TRANS GROUP #1 Marquez, Datinggaling, Daya EDITORS

1. Units of Measure
2. Reagents
3. Clinical Laboratory Supplies
4. Centrifugation
5. Laboratory Mathematics and Calculations
6. Specimen Considerations

LEGEND
Remember Lecturer Book Previous Presentation
Trans

OBJECTIVES:
At the end of the lecture, the student should be able to:
 Convert results from one unit format to another using the SI
and traditional systems.
 Describe the classifications used for reagent grade water.
 Identify the varying chemical grades used in reagent preparation
and indicate their correct use.
 Define primary standard and standard reference materials.
 Describe the following terms that are associated with solutions and,
when appropriate, provide the respective units: percent, molarity,
normality, molality, saturation, colligative properties, Redox
potential, and conductivity.
 Describe two ways to calibrate a pipetting device.
 Identify the preanalytic variables that can adversely affect
laboratory results as presented in this chapter.

I. CLINICAL CHEMISTRY

 Clinical – comes from the greek word kline, meaning “bed”

 Chemistry – is a basic science that utilizes the specialty of
chemistry to study human beings in various stages of health and
disease
 It is an applied science when analysis are performed on body
fluids or tissue specimens to provide information for the diagnosis
or treatment of disease.

II. UNITS OF MEASUREMENT

Système International d'Unités (SI)
 It consists of seven independent base units, each unit is represented
by a symbol.
 Adopted internationally in 1960
 It is devised to provide the global scientific community a
uniform method of describing physical quantities.

A. UNITS

Length meter (m)

Mass Kilogram (kg)
Time Second (s)
Quantity of substance Mole (mol)
Electric current Ampere (A)
Temperature Kelvin (K)
Luminous Intensity Candela (cd)
Table 1.01 (Units of Measurement)

NOTES TO REMEMBER
 It has been recommended that analytes be reported using moles of
solute/volume of solution (mmol/L)
 Enzyme units are given as the international Unit per liter (U/L),
although katal unit has been adopted previously as SI unit, its use is
limited
 pH scale is retained for measurement of hydrogen ion concentrations
 Liter is used as the reference volume
 SI unit is used because compounds react on a molar basis, and
expressions of amounts of substances in such terms allows for a
better understanding of the relative proportion of compounds.

MLS109 Basic Principles of Clinical Chemistry CLINICAL CHEMISTRY

 OUTLINE
III. REAGENTS
 Most instrument manufacturers make the reagents in a ready-to-
use form or “kit” where all necessary reagents and respective
storage containers are prepackaged as a unit requiring only the
addition of water or buffer to the prepackaged components for
reconstitution.

A. Chemicals
1. Analytical Reagents
 Suitable for use in most analytical laboratory procedures
 Impurities must be stated
2. Ultrapure Reagents
 Chemicals that have been put through additional purification steps
 Used in procedures such as chromatography, atomic
absorption, immunoassays, molecular diagnostics and other
techniques that may require extremely pure chemicals
3. Chemically Pure Reagents
 Indicates that impurity limitations are not stated
 Preparations of these chemicals is not uniform
 Not recommended for clinical laboratories unless further
purification or reagent blank is included
4. USP and NF grade
 Used to manufacture drugs
 Based on the criterion of not being injurious to individuals
 Chemicals are pure enough for use in most chemical
procedures USP – United States Pharmacopeia, NF –
National Formulary
5. Technical/Commercial Grade
 Used for manufacturing
 Should never be used in the clinical laboratory

IV. REFERENCE MATERIALS

STANDARD
 A substance or solution in which the concentration is determined
 It is used in the calibration of an instrument of method

A. Types of Standard – Atomic Weight Standard

 Pure to the last molecule
 Grade A (IUPAC)

B. Ultimate Standard
 Grade B (IUPAC)

C. Primary Standard
 Highly purified, concentration is exactly known
 At least 99.98% purity
 Used in clinical laboratories
 Grade C (IUPAC)

D. Working Standard
 <0.05% particulates, 99.95% purity
 Grade D (IUPAC)

E. Secondary Standard
 Substance of lower purity
 Grade C (IUPAC)

V. WATER SPECIFICATIONS
WATER
 Considered as the universal solvent
 It is the most frequently used reagent in the laboratory

