test will happen hematoma to the patient
● Includes the acquisition of the request, handling and 5. Improper storage of specimen- not proper
transportation of the specimen, and patient preparation temperature for certain specimen, improperly sealed
such as fasting specimen.
● Difficult to monitor and control 6. Proper equipment for extraction- (e.g. proper tube,
○ Some of these factors occur outside the laboratory. needles, syringes)
○ Example is Patient Preparation (Fasting): There is
no way to monitor if they really followed the correct
fasting time, the only way to know is to ask the ANALYTICAL PHASE
patient. ● Easily controlled compared to pre-analytical
○ Nutritional Status: It is vital because there are some
● Happens inside the laboratory
tests that would rely on this.
○ Alcohol intake, Smoking, Exercise, Stress, Sleep,
Posture are also examples of factors that are difficult ● Water quality
to monitor and control 1. Interfering substances used during testing
2. Use tap water and unfiltered water
ANALYTICAL
● Calibration of equipment, machine and reagents
● During the analysis of the sample ○ Calibration means equipments and
● Includes all factors related to the test factors or the test
machines are working properly
process itself; Actual procedure of the experiment
● Stability of electrical power
● Primarily dependent on the instrumentation, the reagents
and Medical technologist are involved in the procedure. ○ Machines may require their own power source
○ Better control ■ No extension cords
○ Analytical involves actual measurements (e.g. test ■ Should have their own supply of power
procedures) ● Thermostatically controlled instruments
○ Quality control - everything involved in analytical Instruments should be inside a cold room have their
phase
own air-conditioning unit to supply the correct
temperature
POST-ANALYTICAL If not, the results will fluctuate and incorrect
● All the steps after testing; Starts when the result is ● Reagent and kits expiration date
generated ○ Do not use materials beyond expiration date
● Everything done after the result is considered
post-analytical. POST ANALYTICAL PHASE
● Consists of the recording and reporting of the patient Involved in releasing area
data (e.g. write the results in the log book, print, report) ● Test reporting
1. Wrong patient ID where result was given to wrong
patient

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 2 --
 2. Illegible report where handwritten is not understood
causing misinterpretation
3. Delay on the release of report
4. Transcription error - Encode different results in the
result form
5. Excessive turn-around time (TAT)

● Test interpretation - how the medical technologist

interprets the result as it comes out.
1. Previous values not available for comparison
2. Did not double check
3. Incorrect reference value

REMEMBER:
Quality assurance is the process responsible for maintaining
the integrity of everything in the laboratory. Quality Assurance
in place produces a Quality system that will be adapted in the
laboratory making it a quality laboratory. Quality laboratory
will produce correct results that are given to the right patient
in a very timely manner. With all of these in place it would
reassure that there is QUALITY IN SYSTEM

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 3 --
 CLINICAL CHEMISTRY (MTCC1) - LECTURE
MODULE 4: QUALITY CONTROL

○ Essentially, we need to perform QC protocols

by running samples with known concentrations.
Learning Objectives:
These are known as control solutions.
● Discuss the principles of Quality Control
○ Kapag alam natin ang concentrations ng known
● Classify Errors in Quality Control
samples, we can predict of the given laboratory
● Identify Materials in Quality Control
methodology is accurate and (?? 15:04).
● Understand the importance of Quality Control
○ Why? Kasi alam natin, for example, alam natin
Topic Outline:
na that particular analyte contains 100 mg/dL of
● Parameters of Quality Control
glucose. So, we know that the laboratory
● Quality Control Materials
methodology is adherent to quality control
● Errors in the Clinical Laboratory
protocols because we can target the known
● Measures of Diagnostic Efficiency
concentration of control solutions.
● Quality control is A COMPONENT
○ An essential component of Quality Assurance
INTRODUCTION
System
It is quite important for us to understand these ○ Quality control is a system that ensures
concepts because in achieving quality control, we have accuracy and precision.
to always look at the importance of quality control, ○ How do we encounter and achieve this? By
especially when dealing with health services and the running QC reagents in every series of
health of the individual. In order for us to provide quality measurements
health care as medical technologists, we have to ensure
PARAMETERS OF QUALITY CONTROL
that all of our procedures are literally in control. Our
techniques and methodologies are of the highest What are the given parameters of quality control?
quality. To achieve this, we must employ quality control ● Syempre, meron tayong kailangan mafulfill na
procedures in a given clinical laboratory. characteristics ng isang maganda quality control
protocol. These are the following:
What is Quality Control?
● Quality control is A SYSTEM
SENSITIVITY
○ It is a system that is currently being employed in
order to attain the high quality standards that a ● Described as the ability of an analytical machine
given institution aims to achieve. to measure the smallest concentration of the
○ How? - It ensures Accuracy and Precision in the analyte of interest.
laboratory by including QC reagents in every ● For example, meron tayong isang litro ng tubig,
series of measurements. and we are going to add 1 milligram of glucose
○ RED VELVET and BLACKPINK as materials: into that 1 liter of water. Para masabi natin na a
■ Materials that are constantly being exposed person has an extremely sensitive taste buds is
to QC protocols. In order for them to kapag kaya niyang lasahan yung 1 milligram of
achieve accuracy and precision, they need sugar in that 1 liter of water.
to undergo a series of quality control ● That is how we describe sensitivity in the
procedures. context of determination of analytes of interest.
■ With this, isinasagawa natin ang
pag-execute ng quality control. These things Siguro, pwede nating example dito sa sensitivity para
have to undergo a system. mas maintindihan natin:
● Quality control as A PROCESS
○ Ensuring that analytical results are corrected by ● Tumor markers or cancer analytes: We have to
testing known samples (control solutions) that measure even the smallest concentration of the
resemble patient samples analytes of interest in order for us to detect the
presence of a condition of cancer. However,
since we are dealing with the context of cancer

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 1 --
 diagnosis, we also need to have a highly specific
method for the detection of the tumor marker or
the substance that pertains to the presence of
cancer. Generally speaking, if we are dealing
with tumor markers or cancers, the analytical
method should be (extremely) sensitive and
(extremely) specific.
Alongside your course on clinical chemistry,
maeencounter niyo yung terms like “the most sensitive
test” or “the most specific tests.” So, para mas
ma-understand natin na bakit siya tinawag na most
sensitive or most specific test? It is in the context of
detecting those substances.
(photo above) In the context of disease diagnosis, we
have here 10 individuals and upon testing these 10
individuals, for example, in the context of COVID-19
disease, we have here 8 people that tested positive for
the presence of a disease. In this example, 8 out of 10
persons tested positive for this COVID-19 disease. We
can say that this analytical instrument that we have has a
sensitivity of 80%. In this situation, ano ngayon yung
attribute na maidedescribe natin dito sa dalawang
particular patients na ito?
We have here two patients that have the disease, but
were not able to be detected in your analytical
instrument. This is a phenomenon called
FALSE-NEGATIVE RESULT OR TESTING. Again, meron
silang sakit pero hindi sila nagkaroon ng positive result.
Sensitivity is an excellent characteristic that we must
have for screening tests.
For the context of clinical chemistry, let's say for
example na ang isang patient ay may diabetes mellitus
(type 2). What are the possible factors that may lead to
false negative results? Madami po. Perhaps there is an
issue with the analytical instrument, patient
preparations, or masyadong extensively long yung
fasting preparations ng patient. Instead na makakita ka
ng pathologically abnormal na fasting blood sugar result,
dahil nga sobrang haba ng kanyang fasting preparations,
Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.
“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 2 --
nag-normalize ang kanyang blood sugar. So, that is a
potential phenomena for a false-negative result.
AGAIN, SENSITIVITY: ability of the test to identify
persons with a disease.
SPECIFICITY
The next parameter in the context of disease diagnosis
is Specificity.
(photo above) Meron ngayon tayong 10 people ulit.
These 10 patients are able to be ruled out of a disease.
So, what does that mean? Itong specificity na ito, this
pertains to the ability of an analytical instrument or
a laboratory methodology to identify persons
without a disease.
So, 7 sa ating sets of patients right here, tested negative
for SARS-COV 2 infection. We can pertain to this
specificity at the rate of 70%. How about yung 3 people
na meron tayo sa ating set of individuals. 3 patients do
not have the disease, but they still tested positive
anyway.
This is a phenomenon called FALSE-POSITIVE. Wala
silang sakit (3 patients), but for some reason, nagkaroon
sila ng positive result sa ating specific testing.
NOTE: It is very important that we can discern and
describe specifically the parameter of sensitivity and
specificity both in the context of analyte detection,
analyte concentration, and in the context of disease
detection or diagnosis.
● This is the ability of the analytical machine to
measure the only analyte of interest.
● A simple example for this one is that if we try to
combine sugar with salt. Pareho silang color
white and same silang granular in terms of their
consistency. In the naked eye, you will see that
salt and sugar are just the same. However, if you
are specific in identifying the sweet taste of
sugar, then you can determine the presence of
sugar in that combination of salt and sugar.
● To give you a demonstration or a stronger idea
on specificity and sensitivity, we are referring to
 sensitivity and specificity in the context of
analyzing chemical substances.
● These two descriptions on sensitivity and
specificity pertain in the context of analysis of
analytes or substances in the human
specimens.
ACCURACY
Accuracy – refers to the nearness or closeness of a
tested value to the true or target value. It is the ability of
an analytical instrument to attain a close or near value to
the true value of a given analytical instrument.
Example: Fasting Blood Sugar is 110 mg/dl. An
analytical instrument can be regarded as accurate if the
Assayed Value is 109 mg/dl or 111 mg/dl. The assayed
value must be near or close to the targeted value.
PRECISION
Precision – ability of an assay to give repeated results
on the same sample that agrees with one another.
Example: The known Value of Fasting Blood
Sugar is 110 mg/dl. The Assayed values are as follows:
108 mg/dl, 109 mg/dl, and 110 mg/dl and these values
should agree with one another provided that these
particular values come from a single specimen or
person in order to say that the instrument is precise.
What if the instrument is imprecise?
The FBS known value is 110 mg/dl and the Assayed
Values are followed by 200 mg/dl, 174 mg/dl, and 300
mg/dl and so on. All of these values do not agree with
one another but these values came from the same
patient thus we can say that the instrument is imprecise.
REPEATABILITY
Repeatability - repeated measurements
unchanged conditions show the same result.
Example: There is a person, he likes to eat
hamburgers and we know that eating too much
hamburger can impact our heart negatively. This person
is referred to by the physician to get himself tested for
cholesterol values.
Scenario: Med technologist 1 (Ma’am Shindle)
performed the blood cholesterol procedure using
method letter A and this method requires the instrument
letter A. After she performed, she got 350 mg/dl
cholesterol value. If Ma’am Shindle repeatedly performs
Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.
“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
the procedure and gets the same values or levels we
can say that the parameters of repeatability were met.
REPRODUCIBILITY
Reproducibility- repeated measurements under
changed conditions show the same results.
Example: Same scenario with repeatability but
with different medical technologists and methods used.
Med Technologist 2 (Ma’am Ivy) performed the blood
cholesterol levels. Instead of using the same method
used by Ma’am Shindle, she used method B for the
analysis of blood cholesterol levels. So both of these
Medical Technologists are using different laboratory
methodologies but both analyzing single analyte (blood
cholesterol levels) which are also being obtained from a
single person. How can we know that the quality control
is adherent to the reproducibility? – If both the medical
technologist’s results agree with one another (same 350
mg/dl values obtained).
What if they repeat the lab result? Ma’am Shindle
obtained 352 mg/dl and Ma’am Ivy obtained 353 mg/dl.
If one could notice, the med technologists are different,
the methodologies as well but still their results agree
with one another. With this, we can say that the
parameters of quality control precision, accuracy,
repeatable and reproducible are manifested.
QUALITY CONTROL MATERIALS
What are Quality Control Materials?
● Specimens that are analyzed for QC purposes
● Specially prepared
● Commercially available
● Has known values
● Used to check for accuracy
● AKA Control Solutions or Control Products
under Example: We have a quality control reagent or
matrix. In order to know if the matrix machine is high
quality, the laboratory should be given a quality control
solution that is appropriate to that matrix brand. Dapat
same brand oki ^_^
Matrix - a brand of automated chemistry analyzer
CHARACTERISTICS OF A GOOD QC MATERIAL
1. Resembles Human samples
● They are liquid, pale yellow especially when the
quality control material is in the Chemistry
section of the laboratory.
● Human Serum: Pale Yellow
-- Page 3 --
 ● Hematology Section
○ Whole Blood for CBC and Platelet count -
Expect for QC material is similar to whole
blood.
○ Whole blood is not coagulated, liquid state,
and mixed.
● Biochemical attributes
○ QC Material should simulate human samples
in terms of its contents and concentration.
○ Because in our body, we have normal and
abnormal conditions. We have abnormally
high and low laboratory results.
○ In order for us to have good quality control
material, our QC Material should have levels
appropriate for abnormally low, high, and
normal ranges of quality control materials.
● In some chemistry methodologies, there are
some QC materials that can be reflected as
positive or negative. It depends on the analytes
being tested in the laboratory methodology.
2. Available in sufficient quantities for at least 1
year
3. Stable over the period of availability
● Context of Finance: We want that our QC
materials are budget friendly but would generate
a good amount of profit.
● Mas mataas dapat profit kesa sa expenses.
● Good: High profit, less expenses, less wastage.
● We have to assess if the laboratory services that
we are offering gerates that much profit.
● Quality control is a system and process and it
requires money.
● If high expenses, then less profit then we need
to have other alternatives in able to provide us
more profit and unnecessary expenses.
4. Available in convenient vial volume and cost
effective
5. Vary minimally in concentration and composition
from vial to vial.
- Example: Vial 1 (100 mg/dL) hindi naman pwede
na yung 2nd vial may 20 mg/dL of glucose.
- That means there is inconsistency with the
substances or concentration of control solution
and that is not an ideal characteristic of good
QC Material
- They should be stable
- If there is a variation in terms of concentration it
should only be minimal.
6. Should span the clinically important range of the
analytes concentration
- The qc material is capable to provide normal
and abnormal concentration of the chemical
analyte of interest
Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.
“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 4 --
-
Dapat practical ranges and dapat i produce or
simulate ng QC Material.
- Hindi naman pwede na normal range ng FBS is
80-120 mg/dL then QC Material 10-20 mg/dL.
Hindi siya akma or consistent.
- It should be fit to the clinical important range of
analyte concentration.
7. Should not have any communicable diseases
- Blood transmittable disease - Before, QC
material can exist or derived from human or
animal samples.
SOURCES OF QC MATERIALS
It can either be Human obtained samples, and Animal
obtained samples:
● Home-Made materials
○ 100 patients: We can collect their serum
samples. Take note that, they share similar
characteristics with one another and they are
normal.
○ They can pull their serum samples and their
samples can be made as quality control
materials
● Disadvantage: Difficult to minimize infectious
disease. Ex: HIV, Hepa B and C etc.
○ Difficult to minimize because we didn’t test
them prior to collection. You are at risk of
nosocomial infections.
● More susceptible to deterioration and contamination
○ Because they are obtained from blood samples
and obtained through various methodologies of
blood collection.
○ We have variable techniques that can influence
the integrity of human samples.
● Separate control pools for specific analytes
○ Ex: Special tests - Cancer markers
○ Patient diagnosed with colorectal carcinoma
and we don't have QC Material provided by the
service provider for carcinoembryonic antigen
(CEA). What we will do is to collect serum of the
patients that are diagnosed with colorectal
carcinoma provided that we know the
concentration of CEA tumor marker to their
blood sample. Then we can utilize their serum
samples as QC material for CEA.
○ Note: In order for us to have QC Material, we
must have a known concentration of analyte of
interest in order for it to be classified as a
control.
○ Ex: CEA + Alpha-fetoprotein (AFP) + Prostate
specific antigen (PSA). But it's quite difficult to
perform this to people with CEA, AFP, and PSA
because we need to find na tao na may ganyan
siya lahat na tatlo.
 ○ Challenging to perform a whole sample for
specific analytes. Because we must know
specifically the concentration of special
chemical analytes present in their serum
samples
Better alternative:
Bovine-based QC Material is most commonly used and
preferred samples for routine QC protocols or runs.
○ It is lower in price than human-derived.
○ Stability is same with human-derived
○ Disadvantage of Animal Derived QC material:
Not good for immunochemistry and dye-binding
techniques for albumin and bilirubin.
■ Chemical composition of albumin between
humans and animals are not the same.
■ Constituents of bilirubin are different
between animals and humans.
■ It is direct to the point difference between
human derived quality control material and
animal derived quality control material
■ If we are going to pertain with the use of
Immunochemistry - combination
immunological techniques executed with
chemistry techniques. In Immuno-sero,
there are certain immunoserological
methods that utilize anti human globulin
reagents.
■ Animals do not have human-derived antigen
and antibodies. Hindi siya mag react if we
employ animal derived quality control
material in determining the functionality or
quality control process involved in
immunochemistry methods.
■ Right now we are using animal derived
control materials in the clinical laboratory
TYPES OF QC MATERIALS
COMMERCIAL: ASSAYED
● More expensive, has established control value, most
useful for infrequent tests.
● E.g., thyroid hormone test, tumor marker test (both
are special kind of test)
COMMERCIAL: UNASSAYED
● With target values, appropriate for frequent tests,
mostly used.
● E.g., lipid profile, fasting blood sugar, kidney
function test, liver function test, SGPT, SGOT
HOMEMADE
● “In-house”, Pooled sera collected in the laboratory
● Preserved in small quantities for daily use
Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.
“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
● They are not routinely performed anymore in the
laboratory, but there some instances na ginagawa
pa rin ang pagbuo ng quality control materials
obtained from pooled serum
VARIATIONS OF QC MATERIALS
a. LYOPHILIZED
● Freeze-dried
● Powder form
● Reconstitute with water or specialized diluent
before used
- Be careful in reconstituting QC materials
with warm water. As much as possible, we
try to utilize triple distilled water in the
reconstitution of lyophilized QC material.
- Why is tap and mineral water not allowed?
Because they already have particulate or
substances
- Triple distilled water is the best to avoid the
addition of unnecessary substances or
particles in reconstitution of lyophilized QC
material.
of ● Standardize reconstitution step
- Depends on the type of QC material that is
used.
- E.g., A QC material in a 10 mL amber bottle vial.
In order for us to reconstitute that QC material,
we need to use triple distilled water.
- It is important that we follow proper instructions
in the reconstitution of the lyophilized sample.
- Why do we study the different types of
pipettes? This is because if we use a wrong
pipette in reconstituting QC material, the
integrity of the QC material itself is
compromised along with the known
concentrations of the QC materials.
- When the reconstitution is wrong, the QC
material is wrong, the quality is also wrong.
b. LIQUID
● Does not require reconstitution
● More expensive (because it is READY TO USE)
● Contains additives or preservatives
- Can sometimes affect the quality control
procedure of our samples and laboratory
instruments.
- That is why economically speaking,
lyophilized QC material is more preferred.
When the powder form or freeze dried QC
material continue to still be in powdered
form and freeze dried, then we can
guarantee that the lyophilized sample
material is stable for 1 year. And when
reconstituted with warm water or
-- Page 5 --
 specialized diluent before use, then we can For example, we have a calibrator for glucose, the said
easily process the collection of quality calibrator only has one chemical to tell if the machine
control materials and the addition of quality can only identify glucose.
control materials in the analytical
instrument. “In control, It really simulates the condition of human
● Error due to matrix problems samples. Multiple concentrations are detected as
long as we know the known concentration of those
Examples of Quality Control Materials analytes of interest.”

For example, beta-hcg. A QC material contains b-hcg

and the negative control does not contain the b-hcg or
the analyte of interest. Moreover, through control we can
observe that a machine can detect abnormal and normal
values. Say for instance, a machine can detect glucose
levels greater than 120 mg/dL which is abnormally high,
or the glucose values of the abnormally low QC material
Lypocheck Liquicheck can be detected by the analytical instrument that we are
In powder form; In liquid form; has to be transferred using.
has to be to the sample cuvette. ERRORS IN THE CLINICAL LABORATORY
reconstituted in Why do we need to put the QC
triple distilled material in the sample civette? This Clerical errors
water; requires 5 is because QC materials are run or - Also known as pre-analytical errors
mL of triple tested as if they are patient samples. - Examples are: Handwritten labels and
distilled water So it makes sense that we are going request forms. (e.g., handwriting of the MT is
since there is a to load the content of this QC not legible, erasure of the labels, wrong
5mL H2O written material in a sample cuvette, in information about the patient written on the
on the bottle order for us to perform our quality labels and forms).
control check/ run. - These clerical errors are dangerous. There
are a lot of factors that can contribute to clerical
errors. Thus it is important for us to
CONTROLS AND CALIBRATORS troubleshoot them to avoid further errors.

These are commonly misinterpreted by MTs Analytical errors

CALIBRATORS CONTROLS - Experimental errors
- Errors that are happening during the test
- A solution with a - A specimen or a proper itself (solely focused on ANALYTICAL
known concentration solution which is PHASE)
of an analyte. analyzed solely - Can be Random or Systematic
- Used to adjust the for quality control
instrument, kit, test purposes, not
system in order to calibration.
standardize the - Can exist as a
assay. Positive control,
- Sometimes called the Negative control
STANDARD - Can also exist as
Normal,
Abnormal high,
Abnormal Low

“In the calibrator, a single concentration of an

analyte is detected.”

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 6 --
 E.g., to obtain accurate bilirubin results we must follow
RANDOM ERRORS SYSTEMATIC ERRORS
appropriate specimen collection such as wrapping it in a
carbon paper. Now, let’s say that the MT forgot to wrap
● Due to chances ● Predictable errors
it in a carbon paper, this is a case of random error due to
● Inconsistent that happen all
shortcomings of the medical technologist in question.
chances the time
● Affects precision ● Remains
SYSTEMIC ERRORS
(Precision is the constant
- Poor accuracy
ability of an ● Affects accuracy
- Definite cause
analytical
- Reproducible (errors are encountered
instrument to
frequently like faulty instruments or expired
produce results
reagents)
that agree with
E.g. swapping the wrong reagent in a specific machine,
one another
this will lead to a systematic error because you are
provided that
constantly providing inaccurate results over and over
these results are
again.
tested from a
single specimen. MEASURES OF DIAGNOSTIC EFFICIENCY
There is a degree
● 2x2 Table
of agreement or
● Measures of Diagnostics
consistency in
● Reference Limits
precision)
2X2 TABLE
For example, we are going to perform a test and we
have noticed that the results do not agree with one Columns: presence or absence of disease
another. It is not repeatable or reproducible. This Rows: positive or negative results of a given test
scenario is attributed to a random error. methodology

If it is a systematic error, the error remains content and

2X2 TABLE DISEASE
predictable. For example, we have executed a test and
the true value was not achieved. If the run tests are TEST + -
consistently inaccurate, this scenario is attributed to a
systematic error. + TP FP

Possible causes of Random errors - FN TN

- Specimen problems (for example an MT
does not use the proper anticoagulant of TP: true positive FN: false negative
choice, let’s say Sodium and Potassium test. FP: false positive TN: true negative
Sodium test is usually employed using a red top
colored tube and if you are going to use the If we are to identify the presence of a disease and the
EDTA tube the results will be falsely elevated.) test shows a positive result this refers to a true positive
- Electrical interferences (for example there is result. If a patient does not have the disease but it still
a sudden black out, random errors are usually
shows positive anyway, we refer to this as a false
associated with chance and human errors)
- Instrument instability positive result. If a patient has a disease but the test
fails to have a positive result or the test yields a negative
Possible causes of Systematic errors result, we are dealing with the false negative result.
- Instrument bias Lastly, if the patient does not have a disease and tested
- Reagent lot bias negative with the result we are dealing with a true
- Calibration bias
negative result.
RANDOM ERRORS
- Poor precision How do we apply our 2x2 table in determining the
- Non-specific causes parameters of quality control measures of diagnostic
- Not reproducible efficiency:

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 7 --
 - We just have to compute the sensitivity, - The % of patients who do not have the disease
specificity, the positive predictive value and the and tested negative.
negative predictive value.
In order for us to compute, we have to remember first
Formula:
the designations of true positive, false positive, false 𝑇𝑁 𝑑
negative, and true negative. 𝑇𝑁 + 𝐹𝑃 𝑑+𝑏

2X2 TABLE DISEASE

Positive Predictive Value (PPV)
TEST + - - % Positive Tests that are true positives
- Probability that a positive test indicates disease
+ a b
- Layman’s Term: gaano katotoo ang ating
- c d positive result mo sa ating analytical
methodology.
a. TP = Patient with disease; tested positive
b. FP = Patient without disease; tested positive
c. FN = Patient with disease; tested negative Formula:
d. TN = Patients without disease; tested negative 𝑇𝑃 𝑎
𝑇𝑃 + 𝐹𝑃 𝑎+𝑏

MEASURES OF DIAGNOSTICS
We are going to use the 2x2 table in order for us to Negative Predictive Value (NPV)
compute the: - % Negative Tests that are True Negative
● Sensitivity - Probability that a negative test indicates the
● Specificity absence of disease.
● Positive Predictive Value - Layman’s Term: gaano katotoo and negative
● Negative Predictive Value
result mo sa ating analytical methodology.

2X2 TABLE DISEASE

Formula:
TEST + - 𝑇𝑁 𝑑
𝐹𝑁 + 𝑇𝑁 𝑐+𝑑

+ TP (a) FP (b)
LET’S TRY:
- FN (c) TN (d)
We have 100 individuals suspected to have a disease
Sensitivity and we have 1000 individuals suspected to not have the
- This is in the context of disease diagnosis. Don’t disease. Out of the 100 patients, 60 of them with the
forget the context of sensitivity in analyte disease tested positive, while 40 tested negative. For
detection and disease diagnosis. our 1000 patients without the disease, 300 patients
- The % of patients who have the diseases and tested positive, while 700 tested negative.
tested positive.
2X2 TABLE DISEASE
- Ability of the test to generate true positive
results than false negative. TEST + -
(100) (1000)
Formula:
+ 60 300
𝑇𝑃 𝑎
𝑇𝑃 + 𝐹𝑁 𝑎+𝑐
- 40 700

Specificity
Computation:

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 8 --
 Sensitivity: Specificity: Correlate it to With a rating of
𝑇𝑃 𝑎 𝑇𝑁 𝑑 sensitivity… 94.60%, can we say
𝑇𝑃 + 𝐹𝑁 𝑎+𝑐 𝑇𝑁 + 𝐹𝑃 𝑑+𝑏
that the ability of this
60 700 If we have a PPV as low instrument or
= 60% = 70%
60 + 40 700 + 300 as 16.67%, are we methodology is
confident that this reliable?
Interpretation:
analytical method is - Yes, the test is
The laboratory The ability of an reliable? reliable. Your
methodology is capable analytical instrument or - No, because this confidence level
of identifying patients a laboratory does not provide us that your negative
without a disease to a methodology to identify a good confidence results are true
degree in which it has a individuals without the level in terms of its negative, with a
sensitivity rate of 60%. disease, it has a positive predictive rating of 94.60%.
----------------- specificity rate of 70%. value. It is too low - In the context of
The sensitivity of the which means that specificity, we are
analytical instrument 16.67% is the able to identify
refers to the ability of the probability of the 60 patients without the
instrument to detect patients that have disease. If you’d be
patients with a disease, it positive results, able to identify
has a sensitivity rate of maybe because patients without the
60%. yung iba dito is disease and we
totoong may sakit come up with a
Is our sensitivity and specificity high? talaga. Yung mga negative predictive
- The specificity and sensitivity would depend on the nag-positive is baka value of 94.60%
type of the analysis, and would depende of the type hindi pa talaga siya then, we can have
of disorders that we are diagnosing. ganun ka-positive a high confidence
2X2 TABLE DISEASE kasi mababa yung level that patients
ating positive who tested
TEST + - predictive value. negative are true
(100) (1000)
negative.
+ 60 300 There is an established
low probability that the
- 40 700 patients who tested
positive in this test are
indeed true positive.
Computation:
***Positive predictive value and negative predictive value
PPV: NPV: correlated with sensitivity and specificity.
𝑇𝑃 𝑎 𝑇𝑁 𝑑
𝑇𝑃 + 𝐹𝑃 𝑎+𝑏 𝐹𝑁 + 𝑇𝑁 𝑐 +𝑑
RECAP ( 2x2 TABLE )
60 700
60 + 300
= 16.67% 40 + 700
= 94.59% or
94.60%

Interpretation:

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 9 --
 (MEASURES OF DIAGNOSTIC EFFICIENCY)

● TP/ TP+FN = SENSITIVITY

● TN/ TN+FP= SPECIFICITY
● TP/ TP+FP = POSITIVE PREDICTIVE
VALUE (PPV)
● TN/ FN+TN= NEGATIVE PREDICTIVE
VALUE (NPV)
● TP+TN/TP+FP+TN+FN=ACCURACY

EXAMPLE (ACCURACY RATE)

Formulas:

● False Positive (%)

𝐹𝑃 𝑏
𝑇𝑃+𝐹𝑃
𝑜𝑟 𝑎+𝑏

● False Negative (%)

𝐹𝑁 𝑐
𝑇𝑁+𝐹𝑁
𝑜𝑟 𝑐+𝑑
𝑇𝑃+𝑇𝑁
𝐴 = 𝑇𝑃+𝑇𝐹+𝑇𝑁+𝐹𝑁 Note: Don’t forget to multiply it by 100
60+700
𝐴 = 60+300+700+40 ↑ Sensitivity Low False Negatives - High NPV
760
𝐴 = 1100 High sensitivity is usually demonstrated by having a
low rate of false negatives and high negative
𝐴 = 0. 6909𝑥100 predictive value.

𝐴 = 69. 09% ↑ Specificity Low False Positives - High PPV

Note: Don’t forget to multiply it by 100 Our laboratory instrument or methodology has high
specificity if it has a low rate of false positives and
With this level of accuracy, pwede ba siyang high percentage of positive predictive value.
maclassify as a highly accurate laboratory method?
↑ Prevalence High PPV, Low NPV
● No, hindi pwede basta-basta i-identify as
accurate or precise because NEED pa We have a high prevalence predictive value and low
i-analyze in conjunction with the other negative predictive value.
quality control parameters.
● Accuracy is 69.09% pero namention before this Prevalence - refers to the frequency of a disorder
na meron issue with sensitivity and being detected in a given population or community.
specificity. In order for us to obtain an accurate ● If many are being diagnosed with the disease,
result or ideally accurate results dapat a laboratory instrument or methodology has
magkaroon ng sensitivity of at least 95%, the opportunity to have an increased positive
same with specificity. predictive value.
● But with these types of tests, dapat mameasure ● If many are sick, provided that the laboratory
in conjunction with other parameters of instrument has no issues with the specificity
diagnostic efficiency, kung totoo ba na accurate and sensitivity, we will have a high positive
or kung totoo ba na precise yung laboratory predictive value.
instrument.
↓ Prevalence Low PPV, High NPV
FALSE RESULTS

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

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-- Page 10 --

If, in a given population, only a few are sick, we will
have a low positive predictive value and high negative
predictive value.
For example:
● When 100 healthy participants (without disease or
illness) are subjected to analytical testing and
yield negative results, then we can see that the
analytic instrument’s negative predictive value is
high.
● This is because it has been established; that the
test is able to identify patients without the
disease.
● If there is the detection of patients without any
disorders, and at the same time supported by the
negative result produced by our analytical
methodology, we have a high negative predictive
value.
REFERENCE LIMITS
What are Reference Limits?
● It is referred to as Reference Interval or Value.
○ Back in the days, it was called the “normal
values”.
○ But in the context of today’s situation, “normal”
is an understatement. We have different genetic
make-ups, geographical distributions, or
ethnicities.
○ So it is just appropriate to use the term
“reference interval”.
● Usually, there are values for a healthy population
that represent 95% Central Tendency. (PPT: 95%,
video: 75%)
● Obtained by observation or measurement of a
particular type of quantity on a reference interval.
○ For example: We have utilized 100 Filipinos from
whom we will be obtaining a reference interval
for cholesterol. That group of Filipinos will be
the reference group in order for us to obtain a
reference interval.
● Ideally, the laboratory should have age and sex
stratified reference values on population.
○ For example: For the analysis of creatinine, we
should have 100 males at the age of 20 years
old and greater; or perhaps, 100 female
patients/volunteers in the range of 20 years old
and greater.
○ We have a selected sample from a given
population, and at the same time, we are
obtaining their values which will be the
reference interval or value.
Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.
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-- Page 11 --
● To derive reliable estimates, at least 120 individuals
should be tested in each age and gender
categories.
○ Notice, we have reference values for males and
females, and in some journals and books, there
are values that are used in pediatric patients or
geriatric patients.
● However, only 20 reference intervals need to be
sampled for analysis on the test instrument. If the
laboratorian determines that the test instrument and
test subject population are similar to those
described in the manufacturer’s package insert.
○ For example: If Erba (brand) says that you need
at least 20 Filipinos, that is already enough to
have a reference interval. You don’t have to
replicate the testing of 120 individuals.
○ You just need to have 20 reference intervals
from 20 participants if the test instrument and
test subject population are similar with the one
used by the service provider.
Factors to Consider:
Age Pregnancy
Sex Lifestyle Choices
Medications Body Weight
Physical Activities Biological Rhythms
● We have multiple factors to consider.
○ Ideally, in the Philippines, since it is a diverse
country, the reference intervals we use are
suited for the Filipinos and are also performed
by the Filipino scientists. But with our current
standards, we are using the Western reference
values.
○ Once you are in your internship or further
studies, almost all references are from the
Western countries, specifically, the United
States. You will observe that the reference
intervals are obtained from testeed Americans.
○ Since there is an obvious difference in ethnicity
(they are Caucasians and we are Asians), and a
drastic variation in terms of their lifestyle
choices, body weight and biological rhythms,
this will be an issue for us if we are going to
apply these to Filipinos.
○ This is the challenge for Filipinos: To try and
obtain reference intervals that are suited for the
Filipino people.
 CLINICAL CHEMISTRY (MTCC1) - LECTURE
MODULE #5: GLUCOSE METABOLISM AND REGULATION

Steps:
GLUCOSE METABOLISM ○ Digestion of the complex carbohydrates (e.g.
starch & glycogen) facilitated by pancreatic
We have that in order to live, organisms rely on the
amylase and salivary amylase (enzymes for
oxidation of complex organic compounds like
carbohydrates, proteins and lipids to obtain energy. Of breaking down glycolytic box of these
these three possible sources of energy, carbohydrate in carbohydrate polymers)
the form of glucose serves as the primary source. ○ From either short branched polysaccharides or
CARBOHYDRATE CLASSIFICATION disaccharides, these substances are broken
down to their simplest form with the help of
● Monosaccharide - single sugar molecule
various enzymes.
○ Glucose, Fructose, Galactose
■ This process of conversion usually occurs
● Disaccharide - two sugar molecules linked
○ Maltose, Sucrose, Lactose in the intestines specifically in the
● Polysaccharide - many sugar molecules linked duodenum
○ Starch, Glycogen, Cellulose ● Once they are in the form of monosaccharides, they
○ Most of the carbohydrates that we ingest are are ready to be absorbed in the intestine and travel
the complex type, the starch, glycogen and into the tissue cells.
cellulose.
● Simple sugars: 1 glucose, 1 fructose, and 1
■ Cellulose - not digested by our body.
○ Polysaccharides cannot be used up by the body galactose.
unless they are converted in the form of ○ Our focus will be on 2 glucose (the one with the
glucose. red circle above)
● It is important to know that these polysaccharides, ○ Glucose is the only carbohydrate directly
disaccharides and some monosaccharides like involved in energy production and later on
fructose and galactose, cannot be used by the body stored as glycogen.
in this form. They have to be converted or
○ Fructose and galactose need to be converted
metabolized to become glucose
○ Glucose - primary source of energy into glucose for them to be used by the body

CELLULAR METABOLISM

● The ultimate fate of glucose in the body is to

generate energy
● In the process, it will be completely broken down
into carbon dioxide and water
○ Glucose Metabolism - the process in which the
Refer to the table:
glucose is converted into energy for cellular
● Several enzymes are involved in the hydrolysis of
utilization. This energy is usually in the form of
sugar.
ATP.
● Also called glycolysis, the term used for the
breakdown of glucose into energy for cell use.

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

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-- Page 1 --
 Two things to remember about Krebs cycle
GLUCOSE METABOLISM
1. Requires oxygen – Aerobic glycolysis
2. Gluconeogenesis - in which other substances such
as glycerol, amino acids, and fatty acids can enter the
cycle can be converted into glucose-6-phosphate. This
is the production of glucose from non-glucose sources
○ Aside from carbohydrates, there are also fatty acids
and proteins entering the krebs cycle that can
generate energy.
ANAEROBIC GLYCOLYSIS

1. First step in the glucose metabolism is for the

glucose to be converted to glucose-6-phosphate
in the presence of enzyme hexokinase
2. Once it becomes a Glucose-6-phosphate it can
either go to the Embden Meyerhof Pathway to
produce energy or go to the Hexose
Monophosphate shunt to produce NADPH
3. If the cell’s energy requirement is being met, the
substance can be stored in the form of Glycogen
through the process called Glycogenesis
Another way to metabolize glucose under the Embden
EMBDEN MEYERHOF PATHWAY Meyerhof Pathway

● Important in cells that has energy requirements but

● Embden Meyerhof Pathway is the most important
in terms of the generation of ATP
● When the carbohydrate glucose enters this pathway
it is broken down into pyruvate as you can see in
the diagram.
● Once it is in the pyruvate it can now enter the
tricarboxylic acid cycle or what we call as the
Krebs cycle.
has insufficient oxygen supply such as in the
muscles
● Only a lesser amount of energy is created.
● Anaerobic: 2 ATPs; Aerobic 38 ATPs
● Glucose is not converted to its final product which
is CO2 + H2O + ATP. Instead, there will be lactic
acid production which will diffuse in the muscles
and the liver later on.
HEXOSE MONOPHOSPHATE SHUNT
● An alternative pathway for glucose is for it to enter
the hexose monophosphate shunt
● AKA Pentose Phosphate Pathway
● It does not produce energy unlike in the Embden
Meyerhof Pathway. However, its products are useful
in biosynthetic reactions.
● Alternative pathway for glycolysis.
● In this method, the oxidation of glucose leads to the
formation of NADPH

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

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-- Page 2 --
 ○ NADPH is a reduced form of NADP
○ NADPH is one of the products produced in the
Hexose Monophosphate Shunt
■ It serves as protection of the cell against
oxidation and free radicals. Without it, the cell
can be destroyed and could lead to its death
● Another product that is important is the
Ribulose-5-Phosphate
○ This will be used in the synthesis of nucleotides
and nucleic acids.
GLYCOGENESIS
The conversion of Glucose to Glycogen is called
● The Glucose will be converted into Glucose
6-Phosphate, the Glucose 6-phosphate will be
further metabolized and will become Glucose
1-Phosphate. Glucose 1-phosphate is converted
into UDP Glucose or the Uridine Diphosphoglucose
and will be further converted into glycogen with the
help of the enzyme Glycogen synthase.
● Glycogen will indefinitely stored in the LIVER and
will be use if the body needed more glucose
● Glycogen, when catabolized, will lead to higher
glucose level in the blood which will be delivered to
the cells for the energy requirement.
GLYCOGENOLYSIS
The conversion of Glycogen to Energy through
Glycolysis Is called Glycogenolysis.
Glycogen > Glucose > Energy
● Even without eating for days, the body still
continues the metabolic processes because of the
stored glucose called Glycogen that is converted
back to Glucose 1-Phosphate through the enzyme
Glycogen Phosphorylase and will be further
converted to Glucose 6-Phosphate and will undergo
Glycolysis to produce energy for metabolic
processes.
Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.
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-- Page 3 --
SUMMARY OF GLUCOSE METABOLISM
● The principal pathway for glucose metabolism is the
Embden Meyerhof pathway. Glucose and other
substrate like protein, and as well as fatty acids use
the tricarboxylic or krebs cycle to produce energy
● Hexose Monophosphate Shunt is a side pathway
that produces NADPH. NADPH is the substance
that would help protect the cells against oxidation.
● Glycogenesis stores the excess glucose in the Liver
for later use. Liver as well as the muscle are the
major tissues that involve carbohydrate storage.
Once supply of glucose is low, catabolism of
glycogen begins in order to increase the blood
glucose level.
GLUCOSE REGULATION
● Blood glucose is maintained by the body within a
narrow range
● The tight regulation is referred to as glucose
homeostasis
● It involves the action of various hormones that will
either lower the blood glucose level or raise it
depending on the need of the body
● During overeating or starvation, the body has to
respond in such a way to regulate the excess or
deficiency of glucose we take in.
● Glucose: primary source of energy of the body
● Glucose Regulation: Rate of glucose entering =
Rate of glucose removal
○ Plasma glucose concentration is a full function
of the rate of glucose entering the circulation
balanced by the rate of glucose removal from
the circulation
● Circulating glucose is derived from:
1. Intestinal absorption during fed state - gastric
emptying
■ During fed state the gastric emptying is
the major determinant of how quickly
glucose appears in the circulation
2. Glycogenolysis (hepatic process) = breakdown
of glycogen
 3. Gluconeogenesis: formation of glucose Proinsulin
primarily non-glucose sources during fasting ● Contains 3 distinct chains:
state ○ A Chain
● Glucose is primarily used for energy when it enters ○ B Chain
the body. But during fasting, glucose is supplied to ○ C Peptide Substance
the extracellular fluid from the liver through the ■ When there is an increase in body glucose
breakdown of glycogen. level, the C peptide dissociates from its
● If fasting is further prolonged like in the cases of main structure leaving the insulin in its
starvation, glucose is synthesized from other active form (Image above)
sources through gluconeogenesis.
○ Fat cells are then metabolized to glucose
○ Fat cells are used to generate glucose and later
on energy
○ This is the main principle why we get thinner
when we reduce our food intake
○ When there is less glucose and glycogen is
being depleted, the body begins to metabolize
the stored fat and convert them to glucose
HORMONES
● Chemical messengers that regulate the breakdown
and building up of glucose
● Insulin, Glucagon, Epinephrine, Growth Hormone,
Cortisol
○ Affects blood sugar level
○ Keeps blood sugar in healthy individuals within
a range of 70-99 mg/dL OR 3.92-5.04 mmol/L
■ Conversion factor: multiply by 0.056 ● Main action: Decrease circulating glucose level in
(conversion of mg to mmol) blood by transporting it inside the cells
● The insulin receptors present in the cell bind with
INSULIN
the insulin. This binding opens up the glucose
● Most known hormone that regulates the blood channel.
sugar level ● As the glucose moves into the cells, it lowers the
● Until recently, it is the only known pancreatic cells circulating glucose, causing a decrease in blood
hormone to lower blood glucose concentrations sugar level.
● Comes from the beta cells of the islets of ● Thus with this action, insulin is considered to be a
langerhans cells in the pancreas hypoglycemic agent.
● The substance is stored in its inactive pro-hormone
precursor called proinsulin.

● Insulin serves as a key and without it, glucose

channel will not open and the glucose will remain in
the circulation

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

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-- Page 4 --
● In patients with DM where insulin is insufficient or
GLUCAGON
absent, many of the glucose molecules remains
outside while cells are deprived of energy
● Causes high blood sugar but despite the sugar
levels, they feel a lack of energy because it is not
utilized.

ACTION OF INSULIN
● Cellular uptake of glucose
● Promotes glycogenesis
● Promotes lipogenesis
○ Conversion of carbohydrates to fatty acids
● Promotes glycolysis
○ Metabolism of glucose to pyruvate for energy
production
● Reduces blood glucose level by inhibiting
glycogenolysis

AMILYN ● Glucagon is the hormone that increases the glucose

● Newly recognized hormone co-expressed and level. It is produced also by the islets of langerhans
co-secreted with insulin by the pancreatic beta cells and the alpha cells are the ones responsible for its
● Released in response to nutrient intake and found to production
be deficient in patients with type 1 and type 2 DM ● Glucagon is released during stress and fasting.
● Works with insulin to coordinate the rate of glucose ● It stimulates the glycogenolysis in the liver, and
appearance in the circulation increases the gluconeogenesis which is known as
● Suppresses postprandial glucagon secretion the formation of glucose from non-carbohydrate
● Slows gastric emptying sources.
● When there is low blood glucose level, glucagon will
Amilyn works by regulating the glucose appearance be released from the pancreas and stimulate the
from endogenous (liver-derived) and exogenous breakdown of glycogen. This breakdown then raises
(meal-derived) sources. the blood sugar level. Because of this action,
glucagon is called the hyperglycemic agent.
● Insulin and glucagon have opposing actions. Thus
INCRETIN HORMONES making insulin, the hypoglycemic hormone.
● L cells - Intestines ADRENAL GLANDS
● Intestinal peptides are also important for hormonal
regulation. These are called incretin hormones ● Secrete hormones that regulate the glucose
● GIP ( glucose-dependent insulinotropic peptide) homeostasis which include:
○ Stimulates insulin secretion and regulates fat ○ Epinephrine (released from medulla)
metabolism ■ Release is stimulated by physical or
● GLP 1 (glucagon-like peptide 1) emotional stress which is known as the
○ Enhances insulin secretion “fight or flight response”
○ Suppression of postprandial glucagon secretion ■ Acts against insulin release
○ Slows gastric emptying ■ Increase glycogenolysis
○ Promotes beta cell health ■ Promotes lipolysis
■ Its significant and increasing interest is its ○ Glucocorticoids (released from adrenal cortex)
role in the preservation of beta cell function ■ Increase blood sugar by decreasing
and proliferation intestinal entry into the cell
■ Increase gluconeogenesis
■ Increase lipolysis
● All together, they raise the blood sugar level

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 5 --
 PITUITARY GLAND OTHER HORMONES
● Hormones released by the pituitary gland also ● Thyroxine
regulates the blood glucose ○ Released by the thyroid gland
● Adrenocorticotropic Hormone (ACTH) ○ Raises blood sugar by increasing
○ Stimulate the adrenal glands to secrete its glycogenolysis, gluconeogenesis, and the
secretion which raises the blood glucose level absorption of glucose in the intestine

● Somatostatin
○ Comes from the pancreas, but is released by
the delta cells
○ In terms of glucose regulation, its main action is
to inhibit the action of insulin
● Growth hormone
○ Has regulatory function to glucagon, growth
○ Acts on the liver to stimulate gluconeogenesis
hormone, and other hormones.
○ Acts on adipose tissues to stimulate lipolysis
○ Acts in contradictory to the action of insulin

● In summary, insulin, amelene, and incretin

hormones are hypoglycemic agents. While
glucagon, somatostatin, ACTH, growth
hormone, epinephrine, cortisol, and thyroxine
work to raise the blood glucose level.