METHODS OF FILTRATION – A. FILTRATION

 Passage through a filter or other material that prevents passage
of certain molecules, particles or substances
 Can remove particulate matter from municipal water supplies
before any additional treatments

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 MLS109 Basic Principles of Clinical LE TRANS
 Used as pre-treatment  0.2 micron filter
 Specialized filters  Activated carbon
 Activated carbon
 Removes chloride and organic material  Type II
 Submicron filter  Used for most laboratory determinations
 0.2 microns  Microbial content ≤ 103 cfu/mL
 Removes all particles that are larger than its size  Silicate content ≤ 0.1 mg/L
 Limitation  Type III
 Only particles that are larger than the filter size are not allowed to  For most qualitative measurement
pass through; therefore, smaller particles can still contaminate  For urinalysis, parasitology and histology
the filtrate.  For washing glasswares not requiring Type I or II reagent grade
water (RGW)
B. Distillation  Silicate content ≤1.0 mg/L
 CO2 free water
 The process of vaporizing and condensing a liquid to purify  Obtained by boiling Type II RGW
or separate a volatile substance from a non volatile
 Used if CO2, amonia, or O2 may affect analysis
substrate
 Limitations
VI. LABORATORY EQUIPMENT – GLASSWARE
 Carryover of volatile impurities and entrapped water droplets may
A. Borosilicate
contain impurities such as:  General purpose, for test tube and disposable glassware
 Volatiles  Most common type encountered in volume measurement
 Sodium, potassium, magnesium, carbonates and sulfates
 With high thermal resistance and low alkali content
 Should not be heated above strain point because rapid cooling
C. Ion Exchange
cracks the glass easily when heated again
 Examples:
 A process that removes ions to produce mineral-free water
 Pyrex – strain point 512 °C
 Accomplished by passing feed water through columns containing
 kimax
insoluble resin polymers that exchange H+ and OH- ions for the
impurities present in ionized from in the water
B. Alumina-silicate
 Limitations
6x stronger than borosilicate
 Only ions are removed, therefore it is neither pure nor sterile  Strengthened chemically rather than thermally
 Resist clouding and scratching better
D. Reverse Osmosis  Only half of the thermal resistance of borosilicate glass
 Example : corex
 A process in which water is forced through a
 Used for
semipermeable membrane that acts as a molecular filter
 Graduated cylinder
 Removes 95-99 % of organic compounds, bacteria and other
 Thermometers
particulate matter and 90-97% of all ionized and dissolved
minerals  Test tube for high speed centrifugation
 Limitations
C. Low Actinic Glassware
 Does not efficiently remove gasses
 High thermal resistance
E. Ultraviolet Oxidation  With red color to permit adequate visibility of contents yet give
maximum protection from light
 Used for photosensitive materials like bilirubin standards
 Uses ultraviolet radiation at the biocidal wavelength of 254 nm
 Eliminates many bacteria and cleaves many ionizing organics
D. Corning Boron Free Glass
that are then removed by deionization
 Highly resistant to alkali
 Limitations
 Soft, poor heat resistance
 Requires further purification methods

TYPES OF WATER – A. DISTILLED

 Purified to remove almost all organic materials

 Distillation does not remove volatile organic impurities
 Water may be distilled more than once to remove impurities
B. Deionized
Produced from distilled water using either an anion or a cation
exchange resin followed by replacement of the removed particles  HPLC
with hydroxyl or hydrogen ions respectively  Measurement of nanogram or subnanogram concentration
 Generally, it is purified from previously treated water such as  Tissue or cell culture
pre- filtered or distilled water  Final rinsing of glassware
 Microbial content ≤ 10 cfu/mL
C. Reagent Grade  Silicate ≤ 0.05mg/L
 Water is passed through
 Type I
 Used for the procedures that require maximum water purity
 Preparation of standard solutions
 Ultra micro chemical analysis
 Enzyme analysis
 Immunoassays
 Fluorescence quantitation

CLINICAL 2
 VII. LABORATORY EQUIPMENT – PLASTICWARE
MLS109 Basic Principles of Clinical A. Polyethylene LE TRANS
 For inexpensive, disposable tubes
 Used for storage of alkaline solutions