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

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-- Page 6 --
 CLINICAL CHEMISTRY (MTCC1) - LECTURE
MODULE 6: PROCEDURES, SPECIMEN CONSIDERATIONS, AND STORAGE ASSOCIATED WITH GLUCOSE ANALYSIS

interfering substances that may have been

introduced during the collection.
HANDLING AND COLLECTION
○ However, in some cases, especially where the
In the clinical practice in Clinical Chemistry, we specimen will not be used solely for the
utilize a variety of specimens that we could be using in diagnosis of certain diseases, capillary
the diagnosis of several diseases. puncture/specimens could be carried out for
Usually, in the diagnosis of carbohydrate-related certain procedures.
diseases, specifically Diabetes, the standard clinical ● Serum should be SEPARATED after collection; not
specimen that is often utilized solely for the diagnosis of longer than 30 minutes.
this disease is the VENOUS PLASMA. ○ Upon collection of the desired specimen, serum
● Venous plasma pertains to the liquid portion of the or plasma should be separated from the packed
unclotted blood or the portion of the blood that has RBCs, not longer than 30 minutes (lifted from
been placed in a vacutainer tube that contains an Henry’s; Bishop: 1 hour).
anticoagulant. ○ Separation of serum and plasma should be
● If you could remember in the vacutainer tubes, there carried out immediately to prevent substantial
is what we call additive and anticoagulant. loss of glucose by cellular fractions.
In Clin Chem, we often utilized vacutainer solely for the ● If serum is in contact with cells for longer than 30
testing of glucose is the one with gray top. minutes, add preservatives such as Sodium
● The gray top contains an additive and Fluoride.
anticoagulant. ○ In some instances where in the test could not
● The additive found in the gray top is SODIUM be carried out immediately, if the serum would
FLUORIDE and the anticoagulant is POTASSIUM still be in contact with the cells for longer than
OXALATE. 30 minutes, a preservative could be added such
as sodium fluoride.
○ Sodium Fluoride - an additive that will help
Additive: AC: Potassium Color: Gray
decrease the glycolysis that could happen
Sodium Fluoride Oxalate
during the contact time between the liquid and
The additives serve as an additional cellular portion of the blood.
component that prevents the glycolysis of the cells that ● Results remain clinically acceptable even after a
will interfere further for the testing of glucose. The delay of up to 90 minutes before separation of
anticoagulant prevents the blood from clotting thus serum and cells.
yielding the specimen plasma. ● If whole blood is refrigerated, 2 mg of sodium
In testing glucose, we could use either fluoride per mL of whole blood [2 mg NaF/mL of
venipuncture or capillary puncture. However, in most whole blood] prevents glycolysis for up to 48 hours.
cases, we prefer the use of venous plasma as this will ○ Fluoride - has a little effect on the reduction of
provide us clearer and more accurate results compared glycolysis within the first hour of storage and
to that of the capillary because fasting glucose in whole may not reach complete inhibition until 4 hours
blood collected via capillary puncture is lesser in 11 to of storage.
15% compared to serum or plasma.
Processing of this specimen, regardless of the analyte
GENERAL SPECIMEN CONSIDERATIONS, and test requested, should be carried out immediately.
HANDLING, AND COLLECTION
Room temperature (20-25 degrees Celsius)
● Fasting glucose in WHOLE BLOOD collected via ● Decrease by 7 mg/dL/hour if left uncentrifuged
capillary puncture is 11-15% lower than in and separated d/t cell glycolysis
serum/plasma. ● In storage there is a notable changes in the glucose
○ Recall: A specimen collected under capillary levels so the glucose levels decrease and that is
puncture could have an erroneous result or dependent if this specimen is stored under room
lesser accuracy as these are prone to temperature or refrigerated temperature
contamination from tissue fluids or other

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 1 --
● Room temperature there is a decrease of 7 milligram
per deciliter per hour
● If the blood is left uncentrifuged and not separated
due to cell glycolysis
Refrigerated Temperature (4 degrees Celsius)
● Decrease by 2 mg/dL/hr
● In 4-8 degrees Celsius there is a decrease of 2
mg/dL/hr noted in the glucose levels
● Decreases are observed because the liquid portion
of the blood is left unseparated from the cellular
fraction. If they were separated immediately then
there will be no effect from the glucose
concentrations.
Plasma glucose concentration INCREASES WITH
AGE:
● Because of metabolism. As we age some system
found in the body or part of the metabolism tends to
get slower thus, contributes to the slow efficiency of
the system to metabolize certain analytes.
● Contributing to the piling up of the concentrations
found in the body upon measurement.
○ Fasting glucose: 2 mg/dL/decade
○ Postprandial: 4 mg/dL/decade
○ Glu Challenge: 8-13 mg/dL/decade
● Others specimens that are measured for glucose
levels that aids in different diagnosis/monitoring
○ Aside from blood itself, we have variety of
specimens that we could utilize in testing
glucose or measuring the glucose concentration
levels that will aid in different diagnosis or
monitoring of certain conditions be itb
pathological or going to the normal homeostasis
■ Cerebrospinal fluid
■ Peritoneal fluid
■ Synovial fluid
■ Urine - used but not for diagnosis; often
always used for monitoring or in correlation
with certain diseases but never for
diagnosis alone.
■ Blood:
➢ Venous serum/plasma - for diagnosis
➢ Venous whole blood - HbA1c; from
EDTA treated vacutainer tube for HbA1c
➢ Capillary whole blood - monitoring;
usually see from POCT devices or the
one utilizing the prick method; often
always used for monitoring but not for
diagnosis purposes
Usual patient preparation: 8-10 hours
(Bishop)
Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.
“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
● In clinical practice: 6-8 hours, some hospitals
require patients only 6-8 hours of fasting time that
alone would be for glucose. If there is another test
to be carried out, the fasting hours would be
adjusted.
Glucose testing with FBS request: Preferably tested
in the morning, basal state collection (Diurnal
variation) and without vigorous activities
● Preferably tested in the basal state, because diurnal
variation would have an effect on the testing.
Vigorous activities may also interfere with the results
of glucose testing.
SOURCES OF ERROR PRIOR TO TESTING
There will be some instances wherein you will
encounter errors prior to testing that are brought by
several factors that could be observed when you had a
result of an increased level or decreased level.
INCREASED DECREASED
● Underfast ● Overfast
● Smoking ● Lipermis
● Alcohol intake ● Gross hemolysis
● Increased uric acid levels
● Strenuous activities
● Hemoconcentration - have
a delusional effect on the
glucose test
TERMINOLOGIES
HYPERGLYCEMIA
● Increased in plasma glucose levels (≥126 mg/dL)
● Can be caused by an imbalance in hormones
(insulin, glucagon, growth hormone)
● Some instances that contribute to hyperglycemia
include more units of glucose circulating in the
blood, or the patients have an extreme intake of
carbohydrate-rich foods so there could be a
condition associated with such.
HYPOGLYCEMIA
● Involves decreased plasma glucose levels which
can be caused by several factors (drugs, hormonal,
pre-existing disease)
● Post Absorptive Hypoglycemia (Fasting)
fasting ○ Brought by fasting
● Post Prandial Hypoglycemia (reactive)
-- Page 1 --
 ○ Caused by drugs or pre-existing diseases
Impaired glucose 2-hr PG 140-199 mg/dL
tolerance (7.8-11.1 mmol/L)
Levels for Hypoglycemia
● 65-70 mg/dL Provisional glucose 2-hr PG ≥ 200 mg/dL
○ Initiates release of glucagon tolerance (≥11.1 mmol/L)*
● ≤60 mg/dL
○ Suggestive of hypoglycemia PG, plasma glucose
● 50-55 mg/dL *must be confirmed
○ Observable symptoms appear such as
dizziness, fainting, etc.
DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS
1. HbA1C ≥6.5% using a method that is NGSP
IMPAIRED FASTING GLUCOSE certified and standardized to the DCCT assay*
● Characterized by fasting blood glucose 2. Fasting plasma glucose ≥126 mg/dL (≥7.o mmol/L)*
concentration between normal and diabetic values 3. 2-hour plasma glucose ≥200 mg/dL (≥11.1 mmol/L)
● FBS: 100 - 125 mg/dL during an OGTT*
● Usually coined when the patient has underwent FBS 4. Random plasma glucose ≥200 mg/dL (≥11.1
mmol/L) plus symptoms of diabetes

IMPAIRED GLUCOSE TOLERANCE NGSP - National Glycohemoglobin Standardization

● Characterized by fasting blood concentration less Program
than those required for the diagnosis of diabetes, *In the absence of unequivocal hyperglycemia, these
but the OGTT value is between normal and diabetic criteria should be confirmed by repeat testing on a
values different day. The fourth measure (OGTT) is not
● 2-HR OGTT: 140 - 199 mg/dL recommended for routine clinical use
● Coined when px had underwent OGTT

GUIDELINES

GLUCOSE DIAGNOSTIC LEVELS

GLUCOSE DIAGNOSTIC LEVELS
● These levels determine glucose status of patient
depending on the test performed
CATEGORIES OF FASTING PLASMA GLUCOSE

Normal fasting glucose FPG 70-99 mg/dL

(3.9-5.5 mmol/L)

Impaired fasting FPG 100-125 mg/dL

glucose (5.6-6.9 mmol/L)

Provisional diabetes FPG ≥126 mg/dL

diagnosis (≥7.0 mmol/L)*

FPG, fasting plasma glucose

*must be confirmed

CATEGORIES OF ORAL GLUCOSE TOLERANCE

Normal glucose 2-hr PG ≤140 mg/dL

tolerance (≤7.8 mmol/L)

Alfonso, A. | Aquino, R. | Bagtas, R.| Cunanan, K. | Cortez, M. | De Jesus, F | Del Rosario, J. | Dizon, M. | Garcia, M. | Labasan, A. | Mamaril, S. | Mendoza,
M. | Namocatcat, K.| Ong, S.| Policarpio, M. | Pornobi, A. | Quizon, Q. | Talavera, S. | Tantamco, L. | Yco, M. | Yturralde, K.

“Unless otherwise noted, this trans is for everyone’s use, credit goes to the rightful owners. Padayon future RMT!”
-- Page 1 --
GENERAL SPECIMEN CONSIDERATIONS, HANDLING AND
COLLECTION
1 Standard Clinical Specimen: Venous Plasma
Additive: AC: Potassium Color: Gray
Sodium Fluoride Oxalate
• In the clinical practice in clinical chemistry
we utilize variety of specimens that we
could be using in the diagnosis of several
diseases.
• Usually in the diagnosis of carbohydrate-
related diseases, specifically the diabetes,
the standard clinical specimen that we
often utilize solely for the diagnosis of this
disease is the venous plasma.
• The venous plasma pertains to the liquid
portion of the unclotted blood or the portion
of the blood that has been placed in a
vacutainer tube that contains an
anticoagulant.
• If you remember from the vacutainer tubes,
there is the term that we call additive and
anticoagulant.
• In the clinical chemistry the often vacutainer
that we use solely for the testing glucose
testing is the gray top.
• The gray top contains an additive and an
anticoagulant. The additive found in the
gray top is sodium fluoride and the
anticoagulant found in the gray top is
potassium oxalate.
• The additive serves as an additional
component that prevents the glycolysis of
the cells that will interfere further for the
testing of glucose.
• ANTICOAGULANT: The potassium oxalate
prevents the blood from clotting thus
yielding the specimen plasma.
• In testing glucose we could use either
venipuncture or capillary puncture.
However in most cases, what we prefer in
the clinical practice is the use of venous
plasma as this will provide us a clearer and
a more accurate results compared to that
of the capillary puncture.
Why is that so?
2 Fasting glucose in WHOLE BLOOD collected via
capillary puncture is 11-15% lower than in
serum/plasma
• If you also recall from the basics of blood
collection, a specimen collected under
capillary puncture could have an erroneous
results or lesser accuracy as these are
prone to contamination from tissue fluids or
any other interfering substances that may
have been introduced during the collection.
• However, in some cases especially in cases
where in the specimen will not be used
solely for the diagnosis of certain diseases,
capillary specimens could be carried out for
certain procedures.
3 SERUM SHOULD BE SEPARATED AFTER COLLECTION
(NOT LONGER THAN 30 MINUTES)
• Upon collection of the desired specimen,
serum or plasma should be separated from
the cells from the packed red cells itself not
longer than 30 minutes.
• This 30 minutes is lifted from henry's and if
you could see in your bishop, it would tell
you that an hour is okay for the separation.
• Separation of serum and or plasma should
be carried out immediately as this will
prevent substantial loss of glucose by
cellular fractions.
4 If serum is in contact with cells for longer than 30
minutes, a preservative may be added such as
SODIUM FLUORIDE.
• Sodium Fluoride – an additive that will help
decrease the glycolysis that could happen
during the contact time between the liquid
portion and cellular portion of the blood.
5 Results remain clinically acceptable even after a
delay of up to 90 minutes before separation of
serum and cells.
6 If whole blood is refrigerated, 2 mg of Sodium
Fluoride per mL of whole blood prevents glycolysis
for up to 48 hours.
• Fluoride – has a little effect on the reduction
of glycolysis within the first hour of storage
and may NOT reach complete inhibition
until 4 hours of storage.
o That’s why what we always
encourage is that processing of the
specimen (regardless of the analyte
and the test that is requested)
should be carried out immediately.
7 Room Temperature (20-25C)
• for storage considerations where the blood
is stored in RT for 20-25C, there is a
noticeable DECREASE of 7 mg/dL/hour if left
uncentrifuged and separated due to cell
glycolysis
8 Refrigerated Temperature (4C)
• if the blood is refrigerated wherein it has a
temperature of 4 or within the range of 4-
8C, there is a DECREASE of 2 mg/dL/hour
Note: these decreases are observed because the
liquid portion of the blood is left unseparated from
the cellular fraction(?). If we have separated them
immediately, then there will be NO effect on the
glucose concentrations.
9 Plasma Glucose INCREASES WITH AGE
• this is because of our METABOLISM – as we
age, some system found in the body or part
of the metabolism tends to get slower, thus
it contributes to the slow efficiency of the
system to metabolize certain analytes,
contributing to the piling up of the
concentrations found in the body upon
measurement.
Fasting glucose 2 mg/dL/decade
Post prandial 4 mg/dL/decade
Glu challenge 8-13 mg/dL/decade
10 Other Specimens that are measured for Glucose
Levels that aids in different diagnosis or monitoring
• Cerebrospinal Fluid (CSF)
o These are used to differentiate
meningitis
• Peritoneal Fluid
• Synovial Fluid
o It can be used to differentiate
bacterial infections from others
• Urine
o It is not used for diagnosis
 o It is often used for monitoring or in
correlation with certain diseases
• Blood
o Venous Plasma or Serum – for
diagnosis
o Venous Whole Blood – for HbA1c
▪ It usually comes from
EDTA-treated vacutainer
tube
o Capillary Whole Blood – for
monitoring
11 Usual patient preparation: 8 – 10 hours fasting
according to Bishop
• Clinical Practice: 6 to 8 hours fasting for
Glucose
o If there are other tests to be carried
out, the fasting hours could be
12 Preferably tested in the morning, basal state
collection (diurnal variation) and without vigorous
activities
• If the patients have FBS request, it is
preferably tested in the morning, wherein
the patients are on their basal state
collection as the diurnal variation would
have an effect in the testing
• The patients are also restricted from
vigorous activities because these may
interfere with the glucose level
determination
SOURCES OF ERRORS PRIOR TO TESTING
INCREASED DECREASED
• Underfast • Overfast
• Smoking • Lipemia
• Alcohol Intake • Gross Hemolysis
• Increased Uric Acid
Levels
• Strenuous Activities
• Hemoconcentration
Notes:
• Hemoconcentration may have delusional effect
on the glucose testing
TERMINOLOGIES
HYPERGLYCEMIA
• Is an increase in plasma glucose levels (≥126
mg/dL of glucose in the blood)
• Can be caused by some several factors:
o an imbalance in hormones (insulin,
glucagon, growth hormone)
o some instances that also contributes to
hyperglycemia is that perhaps some
patients may have extreme intake of
carbohydrate-rich foods, so there could
be a condition associated for such
HYPOGLYCEMIA
• involves decreased plasma glucose levels which
can be caused by several factors (drugs,
hormonal, pre-existing disease)
• Post Absorptive Hypoglycemia (Fasting)
o Brought by fasting itself
• Post Prandial Hypoglycemia (Reactive)
o Usually caused by drugs or pre-existing
diseases
• Can be defined by the levels that the patient has
been experiencing
o 65-70 mg/dL concentration of glucose
▪ Initiates release of glucagon
o ≤60 mg/dL
▪ Suggestive of true hypoglycemia
o 50-55 mg/dL
▪ Observable symptoms appear
(dizziness, fainting, and such)
IMPAIRED FASTING GLUCOSE
• Characterized by fasting blood glucose
concentration between normal and diabetic
values
• FBS – this is usually coined when the patient had
underwent FBS, so the FBS levels is between 100-
125 mg/dL
• As you know hyperglycemia is characterized by
126, so yung levels between 100-125 is called the
impaired fasting glucose
IMPAIRED GLUCOSE TOLERANCE
• Coined when the patient had underwent OGTT
or the Oral glucose tolerance test
• It is characterized by fasting blood concentration
less than those required for the diagnosis of
diabetes, but the OGTT value is between normal
and diabetic values
• 2nd HR OGTT: 140-199 mg/dL
GUIDELINES
GLUCOSE DIAGNOSTIC LEVELS
These levels determine glucose status of patient
depending on the test performed.
CATEGORIES OF FASTING PLASMA GLUCOSE
FPG 70-99 mg/dL
Normal fasting glucose
(3.9-5.5 mmol/L)
Impaired fasting glucose
FPG 100-125 mg/dL
(5.6-6.9 mmol/L)
FPG ≥ 126 mg/dL
Provisional diabetes diagnosis
(≥ 7.0 mmol/L)a
FPG = fasting plasma glucose
aMust be confirmed
CATEGORIES OF ORAL GLUCOSE TOLERANCE
Two-hour PG ≤ 140 mg/dL
Normal glucose tolerance
(≤ 7.8 mmol/L)
Two-hour PG 140-199 mg/dL
Impaired glucose tolerance
(7.8-11.1 mmol/L)
Two-hour PG ≥ 200 mg/dL
Provisional diabetes diagnosis
(≥ 11.1 mmol/L)a
PG = plasma glucose
aMust be confirmed
DIAGNOSTIC CRITERIA FOR DM
1. HbA1c ≥ 6.5% using a method that is NGSP
certified and standardized to the DCCT assaya
2. Fasting plasma glucose ≥ 126 mg/dL (≥ 7.0
mmol/l)a
3. Two-hour plasma glucose ≥ 200 mg/dL (≥11.1
mmol/L) during an OGTTa
4. Random plasma glucose ≥ 200 mg/dL (≥11.1
mmol/L) plus symptoms of diabetesa
HbA1c, hemoglobin A1c
NGSP. National Glycohemoglobin Standardization
Program
OGTT, oral glucose tolerance test
aIn
the absence of unequivocal hyperglycemia, these criteria
should be confirmed by repeat testing on a different day. The
fourth measure (OGTT) is not recommended for routine clinical
use.
7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
LIPIDS
LIPIDS
GENERAL DESCRIPTION
● Lipids – very necessary for us to understand
and learn as MT because it is one of the
biomolecules that we check in the lab
(Lipid profile)
o There are many underlying diseases
and disorders that are connected
with Lipids
o As a medical practitioner or MLS, we
need to understand all the
structures, function, and metabolism
involved in lipids
● commonly referred to as FATS
● soluble in ORGANIC SOLVENTS like
chloroform and ether.
STRUCTURE
● Contains Carbon, Hydrogen, and Oxygen
● Composed mostly of C-H bonds
● Non-polar
o It is insoluble in water and blood just
like mixing water and oil
o They never mix with water and
blood
● Requires lipoprotein for its transport
mechanism and for the circulation in blood
o Lipoprotein – used by lipids for
transportation
▪ If an object is insoluble, it will
not interact with blood. But
because of lipoproteins,
there is metabolism and
regulation of lipids in the
body.
FUNCTION
● Source of energy
o Lipids are rich source of energy and
an efficient way for the body to
store excess calories
▪ Ex. You eat a lot of food but
not all being eaten is
absorbed or used as a fuel of
the body. What happens is it
is being stored in the adipose
tissues which is why the first
change being seen in a
person who eats a lot of
food is they get fat because
the food was converted into
lipids
▪ It is the source of energy
because when one is in its
fasting state or one is on a
diet, he or she remains alive
TRANSFORMERS
Clinical Chemistry 1 (LEC)
because of the storage of
energy in a form of lipids
● Play an important structural role in cells
o Lipids is part of our cell membrane in
the form of phospholipids
● Lipids are also precursors of steroid
hormones, prostaglandins, leukotrienes, and
lipoxins.
FORMS OF LIPIDS
FATTY ACID
● Fatty acid
o simplest lipid in terms of structure in
the body
o linear chains of C-H bonds that
terminate with a carboxyl group
(-COOH)
■ No rings or any structure just
a linear chain of
Carbon-Hydrogen bonds
■ Terminal group is the
carboxyl group (-COOH)
○ In Plasma: Small amounts exist in the
free form
o In plasma, only relatively small
amounts of fatty acids exist in free or
unesterified form
o Usually, it is bound to albumin
o Majority of FA in the plasma are
found as constituents of triglycerides
and phospholipids
Question: Why is fatty acid not a part of the lipid
profile being tested? – Because only small amount
is being seen in the plasma and is usually bound to
other molecules
FUNCTION
● It is a building block of triglycerides (TAG)
and phospholipids.
o So, sabi ko nga kanina sa inyo
majority of the amount of fatty acids
in our plasma bound or constituents
of triglycerides & phospholipids.
● It is also a source of our metabolic energy.
o Mamaya mas maiintindihan niyo if
we are already tackle metabolism
of lipids.
CLASSIFICATION
Based on the number of:
A. C ATOMS
B. C=C BONDS
● So, for fatty acids we have classifications or
we classify fatty acids based on their
carbon atoms and carbon to carbon
bonds.
1
7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
CLASSIFICATION BASED ON C ATOMS:
1 Short Chain
● Comprises 4 to 6 carbon atoms
o So, bibilangin niyo lang yung
carbon niya so kapag may
nakita kayong picture
bibilangin niyo kung ilan ang
carbon niya. Kapag 4 to 6
carbon atoms it means short
chain fatty acid siya.
2 Medium Chain
● Comprises 8 to 12 carbon atoms
3 Long Chain
● More than 12 carbon atoms
o Basic Biochemistry lang ito. Short
chain fatty acid comprises 4 to 6
carbon atoms. Medium chain fatty
acid comprises 8 to 12. While long
chain fatty acid 12 or more than 12
carbon atoms.
o Kung mapapansin niyo they are
only or the carbon atoms of a lipid
or a fatty acid has even numbers
—4, 6, 8, 12 diba yung mga numbers
ng carbons. It is because the
synthesis of even chain fatty acids
synthesis is done by assembling
Acetyl CoA precursors.
o Mamaya mas maiintindihan niyo
siya because the segments of each
2 carbons in length resulting in the
fatty acids have even numbers or of
carbon atoms.
o So, sa fatty acids hindi pwedeng
magkaroon ng 13 carbons or 19
carbons laging even numbers yung
nilalaman ng kayang carbon. Bakit?
It is because of the assembly of
Acetyl CoA precursors.
So, I will give you an example of fatty acids:
● Kung titingnan niyo linear lamang siya wala
na kayong makikitang pabilog-bilog diyan
or wala na kayong makikitang kung
ano-ano pang structure linear lang siya.
Yung sinasabi ko kanina sa inyo the –COOH
TRANSFORMERS
Clinical Chemistry 1 (LEC)
group or the carboxyl ang kanyang
terminates or it ends in carboxyl group.
● They are all fatty acids and they all end in
carboxyl group.
● Kung titignan niyo (Figure A) 1, 2, 3, 4 mga
even numbers ang ating mga carbons sa
lipids. It is because of the assembly of
Acetyl CoA Precursors.
● Now, you can identify kung ano ang long,
ano ang short at kung ano ang medium
fatty acid bibilangin lang natin ang ating
mga carbon sa structure.
CLASSIFICATION BASED ON C=C BONDS
1 SATURATED
● It has no double bonds.
● Kapag sinabing saturated
wala siyang double bonds.
2 UNSATURATED
● It has double bonds.
● Ang ating unsaturated ay
merong dalawang klase. It
has monounsaturated and
polyunsaturated.
2.1 Monounsaturated
● It has one double bond.
● Isa lang ang kanyang
double bonds.
2.2 Polyunsaturated
● It has 2 or more double bonds.
3 KINDS OF FATTY ACIDS
● Letter A: Stearic acid
o Saturated fatty Acids = Absence of
double bonds
● Letter B: Oleic Acid
o Unsaturated fatty Acids = presence
of double bonds
o What type of unsaturated FA?
▪ Monounsaturated fatty acids
(1 double bond is present in
the structure)
● Letter C: Linoleic Acid
o Unsaturated fatty Acids = presence
of double bonds
2
7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
Clinical Chemistry 1 (LEC)

o What type of unsaturated FA? nangyare yung hydrogen ay

▪ Polyunsaturated fatty acids nasa opposite side. This two
(2 double bond is present in hydrogens has already far
the structure) apart as they can get. So
wala ng repelling
● How do we classify our fatty acids? nangyayare di na siya
1. number of carbons nagbend. Nanatili siya in
2. number of carbon to carbon double linear formation.
bonds of the structure ▪ It is because of the special
● Fatty acids- have a structure of C-H bond orientation of double bonds
trans FA do not bend and
CIS AND TRANS FATS have a physical properties
more similar to saturated FA.
Eto yung tinatawag na SOLID
at room temperature