B. Polypropylene
 Withstands higher temperature
 Becomes discolored by solvents

C. Polycarbonate
 Stronger, more heat resistant
 Lower chemical resistance
 For centrifugal tubes and graduated cylinders
 Autoclavable

D. Teflon
 Almost chemically inert, very heat resistant
 For stopcocks, stirring bars, cap liners and tubings
 Autoclavable

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 MLS109 Basic Principles of Clinical LE TRANS
E. Polyolefins  They consist of cylindrical bulb joined at both ends to a narrower
glass tubing
 Strong and resistant to elevated temperature  A calibration mark is etched around the upper section of the tube
 Autoclavable  The lower delivery tube is drawn out to a gradual taper.

F. Polyvinyl Chloride

 Soft and flexible

 Used to construct tubings

VIII. ANALYTICAL BALANCE

A. Mechanical Balance
 Double pan analytical balance
 Two pans of equal mass suspended from ends of a beam
supported at its center by a knife-edge fulcrum
 Useful for weighing to the nearest 0.1 gram
 Not to be used for measurement of standards
 Single pan analytical balance
 Substitution balance; weights are remove from the side of the
beam holding the unknown mass until equilibrium is restored
 Unequal arms; single pan, provides greater precision and
accuracy
 Digital readout to 0.01 mg
 Used for measuring primary standards, small amount of
chemicals and for gravimetric calibration of pipets

Single Pan Analytical Balance

 Top loading balance
 Used for weighing larger amounts of chemicals for buffers, saline
solution, etc.
 Faster and easier but not precise

B. Electronic Balance

 Have built-in provisions for tarring, so that the weight of the

container can be subtracted
 They have a single pan, top loading or analytical, fast and can be
computer interfaced
 Operate on the principle of electronic force compensation.

IX. VOLUMETRIC SAMPLING AND DISPENSING

PIPETTE
 Used in the transfer of a volume of liquid from one container to
another
 Types of pipettes
 Transfer pipet
 Measuring/Graduated pipet
 Micropipettes

A. Transfer Pipette

 Delivers fixed volume (more accurate and precise)

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 MLS109 Basic Principles of Clinical LE TRANS
I. VOLUMETRIC PIPET
 Used for non-viscous fluids
 Standard, reagents, dilution liquids
 Plasma, serum, urine
 Drains by gravity by placing tip vertically against the
side of accepting vessel but not touching the liquid
 Distinguished by bulb-like enlargement partway up the
stem
 Has the highest degree of accuracy and precision
 TD Pipette (TD)- To Deliver
 Drains freely with the pipette touching the inner
surface of the receiving vessel
 Calibrated for the volume delivered
Do not attempt to wash out the film that
adhere to the inside of the glass surface
 Calibrated to blow out
Remaining liquid after free delivery must be
blown out and added to the initial volume
Indicated by opaque ring near mouthpiece
of the pipet.
 TC Pipette (TC)- To Contain
 Calibrated for the total volume of liquid held in
the pipette
 Must be washed out completely for the delivery
of complete volume
 Most micropipettes in the range of up to 0.5mL
are calibrated “to contain”

Volumetric Pipette

II. OSTWALD-FOLIN PIPETTE

 Similar to volumetric pipette but have their bulb closer to
the delivery tip
 Used for viscous fluids like whole blood
 With etched rings at the top = blow out, TD

Ostwald-Folin Pipette

B. Measuring/Graduated Pipette
 Delivers variable volume
 Long, cylindrical tubes drawn out a tip and calibrated in
uniform fractional volume measurement
 Principally used for measurement of reagents and not
considered sufficiently accurate for measuring samples and
calibrators.

TYPES OF MEASURING/GRADUATED PIPETTE

 Serologic Pipette
 Has graduated marks up to the tip
 Has etched rings
 Calibrated to blow out
 Has larger orifice than Mohr Pipette
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 MLS109 Basic Principles of Clinical LE TRANS

A. Calibration
 Done every 6 months
Class A pipettes do not require recalibration

GRAVIMETRIC METHOD
 Principle is that weight of water delivered by the pipet is equal to
the volume of liquid delivered
 Precision
 Determined by pipetting 10 aliquots of water, weighing the
aliquots and calculating the mean and the standard
Serologic Pipette deviation.
 Accuracy
 Mohr Pipette  Determined by correcting the mean for temperature (20 –
26C) and comparing the observed value to the expected value
 Calibrated between two marks of the stem  It should not deviate more than 5% of the stated value
 Graduations do not go all the way to the tapered tip
 Accuracy is greatest at full volume SPECTROPHOTOMETRIC METHOD
 Requires controlled delivery of the solution between calibration  Principle is that a colored compound of known absorptivity is
marks. diluted into 10 test tubes using the pipet being checked
 The absorbance of each tube is obtained using measurements
from a spectrophotometer
 Precision
 Determined by calculating the standard deviation of the aliquotes
 Must be less than 5%
 Accuracy
Measuring/Graduted Pipette  Determined by comparison to a reference solution

X. MICROPIPETTE B. Centrifugation
The process using centrifugal force to separate the lighter portions
of a solution, mixture or suspension from heavier portions of a
 Pipettes used for measurements of microliters of volume
solution
 Piston operated, positive displacement
 Used to
 Uses disposable tips and detipers
 Remove cellular elements from biological fluids to provide cell-
free specimen for analysis
 Blue Tip – 100 to 1000 uL or 1mL
 To concentrate cellular elements for microscopic examination
 Yellow Tip – 2 to 200 uL
 RPM
 White Tip – 0.5 to 10 uL
 Revolutions/rotations per minute
 Speed of the centrifuge
 Checked and calibrated using a tachometer
 RCF
 Relative centrifugal force
 Force that is generated by the centrifuge that is required to
separate substances in a solution
XI. QUALITY CONTROL AND PREVENTIVE MAINTENANCE

 Routine  Formula
 Rinse immediately after using  RCF = 1.118 x 10-5 x r x (rpm)2
Wash with low alkali, non-ion detergent  Bromsulhalein Dye
 Rinse with tap water, then 3-5x with Type I water  used to check random pieces of residual alkaline detergent
 Final rinse should have a pH of 5.5 – 5.7  Pink = incomplete removal of detergent
 For Blood Clots
 Soak in 1 0% NaOH
 For New Pipettes
 Soak in 5% HCl or 5% nitric acid
 For Grease
 Soak in
 Any organic solvent
 50% KOH or Contrad 70
 For Permanganate Stain (according to ser wel color violet „to
hehe)
 Soak in 50% HCl; or
 1% FeSO4 in 25% H2SO4
 For Bacteriologic Glassware
 Soak in 2-4 % cresol solution; followed by
 Autoclaving and thorough washing
 For Heavy Metal Analysis
 Rinse in dilute acid to remove metal ion contaminant
 Dichromate - H2SO4 cleaning solution
 1M HCl or HNO3

CLINICAL 6
 MLS109 Basic Principles of Clinical LE TRANS
 Where : r = radius in cm from the center of the head to the
bottom of the tube shield or bucket

TYPES OF CETRIFUGES
1. Horizontal Head Centrifuge
 Swinging bucket type
 The centrifuge tubes are held in a vertical position when not
moving but are horizontal when the centrifuge is fully in
motion
 Generate low speeds only but can produce a tighter pellet of
precipitate or clotted cells at the bottom of the tube
 Generates a lot of friction

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 MLS109 Basic Principles of Clinical LE TRANS
 In addition to venipuncture, blood specimens can be collected using a
The major difference between plasma and serum is that skin puncture technique ( hell stick, finger stick)
serum does not contain fibrinogen and some potassium is
released from platelets.

Properly balance Cetrifuge. Colored Circles represent

counterbalanced positions for sample tubes.