Saturated FA Solid at RT Oil from animals

Unsaturated Fluid/liquid in Ex. Vegetable

● Cis and Trans Fat are usually seen in FA nature oil, olive oil
unsaturated fatty acids
o Cis form (bent)
▪ Usually unsaturated FA can
▪ Picture: Both hydrogen
be seen in vegetable oil
atoms on the same side of
(kahit malamig di sila
the C-C double bonds which
namumuo) unlike the oil na
causes bent in molecular
galing sa taba ng baboy or
structure
ung oil na galing sa animals
▪ The bent increases the
namumuo muo.
space that unsaturated fatty
▪ Trans form are not commonly
acids require when packed
found in nature. However,
in a lipid bilayer which
they are present in our diet
requires to a fluid FA
because of the chemical
because they do not ask
hydrogenation treatment
readily cells associated and
used in food processing.
packed together tightly
▪ Trans fats are usually seen in
▪ So, ang ating mga cis form
foods because it has a
or yung mga bent form na
capability to hydrogenate. If
tinatawag, they are
hydrogenation occurs “hindi
associated with more fluid
agad nasisira ang pagkain”.
FA kase they are not
Usually trans fats can be
compact with each other.
seen in processed food.
Technically speaking they
▪ Studies have shown that the
are not tightly packed
consumption of trans FA
together, ang tendency
increases the risk of CHD.
medyo fluidy siya compared
▪ Trans fat is not healthy in our
to our trans form or straighter
body. Its adverse effect is
form of FA. Nangyare yan
attributed to the elevation of
because the two
LDL (bad cholesterol)and
neighboring hydrogen repel
lower the HDL (good
each other, causing the
cholesterol)
carbon chain to bend.
▪ If there is an increase in LDL
○ Trans form (straighter)
in our body, it will build up in
▪ Also known as “trans fats”
our arteries (blood
▪ It is a hydrogen atom on the
vessels)and it will increase
opposite side of the C-C
the risk of heart disease or
double bonds.
stroke.
▪ So, makikita sa picture may
double bonds, kaso ang

TRANSFORMERS 3
7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
▪ Trans fat is used in food
processing to treat the
spoilage of food.
▪ Cis form is more healthy than
the trans fat.
● Fatty Acids- is the simplest form of lipids
when it comes to structure. Di tayo
nakakakita ng free FA sa ating circulation
because it is always bound to albumin and
majority of our FA is bounded with
phospholipids and TAG.
TRIGLYCERIDES
● Also called as Triacylglycerol
● Triglycerides – old name of Triacylglycerol
● They are called as such because they
contain THREE FATTY ACID molecules
attached to ONE molecule of GLYCEROL
● The reason for the bond between glycerol
and the three fatty acids is due to ESTER
BONDS
● No charged group or polar hydrophilic
groups in the triglyceride structure
● NEUTRAL LIPID - It is considered a neutral
lipid because it has no charge
● Very hydrophobic meaning there are no
charges or polar hydrophilic groups, making
it very virtually water insoluble
● Because it has a HYDROPHOBIC and
HYDROPHILIC side, the tendency is that it
becomes neutral
● Most of the triglycerides came from plants.
(Ex. Oils)
○ It is rich in polyunsaturated fatty
acids
● Some come from animals
○ It is rich in saturated fatty acids
○ Usually SOLID at room temperature
NOTE: Magkaiba ang oil sa fat. Ang OIL iyan ang
mga plant sources natin. Under sila sa lipids pero
magkaiba si oil at si fats. Si OIL, ito yung mga rich sa
polyunsaturated and usually ang source niya is
plants. For animal sources naman, it is rich in
saturated fatty acid and usually ito yung mga solid
sa room temperature. Sila yung tinatawag natin na
FATS talaga.
Triglyceride Structure
TRANSFORMERS
Clinical Chemistry 1 (LEC)
● Color Yellow – Fatty acids
● Color Green – Glycerol
● Ester Bonds (encircled in red)– connect the
fatty acids and glycerol
FUNCTIONS
● Storage form of lipids (Main Function)
○ Stored inside the fat cells
(adipocytes) in the form of
triglycerides.
○ Yung mga nakikita niyong
taba-taba o bilbil, adipocytes cells
and laman niyan. Sa loob ng mga
cells na iyan na adipocyte cells, ang
laman niyan ay triglycerides
○ If we no longer have a source of fuel
or energy in our bodies, diyan
unang kukuha ang ating katawan
sa triglycerides na naka-store sa
ating adipocytes
○ Constitutes 95% of stored fats is
stored triglycerides, and it can be
utilized during fasting states
between meals
○ Kaya kung minsan, kahit kumakalam
na ang sikmura, kahit na todo diet
ka, nakakakilos ka pa din o kaya mo
pa magbuhat. It is because meron
pa tayong source of energy and it is
in form of lipids to be classified
specifically under triglycerides.
WHERE DO WE GET IT? Nakatago
siya sa ating adipocytes.
○ Kaya nga kapag nagpapapayat or
nagda-diet ka, pumapayat tayo
kasi nga nagagamit na yung mga
triglycerides sa loob ng adipose cells
natin.
● Integral part of the cell membrane
● Insulation
● Shock absorber
○ Ex. Nabangga ka sa lamesa at
natusok ang tiyan, pero until now
okay ka pa din o okay pa ang liver
mo (kunware liver part ang
natamaan). Okay ka pa din it is
because of triglycerides.
○ Our visceral fats are made from
triglycerides as well.
○ Visceral Fats – adipose tissues
around our organs. Its main purpose
is to cushion and to shield our vital
organs in the body.
Kung bakit nakakagalaw sila
maayos sa ating katawan, kung
bakit ang heart nakakapag pump
ng maayos na hindi nagagasgas,
because meron tayong visceral fats.
4
7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
Clinical Chemistry 1 (LEC)

○ Subcutaneous fats – fat under the

skin. It protects and insulates the
Polar hydrophilic faces toward aqueous
body. head environment
○ Kaya nga yung medyo chubby
chubby na tao, hindi sila ganoon Fatty acids faces inward away from the
kadali malamigan because water in a perpendicular
mayroong insulator sa ating position with respect to the
katawan (triglycerides). Pero yung lipid surface that we have.
mga buto’t balat, payat, at slim ● Naming depends on the phospholipid head
nilalamig agad kasi wala naman present.
insulator kaya yung cold
temperature narating agad yung
nerve endings nila. Unlike sa mga
chubby na maraming triglycerides
sa katawan, madami subcutaneous
fat na tinatawag meron pa silang
insulator o pinapa-init pa nila before
sila lamigin ng husto.

PHOSPHOLIPID
● Similar in structure to triglycerides except
that they only have two esterified fatty
acids.
● The third position on the glycerol backbone
instead contains a phospholipid head
group.
● Has glycerol and fatty acid but in
triglycerides it has three fatty acids while in
phospholipid it has only two fatty acids.
● In the third position instead of fatty acid, it
will be a phospholipid head group.
● PHOSPHOLIPID HEAD GROUP
o Hydrophilic in nature
▪ That is why phospholipid is in
the cell membrane. It has
hydrophobic tails and a
hydrophilic head.
o Examples of phospholipids: Choline,
inositol, inositol phosphates, glycerol,
serine, ethanolamine.
o The various types of phospholipids
are named based on the type of
phospholipid heads present in the
structure.
● Normally, the phospholipids contain 14-24
carbon atom long with one fatty acid
containing saturated or unsaturated fatty
acid.
● Because phospholipids contain
hydrophobic acid chains and hydrophilic
head group, they are defined as
amphipathic lipid molecules and such are
found in the surface of the lipid bilayer.
● The structure shown looks like a triglyceride
which has one glycerol. The only difference
is that fatty acid in the third position has a
phospholipid head group.
● In this case, choline is the phospholipid
head group given in the structure being
shown.
● These are amphipathic lipid molecule
FUNCTIONS
PHOSPHOLIPID BILAYER
● Important part of the cell membrane
LUNG SURFACTANT
● It allows effective gas exchange and
prevents alveolar collapse during
exhalation.
● Usually used for LS RATIO.
o Used to check if the lungs of the
baby is healthy. Checking of its
phospholipids or rather called
lecithin or sphingomyelin.
o Lecithin and sphingomyelin are in
phospholipids in nature.
o If the ratio is okay, therefore the
lungs are healthy and can bear the
respiration (utilized even if the baby
is still in the womb).
o Indication if the baby can still survive
inside the womb.
o Phospholipid in nature is being used
(participates in cellular metabolism
and blood coagulations).

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STRUCTURE, FUNCTIONS, & METABOLISM
● Polar hydrophilic head faces toward the
aqueous environment. Whereas the fatty acid (tail)
faces inward away from the water in a
perpendicular position with respect to the lipid
surface that we have.
PHOSPHOLIPID BILAYER STRUCTURE
● Fatty acid (tail) orients themselves away
from the waters because it is hydrophobic.
● Glycerol and phosphate heads are
attracted to water because it is hydrophilic.
● Remember, phospholipids are the same
with the triglycerides. The only difference is
that phospholipids only have two fatty
acids, one glycerol, and one
phosphate/phospholipid head group.
● For the functions it will be phospholipid
bilayer and lung surfactant.
CHOLESTEROL
● Unsaturated steroid alcohol containing four
rings (A, B, C, and D), and a single C-H side
chain tail.
● Contains a hydroxyl group (-OH GROUP) in
the A-ring which is the only hydrophilic part
of cholesterol.
● Almost exclusively synthesized by animals
● NOT A SOURCE OF FUEL OR ENERGY (not
readily catabolized by most cells or not
rapidly broken down into smaller pieces).
● Therefore, it increases the risk of different
diseases. Increased cholesterol is
dangerous for the patient.
● Have four steroid ring
● – OH group is found on the A ring (red
circle)
TRANSFORMERS
Clinical Chemistry 1 (LEC)
● Cholesterol is almost exclusively synthesize
by animals
o Which is why cholesterol is acquired
in our dietary or lifestyle depending
on what food we eat.
● It is not readily catabolize therefore not a
source of fuel
FUNCTION
● Part of the Cell Membrane
● Can be Converted to Primary bile acids
o Primary bile acids are necessary to
break down other macromolecules
such as bilirubin.
● Precursor to steroid hormones & Vit. D3
o Vitamin D is not only acquired
through sunlight and supplements. It
can also be a precursor from
cholesterol.
DIAGNOSTIC SIGNIFICANCE
● Evaluate risk for atherosclerosis, myocardial,
and coronary arterial occlusions
● Essential in the diagnosis and management
of lipoprotein disorders
● Used to monitor effectiveness of lifestyle
changes and stress management
● As seen in the pic, cholesterol can be
Vitamin D
● A small amount of cholesterol, after being
converted to 7-dehydrocholesterol, it can
be transformed to Vitamin D3 in the skin by
irrigation from sunlight
● Can be converted to steroid hormones
such as:
o Progesterone
o Glucocorticoids
o Mineralocorticoids
o Androgens
o Estrogens
● Can be converted to primary bile acid in
the liver such as:
o Cholic acid
o Chenodeoxycholic acid
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STRUCTURE, FUNCTIONS, & METABOLISM
Clinical Chemistry 1 (LEC)

▪ Promotes fat absorption in LIPOPROTEINS

the intestine by acting as a
detergent LIPOPROTEINS
GENERAL DESCRIPTION
FORMS ● Sabi ko nga kanina sa inyo, kung lipids
CHOLESTEROL (CHOLESTERYL) ESTER/ESTERIFIED alone hindi niya kaya na ma-synthesize ng
● It has ester bond maayos, hindi natin yan kayang marating
● Comprises 60% to 70% of the total or mametabolize ng maayos. Like for
cholesterol example, ako nasa Mexico ako, hindi ko
● Hydrophobic kayang magawa yung dapat kong gawin
o Because cholesterol is bound to sa AUF kung walang vehicle or
fatty acids. transportation. Kailangan ko ng
● Termed as “esterified” due to presence of jeep/sasakyan para makapunta sa AUF
fatty acid ring on one of its four rings para magawa ko yung mga functions na
o Type of bond present when there is meron ako.
a fatty acid is the ester bond. ● Ganoon din si lipids, maraming function si
● Neutral lipid lipids sa ating katawan at hindi niya ‘yon
o Means that there is no charge magagawa without its transport
▪ Since it has positive and mechanism or its vehicle (bus ng ating
negative ending leading to lipids) 🡪 lipoproteins
being neutral. ● It is made of proteins and lipids

FREE/UNESTERIFIED CHOLESTEROL STRUCTURE

● 30% to 40% of total cholesterol in circulation ● Shape: Typically spherical
● Cholesterol ring only ● Size: 10 to 1,200 nm
o A, B, C, D ring only ● Composed of lipids and apolipoproteins
o No fatty acid (proteins)
o No ester bond

Additional Info:
● Cholesterol is not a source of fuel because
it is not readily catabolized by the body.
Thus, cannot be used as a source of
energy.
● Notice that our source of energy such as
carbohydrates have a simple (linear)
structure.
● Cholesterol has a complex structure for
having four rings.
● A High cholesterol level is dangerous since it
is not readily catabolized by the body.
o Kasi paikot ikot lang sa circulation
Surface
● Primarily found in the surface of lipoprotein
(this is just a single monolayer) is the
amphipathic cholesterol and the
phospholipid molecules
● Found in the central or core region is the
hydrophobic or the neutral triglyceride and
cholesteryl ester molecules
● So, primarily located on the surface of the
lipoproteins are the apolipoproteins
o bean-shaped molecules
phospholipids and cholesterol

Core region neutral triglycerides and

cholesteryl ester

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7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
Clinical Chemistry 1 (LEC)

● Hydrophobic core or central region

o usually cholesterol ester, free FA, The larger lipoprotein particles have
and TAG correspondingly larger core regions and, therefore,
● Sa loob, dito niya tinatago and ating mga contain relatively more triglycerides and cholesteryl
esters. The larger lipoprotein particles also contain
lipids or yung tinatawag na hydrophobic
more lipid relative to protein, thus, are lighter in
core (transportation) density.

kahit na mas malaki si chylomicrons, pero yung

kanyang density, maliit lang because the larger
lipoprotein molecule also contain more lipid
relative to protein, and thus, lighter in density. In
chylomicrons, mas marami yung lipids kesa sa
protein kaya when it comes to density, mas lighter
siya compare to HDL.

BUOYANT DENSITY AND ELECTROPHORETIC MOBILITY

FUNCTION
● Delivery of fuel to peripheral cells
● Lipids: source of our fuel/energy – di natin
yan magagawa if there is no lipoprotein,
and they are the delivery of fuel or yung
tinatawag nating mga grab/food panda
● Kailangan natin ng fuel yung mga cells,
tissues, organs natin at sumasakay and
ating lipids sa lipoprotein. Yung lipoprotein
idedesignate niya sa bawat organs sa
bawat peripheral cells para manatiling nag
fufunction ang ating mga organs
● For the size of lipoprotein, it correlates to the
lipid contents. So kung mas malaki si
lipoprotein, mas maraming lipid yung
kaanyang nadadala.
● A larger lipoprotein has a correspondingly
larger core region.
● The larger lipoprotein particles also contain
more lipid relative to protein, and thus,
lighter in density.
How do lipoprotein separate?

● Separation of lipoprotein is based on

buoyant density depending on the ratio
between the lipid and the protein present in
CHARACTERISTIC OF THE MAJOR HUMAN them.
LIPOPROTEIN ○ So kung napapaansin nyo, ang
CHARACTERISTICS CHYLOS VLDL LDL HDL ating nauuna ay si chylomicrons.
Density (g/mL) <0.93 0.93 - 1.019 1.063 ○ Buoyancy - property of a molecule if
1.006 - - 1.21 it will float.
1.063 ■ Kung mapapansin niyo, after
Molecular weight (0.4-30) (10-8 2.75 (1.75 niyo kumain, magpakuha
(kD) x10^9 0) x10^ - 3.6) kayo ng dugo, tapos
x10^ 6 x10^ icentrifuge niyo siya, yung
6 5 serum na makukuha niyo,
Diameter (nm) 80-1,200 30-80 18-30 5-12 creamy or milky siya na
Total lipid (% by 98 89-96 77 50 puti-puti, lahat iyon,
weight) chylomicrons (chylos
Triglycerides (% 84 44-60 11 3 specimen). may tendency
by weight) na magfloat iyan kasi nga
Total cholesterol 7 16-22 62 19 iyong buoyant density or
(% by weight) yung buoyancy nya ay
mababa, compare kay HDL.
Extra notes: ● Electrophoresis - used to separate
The size of the lipoprotein particles correlates with its lipoproteins. (a.k.a electrophoretic mobility)
lipid content. So if mas malaki si lipoprotein, ibig
sabihin, mas malaki rin ang lipids na mapapasok
niya sa loob niya. Kung mas malaki si bus, mas
maraming tao. Kung mas malaking lipoproteins,
mas malaki or mas maraming lipids ang kanyang
maitatransport. Like for example, tignan natin yung
sa diameter. Makikita niyo si chylomicrons ang
pinakamalaki, ang pinakamaliit si HDL. So
technically speaking, Si chylomicrons, mas marami
siyang lipid contents sa loob, mas marami siyang
nabibitbit, compare kay HDL.

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7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
Clinical Chemistry 1 (LEC)

CHARACTERISTIC OF THE MAJOR HUMAN

APOLIPOPROTEINS
APOLIPO MOLECU PLAS MAJO FUNCTIO
PROTEINS LAR MA R N
WEIGHT CON LIPOPR
(kD) C. OTEIN
(mg/ LOCATI
dL) ON
Apo A-I 28,000 100 - HDL Structural,
200 LCAT
activator,
ABCA1
lipid
acceptor
Apo A-II 17,400 20-50 HDL Structural
Apo A-IV 44,000 10-20 Chylos, Structural
VLDL,
HDL
● Eto yung origin niya, si chylomicrons, hindi
Apo 5.4 × 105 70 - LDL, Structural,
niya kayang tumakbo, magi-stay lang siya 125 VLDL LDL
B-100
diyan kasi nga ang dami niyang bitbit na receptor
lipids, tsaka malaki siya. like for example, ligand
magpaunahan kayong tumakbo ng slim at Apo B-48 2.6 × 105 <5 Chylos Structural,
remnant
ng medyo obese, sino kaya ang mauuna receptor
sa kanila? Kaya kapag may lipoproteins ligand
result na, iyong mga hindi gumalaw, ang Apo C-I 6,630 5-8 Chylos, Structural
tawag sa kanila ay chylomicrons. VLDL,
HDL
o HDL (alpha - lipoproteins) Apo C-II 8,900 3-7 Chylos, Structural,
VLDL, LPL
▪ Siya ang pinaka mabilis
HDL cofactor
tumakbo towards the
positive charge. Apo C-III 9,400 10-12 Chylos, Structural,
VLDL, LPL inhibitor
▪ have the highest protein HDL
content. Apo E 34,400 3-15 VLDL, Structural,
▪ handles more HDL, LDL
apolipoproteins, and LDL receptor
ligand
proteins are (?) 01:04:02
o LDL (beta - lipoproteins) Apo(a) (3–7) × <30 Lp(a) Structural,
105 plasminog
o VLDL (pre-beta-lipoproteins) en inhibitor
● It is based on the movement of lipoprotein,
and the nature of the apoprotein APOLIPOPROTEINS
associated with the lipoproteins. APO A-1
● Major protein of HDL
APOLIPOPROTEINS
● Frequently used as a measurement of the
● help MAINTAIN the structural integrity of
LIPOPROTEINS amount of antiatherogenic HDL present in
o Iyong structure ni lipoproteins, sa plasma
surface niya, ang laki laki ni
apolipoproteins APO B
● Serves as a ligands ● Large protein with a molecular weight of
o for cell receptors approximately 500 kD
● Serves as activators and inhibitors of our ● Usually seen in LDL, VLDL, and chylomicrons
lipoproteins ● Two forms: APO B-100 & APO B-48
● it contains AMPHIPATHIC HELIX
o It has the ability to bind the
lipoproteins in our lipids. Location Description
o It has protein segments, which are
arranged with coils. APO found in LDL a ligand receptor or
● These are the hydrophobic amino acids B-100 & VLDL ligand for LDL receptor,
that interact with lipids, and whereas the therefore it is critical in
part of the helix containing the hydrophilic the uptake of LDL by
amino acid faces away from the lipids and cells
towards the aqueous environment.
APO found in first 48% or half of the
B-48 chylomicrons APO B molecule

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7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
Clinical Chemistry 1 (LEC)

particles. This is now recognized by

proteoglycans and receptors in the liver
APO (a) facilitating their uptake.
● Plasminogen-like protein that is found in a ● Chylomicrons are responsible for the
proatherogenic lipoprotein, called triglycerides to hepatic and peripheral cells.
lipoprotein (a) (Lp(a))
o antiatherogenic lipoprotein is the HDL VERY LOW DENSITY LIPOPROTEIN (VLDL)
● APO (A) is covalently linked to APO B-100 ● Composed of APO B-100, APO E, APO Cs
● Like chylomicrons they are rich in
APO E triglycerides, but it is produced primarily in
● Usually seen in LDL, VLDL, and HDL the liver
● serves as a ligand for the LDL receptor and ● Transport endogenous (Hepatic-derived)
the chylomicron remnant receptor triglycerides from the liver to peripheral
● Isoforms: APO E2, APO E3, APO E4 tissue
● Affects the lipoprotein metabolism because o Major carrier of endogenous or
they differ in the ability to interact with LDL hepatic-derived triglycerides
receptors. o Endogenous and exogenous are
different, exogenous are the
triglycerides that comes from what
we intake, while endogenous are
MAJOR LIPOPROTEINS
the triglycerides that are from the
CHYLOMICRONS
liver
● The usual apolipoprotein seen in
o Remember: chylomicrons bring
chylomicron is APO B-48
triglycerides to the liver? And hindi
● Largest & the least dense lipoprotein
naman pede na forever na si liver
particle
ay maraming hawak na
o because of the large size, they scatter
triglycerides, tendency is that the
light which account for the turbidity or
VLDL will get the triglycerides from
milky appearance of post-prandial
the liver and deliver it to the
plasma
peripheral tissue.
o because they are so light, they float
o Excess intake of dietary
readily on the top of the plasma when
carbohydrates, saturated fatty acid
stored overnight at 4 deg Celsius and
and trans fatty acid – enhances
form creamy layer
hepatic synthesis of triglycerides –
● 1,200 nm in diameter
which in turn increases VLDL
● Produced by the intestine
production
● Packed with absorbed dietary lipids and
● If the liver has too much carbohydrate
apolipoproteins
intake, saturated fatty acid intake by the
● Principal role of chylomicrons is to transport
patient it will lead to the increase
exogenous (dietary) triglycerides to hepatic
production of VLDL
and peripheral cells
● Ang ginagawa ng VLDL para mabawasan
o Exogenous/Dietary pathway
ang triglyceride sa liver, kukunin niya ang
■ lipids we get from the food
triglyceride from the liver and idedeliver
we intake. The food that we
niya sa peripheral tissue (this is what we call
ate will go to the intestine
the endogenous pathway)
which produces
chylomicrons, so that the
LOW DENSITY LIPOPROTEIN (LDL)
triglycerides that came from
● Contains APO B-100
the food will be transported.
● More cholesterol rich than other APO B
The triglycerides will be
containing lipoproteins
transported to the liver and
● Major end product of VLDL catabolism
peripheral cells through the
o They form as consequence of the
help of chylomicrons.
lipolysis of VLDL
● Once chylomicrons enter in circulation,
● Synthesize in the liver
triglycerides and cholesteryl esters in the
● Transport cholesterol to the peripheral tissues
chylomicrons are rapidly hydrolyzed by
o LDL is readily taken up by cells via LDL
lipases and within a few hours, this will
receptors in the liver and peripheral
transform into chylomicron remnant
cells

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7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
Clinical Chemistry 1 (LEC)

● In addition, because LDL particles are o Spherical Type of HDL

significantly smaller than VLDL and
Spherical Type of HDL
chylomicrons, they can infiltrate into our
extracellular space of the vessel wall where HDL 2 and HDL 3
they can be oxidized and taken up by the ● Separated because of the difference in
macrophages through the various scavengers’ density.
receptors. Which results to an ending that ● can only be found in sphere shaped HDL
macrophages take up too many lipids, it
becomes filled in the intracellular lipids, and HDL 2 Larger in size, less dense and
drops and turn into foam cells which can be richer in lipid than HDL 3.
predominant in cell type of fatty acid streaks
and early precursor of atherosclerotic plaques HDL 3 Smaller but more dense
compared to HDL 2.
o Tendency that foam cells stay in the
blood vessel, that is why it is said earlier
More efficient in the delivery
that it is a precursor of atherosclerotic of lipids to the liver.
plaques, because this is where it starts
na ‘mamuo (mabuo)’ yung mga
● Reverse Cholesterol Transport:
plaques in the blood vessel, tendency
o Transport excess cholesterol from
nagkakaroon ng high blood pressure
the tissues and return it to the liver
ang patient. That is why it is LDL is called
● Antiatherogenic lipoproteins
as bad cholesterol
● Highly Heterogeneous
o LDL = bad cholesterol (because it can
o It is separable as many as 13/14
infiltrate in the extracellular spaces)
different subfractions
● Turn into foam cells (early precursor of
atherosclerotic plaques)
Note:
● LDL subclass differ largely in content of core ● If HDL is at the normal/reference range,
lipids, the smaller particles are dense and have they will have a lower risk of having Heart
relatively more triglycerides than cholesteryl Disease
esterase
● For the research methods, the usually used MINOR & ABNORMAL LIPOPROTEINS
research method is called Beta quantification INTERMEDIATE DENSITY LIPOPROTEIN (IDL)
(used quantification specifically for LDL) ● VLDL Remnants/Subclass of LDL
● ↑ LDL level = prone to disease o Product of VLDL catabolism or can
● because LDL are the reason kung bakit be converted to LDL
nagkakaroon ng plaques, and if our blood o Usually, VLDL remnants are called as
vessel has too much plaques it can result to IDL
stroke or cardiovascular diseases and others ● Transient form of VLDL to LDL
like thrombotic diseases because of too ● In Plasma:
much atherosclerotic plaque it will cause o Normal: Not Present
injury to the blood vessel. ▪ The triglycerides and
cholesterol contents of IDL
HIGH DENSITY LIPOPROTEIN (HDL) are intermediate between
● “Good Cholesterol” those VLDL and LDL
● Smallest and most dense lipoprotein ▪ Normally, the conversion of
particle VLDL proceeds efficiently,
● Synthesized by the Liver and Intestine that appreciable quantities
● Disk or Spherical shaped of LDL usually do not
o Disk Type of HDL accumulate in the plasma
▪ Discoidal HDL typically after an overnight fast.
contains 2 molecules of ▪ Thus, IDL are not typically
APO A-1 which forms a ring normal in the plasma.
at the central lipid bilayer. o Dysbetalipoproteinemia: Present
o Believed to represent a newly ▪ Rare inborn error of
secreted HDL. metabolism
▪ Most active form in ▪ Elevated levels of IDL that
removing excess cholesterol can be found in the plasma
from the Peripheral Cell.

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7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
▪ due to an abnormal form of
APO E, that delays the
clearance of IDL which still
circulates inside of the body
instead of being converted
to LDL.
● Individuals with this disorder are at a
significant risk of Peripheral Vascular
Disease and Coronary Artery Disease
SINKING PRE-β LIPOPROTEIN/Lp(a)
● This is our Pro-atherogenic Lipoprotein.
● Anti-atherogenic will be HDL.
● This is what we called the Sinking pre- Beta
Lipoprotein due to the electrophoretic
mobility same as the VLDL but density like
VLDL.
● It contains 1 molecule of APO (a) and it is
linked with the APO B-100 by a single
disulfide bond.
● Malalaman mo agad na siya ay Lp (a)
kapag sa kanyang surface na may
apolipoprotein siya na APO (a), tapos itong
APO (a) na ito is linked to the APO B-100 by
the single disulfide bond.
● So, this is like LDL- like particles.
● It is Heterogeneous in both size and density
and Lp (a) is larger than LDL and has higher
lipid content and slightly lower density.
● Plasma levels of Lp (a) vary among the
individuals in the general population but
remain relatively constant with an
individual.
● Lp (a) appears to be poorly cleared by the
LDL receptor but the kidney has been
postulated as the site of removal.
● Elevated levels of Lp (a) may indicate an
increased risk of premature coronary heart
disease (CHD) or Stroke.
● Again, LDL- like particles and elevated
levels may indicate increased risk of
premature coronary heart disease (CHD) or
Stroke. It can be removed by our kidney, so
some studies showed that Lp (a) lipoprotein
can be removed by our kidney.
● Again, plasma levels of Lp (a) may vary and
it is may vary because different individual of
course hindi constant or hindi naman pare-
pareho ang individual, sometimes may
mga nakikita silang Lp (a), pero yung
elevated levels nito can be risk or can be
indicator of risk premature CHD and stroke.
LIPOPROTEIN X/LpX
● Next, will be LpX or yung tinatawag na
Abnormal Lipoprotein, so agad-agad pag
nakita niyo na itong lipoprotein X,
agad-agad isipin niyo na, na Abnormal
TRANSFORMERS
Clinical Chemistry 1 (LEC)
lipoprotein and it present with the patients
suffering with Biliary cirrhosis or Cholestasis
and in the patients with mutation in Lecithin
Cholesterol Acyltransferase (LCAT).
Nagkaroon ng mutation sa LCAT enzyme
ang tendency natin si kidney
nakapagproduce ng LpX and usually
nangyayari ito sa may mga Biliary cirrhosis
or Cholestasis. LpX ang napoproduce nila
kasi natamaan ang action ni enzyme, ang
tawag sa enzyme in Lecithin Cholesterol
Acyltransferase (LCAT).
● So different from the other lipoproteins in
the endogenous pathway due to the lack
of APO B-100
Components
Lipid Free cholesterol and
phospholipids
Protein APO C and albumin
● By the way, LpX is mainly removed by the
reticuloendothelial system of the liver and
the spleen. Kung kanina si Lp (a) can be
removed by our kidney, LpX can be
removed by the reticuloendothelial system
of the liver and the spleen.
FLOATING β LIPOPROTEIN/β-VLDL
● Kung kanina meron tayong tinatawag na
the Sinking pre- Beta Lipoprotein, ngayon
naman may tinatawag tayong na Floating
Beta lipoprotein, also, abnormal lipoprotein
and accumulates in
Dysbetalipoproteinemia.
● It is richer in cholesterol than VLDL and
apparently results from Defective
Catabolism of VLDL. Kaya tinatawag siyang
na β-VLDL, it usually results from the
defective catabolism of VLDL.
● Found in the VLDL density range but
migrate electrophoretically with or near the
LDL. Again, found density katulad ni VLDL,
pero electrophoretically near the LDL.
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STRUCTURE, FUNCTIONS, & METABOLISM
METABOLISM
METABOLISM
● We have four (4) lipid metabolism pathways
in our body.
ABSORPTION
Sample scenario: imagine you are eating
samgyupsal and we all know na rich in lipids yung
pagkain na yon. Our intestines, of course, will
absorb our lipids; magkakaroon ng absorption
pathway.
NOTE: Absorption can happen because of our
peristalsis.
● Peristalsis is the contraction or constriction of
our intestines
o Kung mapapansin niyo
nanonood kayo kung paano
nagkakaroon ng digestion or how
our digestive system works, makikita
niyo yung ating mga intestines
nagcocontract, nagcoconstrict, tas
biglang nageexpand; ang tawag
doon ay peristalsis
o Every time na nagakkaroon ng
peristalsis or paggalaw sa ating
intestines, nagkakaroon ng
absorption. Ang tawag doon ay
absorption pathway. Ina-absorb ng
instestines ang ating mga nutrients.
Tapos yung mga hindi na-absorb, of
course, ilalabas natin (magiging
stool). At lahat ng mga na-absorb
na lipids ni intestines ay mapupunta
sa exogenous pathway.
EXOGENOUS
● Dietary cholesterol and fatty acids that are
absorbed, dadalhin ni chylomicrons.
o Chylomicrons are secreted by our
intestines.
TRANSFORMERS
Clinical Chemistry 1 (LEC)
● With the help of LPL, itong si chylomicrons
dadalhin niya yung mga triglycerides and
dietary cholesterol natin sa ating liver.
● Yung lipoprotein na tumutulong for this
pathway is the chylomicron.
● For the liver, naipasok na yung mga dietary
cholesterol and triglycerides na nadala na
ni chylomicrons, nagkaroon na ng
chylomicron remnants since na break down
na into smaller pieces si chylomicrons;
nagkaroon na ng chylomicron remnants
dahil napunta na siya sa liver. Then it will
proceed to endogenous pathway
ENDOGENOUS
● VLDL
o Magdadala siya ng mga
triglycerides and mga cholesterol
esters si VLDL.
o With the help of the LPL (lipase
enzyme), ma-coconvert siya into
LDL.
o Itong LDL na ito, dadalhin niya ang
mga cholesterol and triglycerides sa
ating peripheral cells.
● Dadalhin niya sa mga peripheral cells; ang
tawag doon ay endogenous pathway.
o Yung mga cholesterol and
pag triglycerides na galing sa liver,
dadalhin sa mga peripheral cells
with the help of our lipases
REVERSE CHOLESTEROL PATHWAY
What if may mga sobra or hindi naman nagamit?
● Instead na pakalat-kalat yan sa ating blood
vessels or sa ating circulation, with the help
of our lecithin and lecithin-cholesterol
acyltransferase (LCAT), magkakaroon ng
HDL.
o Itong HDL na ito, kukunin niya yung
mga peripheral or yung mga excess
cholesterol sa peripheral cells;
kukunin niya at ibabalik niya sa liver
— ang tawag sa transport na iyon is
reverse cholesterol transport
(pathway).
● Kapag mababa si HDL, itong mga sobra or
excess cholesterol and triglycerides ay
mag-stay nalang sila sa ating mga blood
vessel.
o Magkakaroon ng blockage or
occluded blood vessels.
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7 LIPIDS & LIPOPROTEINS-GENERAL DESCRIPTION,
STRUCTURE, FUNCTIONS, & METABOLISM
Clinical Chemistry 1 (LEC)

SUMMARY:
Kakain ka muna, a-absorb ni intestine, at ang lahat
ng na-absorb ni intestine, kukunin ni chylomicrons,
dadalhin kay liver.
At syempre, si liver hindi naman pwede na take
lang ng take ng lipids. At dahil hindi rin naman
pwede na walang source of energy ang ating
mga peripheral cells at ang ating mga organs,
ipapalaganap niya ang mga triglycerides and
cholesterols na iyan.

Nagamit si VLDL and it will be further converted to

LDL.
Itong sina LDL, magbibigay ng cholesterol and
triglycerides sa ating mga peripheral cells.

Yung mga excess or yung hindi naman nagamit na

cholesterol, with the help of our LCAT, kukunin ni
HDL at ibabalik kay liver.

Again, for the lipid metabolism pathway we have

the absorption pathway, exogenous pathway,
endogenous pathway, and the reverse cholesterol
transport pathway.

NOTE (FOR ENZYMES):

● Lipoprotein lipase (LPL)
o It hydrolyzes the VLDL,
maco-convert siya into LDL.
o -Kasama rin siya sa ating exogenous
pathway, bakit? Kasi itong
chylomicrons, i-hydrolyze niya,
magiging chylomicron remnants, at
dadalhin niya sa liver with the help
of the LPL.

ENZYMES INVOLVED IN LIPID METABOLISM

LIPOPROTEIN LIPASE
● Hydrolyzes TAG in lipoproteins, and released
of fatty acids and glycerol

HEPATIC LIPASE
● Hydrolyzes triglycerides (TAG) and
phospholipids from HDL going back to the
liver
● Hydrolyzes lipids on VLDL and IDL when
there is insufficient source of energy

LECITHIN CHOLESTEROL ACYL TRANSFERASE

● Catalyzes the esterification of cholesterol
from HDL
● Enables HDL to accumulate cholesterol as
cholesterol esters

ENDOTHELIAL LIPASE
● Hydrolyzes phospholipids and TAG in the
HDL.