2. Angle Head Centrifuge

 Has a fixed 25 to 52 degree angle at which tubes are held
during centrifugation
 The sediment packs at an angle bit not as tightly as with a
horizontal head centrifuge
 Since the sediment is formed at an angle and not tightly
packed, decantation is not recommended
 Less prone to heat build up due to air friction than horizontal
head centrifuge heads

Samples should be analyzed within 4 hours to minimize the

effects of evaporation, samples should be properly capped and kept away from areas of rapid airflow, light, and heat. If testing is to occur after that time, sam

3. Ultracentrifuge
 Centrifuge head is held at a fixed angle but generates tight
sediments due to high speeds generated
 Generates the highest speeds
 Refrigerated to reduce the heat generated by the friction due to
high centrifugal speeds
 Especially used for lipoprotein separation since refrigeration
enhances separation of lipoproteins

C. CENTRIFUGATION – QC and MAINTENANCE

 Timer and speed to be checked every 3 months

 Speed is checked using a tachometer
 Timer is checked using a stopwatch
 Routinely cleaned everyday
 Must be cleaned immediately if there are spills

XII. SPECIMEN CONSIDERATIONS

 The process of specimen collection, handling, and processing

remains one of the primary areas of pre-analytic error.

A. Phlebotomy or Venipuncture

 23- or 21- gauge needle , routinely used needle

 IV infusion set or butterfly is used when veins are fragile, small, or
hard to reach or find
 Sites adjacent to IV therapy should be avoided; however if both
arms are involved in IV therapy and the IV cannot be discontinued
for a short time, a site below the IV site should be sought.
 The initial sample drawn (5mL) should be discarded because it is
most likely contaminated with IV fluid and only subsequent sample
tubes should be used for analytic purposes
CLINICAL 8
 MLS109 Basic Principles of Clinical LE TRANS

B. Plasma
 Liquid portion of anticoagulated blood that has been centrifuged or
has sit for some time

C. Serum
 Liquid portion of coagulated blood
 It is important that serum samples should be allowed to clot
completely (≈20 minutes) before being centrifuged.

D. Arterial Blood
 Used in measuring blood gases (partial pressure of oxygen and
carbon dioxide) and pH
 Syringes containing heparin anticoagulant are used instead of
evacuated tubes because of the pressure in an arterial blood
vessel

E. Other Body Fluids

 Cerebrospinal fluid
 Paracentesis fluids
 Pleural
 Pericardial
 Peritoneal
 Amniotic fluid
 Urine

F. Hemolysis
 Ruptured or destruction of RBC

G. Icterus
 Increased bilirubin pigment

H. Lipemia
 Increased lipids

XIII. FACTORS AFFECTING COMMON SERUM CONSTITUENTS

A. Prolonged Torniquet Application
 Increased:
 Cholesterol
 Albumin
 Potassium
 Ammonia
 Lactic Acid
B. Posture – Supine to Standing
 Increased:
 Albumin
 Cholesterol
 Calcium

C. Hemolysis
 Increased:
 Potassium,
 Lactate Dehydrogenase (LDH)
 Alkaline Phosphatase (ALP)
 Acid Phosphatase (ACP)
 Creatine Kinase (CK)
 Aldolase
 Phosphate
 Magnesium
 Ammonia
 Iron