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TRANSFORMERS
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INTRODUCTION
LIPID ANALYSIS
• Why is lipid analysis performed?
o To identify the risks that is
associated with abnormalities of
your lipids and lipoprotein
metabolism.
CARDIOVASCULAR RISK
• Lipid analysis provides assessment on
cardiovascular (CVD) risk.
▪ Classically, this is associated better
if we can identify certain
lipoprotein levels in the lipid panel
or lipid profile on our clients or
patients.
▪ It has been enculturated in our
practice that we regard at LDL as
the bad cholesterol and HDL as
good cholesterol, but as we
progress with the study or with the
topics, you will realize that these
elevations of LDL and decline of
HDL are far more complex.
▪ There are other situations that may
influence the values of these
lipoproteins and also, we must put
in consideration the levels of total
cholesterol, total triglyceride
concentrations that is circulating
the human body.
▪ As you recall the previous module,
aside from the lipid panel, we also
have the total cholesterol and
triglyceride that may be associated
to familial disorders.
DETERMINING CUT-OFF VALUES
• Characterization of CVD risk according to
population
▪ To determine the cut off values of
cardiovascular risk according to
population.
▪ If we are to consider the geographic
distribution in the cultural practices of
the people in our country, not just in our
country but also in the world, we can
associate cardiovascular (CVD) risks on
a much higher degree on the western
hemisphere on our planet. Simply
because of their manner of eating since
the western countries are leaning
towards more on high protein, fat, and
salt diet. While at the same time, the
Asian part of our country most
especially here in the Philippines and
our neighbor countries as well as Japan,
and South Korea, we have good
practices in terms of diet and food
choices like we incorporate tons of fruits
and vegetables, fish protein.
▪ This significantly reduces our risk for
cardiovascular disorders but upon
further consideration, this is not the
actual factor that we must put into
TRANSFORMERS
Clinical Chemistry 1 (LEC)
consideration, we must also put into
consideration other predisposing factor,
perhaps, there is a history of
hypertension in the family, as well as
type II diabetes. We are also prone to
developing cardiovascular disorders
attributed to dysregulation of lipid and
lipoprotein processes in the human
body.
• Determined by NCEP (National Cholesterol
Education Program)
STANDARDIZATION OF TESTS
• For research laboratories performing
population and intervention studies.
o Each country or each group of
people have its own characteristics
so one reference range will not
apply to another. It is important that
we establish or standardize the test
that is appropriate with our target
population.
REVIEW
REFERENCE RANGE
ATP III CLASSIFICATION FOR LDL CHOLESTEROL
(mg/dL)
<100 Optimal
100-129 Near Optimal / Above
Optimal
130-159 Borderline High
160-189 High
≥ 190 Very High
*NOTES:
• Let’s have a recap or a review on lipid
metabolism, as mentioned by NCEP
(National Cholesterol Educational
Program), that we identify certain levels
of our lipid panels that will be considered
significant in order for us to identify the
cardiovascular risk and the corrective
measures that is associated with these
particular levels or concentrations of lipid
panels in the body of our patients.
• For the total LDL cholesterol, for the low
LDL cholesterol or other succeeding
cholesterol is pertaining to mg/dL
concentration.
• Low density lipoprotein is regarded as
most cholesterol rich and the most likely
to develop Atherosclerotic blocks or
blockages in the blood circulation.
ATP III CLASSIFICATON FOR TOTAL CHOLESTEROL
(mg/dL)
<200 Desirable
200-239 Borderline High
≥ 240 High
*NOTES
• This is expressed in mg/dl
• In here, we are pertaining again to a high
risk for developing cardiovascular
diseases
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 LIPIDS AND LIPOPROTEIN
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ATP III CLASSIFICATON FOR HDL CHOLESTEROL
(mg/dL)
≤ 40 Low
≥ 60 High
*NOTES:
• This is expressed in mg/dl
• In terms of HDL-C concentrations, we are
pertaining to high cardio-protective
property of HDL-C
o In simpler terms, the higher the HDL
is, the better the cardiovascular
health is.
o The higher the HDL-C, the lower is
the development of cardiovascular
risk
• HDL can be elevated by eating high levels
of unsaturated fats
o This is usually associated with fish
fatty acids such as omega-3
o Healthy plant-derived lipids or fats
o Complete avoidance
significantly reduced intake of
animal-derived fat and oils
▪ Because HDL-C would really
clean up the LDL-C that is
blocking or is accumulating
in the blood circulation of
patients.
▪ By having an increased in
fish protein that contains
high concentrations
omega-3 fatty acids, and
significantly cutting off
animal fat, trans fat, and
saturated fat in our diet, we
are effectively elevating our
HDL-C concentrations
ATP III CLASSIFICATON FOR TOTAL TRIGLYCERIDES
(mg/dL)
<150 Normal
150-199 Borderline High
200-499 High
≥500 Very High
• Recap: Triglycerides (TAG) are originally
diet-derived and some of them are
derived from the subcutaneous fat we
already have in the body since TAG is the
main component of the storage fat in our
body
o We can directly correlate this, the
elevation of TAG, to obesity and
increased adiposity or increased
concentration of adipose tissues in
subcutaneous area of our body.
AMERICAN HEART ASSOCIATION: CHILDREN > 2
YEARS OF AGE WITH FAMILY HISTORY OF
DYSLIPIDEMIA OR PREMATURE CHD
TOTAL LDL-
CATEGORY CHOLESTEROL CHOLESTEROL
(mg/dl) (mg/dl)
Acceptable <170 <110
TRANSFORMERS
Clinical Chemistry 1 (LEC)
Borderline 171-199 111-129
High ≥200 ≥130
• have pre-disposing factor for development
of dyslipidemia or premature cardiac
health diseases, we may observe or may
have a more in-depth analysis on the
concentrations of total cholesterol and
LDL-C
*Please take note of this following values this will
come in very significant usage in the next modules
to come.
PRE-ANALYTICAL CONSIDERATIONS
PRE-ANALYTICAL CONSIDERATIONS FOR LIPIDS AND
LIPOPROTEIN ANALYSIS
• Prior to analysis of lipid and lipoprotein
metabolism, we have to consider some pre-
analytical considerations, because it is
or imperative that we prepare our patients
prior to having their lipid panels checked
• It would also provide the medical
technologists and the physician some high
degree of confidence knowing that the
patient is properly instructed with pre-
analytical considerations (fasting
preparations)
• Lipid profile requires to have a fasting state
of at least 12 hours prior to venipuncture
of BIOLOGICAL VARIATIONS
• Cholesterol levels begin to rise with age in
both sexes
• Women < Men (except in childhood or after
early 50s)
o In childhood or after early 50s, men
tend to have a higher cholesterol
concentration.
o As women approach the middle age
or the old age, they become highly
predisposed to having cholesterol
levels that are in significantly
elevated values. This is because of
the influence of the hormones in the
woman’s body
• Seasonal variation (high cholesterol intake in
winter/cold seasons)
o High cholesterol intake occurs in
winter/cold seasons
o This is simply due to the fact that
cholesterol, or fats in general, are
good insulating
molecules/substances that are
naturally occurring in the human
body
o Fat itself, or the muscles covered in
subcutaneous fat, would really put
the body in a high temperature state
o It keeps us warm, cozy, thus
protecting us from cold seasons.
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• Dietary intake and Medications (oral
contraceptives, post-menopausal
estrogens, and anti-hypertensives)
FASTING: 12 HOURS PRIOR TO VENIPUNCTURE
• Chylomicrons (CM) in post-prandial plasma
resulting to elevated triglycerides
• Chylomicrons are lipoproteins, they are lipid
carriers, and they possess significantly
elevated dietary triglycerides.
• CM are typically cleared within 6-9 hours
• Presence of the CM after 12 hours fasting is
considered abnormal.
o Chylomicrons will be delivered to the
liver via endogenous pathway, in
order for it to be converted into VLDL.
o By having chylomicrons still
accumulating in the blood
circulation even after 12 hours of
fasting, there is a significant
abnormality on the lipid and
lipoprotein metabolism.
o Chylomicrons are triglyceride-rich
because they are lipid transport
carriers (proteins) that deliver dietary
triglycerides to the liver.
NON-FASTING ANALYSIS OF LIPIDS
• Total cholesterol and High-Density
Lipoproteins analysis can be performed
using random blood sampling
• Analysis of Triglycerides and Low-Density
Lipoproteins would require patient fasting.
o Remember that fasting has little
effect on total cholesterol levels. But
high HDL can be a few milligrams per
deciliter (mg/dL) lesser than the
typical fasting levels.
o Let us put into consideration in
analyzing triglycerides and LDL that
12 hours of fasting is still the
recommended time preparation for
clinical diagnosis and
epidemiological studies.
o Post-prandial presence of
chylomicrons can contribute to LDL
miscalculation. Because if CM are
abnormally circulating in the body,
that can significantly affect the
triglycerides level as well. Remember
that CM are triglyceride rich.
o Later on, if we try to compute for the
LDL estimation, you will appreciate
the significance the appropriate
fasting preparation.
o Again, post prandial presence of CM
can contribute to LDL miscalculation
which can later on can result to
misclassification of cardiovascular
risks.
TRANSFORMERS
Clinical Chemistry 1 (LEC)
• What a heart-warming meal to eat in cold
seasons!
o If we reside in Tagaytay or Baguio,
it’s quite delicious to indulge on
bulalo, sinigang or all of those
Filipino delicacies that deals with hot
and warm meals with tons of savory
soup with floating fats on it. So, it
quite scrumptious/indulgent, most
specially when you’re going to eat
these on cold season. As I’ve
mentioned earlier that its normal for
human being to have this kind of
variation in diet influenced by the
environment because, again, fats
keep us warm, fats help us reserve
energy.
• We have the oral contraceptive pill (OCP)
that alters the lipid panels of women.
o In the context of endocrinology and
toxicology, clinical chemistry 3, you
will later on realize that estrogen and
progesterone are sex hormones that
regulate the fertility and the body of
the woman as she approaches
puberty. If she approaches puberty,
these hormones will promote fat
deposition on the body of the
woman.
o That is a simple demonstration on
how sex hormones naturally affect
the fat distribution and fat
metabolism of women as they
approach puberty. Most specially if
they are going to become pregnant.
As you can see most women that we
encounter begin to have an
increased level of adiposity, its simply
because that, there is a
development of new life in the
womb of the woman, and it requires
the body to store reserve energy in
form of fatty acids, triglycerides that
are stored in the adipose tissue.
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 LIPIDS AND LIPOPROTEIN
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o It’s normal for women—most
specially if they become pregnant,
to gain some weight, and gain some
subcutaneous fat.
o That is all in the context of normal
hormonal effect of estrogen and
progesterone in the human body.
• What if we are to consider the use of oral
contraceptive pills?
o Since they are synthetically derived
sex hormones, even without
pregnancy, even without the
change of puberty, the oral
contraceptive pill will just alter the
fertilization cycle of a woman. Those
circulating estrogen, can eventually
alter the adiposity, the
metabolism of the woman, that is
under the prescription of oral
contraceptive pill.
STANDING TO SITTING POSITION
• Extravascular water passes through blood
vessels
• Dilution of non-diffusible plasma
constituents
• 10% Decrease of TC, HDL, LDL, apo A-I and
apo-B after 20 minutes of recumbence
o Do not forget.
• >50% Decrease of TG, in a standing patient
who sits
• Changes in body posture reversibly affects
blood levels. (Sitting to Standing Position)
o It is quite obvious that if there are
sudden changes in the body that is
positioning that can affect our
biochemical concentrations.
• National Cholesterol Education Program
(NCEP) recommends 5 minutes of sitting
prior to venipuncture
o It is advised by the NCEP that the
patient observes 5 minutes of sitting
prior to venipuncture. It’s very
simple, 5 min of sitting prevents
hemoconcentration of lipid panels.
PROLONGED VENOUS COLLECTION
• Caused by posture and prolonged
application of torniquet
• Results to 10-15% increase of cholesterol
levels
o Another artefactual/false elevation
of total cholesterol levels.
VENOUS AND CAPILLARY BLOOD SAMPLING
• Capillary blood values are generally lower
than its venous blood level counterparts.
PLASMA AND SERUM LEVELS
• Plasma and serum for determination of TC,
TG, HDL, and LDL levels.
• Plasma is preferred for ultracentrifugation
and electrophoretic methods.
• Separated plasma stored at 4ºC for
analysis.
TRANSFORMERS
Clinical Chemistry 1 (LEC)
PROTEIN AGGREGATION
• Occurs in anticoagulated plasma that is
stored in refrigerator temperature for a few
days or frozen for longer periods. Protein
aggregation occurs less commonly in
serum.
CHOICE OF ANTICOAGULANT
• Ethylenediaminetetraacetic acid (EDTA)
o Preferred anticoagulant
• Heparin
o Little effect on lipid concentrations
but affects electrophoretic mobility.
• Citrate anticoagulants
o Falsely decreased plasma lipid and
lipoprotein concentrations.
STORAGE
fat • Frozen samples
o Can be used for TC, TG, HDL, and
apolipoprotein analysis
• Frozen samples
o Not recommended for
ultracentrifugation methods
▪ This is because triglyceride-
rich lipoprotein molecules
cannot withstand freezing
temperatures.
▪ So, if you are to consider from
the previous modules, which
among the lipoproteins are
regarded to be triglyceride-
rich? That would be your
CHYLOMICRONS.
▪ Another example of a
triglyceride-rich lipoprotein is
your LDL, if we are to
compare this to its
counterpart, HDL.
▪ We have various lipoproteins
that can be significantly
affected by freezing.
Freezing can result to
erroneous ultracentrifugation
methodologies.
• Do not forget the storage conditions for
long term and short-term storage.
• -70ºC
o Long-term storage
• -20ºC
o Short-term storage
LIPID MEASUREMENT
OVERVIEW
CHOLESTEROL METHODS
• Extraction
• Acidification
• Enzymatic methods
TRIGLYCERIDES METHODS
• Triglyceride hydrolysis and glycerol analysis
• Enzymatic methods
LIPOPROTEIN METHODS
• Ultracentrifugation
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 LIPIDS AND LIPOPROTEIN
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• Electrophoresis
• Polyanion precipitation
APOLIPOPROTEIN METHODS
PHOSPHOLIPID METHODS
MISCELLANEOUS METHODS
• Standing Plasma test
CHOLESTEROL METHODS
CHOLESTEROL CHEMICAL METHODS
• Cholesterol methods usually employ two (2)
techniques: chemical and enzymatic
methods.
• Traditional approaches to knowing lipid
panels begin with Total Cholesterol Analysis.
o Remember that in total cholesterol
(TC), if you’re going to subtype or
subclass the TC analysis, we are
referring to the totality of lipid
molecules that has cholesterol in it.
o As definition implies, you have your
low-density lipoprotein cholesterol
(LDL-C) and high-density lipoprotein
cholesterol (HDL-C).
o In general terms, we are measuring
for the totality or the completeness
of overall cholesterol contents of our
blood sample.
• Strong acids are used to produce a
measurable color compound.
• Principle for chemical cholesterol methods:
dehydration and oxidation of cholesterol to
form a colored compound
• Addition of organic solvents
o For partial or complete compound
extraction for increased specificity
• Reference Assay (CDC): Modified Abell-
Kendall Method (Henry) or Abell Levy and
Brodie Method.
o It is important to remember that the
Reference assay as prescribed by
Centers for Disease Control and
Prevention (CDC) is may utilize to a
Modified AbelL-Kendall Method
(Henry) or Abell Levy and Brodie
Method.
• Hexane Extraction after Alcoholic KOH
hydrolysis, followed by Liebermann-
Burchardt color reagent
o To further explain the process of the
Abell Levy and Brodie Method or
Modified Abell-Kendall Method
involves the use of Hexane as an
extracting reagent after we subject
the specimen Alcoholic Potassium
hydroxide (KOH) hydrolysis, followed
by Liebermann-Burchardt color
reagent.
COLOR DEVELOPERS MIXTURES
• Glacial Acetic Acid
• Acetic Anhydride
• Ferric Chloride
TRANSFORMERS
Clinical Chemistry 1 (LEC)
• Concentrated Sulfuric Acid
LIEBERMANN-BURCHARDT REAGENT
• Sulfuric Acid
• Acetic Acid
• Acetic Anhydride
• So, it is worth mentioning to say the essence
of the Liebermann-Burchardt reaction. So,
the end product is Cholestadienyl
Monosulfonic Acid.
• END PRODUCT: Cholestadienyl
Monosulfonic Acid
• Green end color
SALKOWSKI REACTION
• refers to another chemical method that we
employ cholesterol.
• END PRODUCT: Cholestadienyl Disulfonic
Acid
• Red End Color
PRECAUTIONS
• What are the precautions we need to
anticipate if we are to perform cholesterol
chemical methods?
• No to hemolyzed specimens
o The phlebotomist or the medical
technologist should practice good
blood extraction methods.
o The tourniquet should not be applied
too long.
o The appropriate gauge is also
applied or given to the patient with
accordance to the veins or structure
of the patient blood vessels.
o Why do we reject hemolyzed
specimens?
▪ Because hemolysis can result
to falsely increased
cholesterol levels.
• No to icteric specimens
o When we say icteric, this refers to
serum specimens that have
significantly elevated bilirubin
concentrations.
o The bilirubin may result to 5-6 mg
percent (%) increase in cholesterol
mg bilirubin above the normal
concentrations. That can result to
falsely elevated cholesterol
concentrations. The appearance of
your bilirubin in very icteric
specimens.
• Avoid water contamination
o That can affect with the precise and
accurate development of the color
of our chemical methods for
cholesterol determination.
o Since we are dealing with chemical
reactions, these chemical reactions
are observed through time. So, it is
important that we are kin to
observing the appropriate timing or
monitoring of chemical reactions. If
we miss the correct timing or time
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 LIPIDS AND LIPOPROTEIN
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frame, for the reading of our
cholesterol chemical methods, we
are most likely to have an erroneous
laboratory interpretations
cholesterol methods.
• Precise and Accurate timing for color
development
ONE STEP METHOD
• Colorimetry (C) (Pearson, Stern and Mac
Gavack)
TWO STEP METHOD
• (C) + Extraction (E) (Bloors)
THREE STEP METHOD
• C + E + Saponification (S) (Abell-Kendall)
FOUR STEP METHOD
• C + E + S + Precipitation (P) (Schoenheimer
Sperry, Parekh and Jung)
• So, just to provide you an overview on how
cholesterol chemical methods are practiced all
throughout the decades. Generally speaking,
we have colorimetric method, extraction
method, saponification method and
precipitation methods.
• Each step for cholesterol determination, a new
technique is added, that's why we have derived
with these 4 step methods.
• The aim of this is to increase the sensitivity and
increase the specificity of the techniques.
• With this, it is quite obvious that the classical
cholesterol chemical methods are quite tedious
and laborious to perform because it involves
multiple steps.
• But the objectives of this is to increase the
specificity of this techniques and in order for that
to happen, we tried to apply color, extract
cholesterol, subject to saponification methods,
and for precipitation methods. So please put
that into consideration.
ISOTOPE DILUTION MASS SPECTROMETRY
• Instrumentation for the determination of
cholesterol via Abell-Kendall Multistep
Chemical Reaction.
• This multistep manual method is
complicated but gives good agreement
with the gold standard method developed
and applied at the US National Institute for
standards and technology.
• The so-called definitive method is
compared to the Abell-Kendall Multistep
chemical reaction
• The Abell-Kendall Method and the Isotope
dilution mass spectrometry are at par with
each other. They agree with one another.
TRANSFORMERS
Clinical Chemistry 1 (LEC)
CHOLESTEROL ENZYMATIC METHOD
• Enzymatic methods measure total
cholesterol directly from plasma or serum.
of o Absence of acids. Strong acids are
not utilized in this method
▪ Because there is no need to
extractor isolate cholesterol
biomolecules from specimen
o But enzymes that has specific nature,
specificity to target specific
functional groups that are present in
the cholesterol molecules
o The use of acids for isolation is no
longer necessary
• Principle: Hydrolysis of Cholesteryl Esters
o Remember: enzymes have their
specificity traits towards its target
substrate hence drastically
diminishes process time.
• Extraction and Precipitation tests are
removed from the processing of sample.
o Results to faster turnaround time
• Enzymes are better suited for automated
chemistry analyzers
• The intensity of the color dye is directly
proportional to the cholesterol
concentration
• With the presence of cholesterol esterase
enzyme, the enzyme catalyzes the ester
group in the presence of water.
• This is referred to as the “hydrolysis of
cholesteryl esters”
• With the hydrolysis of cholesteryl esters, this
chemical reaction would yield 2 products:
o Cholesterol
o Fatty acids
• The cholesterol (product from the first
chemical reaction) will interact with the
presence of oxygen.
• The cholesterol will be converted to cholest-
4-en-3-one that is catalyzed by cholesterol
oxidase in the presence of oxygen.
• Once this oxidase performs its biological
reactions with cholesterol and oxygen
molecules, this reaction will yield to:
o Cholest-4-en-3-one
o H2O2 (hydrogen peroxide)
• At this stage the two cholest-4-en-3-one,
can be read automatically at 240 nm
however it is much time consuming and
difficult to perform
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• We resort to the conversion of Hydrogen
peroxide to its intended chemical end
product
o Because it much easier to determine
the cholesterol concentration.
• The hydrogen peroxide be oxidatively
coupled to 2 chromogenic substrates by
catalysis of oxidase
o 2 chromogenic substrates are:
phenol and 4-aminoantipyrine
o These two compounds will result to
color development
o Results to production of
Quinoneimine dye which is the end
product for the overall cholesterol
enzymatic method chemical
reaction.
o Quinoneimine dye can be read
photometrically at 500 nm.
CHOLESTEROL INTERFERENCES
PLANT STEROLS
• Interferes with Cholesterol Oxidase and
Chemical Methods
• Can be found in patients with β-
sitosterolemia
ASCORBIC ACID
• Reducing agent that competes with
chromogenic substrates.
o Presence of ascorbic acid interferes
with chromogenic substrates.
o If you could recall, we are relying on
the activity of an oxidase enzyme to
facilitate the conversion of your
hydrogen peroxide. It also facilitates
the reaction with chromogenic
substances by adding a reducing
agent onto the mix.
▪ Let’s say the presence of a
reducing agent in the blood
sample of our patient, that
can negatively impair the
oxidation reaction that will
occur on the third cholesterol
enzymatic chemical reaction
▪ Presence of ascorbic acid
can result to a significantly
diminished/decreased
synthesis of quinoneimine
dye.
▪ In effect, that can result to a
significantly decreased total
cholesterol determination if
we are employing enzymatic
methods.
BILIRUBIN
• Has a light absorbance of 500nm
• Can also be oxidized by Hydrogen
Peroxide
TRANSFORMERS
Clinical Chemistry 1 (LEC)
HEMOGLOBIN
• Has a pseudo-peroxidase activity
• Hemoglobin is visually observed
macroscopically
o If we are dealing with grossly
hemolyzed specimens, if the serum
sample of our patient is in a pink or
red hue or color, the presence of
hemoglobin can significantly affect
the total cholesterol concentration
or the total cholesterol enzymatic
methods.
CATALASE
• Presence of catalase that competes with
peroxidase for hydrogen peroxidase
Note: Basic concept of enzymology, if the interfering
substance emulates the chemical reaction, it
facilitates that can result to a falsely elevated
concentration. But if an interfering substance is inn
contradiction to the chemical reaction that the
enzymes is facilitating, then they can result to falsely
declined values of total cholesterol.
TRIGLYCERIDES METHOD
TRIGLYCERIDES ASSAYS/METHODS
• Principle: Hydrolysis of Triglycerides and
Glycerol Measurement
• Here we have a molecule that TAG are 3
fatty acid chains that are linked to a glycerol
head via their ester bonds.
• The objective of Triglyceride Method is we try
to break off the ester bonds
• If we are going to break the ester bonds, we
can liberate the fatty acid contents.
• We have this chemical reaction triglycerides
+ water with the action of lipase, this lipase
will directly hydrolyze the glycerol and fatty
acid chains that are bonded by ester
functioning groups.
• In a normal context, lipase will literally free
the fatty acids from its glycerol head.
• Lipase do not discriminate other mono, di or
triglycerides. As long as the molecule that
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 LIPIDS AND LIPOPROTEIN
8 MEASUREMENT
we have has an ester bond that links a fatty
acid to a glycerol molecule.
• Lipase will cleave that ester bond or that
ester linkage. It does not discriminate from
other mono or diglycerides. Hence, the
presence of these compounds may result to
over estimation of glycerol concentration
and can subsequently be misinterpreted as
falsely elevated triglycerides level.
• As mentioned, it is problematic if there are
presence of monoglycerides or
diglycerides.
• Reference method (CDC) – Modified Van
Handel and Zilversmith.
• For the reference method, as presecribe by
the center for disease control and
prevention (CDC), we have the modified
Van Handel and Zilversmith.
• This involves the alkaline hydrolysis,
chloroform extraction, extract treatment
with silicic acid, color reaction with
chromotropic acid.
• The end point of this chemiical reaction
would be a pink colored solution or a pink
colored compound.
• On the interference of the monoglycerides
and diglycerides, correction requires
substraction of endogenous on esterified
glycerol. However, endogenous on
esterified glycerol is negligible.
• 10 to 20 mg/dL can be an over-estimating
factor for triglycerides. That is caused by the
presence of endogenous on esterified
glycerol.
COLORIMETRIC METHOD (VAN HANDEL &
ZILVERSMITH)
• Colorimetric Method (Van Handel &
Zilversmith)
• For the coloremetric method, for the
tryglerides, we have the classical method of
Van Handel & Zilversmith.
• We have tryglycerides that participates with
a chemical reaction this is facilitated by the
presence of your alcoholic potassium
hydroxide and with this reaction this would
result to the breaking of the fatty acids and
glycerol.
• Glycerol with the action of periodic acid
which is a type of acid that facilitates
oxidation reaction would results to the
production of Formaldehyde.
• Formaldehyde with combination with
chromotrophic acid would result to the
formation of the blue colored compound.
TRANSFORMERS
Clinical Chemistry 1 (LEC)
FLUOROMETRIC METHOD (HANTZSCH
CONDENSATION)
• For the fluorometric method or the Hantzsch
condensation, we have your tryglycerides
again that follows the hydrolysis of
tryglycerides by using the alcoholic
potassium hydroxide and this would yield
glycerol and fatty acid.
• Again, glycerol with the action of periodic
acid that facilitates further in an oxidation
reaction would result to the formaldehyde.
• However, the difference between the
previous chemical reaction and this current
chemical reaction is that formaldehyde is
added to Diacetyl Acetone and ammonia.
And this would result to the production of
Diacetyl Lutidine Compound.
TRIGLYCERIDES (ENZYMATIC) METHODS
• Enzymatic methods for tryglycerides
measurement.
• The enzymatic method for tryglycerides
involve triglycerides with the action of
lipase enzyme would result to the
production of glycerol and fats.
• Glycerol in the presence of ATP or
Adenosine triphosphate will then be
converted by glycerol kinase into two
products.
o Glycerol phophate
o Adenosine diphosphate.
• Now this glycerol phosphate can proceed
to chemical reactions.
Chemical Reaction 1
• In the chemical reaction #1, we have
Glycerol phosphate in the presence of
NADH. Now, this will be facilitated or
catalyzed with Glycerophosphate
Dehydrogenase, this will produce the
chemical products of Dihydroxyacetone
phosphate, NADH and Hydrogen number 4.
• Now, this NADH with the presence of
Tetrazolium dye will be catalyzed by a
Diapharase enzyme. Now this Diapharase
enzyme will produce Formazan and NAD.
8
 LIPIDS AND LIPOPROTEIN
8 MEASUREMENT
• NADH can be read at 340 nm, while
Formazan can be read at 500-600 nm. So,
depending on the reagent availability you
can either read NADH or Formazan.
• In essence, we have multiple options, we
have two options, to determine the
triglyceride contents of the blood of our
patient. You just always need to remember
that in order for us to obtain NADH and
Formazan, the starting compound that we
need to break even further is your Glycerol
Phosphate.
For the second reaction:
• NOTES
• Now for the second reaction for the
conversion of glycerol phosphate, we have
the Glycerol Phosphate in the presence of
oxygen, with the action of
Glycerophosphate Oxidase will then
produce the byproduct of chemical
reaction, the Dihydroxyacetone and
Hydrogen Peroxide.
• Now, Hydrogen Peroxide can be used or
can proceed similarly to the cholesterol
pathway analysis. If you could still recall the
cholesterol enzymatic methods in the
previous discussion that Hydrogen Peroxide
can proceed or is coupled rather with 2
Chromogens, Phenol and 4 aminoantipyrine.
This is facilitated with peroxidase enzymes. In
essence you can also analyze triglyceride
indirectly by using cholesterol pathways.
• The first two chemical reactions have
highlighted the use of Glycerol Phosphate as
the start of material for us to derive necessary
end products of chemical reaction. I would
just like to put an emphasis that the end
product of the chemical reaction if you are
going to use Glycerol Phosphate are the
NADH and your Formazan.
• And on the second reaction the glycerol
phosphate, the end product for that second
reaction is your hydrogen peroxide, that
hydrogen peroxide can be coupled with 2
chromogenic substances and can undergo
the cholesterol enzymatic pathway that is
facilitated by peroxidase enzyme.
TRANSFORMERS
Clinical Chemistry 1 (LEC)
• Now let’s focus on the second compound,
the ADP, this ADP will be used to quantify
your triglycerides.
• How are we going to do that? by combining
the ADP to your Phosphoenol Pyruvate. Now
this Phosphoenol Pyruvate and ADP will be
converted by the Pyruvate Kinase enzyme
into ATP and Pyruvate.
• Now the Pyruvate will be added to your
NADH and your hydrogen, now in the action
of Lactate Dehydrogenase enzyme will
produce lactate and NAD. Now it is worth
mentioning here that the disappearance of
NADH is read at 340 nm. The disappearance
of NADH is attributed to the synthesis of
Lactate and NAD products so we can
measure the level of triglyceride by how fast
or how slow the disappearance of the NADH
is in this particular enzymatic method.
TRIGLYCERIDES INTERFERENCES
PRESENCE OF FREE GLYCEROL
• Glycerol is normally present in blood
circulation at 0.163mmol/L (or 1.5mg/dL)
• This is equivalent to about 14mg/dL of
triglycerides
o Why? Because in enzymatic method
for triglycerides, we are using
glycerol to obtain the startp-up
material – glycerol phosphate.
o The appearance of endogenous
unesterified glycerol can falsely
elevate triglycerides concentration
because glycerol can progress into
triglyceride enzymatic methodology.
PRE-DISPOSING FACTORS
• Uncontrolled Diabetes Mellitus
• Vigorous exercise
• Hyperglycerolinemia
CONTAMINATION
• Glycerol-containing lubricant used in tube
stoppers of some blood collection tubes
MEDICATION
• Ascorbic acid – interferes with
oxidoreductase enzyme reaction.
• Bilirubin – biochemical and spectral
interferences
o Bilirubin provides an intense yellow
color to the serum sample. Affects
the intensity of the color
9
 LIPIDS AND LIPOPROTEIN
8 MEASUREMENT Clinical Chemistry 1 (LEC)

development of triglycerides and

products.
• Gross hemolysis – results to dilution effect of
plasma triglycerides
LIPOPROTEIN ASSAYS

LIPOPROTEIN ASSAYS (HDL AND LDL CHOLESTEROL

METHOD
ESTIMATION OF LIPOPROTEIN
ULTRACENTRIFUGATION
• Method that lies of lipid content and
lipoprotein density.
• Contents of the lipoproteins has an influence
to its density, MW, and behavior on
electrophoretic mobility and
ultracentrifugation.
ELECTROPHORESIS • Now with regards to density as you can see,
• Names of lipoprotein were derived that if we are to apply as plasma with
according to where these substances ultracentrifugation method, we can see that
migrated in electrophoretic regions the High-Density Lipoprotein tends to settle
• Parts of electrophoresis: origin, alpha region, at the bottom of the tube. Followed by the
beta region and gamma region Low-Density Lipoprotein, we have the Very
o Certain lipoprotein will migrate to a Low-Density Lipoprotein, and Obviously we
region. The region where it migrated have the presence of Chylomicrons at the
is also its name. top most portion of our ultracentrifuged
o Ex: Protein migrated into B-region. It plasma.
will be called as Beta-lipoprotein • Lipoproteins with high triglycerides and low
POLYANION PRECIPITATION protein contents tend to have lower
densities.
• Major classes of lipoproteins can form
• So, if we are to observe among these
precipitate complexes with polyanions and
lipoproteins which among these has the
divalent cations.
highest triglyceride contents?
IMMUNOCHEMICAL METHODS
o We have the Chylomicrons that has
• Antibodies specific to epitopes of certain
a total percentage of 80-95% total
apolipoproteins
triglyceride content, so it makes
PARTICL MOBILI DENSITY TAG % CHON % SVEDBE
E TY (kg/l) RG sense that chylomicrons, being
UNITS triglyceride rich tend to have the
Chym Origin <0.95 80- 1-2% >400 least density among them all, and
omicr 95% has the tendency to really float at
ons the top most portion of our
VLDL Pre-β 0.95- 45- 6-10% 20-400 ultracentrifuged specimen.
1.006 65% • Now, if we are to observe which among
IDL β or 1.006- 12-20 these lipoproteins have the lowest content
Pre-β 1.019 of triglycerides?
LDL β 1.019- 4.8% 18-22% 0-12 o Then we are observing/ or we can
1.063 see that the High-Density Lipoprotein
HDL2 α 1.063- is at 2-7% of its triglyceride content
1.125 2-7% 45-55% o So, in effect, if we apply
HDL3 α 1.125- ultracentrifugation methods, the
1.210 High-Density Lipoprotein will
Lp(a) Pre-β 1.045- definitely settle at the bottom of the
1.080 tube.
• Now, in terms of protein content,
• Mobility – Area of the electrophoretic
Chylomicrons is generally the lowest
patterns from which the lipoprotein tends to
containing protein content at 1-2%.
migrate
• While the most protein rich of them all is the
• Agarose gel – Medium used in
High-Density Lipoprotein at a percentage of
electrophoresis of lipoprotein. Facilitated
45-55% of total protein content
with electrical current or charges for
lipoproteins to migrate. • Put this in the context of lipoprotein
circulation in the human body.
➢ So, triglycerides, being derived from
diet is packed in chylomicrons. We
tackled in the previous module, is

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 LIPIDS AND LIPOPROTEIN
8 MEASUREMENT Clinical Chemistry 1 (LEC)

that the presence of chylomicrons is
significantly abnormal if
presence of chylomicrons continues
to persist even after 12 hours of
fasting. So, if there is continuous
persistence of chylomicrons that
contains high levels of Triglycerides,
which may promote or might add a
higher risk of cardiovascular
disorders/diseases.
• Now, if we’re going put this again on the
context of Low-density lipoprotein, let us
focus on the protein content of the low-
density lipoprotein. It has a protein content
that ranges from 18-22% of its total
biochemical composition. This makes your
LDL less dense in comparison to your HDL.
Remember that the LDL cholesterol are the
lipoproteins that migrate and deliver
cholesterol from the liver to the peripheral
tissues of the cells to the blood vessels. It also
makes sense that LDL tends to accumulate
better in the peripheral blood circulation
and in the vasculature of our body. It is
simply attributed to its characteristic
buoyancy, characteristic density, and its
content.
LIPOPROTEIN ELECTROPHORESIS
other types would have the designation of
the pre-beta lipoprotein, or beta-lipoprotein,
which is simply how they migrate to the
electrophoretic regions of agarose gel
electrophoresis.
POLYANION PRECIPITATION
POLYANION REAGENTS
• Heparin Sulfate, Dextran Sulfate,
Phosphotungstate
DIVALENT CATIONS
• Manganese (Mn2+), Calcium (Ca2+), and
Magnesium (Mg2+)
PURPOSE
• Historically, this is done to remove Apo-B
containing lipoproteins prior to HDL
Cholesterol Analysis
o It is worth mentioning that Apo-B in
VLDL and LDL are rich in positively-
charged amino acids.
o These positively-charged amino
acids, will then, preferentially form
complex cells with divalent cations.
In effect, they precipitate. And if they
precipitate, we can now remove
interfering substances and focus on
what remains in the specimen. And
that is the, HDL-Cholesterol.
o So, essentially, the polyanion
reagents in the divalent cations
simply server to remove the
unnecessary lipoprotein constituents
of the blood sample.
o This can effectively analyze the
concentration of HDL. Hence,
elevating or increasing the
specificity of the assay.

PHOSPHOLIPID ASSAYS (LECITHIN AND

SPHINGOMYELIN)

SPECIMEN PHOSPHOLIPID ASSAYS

• Unfractionated Plasma and Fractionated
Plasma
MEDIUM
• Agarose gel- Most commonly used
medium.
COLORING DYES
• Oil Red O, Fat Red 7, Sudan Black B.
• Stains ester bonds in triglycerides and
cholesteryl esters.
• Now, if you have noticed that you are initially
demonstrating the presence of triglycerides
and cholesteryl esters through the use of
these fat-loving coloring dyes. It is also worth
mentioning that phospholipid rich and free
cholesterol-containing lipoproteins
stained poorly with the use of these dyes.
• The designation of the lipoproteins are
dictated by how they are migrating towards
the positively-charged ion/cathode. As you
can see, the a-lipoproteins are initially
consisted of HDL, while the other forms or the
• An example of a miscellaneous topic that
deals with phospholipids in general
• For the sake of example, we shall be dealing
with lecithin and sphingomyelin
ANALYTES OF INTEREST
• Lecithin, Sphingomyelin and Lysolecithin –
accounts for 95% of total phospholipids in
serum
ENZYMATIC METHODS
• Use of Phospholipase D, Choline oxidase
and Horseradish Peroxidase
PURPOSE
• Assessment of Fetal Lung Maturity (FLM)
• Use of Amniotic Fluid as specimen of
are
choice for this purpose
o For the determination of the Fetal
Lung Maturity
o Recap for measuring FLM:
▪ The higher the phospholipids,
the better the prognosis or

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 LIPIDS AND LIPOPROTEIN
8 MEASUREMENT Clinical Chemistry 1 (LEC)

indication that the fetus is MISCELLANEOUS ASSAYS

having a functional, healthy
and mature lungs. MISCELLANEOUS ASSAYS
▪ If there is a deficiency in the STANDING PLASMA TEST
production of lecithin, that • Detection of Chylomicrons (in significant
may put the baby at risk for amounts)
developing respiratory • Uses 2ml of plasma that is placed in a 10x75
distress syndrome. mm test tube.
• It involves overnight standing inside a
refrigerator (4°C, undisturbed)
FATTY ACID ASSAYS • The significant findings that we can
observed in standing plasma cells or rather
FATTY ACID ASSAYS (LIPID AND LIPOPROTEIN standing plasma test involves the
ANALYSIS) appearance of a floating “cream” that is
PURPOSE visually observable.
• Fatty Acid Analysis is said to contribute to Note: The appearance of “floating cream” would
CHD risks suggest the presence of chylomicrons
• Since its fatty acids (FAs) are the building • Appearance of turbid plasma (despite
blocks of fats. In effect, fats, when they are standing overnight of the sample/ the
digested or they are broken down into their plasma) indicates the presence of VLDL
intended compounds or biomolecules, that (Very Low-Density Lipoprotein)
can be easily correlated to CHDs that are
attributed to FA dysregulation and lipid and
lipoprotein metabolic abnormalities.
METHOD
• Gas-Liquid Chromatography – After
Extraction
• Alkaline Hydrolysis
• Conversion of Methyl Esters of
Diazomethane
REFERENCE STANDARD
• Constituents include laurate, myristate,
palmitate, palmitoleate, phytanate,
stearate, oleate, linoleate, arachidate, and
arachidonate
• These are examples of FAs that we can use
for the analysis of FAs.
• We should remember that FAs come in • This picture is not medically accurate but
various forms. There are saturated, please appreciate the presence of
unsaturated FAs, and the molecular chylomicrons in plasma in this EDTA test
structure of these FAs is quite relevant to its tube ☺
associated risk • In reality or in the actual practice of the
• Trans fatty acids or commonly known as standing plasma cells involves the
“trans fats”, even the saturated fats separation of the plasma from the red blood
contribute to a much higher CHD risk. cells.
• This involves having diet that are fried,
eating tons of processed food, that is rich in
trans fats or saturated FA that obviously can
contribute to CHD risk.
• While having a rich intake of unsaturated
FAs, such as, fish oils, fish proteins, plant-
derived oils that can elevate the
unsaturated FAs.

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9 PROTEINS: FUNCTION AND STRUCTURES Clinical Chemistry 1 (LEC)

GENERAL DESCRIPTION ● There are two routes of converting

INTRODUCTION
● Let us go back to our biochemistry while we
are on our first-year level
BASIC BIOCHEMISTRY
● We define protein as polymers of amino
acids that are linked with covalent bonds
through peptide bonds.
o We all know that it is consists of
peptide bonds and it is made up of
amino acid
NITROGEN CONTENT
● Protein contain NITROGEN that sets apart
from pure carbohydrates and lipids
o Again, what makes protein different
from carbohydrates and lipids? It
contains NITROGEN, so other books
write protein as the abbreviation
CHON meaning it is the substance
that contains Carbohydrates,
Hydrogen, Oxygen and Nitrogen.
● And since we have nitrogen content in the
proteins, we have Nitrogen balance
intracellular proteins to free amino acids:
Lysosomal Pathway and Cytoplasmic
Pathway
LYSOSOMAL PATHWAY
● The pathway which degrades extracellular
and some intracellular proteins
CYTOSOLIC PATHWAY
● This is the pathway which is important in
degradation of intracellular protein only
ANABOLISM
● The Central Dogma (DNA to RNA to Protein)
● This refers to the protein synthesis
● *Reading Assignment
TRANSAMINATION
● The central reaction that removes amino
acid nitrogen in the body
● This is under catabolism because it
degrades or disintegrate the protein by
removing amino acid nitrogen

● Nitrogen balance – a balance exist

between protein anabolism or what we call PROPERTIES OF PROTEIN
(protein synthesis) and protein catabolism
PROPERTIES OF PROTEIN
or what we call the (breakdown) of protein
MOLECULAR SIZE
Two types:
● Proteins are macromolecules. It is a
○ Positive Nitrogen Balance – Synthesis
molecule with a molecular mass of
is greater than catabolism meaning,
thousand and more. Because of their size
the intake of nitrogen exceeds their
and differing molecular masses, it is possible
laws as net protein synthesis
to separate them through:
proceeds. This happens usually in
○ Dialysis
pregnant women, growing children,
○ Ultrafiltration
and adults recovering from major
○ Gel Filtration Chromatography
illnesses
○ Density-Gradient Ultracentrifugation
○ Negative Nitrogen Balance – Vice
versa of positive meaning
DIFFERENTIAL SOLUBILITY
catabolism is greater than synthesis.
● Protein solubility is affected by the pH, ionic
It is happening when more nitrogen
strength, temperature, and dielectric
is excreted than incorporated into
constant of the solvent. The solubility of
the body. Example is the excessive
proteins in blood requires a pH on the range
tissue destruction whenever there
of 7.35 to 7.45
are burns, wastingdiseases,
continual high fevers, and starvation CHARGE AND ISOELECTRIC POINT
● Proteins can be positively and negatively
PROTEIN SYNTHESIS AND BREAKDOWN
charged
CATABOLISM
○ Protein is under isoelectric point
● This refers to the protein breakdown
meaning it contains both positive
● The disintegration of protein occurs in the
and negative charge from both
digestive tract, kidneys and particularly, the
ends
liver. Nitrogen elimination begins
intracellularly with protein degradation.
●

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9 PROTEINS: FUNCTION AND STRUCTURES Clinical Chemistry 1 (LEC)

ABSORPTION ON FINELY DIVIDED INERT MATERIALS LEVEL OF GLOBULAR PROTEINS

● These materials offer large surface areas for
the interactions with proteins. These P ● The primary
interactions may be affected by: R structure
○ hydrophobic I represents the
○ absorptive M number and
○ ionic A
types of amino
R
○ molecular hydrogen bonding acids in the
Y
specific amino
SPECIFIC BINDING TO ANTIBODIES, COENZYMES, OR acid sequence.
HORMONE RECEPTORS
● When we say, it
● The unique property of a protein to
is the structure or
recognize and bind to a complementary
it is the level of
compound with high specificity which is the
protein where it
basis for immunochemical assay. Proteins
gives you the
may also be separated by affinity
specific amino
chromatography, in which ligand attached
acid sequence it
to solid medium provides high selectivity.
can be found in
primary structure.
Note: if you remember in Immunology and Serology ● Usually if there is
subject, protein is considered as immunogenic mutation or there
substances. Meaning it has a high sensitivity to bind is alteration in
with the antibody which will cause antigen and the sequence of
antibody reaction. Usually if the foreign object is amino acids, it
made up of protein or it is protein in nature it is will result as the
considered as immunogenic. Because protein has
disease or
sensitivity or specific binding capacity to our
disorder.
antibodies.
STRUCTURES OF PROTEINS ● Some
hematological
STRUCTURES OF PROTEIN diseases happen
FIBROUS PROTEINS when there is
● Mainly structural alteration in the
sequence of the
primary structure.
There is an
alteration in the
sequence of
amino acids.

S ● The secondary
E structures are
C regularly
O repeating
GLOBULAR PROTEINS N
structures
● These are compact and have little space D
A stabilized by
for water in the interior of the molecule,
R hydrogen bonds
where most hydrophobic R groups are
Y between the
present. Most R groups are present in the
amino acids
surface, where they exert substantial
within the
influence on protein such as:
protein.
○ solubility
● Usually, it
○ acid-base behavior
produces:
○ electrophoretic mobility
o Alpha
● For globular proteins, we have four (4)
helix
levels of proteins: primary, secondary,
o Beta
tertiary, and quaternary.
pleated
sheets

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9 PROTEINS: FUNCTION AND STRUCTURES Clinical Chemistry 1 (LEC)

T ● Refers to overall three-dimension

E shape or al configuration.
R conformation of
T the protein
I DENATURATION OF PROTEIN
molecule. This is
A
rather the 3D ● For the structures of proteins, we can
R
overall shape or denature it. So, loss of its native or naturally
Y
three occurring folding is called denaturation.
dimensional Denaturation can be caused by heat,
shape or hydrolysis by strong acid or alkali, enzymatic
dimensional action, exposure to urea or other
conformation of substances, or exposure to ultraviolet light.
protein. ● Again, remember your experiment or your
● So, this procedure in the laboratory, we are
conformation is employing this one to our BB/blood banking
known as the section and Immunology and serology
folds or spatial section, we denature proteins by heating,
relationship of so that it won’t hinder the result of the
the secondary experimentation.
structures to one ● Before employing the principle or the
another technique that we are about to apply, we
first denature the protein.
Q ● Hemoglobin ● Protein has specificity to bind with antibody,
U structure is under coenzyme and hormones receptor. And in
A or is an example that property, it usually interferes with the
T of Quaternary results in our experiment that is why before
E
structure taking some experiments, we are
R
N because it is denaturing proteins. The usual denaturation
A defined as the process that we do is through heating.
R shape or
Y structure that
results from the CLASSIFICATION OF PROTEINS
interaction of
BASED ON FUNCTION
more than one
ENZYMES
protein
● Proteins that catalyze chemical reactions
molecule, or
o These are the
protein subunits.
proteins/chemicals/substances that
Again, Tertiary is
hastens the reactions happening in
the overall
our body. Without proteins/enzymes,
shape of one
digestion will be difficult for us.
protein. For
o Enzymes are protein in nature
Quaternary
● Proteins that are chemical messengers that
Structure, it is the
control the actions of specific cells or
combination of
organs. They affect growth and
two or more
development, metabolism, sexual function,
proteins.
reproduction, and behavior.
● It is held together
by noncovalent
TRANSPORT PROTEIN
bond forces and ● Proteins that transport movement of ions,
the example of small molecules, or macromolecules – such
which are as hormones, vitamins, minerals, and lipids –
hydrogen bonds across a biologic membrane
and electrostatic ● Examples of commonly measured transport
interactions, proteins:
which are part of o Hemoglobin - transport oxygen
the larger protein o Albumin
complex with a o Transferrin – transports ion
precise

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9 PROTEINS: FUNCTION AND STRUCTURES Clinical Chemistry 1 (LEC)

o They are also protein in nature others. It is the prosthetic groups that define
the characteristics of these proteins.
IMMUNOGLOBULINS (ANTIBODIES)
● Proteins produced by B cells (lymphocytes) EXAMPLES:
in the bone marrow that mediate the METALLOPROTEINS Metals + proteins
humoral immune response to identify and GLYCOPROTEINS Glucose + proteins
neutralize foreign objects LIPOPROTEINS Lipid + proteins
● They are also protein in nature NUCLEOPROTEINS Nucleic acid + proteins
MUCOPROTEINS Sugar + proteins
STORAGE PROTEINS
● Proteins that serve as reserves of metal ions ● What is the difference between
and amino acids that can be released and glycoprotein and mucoproteins?
used later without harm occurring to cells o Mucoproteins have a higher
during the time of storage. percentage of sugar than
glycoproteins.
ENERGY SOURCE
● Plasma protein serves as a reserve source of
energy for tissues and muscle.

OSMOTIC FORCE
● First, let us recall our clinical microscopy
subject, as a result, water is absorbed from
the tissue into the venous portion of the
capillary.
● So, water should be circulating in our blood,
the interstitial tissues, those are the water.
They should be circulating inside our venous
or inside our veins but since, without
osmotic force it will be absorbed from the
tissue into the venous portion of the
capillary and when the concentration of
plasma protein is significantly decreased,
the concomitant decreased in the plasma
colloidal osmotic and the pressure resolves
increased level of interstitial fluid and
edema
● This is often seen in renal disease with
proteinuria resulting in decreased plasma
protein because there is a concentration
and swelling of the hands and the feet.
● Protein is really important when it comes to
osmotic force because plasma proteins
function in the distribution of water
throughout the compartments of the body.

BASED ON STRUCTURE
SIMPLE PROTEIN
● Contain peptide chain composed of only
amino acids
● We classify simple protein if only it contains
amino acids and nothing else.

CONJUGATED PROTEIN
● Consist of protein and a non-protein
(prosthetic) group. The prosthetic group is
the non-amino part of a conjugated
protein. The prosthetic group may be lipid,
carbohydrate, porphyrins, metals, and

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10 PLASMA PROTEINS Clinical Chemistry 1 (LEC)

is protein, while all that reside in (a1, a2,

PRE-ALBUMIN beta, gamma) are globulins.

INTRODUCTION

Red – albumin
Blue – a1 & a2
Black – beta & gamma

If changes occur in the pattern, there is a related

disease/disorder.

PRE-ALBUMIN (TRANSTHYRETIN)

• Plasma consists of water, electrolytes,

metabolites, nutrients, proteins, and
hormones.
• The concentration of Total Proteins in human
plasma is approximately 6.0-8.0 mg/dL and
comprises that major part of solids of plasma

PLASMA PROTEINS • Other books use prealbumin while others use

● Most frequently analyzed of all proteins transthyretin, but they are just the same.
● Divided into 2 groups (major measurement • It is named as such because it migrates
of plasma): ahead of albumin in the classic
• Albumin
electrophoresis of serum or plasma protein.
• Globulin (4 major parts)
• Refer to the photo:
✓ α1-Globulins
o The photo shows the normal pattern
✓ α2-Globulins
✓ β-Globulins of electrophoresis
✓ γ-Globulins o Sometimes, a small band before the
• Typical blood panel provides 4 different albumin will be seen, that is the
measurements prealbumin (red arrow)
✓ Total protein ● It can also be separated by high resolution
✓ Albumin electrophoresis (HRE) or the
✓ Globulin immunoelectrophoretic technique
✓ Albumin/Globulin (A/G) Ratio ● This protein is rich in tryptophan
● A low prealbumin level is sensitive marker of
poor nutritional status
▪ When a diet is deficit in protein,
hepatic synthesis of protein is
reduced with a resulting decrease in
the level of protein originating in the
liver, including the prealbumin,
albumin, and globulin
▪ Recall from previous module: some
proteins are synthesized by liver. If
there will be malnutrition, or if there
• One of the analytical techniques employed
will be deficiency of protein intake of
in measuring protein is electrophoresis.
our body, there will be also be
• In healthy/normal individual (no underlying
deficiency or lower synthesis of
disease), it gives this (diagram) pattern of
protein
electrophoresis. All proteins reside in albumin

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10 PLASMA PROTEINS Clinical Chemistry 1 (LEC)

▪ Usually, the one that decreases first is • Indicator of Nutritional status

prealbumin
● Half-life: 2 days
o It has short half-life so it decreases
more rapidly than other proteins
● It is increased on patients receiving steroids
and alcoholism, as well as in chronic renal
failure
• Used to confirm if the specimen is really CSF.
• It is sensitive and highly prognostic marker in
FUNCTION OF PRE-ALBUMIN
cases of cystic fibrosis.
• It is the transport protein for thyroxine (T4)
• Lowest plasma levels of albumin are seen in
and triiodothyronine (T3) for the thyroid
active nephrotic syndrome.
panel
• it binds to with retinol-binding protein to form
a complex that transports retinol (vitamin A)

ALBUMIN
▪ The albumin is synthesized in the liver

• This is a representation of the electrophoresis

pattern of the patient that is suffering from
Nephrotic syndrome.
o The photo above is the normal • As you can see in the graph, there is a
representation of albumin decrease in the albumin levels, and usually,
the given diagnosis for this type of result in
the electrophoresis test is Active Nephrotic
Syndrome.
• The lowest plasma levels can be seen in
active Nephrotic syndrome.

DECREASED CONCENTRATIONS OF SERUM ALBUMIN

• Decreased concentration of the serum
albumin may be caused by the following:
o An inadequate source of amino
acids that occurs in malnutrition and
FUNCTIONS OF ALBUMIN
malabsorption
• Primary function is to bind to various
▪ There is a decrease of
substances in the body
albumin if we have a low
• Responsible for osmotic pressure.
source of amino acids which
o Albumin is responsible for nearly 80
we can get from the food we
percent of colloid osmotic pressure
eat.
of the intravascular fluid, which
▪ Low food intake = low
maintains the appropriate fluid
albumin synthesis.
balance in the tissue.
o Liver disease, resulting in decreased
• Albumin also acts as a buffer pH
synthesis by the hepatocytes
• Albumin is a negative acute phase reactant
▪ Note that the increase in
o There are four binding sites of
globulin that occurs in early
albumin and these have varying
cirrhosis balances the loss of
specificity of different substances.
albumin to give total protein
• Albumin also transports thyroid hormones
and other hormones particularly the fat- concentration within
soluble ones which are iron and fatty acid acceptable limits.

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10 PLASMA PROTEINS
▪ Sometimes, albumin is
decreased in liver diseases
because the hepatocytes
don’t have enough to
synthesize. If that happens,
our globulins become
elevated. Why? It’s because
they want to compensate for
the loss of albumin.
▪ With that being said, there is
a tendency that even if a
patient has liver disease, their
total protein is still normal.
However, their albumin is low
while their globulin is high. This
is why total protein is not the
only one being observed in
the laboratory.
▪ We have the total protein
and albumin/globulin (A/G)
ratio. This indicates that just
because total protein is within
normal values means the
patient is normal overall.
Sometimes, when albumin is
decreased due to liver
disease, our body can sense
that our albumin is too low;
this is when globulin increases
to compensate for the loss of
albumin.
o Protein-losing enteropathy or
gastrointestinal loss as interstitial fluid
leaks out in inflammation and
disease of the intestinal tract as in
diarrhea
▪ Albumin decreases when
there is protein-losing
enteropathy
o Kidney loss to the urine in renal
disease
▪ Albumin is normally excreted
in very small amounts only.
This excess excretion occurs
when the glomerulus no
longer functions to restrict the
passage of the proteins from
the blood(?) as in the
nephrotic syndrome.
▪ We all know that protein is a
macromolecule. Based on
clinical microscopy, there are
nephrons or glomerulus that
function to filter the
molecules entering the
kidney.
TRANSFORMERS
Clinical Chemistry 1 (LEC)
▪ Usually, albumin cannot pass
through. Why? It’s because
it’s a macromolecule. This is
why whenever there is a high
amount of albumin present in
urine, there is something
wrong with the function of
the kidney.
o Skin loss in the absence of the skin
barrier such as in burns or exfoliative
dermatitis
o Hypothyroidism
▪ Albumin also decreases
▪ Albumin transports our
thyroid panel which is the Th3
and Th4.
▪ If there is hypothyroidism,
what else will it transport?
o Dilution by excess
▪ Polydipsia (excessive thirst or
excessive administration of
intravenous fluids)
o Analbuminemia or bisalbuminemia
▪ This is due to mutation
▪ Mutation resulting from
autosomal recessive traits
causing analbuminemia
(absence of albumin)
▪ Bisalbuminemia is the
presence of albumin that has
unusual molecular
characteristics. These are
demonstrated by the
presence of two (2) albumin
bands instead of a single
band and usually seen in
electrophoresis. However,
these are considered rare.
o Redistribution by hemodilution
▪ Increases the capillary
permeability or decreases
the lymph clearance and
▪ In sepsis, there is profound
reduction of plasma proteins
associated with marked fluid
shift
These are the conditions wherein you will see that
albumin is decreased.
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10 PLASMA PROTEINS Clinical Chemistry 1 (LEC)

GLOBULIN ● If there will be deficiency in alpha1-

antitrypsin usually yung sakit nila will be
GLOBULIN Emphysematous pulmonary disease and
● a1, a2, β, and γ fractions Juvenile hepatic cirrhosis.
● Measurement: Total protein (TP) – Albumin =
o This is because its major function is to
Globulin
inhibit protease neutrophil elastase
● Used to detect early cirrhosis
but if there will be antitrypsin
deficiency, neutrophil elastase is
released from the leukocytes and this
neutrophil elastase that is released
by leukocytes to fight infection will
destroy the alveoli eventually.
o Pagnasira na alveoli magreresult siya
into desease which is yung
Emphysematous pulmonary disease.
● It comprises almost 90% of the a1-globulin
band
o Just an FYI, quantitative methods
used to confirm electrophoresis
GLOBULINS
findings are the radio
a1- Globulins • a1-antitrpysin
immunodiffusion and automated
• a1-Fetoprotein
• a1-acid glycoprotein, a1- immunonephelometric assay.
lipoprotein Phenotyping is done using
• a1-antichymotrypsin immunofixation.
• Inter-a-inhibitor
• Gc-globulin a1- Fetoprotein (AFP)
a2-Globulins • Haptoglobulin Ceruloplasmin ● It is synthesized in the developing embryo
• a2-Macroglobulin and fetus then by parenchymal cells of the
β-globulins • Pre-B-Lipoproteins liver.
• Transferrin
• Hemopexin ● Peak: 13th week of gestation/ 7 or 8th month
• β2-microglobulin of pregnancy
• C4
● Used as tumor marker (for hepatic and
• C3
gonadal cancer)
• C1q complement Fibrinogen
• C-reactive protein ● Specimen used: maternal serum and/or
γ -globulins • IgG amniotic fluid; serum (for cancer screening)
• IgA
• IgM o Or sometimes if you want or if you
• IgD intend to measure or to check the
• IgE cancer screening, serum is used. But
if you want disease from the fetus, we
a1 – Globulin will use maternal serum or the
a1- Antitrypsin (AAT) amniotic fluid.
● Anti1 or Alpha1-Antitrypsin or AAT
● It is usually ELEVATED in spina bifida, neural
● It is a glycoprotein.
tube defects, abdominal wall defects,
● It is synthesized in the liver.
anencephaly, presence of twins and
● An acute phase reactant so it elevates general fetal distress.
whenever there is an infection and
inflammation. ● LOW LEVELS in increased risk of Down
● Most important function is the inhibition of syndrome and trisomy 18.
o Low levels will be seen in increased
protease neutrophil elastase so there will be
risk of Down syndrome and trisomy
a mutation of serpina1 gene caused by 18.
antitrypsin deficiency. Neutrophil elastase is ● By the way, AFP has no known function in
released from leukocytes to fight infection adults but in fetus it is known or it promotes
but can be destroyed by alveoli which can protection in any immunological attacks.
lead to emphysema if not controlled by
alpa1-antitrypsin