D. Alcohol
Increased Decreased
Gamma-Glutamyl Transferase
Glucose
(GGT)
Triglycerides

E. Diurnal Variation

CLINICAL 9
 MLS109 Basic Principles of Clinical LE TRANS
Increased in AM Increased in PM c. fractional distillation
Cortisol Acid Phosphatase (ACP) d. centrifugation
Adrenocorticotropic Hormone 6. establishes relationship bet. concentration and absorbance in many
(ACTH) Growth Hormone (GH) photometric determinations
Iron TSH a. beer lambert's law
b. ideal gas law
F. Exercise c. avogadro's number
d. boyle's law
 Increased: 7. process that uses pressure to force water through a semipermeable
 CK membrane
 Aspartate aminotransferase a. commercial grade water
 LDH b. RO water
c. chemical grade water
 Ammonia
d. deionized water
 Lactate
8. weak acids and bases and their related salts
a. conjugate acid
G. Stress b. conjugate base
c. buffer state
 Increased: d. buffers
 Cortisol 9. highly purified chemical that can be measured directly to produce a
 ACTH substance of exacf known concentration
 Cathecolamines a. secondary standard
Increased Decreased b. primary standard
Glucose H. Meals Phosphatase c. commercial grade chemical
Insulin Chloride 10. this water is purified from previously treated water
Ionized calcium a. hard water
Gastrin b. RO water
c. ultrafiltrate
d. deionized water
11. used primarily in manufacturing and should never be used in a
I. Other Factors clinical laboratory
a. technical grade reagent
 Ammonia – cigarette smoking b. organic reagent
 Bilirubin – Light Sensitive c. ultrapure chemical
 Potassium – increased during fist clenching (opening and closing d. analytic reagent
of hand before collection) 12. it is a pressure when a solvent moves through a.semipermeable
 LD – LD4 and LD5 are Cold Labile membrane to establish equilibrium
a. RO
 ACP- pH stabilized at 5.4 for proper storage
b. osmotic
c. vaporized
REFERENCES d. hydrogenated
13. this is excellent in removing particulate matter , microorganism,
Basic Principles of Clinical Chemistry Powerpoint of Sir William Christopher
many pyrogens or endotoxins
C. Salazar, RMT
a. nanofiltration
Chapter 1 – ebook: Clinical Chemistry 8th edition by Bishop.
b. ultrafiltration
c. both
REVIEW QUESTIONS (REQUIRED)
d. none of the above
14. this concentration is less likely to be encountered in clinical
1. this is expressed as percent solution, molarity and normality
laboratories but it is often used in chemical titrations and chemical
a. precision
reagent classification
b. accuracy
a. molarity
c. concentration
b osmolarity
d. conclusion
c. normality
2. this pipet has no graduations to the tip, self draining pipet
d. molality
a. ostwald folin
15. this defines as physical quantity or dimension
b. serological
a. measurement
c. volumetric
b. space
d. mohr
c. volume
3. another method for separating macromolecules from a solvent or
d. unit
smaller substances Answers: 1C 2D 3C 4D 5B 6A 7B 8D 9B 10D 11A 12B 13C 14C 15D
a. filtration
b. centrifugation
c. dialysis END OF TRANSCRIPTION
4. a representation of concentrated/stock material to the final volume of RAK NA ITU 2022!!! \m/
solution
GOODLUCK FUTURE DOCTORS AND RMTs
a. diluent
b. dilutant
c. dilute
d. dilution
5. has a stationary and mobile phase, retardation factor is used
a. filtration
b. chromatography

CLINICAL 10
MLS109 Basic Principles of Clinical LE TRANS

CLINICAL 7
 MLS109 Instrumentation CLINICAL CHEMISTRY

WILLIAM CHRISTOPHER C. SALAZAR, RMT | SEPTEMBER 4, 2020 LE 1TRANS 02

OUTLINE  Low points of the wave

1. Units of Measure  Wavelength
2. Reagents  The distance between the top of one crest and the top of
3. Clinical Laboratory Supplies the next one
4. Centrifugation  Distance from any point of a wave to another identical point to
5. Laboratory Mathematics and Calculations a next wave
6. Specimen Considerations  Frequency
 How often a vibration occurs
 The lower the wave frequency,LEGEND
the longer the wavelength
Remember Lecturer Book Previous Presentation
Trans

OBJECTIVES:
At the end of the lesson, the student would be able to:
1. Understand the principles,
2. Explain correctly proper specimen collection, handling and transport
according to test requested.
3. Use spectrophotometer, UV-Vis and IR spectrophotometer.
4. Discuss the principles and concepts and advantages of automation.
5. Classify the different types of automation used in Clinical Chemistry.
Figure 2 Wavelength

COLORIMETRIC
A. Photometry
 Measurement of the intensity of light
 Light from a light bulb contains the entire visible spectrum. Materials
absorb light at one wavelength and reflect the other parts of the
spectrum
 Reflectance photometry
 Measure light reflected from solid surfaces
 Principle
 Light is directed on unpolished solid surface of sample
 Some light is absorbed, the rest is reflected
 Light reflected is measured with a detector and filter
 The more light absorbed, the less light reflected
 Reflectance is nonlinear; therefore, a microprocessor
is used to transform the data to a linear Figure 3 Short and Long Wavelength
response.
 Used in
 Dipstick reagent pads

Figure 4 Wavelength, Color and Reflected Light

Figure 1: Dipstick Reagent Pads

B. Spectrophotometry
 Involves measurement of the light transmitted by a solution to
 Light determine the concentration of the light absorbing substances in
the
 Electromagnetic radiation with a range of wavelength between
390 (violet) and 770 (red) nanometers (nm), capable of TRANS GROUP #1 Marquez, Datinggaling, Daya EDITORS
stimulating the subjective sensation of light.
 Visible range: approximately 400nm – 700nm
 <400nm – ultraviolet region (UV)
 >700nm – infrared region (IR)
 PARTS OF A WAVE
 Amplitude
 The distance from the midpoint of the crest or trough to the
midpoint of vibration
 Crest
 High points of the wave
 Through
 solution.
 It measures the light intensity in a narrower wavelength.
 Measures the array of lights or radiant energy
absorbed or transmitted.
 Measures the light transmitted by the analyte in order to
determine the concentration of light-absorbing analyte.