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10 PLASMA PROTEINS
α1- acid Glycoprotein/ orosomucoid (AAG)
● It has a low pH (2.7); it has a negatively
charged even in acid solution.
o Alpha 1 acid glycoprotein is a major
plasma glycoprotein and a negative
charged even in acid solution.
o This is a fat that gives its name. This
protein is produced in the liver and is
an acute phase reactant. And the
possibility that AAG regulates
immune response has been
suggested by several findings.
o There are strong similarities in the
amino acid sequence between
AAG and immunoglobulin. If you will
study their structure, AAG can be
similar to the sequence of amino
acids compared to our
immunoglobulin.
● An acute phase reactant.
● CHO and sialic acid
o Usually, AAG structure is a ● It is used as prognostic indicator of survival of
combination of carbohydrates (?)
glycoprotein. Glycoprotein is a
protein with a combination of
glucose or carbohydrates and
proteins. And since it is acidic, it has
sialic acid as well.
● Useful in diagnostic tools in neonates with
bacterial infections.
o Serum AAG levels also provide a
useful diagnostic tool in neonates
with bacterial infections.
● INCREASED: pregnancy, cancer,
pneumonia, rheumatoid arthritis (RA), cell
proliferation.
o It is elevated in stress, inflammation,
tissue damage, acute myocardial
infarction, trauma, pregnancy,
cancer, pneumonia, rheumatoid
arthritis and surgery.
● Normal range is 50-120 mg/dL.
● In the past decades, the binding of drugs to
this plasma protein has extremely or
increasingly become important in regards to
drug action distribution and disposition. If
you will study pharmacology, AAG promotes
a big factor to our drug actions because it
increasingly becomes important to our drug
actions since this is the binding site of the
drug that we intake.
● For the measurements, analytic
measurement usually used for this one is
radial immunodiffusion, immunoturbidity
and nephelometry. So, for immunofixation it
has been used to study inherited variance of
AAG.
a1- Antichymotrypsin (a1-x)
● Member of the serine proteinase inhibitor
family (Catepsin G); acute phase reactant
● Migrates between the a1 and a2 zones;
synthesized in the liver
TRANSFORMERS
Clinical Chemistry 1 (LEC)
● It binds and inactivates prostate-specific
antigen (PSA)
● Can be found in amyloid plaques with
Alzheimer's disease (recent study)
● INCREASED: infection, malignancy, burn,
AMI, Alzheimer’s disease
o Deficiency for this protein has been
associated with liver disease and
mutation has been identified in
patients with Parkinson’s disease and
chronic obstructive pulmonary
disease.
Inter-a-Trypsin Inhibitor
● They are a family of serine protease inhibitors
● Elevations are seen in inflammatory disorders
Gc – Globulin (Group – Specific Component)
● It is a major carrier protein of Vitamin D
patients with significant tissue injury
a2 - Globulin
Haptoglobin
● It is considered also as an acute-phase
protein that is elevated in many
inflammatory diseases such as ulcerative
colitis, acute rheumatic disease, heart
attack, and sever infection.
● Its function is to prevent the loss of
hemoglobin and constituent iron into urine
● And also, it is useful measurement for serially
monitoring patients who have a slow but
steady rate of red cell breakdown.
● And it evaluates the degree of intravascular
hemolysis.
● So, also is INCREASED in stressful conditions
and myoglobinuria
● And DECREASED in intravascular hemolysis
and hemoglobinuria
Ceruloplasmin
● It is a copper-containing protein
● Also, an acute-phase reactant
● And, it imparts the blue color to protein
● It is an indicator of Wilson’s Disease
● So, how can we tell [it is] or the patient is
positive in Wilson’s Disease. We will be having
a DECREASED level of ceruloplasmin in
Wilson’s Disease, malnutrition,
malabsorption, nephrotic syndrome,
Menkey’s kinky-hair syndrome.
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10 PLASMA PROTEINS
● So, usually ceruloplasmin deposits in our eyes
and it is obviously seen in our hair.
● For example, in this one (Kayser Fleisher
Rings – right photo) itong nakikita niyo is a
ceruloplasmin or iron deposit sa ating mata
and what we call in that case is “Kayser
Fleisher Rings” and we also have “Menke’s
kinky-hair syndrome”
a2 – macroglobulin (A2M)
● It is the largest major non-immunoglobulin
protein in plasma and it is synthesized by the
liver.
● It is also the major component of the alpha-
2 band in the protein electrophoresis
● For the structure, just to review it is a
tetramere of four identical subunits that
inhibits protease such as trypsin, thrombin,
kallikrein and plasmin by means of bait
region that can entrap proteinases
producing the accessibility of the
proteinases functional sites particularly the
large molecule.
● In nephrosis, the level of serum A2M may
increase as much as 10x because its larger
size or its large size aids in each retention.
● So, the protein also INCREASES in diabetes
and liver disease. It is also used for
contraceptive medication and pregnancy
INCREASE serum by 20%.
● For the analytical methods, you can use
radial immunodiffusion,
immunonephelometric, enzyme-linked
immunosorbent assay or the ELISA and the
latex agglutination immunoassay.
β – Globulins
β2 – macroglobulin (B2M)
● Major human leukocyte antigen (HLA)
o It is a light-chained end component
of the major human leucocyte
antigen.
● Found on the surface of most nucleated
cells and present in high concentrations of
lymphocytes
● Appears in urine when reabsoption is
incomplete because of proximal
convoluted tube damage
TRANSFORMERS
Clinical Chemistry 1 (LEC)
o Because it is small in size, B2M is
usually filtered in the renal glomerulus
but reabsored and catabolized in
the proximal tubules.
● INCREASED in renal failure, multiple
myeloma, rheumatoid arthritis (RA), systemic
lupus erythematosus (SLE)
o FYI: In patients with Human
Immunodeficiency (HIV+), there will
be a high B2M levels in the absence
of renal failure and indicates large
lymphocyte turnover rate
● This can be measured through analytical
method using immunoassay
Transferrin (Siderophilin)
● A glycoprotein; negative acute phase
reactant
● Transport iron to its storage sites; present in
CSF in small amount
● Its function is to prevent the loss of iron
through kidney
● Deficiency in this protein will result
accumulation of iron in apoferritin or in
histiocytes or precipitates in tissues as
hemosiderin
● INCREASED in hemochromatosis and Iron
Deficiency Anemia (IDA)
● DECREASED in liver disease, malnutrition,
nephrotic syndrome
Lipoproteins
● Complexes of proteins and lipids such as
LDL, HDL, VLDL, CHYLOMICRONS
● Transport cholesterol, triglycerides and
phospholipids.
o Transportation of cholesterol is the
major function of lipoproteins.
Fibrinogen
● It’s one of the largest proteins in the blood
plasma.
● It is synthesized by the liver and classified as
glycoprotein because it is considerable of
having a carbohydrate content.
● On the plasma electrophoresis fibrinogen is
seen distinctly between the globulin and the
Beta globulin and globulin.
● Acute-phase reactant
● The function of the fibrinogen is to forms fibrin
clot when activated by thrombin
● INCREASED in inflammatory disorders,
pregnancy and patients using birth control
pills.
● DECREASED in extensive coagulation.
● For the measurement it can be determined
by using Immunoassay methods such as: RIA
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10 PLASMA PROTEINS Clinical Chemistry 1 (LEC)

(Radioimmunoassay) nephelometry, RID

(Radial Immunodiffusion).

Complement
● Serves as a link to the inflammatory response.
● INCREASED in inflammatory states
● DECREASED in lupus erythematosus, DIC,
malnutrition
● Complements: C3, C4

C–Reactive Proteins (CRP) TROPONIN

● Undetectable in healthy individuals ● Cardiac marker for acute coronary
● Used warning test to persons at risk of syndrome – Troponin C (TnC)
coronary artery disease ● We have 3 different types of troponin:
● An acute phase reactant o TnI, TnC, TnT – troponin complex
● INCREASED in acute rheumatic fever, ● Actin and myosin are structural protein with
myocardial infarction, RA, gout, bacterial regards to their function
and viral infection

IMMUNGLOBULINS
● Or Gammaglobulins
● Synthesized in PLASMA CELLS
o Other proteins are synthesized in
liver except for this one.
● INCREASED in hepatic disease, infections,
toxoplasmosis, cytomegalovirus infections,
rubella, herpes, syphilis, allergic reactions,
multiple myeloma
● If there will be a hepatic diseases hindi
makapag-synthesize ng maayos si
hepatocyte ng albumin, usually ang nag
iincrease will be the globulins. It INCREASES in BRAIN NATRIURETIC PEPTIDE
hepatic diseases para macompensate yung ● Neurohormones that affect body fluid
loss of albumin homeostasis and blood pressure.

FIBRONECTIN FETAL FIBRONECTIN (fFN)

● Main function: for cellular interaction (e.g.,
cell adhesion)
● It is the reason of the adherence of the
placenta to the uterus.
o As seen in the image, the bridging or
the linkage between the placenta
● These are the structures: IgG, IgE, IgD, IgM, and the uterus. That is the fFN.
IgA

OTHER PLASMA PROTEINS

MYOGLOBIN
● Myoglobin is the primary oxygen-carrying
protein (approx. 2% of total muscle protein)
found in striated skeletal and cardiac
muscle
o It is under our structural protein
● ↑ 2-3 hours of onset, peak at 8-12 hours
o Expected in persons with muscle ● It INCREASES in the preterm labor and
dystrophy or crushing injury delivery.
● ↑ Crushing injury and muscle dystrophy

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CROSS-LINKED C – TELOPEPTIDES
● Proteolytic fragment of collagen I
● Biochemical marker of bone resorption.

CYSTATIN C
● Cysteine proteinase inhibitor

AMYLOID
● Fibrous (in structure) protein aggregates
formed from alteration of B (beta) pleated
sheaths.
● INCREASES in:
o Amyloidoses
o Tau proteins – only seen in the CSF
● This is correlated with Alzheimer’s disease.
o Its pathophysiology is, there is the
buildup of plaques (nerve injury in
the nerve endings in the brain =
plaque)
o Usually, there is an increase in
amyloid levels in Alzheimer’s disease
patients.
o Plaques and bindings, to building up
of proteins in the brain causes
Alzheimer’s disease
o One of its symptoms is dementia

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11 NON-PROTEIN NITROGEN COMPOUNDS Clinical Chemistry 1 (LEC)

NON-PROTEIN NITROGEN COMPOUNDS UREA IN THE LIVER

● After it's synthesis in the liver, Urea is carried
NON-PROTEIN NITROGEN COMPOUNDS in the blood and it goes into the kidney.
● The following are the clinically significant
NPNs found within the body

1 UREA
● Having the highest plasma
concentration among all of them

2 AMINO ACID
3 URIC ACID
4 CREATININE
5 CREATINE
6 AMMONIA WITHIN THE KIDNEY
● Having the least plasma concentration ● Within the kidney, particularly in the
glomerulus, urea is readily filtered in the
plasma. Majority is excreted in the urine
while some are reabsorbed by passive
diffusion or passive transport in the renal
tubules.
● The amount of reabsorbed urea depends on
the urine flow rate and extent hydration.

Note: So, most of these compounds results from

the catabolism of proteins and nucleic acids.
They are cleared from the body by the kidney
following glomerular filtration. Generally used to
check for renal function and other conditions.
UREA

UREA
● It is a major excretory product of protein
metabolism.
● Urea has the highest plasma concentration. SUMMARY
● Historic assay used for urea measurement ● Urea is formed in the liver and then it is
based on the determination of nitrogen. carried in the blood
Thus, the term Blood Urea Nitrogen. ● It can go to two pathways:
● Urea N is being used since it is more o 90% of the formed urea is
appropriate. excreted from kidney in the form
of urine
UREA SYNTHESIS
o 10% of the generated urea goes
● Urea is formed in the liver and a process that
into the GIT and skin where it is
involves enzymes. It is produced from the
reabsorbed
amino acids and ammonia, generated
during protein catabolism.

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11 NON-PROTEIN NITROGEN COMPOUNDS Clinical Chemistry 1 (LEC)

CONCENTRATION OF UREA IN THE PLASMA IS ● Then it is transported into the muscles where
DETERMINED BY: in it is converted into phosphocreatine or
● Diet creatine phosphate
o Protein content of the diet
● Protein catabolism
o Rate of protein catabolism
● Renal function and perfusion

URIC ACID

URIC ACID
● Is the primary product of purine nucleic acid
catabolism
● So, creatine loses water, and then creatine
● Sources of purines are from the breakdown
phosphate loses phosphoric acid forming a
of ingested nucleic acids or from tissue
cyclic compound called creatinine
destruction
● Similar to urea, uric acid is also formed in the
liver.
● It is relatively insoluble in plasma
● At a high concentration, it can be deposited
in the joints and tissues, causing painful
inflammation.

AFTER LIVER SYNTHESIS OF URIC ACID

● After synthesis in the liver, the uric acid is also
transported in the kidneys where it will be
filtered at the glomerulus.
● As you can see in the picture 98% to 100%
uric acid is reabsorbed in the proximal
tubules. Small amount is excreted or
secreted in the urine by the distal tubules
and the rest goes to gastrointestinal tract
(GIT) wherein it is degraded by bacterial
enzymes
● Most of uric acid present in the plasma is in
the form of monosodium urate.
● So, the synthesized creatinine is released into
the circulation at a relative constant rate,
● And it is said to be proportional to one’s
muscle mass
● Thus, then removed from the circulation by
glomerular filtration and it excreted in the
urine.
● Small amounts of creatinine are secreted by
the proximal tubule and reabsorbed by the
renal tubules.
● Remember: Plasma Creatinine is inversely
related to your Glomerular Filtration Rate.
● Also, creatinine is commonly used to assess
renal filtration function.

CREATININE

CREATININE AMMONIA
● Creatinine is formed in the muscles and then
AMMONIA
it is secreted into the plasma at the constant
● Ammonia is produced during deamination
rate related to muscle mass.
of amino acids during protein synthesis, the
● Creatine (pic) is generated from arginine,
synthesized AMMONIA is removed from the
glycine, methionine in the liver.
circulation and some of it is converted into
urea within the liver, only a small amount of

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11 NON-PROTEIN NITROGEN COMPOUNDS Clinical Chemistry 1 (LEC)

ammonia is present in the plasma since

free ammonia is toxic.

AMMONIA PRODUCTION
● The production of ammonia comes from
different pathways so from the catabolism
of amino acids by bacterial metabolism into
the lumen of the intestine

● the anaerobic metabolic reaction that

occurs in skeletal muscle during exercise
produces endogenous ammonia

● then also your liver produces ammonia but

this produced ammonia is also consumed
within the liver in order to produce urea.

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12 NPNs: CLINICAL APPLICATION & SPECIMEN CONSIDERATIONS
CLINICAL APPLICATION
INTRODUCTION
● It is important that we understand the clinical
application of these NPN compounds, so
that we may be able to know their
importance and be aware of how we may
guide and instruct the patient of the dos and
don’ts before the sample may be taken
UREA
● Evaluate renal function
● Assess hydration status
● Determines nitrogen balance
● Diagnosis of renal disease
● Verify adequacy of dialysis
● Severe liver damage
✔ Decreased Urea
Performed in protein-free filtrate of whole blood’s
Nitrogen content. Reported in terms of nitrogen
concentration than Urea Nitrogen. Thus, Urea
Nitrogen can be converted into Urea
concentration.
CONVERSION UREA NITROGEN TO UREA
CONCENTRATION
● 2.14 – constant derived from MW of Nitrogen
atom in Urea and MW of Urea
● 2 nitrogen atoms in Urea – (2 * 14 (AW)
● 60 – MW of urea
● 60/28 = 2.14
● Reported in mg/dL (Conventional unit)
CONVERSION FACTOR OF UREA
● 0.357 (SI unit)
SPECIMEN CONSIDERATIONS FOR UREA
1 Plasma serum and urine
● Can be used when measuring urea
● When using urine, since it is more
susceptible to bacterial decomposition
and growth, it must be refrigerated if not
processed immediately
2 No to hemolysis; fluoride, citrated, ammonium
containing anticoagulant
● When using plasma, these should not be
used and at the same time, hemolysis
should be avoided
3 Fasting is not required
● The effect of a single meal will not affect
the urea measurement
4 Can be refrigerated if not analyzed immediately
URIC ACID
● In a clinical setting, uric acid is measured
because it assesses inherited disorder of
purine metabolism such as:
TRANSFORMERS
Clinical Chemistry 1 (LEC)
o Deficiency of Glucoses-6
Phosphatase (G6P deficiency)
o Lesch-Nyhan syndrome
● Confirm diagnosis and monitor treatment of
grout
● Assist in the diagnosis of renal calculi
● Prevention of uric acid nephropathy during
chemotherapeutic treatment
● Detection of kidney dysfunction
SPECIMEN CONSIDERATIONS FOR URIC ACID
1 Heparinized plasma, serum, urine; measuring
uric acid
● When serum is used, serum should be
removed from packed RBCs as quickly
as possible to prevent dilution by
intracellular content. When separated,
the uric acid in the serum is stable from
3-5 days especially when placed in a
refrigerator temperature.
● When using urine as a sample, the urine
should be maintained at the pH 8 or
alkaline pH
2 Fasting is NOT required
● Diet may affect the uric acid
concentration overall, But the recent
meal has NO significant effect.
Therefore, fasting is NOT necessary.
3 Lipemic, Bilirubin, Hemolysis – should be
avoided
● Gross lipemia and hemolysis should be
avoided because hemolysis can result in
a low value due to concomitant
glutathione release.
● Substances may interfere, the results of
uric acid such as bilirubin that can falsely
DECREASED the results.
4 Salicylates and Thiazides
● Drugs such as Salicylates and Thiazides
INCREASE the value of uric acid.
5 EDTA and Fluorides (NOT recommended)
● EDTA or Fluoride additive should NOT be
used for specimens that will be tested by
uricase method.
CREATININE
Clinical application
● Determine the sufficiency of kidney function
o Measurement of creatinine
concentration is used to determine
the sufficiency of kidney function.
● Identification of the severity of kidney
damage
● Monitoring the progression of kidney disease
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12 NPNs: CLINICAL APPLICATION & SPECIMEN CONSIDERATIONS
o It’s also used to determine severity of
kidney damage and monitor the
progression of kidney disease.
● Plasma creatinine concentration is
influenced by protein content of the diet
● Urinary creatinine excretion/creatinine
clearance measures the amount of
creatinine eliminated from the blood by the
kidney, thus, gauging the renal function.
SPECIMEN CONSIDERATIONS FOR CREATININE
1 Plasma, serum, and urine
● When using urine, it should be
refrigerated after collection or frozen if
longer storage (> 4 days) is required.
2 Hemolyzed, icteric, lipemic
● These should be avoided, particularly if
you’re using the Jaffe method.
● Lipemic samples can also produce
erroneous results using other methods.
3 Fasting is not required
● Fasting is not required but high protein
ingestion may transiently elevate serum
concentrations.
4 SOURCE OF ERROR
● Ascorbate, glucose, a-keto acids, and
uric acid
o Increase creatinine
concentration measured by
Jaffe reaction specifically
temperature above 30 degrees
Celsius. This interference is
significantly decreased when
kinetic measurement is applied
depending on the concentration
of reactants and measuring fine
interferences of a-keto acids
may persist using the kinetic Jaffe
method.
o Ascorbate will interfere in
enzyme as well when using
peroxidase as a reagent
● Bilirubin
o It causes negative bias in both
Jaffe and enzymatic methods.
● Cephalosporin, Dopamine, Lidocaine
o Patients taking Cephalosporin
antibiotics may have falsely
elevated results when Jaffe
reaction is used.
o Other drugs have been shown to
increase creatinine results
particularly Dopamine – known
to affect both enzymatic and
Jaffe method.
o Lidocaine causes a positive bias
in some enzymatic methods
TRANSFORMERS
Clinical Chemistry 1 (LEC)
AMMONIA
● Severe Liver Disease
o Ammonia provides useful
information for severe liver disease
o Disturbed ammonia metabolism can
be common
● Hepatic Failure
o Monitors the prognosis and severity
of hepatic failure
o It cannot be directly correlated with
the disease
● Reye’s Syndrome
o Acute metabolic disorder of the liver
o Autopsy findings shows severe fatty
infiltration of the organ where blood
ammonia can be correlated to both
severity and prognosis of the
syndrome
● Inherited Deficiencies of Urea Cycle
Enzymes
o Associated with unexplained
nausea, vomiting, and deterioration
and, (from Bishop: neurological
deterioration associated with)
feeding could be obvious of
ammonia determination
● Hyperalimentation Therapy
● Monitoring of this therapy wherein urine
ammonia can be used to confirm ability of
kidneys to produce ammonia
SPECIMEN CONSIDERATIONS FOR AMMONIA
1 Plasma, Whole blood, or Venous blood
● Careful specimen handling is strictly
important for plasma ammonia assay.
● Whole blood ammonia concentration
increases rapidly following specimen
collection because in vitro amino acids
deamination.
● When using venous blood, it should be
taken without trauma and the blood
should be placed on ice immediately
2 Heparin and EDTA
● Heparin and EDTA, so double
anticoagulants. However, the sample
should be centrifuged at 0 to 4 degrees
C within 20 minutes collection.
● In the plasma, it should be removed
immediately and the assay should be
performed right away and if it is not
possible, it should be frozen. Although it
is stable at -20 degrees C for several
days, assay must be performed
immediately.
3 Store at 0 – 4C within 20 minutes
2
12 NPNs: CLINICAL APPLICATION & SPECIMEN CONSIDERATIONS Clinical Chemistry 1 (LEC)

4 HEMOLYSIS preventing contamination with platelet

● must be avoided because red blood or leukocyte amino acid.
cells may contain 3x more ammonia
than in plasma. Take note: WBC has 100x more aspartic and
glutamic acid than in plasma.
5 SMOKING 3 Avoid Hemolysis
● the patient should refrain from smoking 4 Deprotenization
for several hours before the sample must ● Should be performed within 30 minutes of
be taken. sample collection.
● Analysis should be performed
6 INTERFERENCES (INCREASES AMMONIA IN immediately.
PLASMA) o If not, sample should be frozen at
● ammonium salts, asparaginase, -20 to -40 degrees Celsius.
barbiturates, diuretics, ethanol,
hyperalimentation, narcotic analgesics, URINE
and some other drugs. 1 Random or 24-hour urine
● Urinary amino acid analysis can be
7 INTERFERENCES (DECREASES AMMONIA IN performed on a random specimen for
PLASMA) screening purposes only.
● Diphenhydramine, lactobacillus ● For quantitation, a 24-hour urine
acidophilus, lactulose, levodopa, and specimen, preserved with thymol or
several antibiotics organic solvent, is required.

AMINO ACID
● Provides useful information for genetic
metabolic disorders or aminoacidopathies

AMINOACIDOPATHIES
● a class of inherited errors of metabolism in
which there is an enzyme defect that inhibits
the body’s ability to metabolize certain
amino acid
● abnormalities exist either in the activity of
specific enzymes in the metabolic pathway
or in the membrane transport system for the
amino acids

MEDICAL COMPLICATIONS CAUSED BY

AMINOACIDOPATHIES
● Brain damage due to accumulation of toxic
amino acid for their byproducts in the blood
and tissues.
● Examples are:
o Tyrosinemia
o Phenylketonuria (PKU)

SPECIMEN CONSIDERATIONS FOR AMINO ACID

PLASMA
1 6-8 hours fasting
This should be done to avoid the effect of
absorbed amino acid, coming from dietary
proteins.
2 Heparin
● Sample is collected in a heparin tube,
with the plasma promptly removed from
the cell.
● Taking care not to touch, take away or
aspirate the platelet and WBC layer,

TRANSFORMERS 3