Figure 5. Example of Spectrophotometry

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 MLS109 Basic Principles of Clinical LE TRANS
TYPES OF SPECTROPHOTOMETER  Stray light
 Single-beam spectrophotometer  Any wavelengths outside the band transmitted by
 Simplest type of absorption spectrophotometer the monochromator
 Designed to make one measurement at a time at one specified  Does not originate from the polychromatic light source
wavelength.  Causes absorbance error
 The absorption maximum of the analyte must be known in  Limits the maximum absorbance that a
advance when a single-beam instrument is use spectrophotometer can achieve
 Double-Beam Spectrophotometer  Most common cause of loss of linearity at high analyte
 An instrument that splits the monochromatic light into two concentration
components – one beam passes throughout the sample, and the  Monochromator
other through a reference solution or blank.  Prism
 The additional beam corrects for variation in light source intensity  Wedge-shaped piece of glass, quartz or sodium chloride.
 TYPES:  Can be rotated, allowing only the desired wavelength to
 Double-beam in Space pass through an exit slit.
 Uses 2 photodetectors, one each for the sample beam  A narrow light focused on a prism is refracted as it enters
and the reference beam. the more dense glass.

Figure 6 Double Beam in Space

 Double-beam in Time Figure 8. Monochromator

 Uses only one photodetector and alternately passes the
monochromatic light through the sample cuvet and then
reference cuvet using a chopper or rotating sector mirror.

Figure 9. Process of Monochromator

Figure 7 Double Beam in Time
Parts of the Spectrophotometer
 Light Source
 Provides polychromatic light.
 Must generate sufficient and consistent radiant energy
or power to measure the analyte of interest.
 An intense beam of light is directed through the
monochromator and the sample.
 To give accurate absorbance measurements throughout
its absorbance range, its response to change in light
intensity must be linear.
 TYPES
 Continuum Source Figure 10. Another Example of the Process of Monochromator
 Emits radiation that change in intensity
 Most widely used in the laboratory  Diffraction Gratings
 Examples: o Most commonly used
o Tungsten (halogen / iodide) lamp o Has better resolution than prism
 Commonly used for visible – near infrared region o Made by cutting grooves or slits into an aluminized surface of a
o Deuterium lamp flat piece of crown glass
 Provides UV radiation o Wavelength are bent as they pass a sharp corner
o Xenon lamp  Made up of
 Produces a continuous source of radiation which  Grooves = for refraction
covers both the UV and visible range  Slits = for defraction
 Line Source
 Emits limited radiation and wavelength
 Used in atomic absorption, molecular and
fluorescent spectroscopy
 Examples:
o Mercury and sodium vapor lamps
o LASER – Light Amplification by Stimulated Emission
of Radiation
Figure 11 Diffraction Gratings
 Entrance Slit
 Minimizes unwanted or stray light and prevents the entrance of
scattered light into the monochromator system.

CLINICAL 2 of
 MLS109 Basic Principles of Clinical
Figure 12 Diffraction Gratings
 Filters
 Simplest, least expensive, least precise
 Made by placing semi-transparent silver films on both sides of a
dielectric such as magnesium fluoride
 Produce monochromatic light based on the principle of
constructive interference of waves – light waves enter one side of
the filter and are reflected at the second surface
LE TRANS
 It has a photosensitive material that gives off electron when
light energy strikes it
 Requires external voltage for operation
 Photomultiplier tube
 Most commonly used detector – measures visible and
UV region
 Excellent sensitivity and rapid response (detects very
low level of light)
 Limited to measuring low power radiation because
intense light causes irreversible damage to the
photoelectric surface.
o May burn-out if exposed to room light
 Photodiode
 Not as sensitive as PMT but with excellent linearity
 Measures the light at a multitude of wavelengths but
detects less amounts of light
 Has lower dynamic range and higher noise compared
to PMT
 Most useful as a simultaneous mechanical detector.

 Read-out Device / Meter

 Displays the output of the detection system
 Example
 Galvanometer
Figure 13. Filters
 Ammeter
 Exit Slit
 LED display
 Controls the width of light beam
 Bandpass
 The total range of wavelengths transmitted
 Accurate absorbance measurement requires a bandpass
less than 1/5 of the natural bandpass of the
spectrophotometer
 Spectral purity of the spectrophotometer is reflected by
the bandpass o The narrower the bandpass, the greater
the resolution
 Allows only a narrow fraction of the spectrum to reach the sample
cuvette
 Cuvet
 Holds the solution to whose concentration is to be measured Figure 14. Basic Instrumentation of a Spectrophotometer
 Also called absorption / analytical / sample cell
 TYPES THINGS TO CONSIDER
 Alumina silicate glass Light Shield
 Most commonly used  used to protect cuvette from unwanted light that may cause measurement err
 Can be used in 350 -2000 nm Holmium oxide / didymium filters
 Transmit light effectively at wavelengths >200nm  used to check wavelength calibration of spectrophotometer
 Quartz and Plastic Nickel II Sulfate
 Used for measurement of a solution requiring visible  solution with sharp cutoff wavelength used for detection of stray light
and ultraviolet spectra
 Borosilicate Glass
 For alkaline solutions
 Alkaline solutions should not be left standing in cuvets BEER’S LAW
for long periods because alkali slowly dissolves glass,  States that the concentration of the unknown substance is directly
producing etching. proportional to the absorbed light (absorbance or optical density) and
 Soft Glass inversely proportional to the amount of transmitted light (%
 For acidic solutions transmittance).
 Photodetector  Mathematically establishes the relationship between concentration
 Detects the amount of light that passes through the sample in the and absorbance.
cuvet.
 Detects and converts transmitted light into photoelectric energy  Beer’s Law
 Types of Photodetectors or
 Barrier Layer Cell / Photocell / Photovoltaic cell
 Simplest detector, least expensive, temperature sensitive
 Used in filter photometers with a wide bandpass
 Basic phototransducer that is used for detecting Where:
and measuring radiation in the visible region Cu = concentration of unknown
 Composed of selenium on a plate of iron covered Cs = concentration of standard
with transparent layer of silver Au = absorbance of unknown
 Has a maximum sensitivity at about 550nm and the As = absorbance of standard
response falls off to about 10% of the maximum at 350nm to
750nm  Percent Transmittance
 Phototube  The ratio of the radiant energy transmitted (T) divided by the
 Contains cathode and anode enclosed in a glass case radiant incident on the sample (I).

CLINICAL 3 of
 MLS109 Basic Principles of Clinical LE TRANS
 Measured by spectrophotometers to derive the concentration of 6. Exit Slits
the unknown. 7. Detector
 Requires water and standards to establish thermal
Where It = transmitted light thru the sample equilibrium before measurements.
Io = intensity of light striking the sample
Internal Standards
In actual practice, light transmitted by a blank is substituted for I o  Provides a reference point in sample, control and standards.
 Corrects variations in flame and atomizer characteristics.
 Minimizes the effects of changes in flame temperature and
aspiration rate.
 Acts as a radiation buffer to reduce the effects of mutual excitation.

For Sodium and Potassium Measurement

 Lithium
 Potassium
 Cesium
 Cesium

Figure 15
 Absorbance
 Amount of light absorbed
 Inverse logarithm of transmittance  Lithium and Rubidium - red
 Cannot be directly measured by the spectrophotometer 4. Entrance Slit
 Mathematically derived from % transmittance 5. Monochromator
 Absorbance = abc = 2 – log % transmittance  Isolates the specific wavelength of interest
 Blanking Techniques
 Used to correct for artificial absorbance readings
 Reagent blank
 Corrects the absorbance caused by caused by the color of the
reagents
 The absorbance of reagents is automatically subtracted from
each of unknown reading.
 Sample Blank
 Measures absorbance of the sample and reagent in
the absence of the end product.
 Corrects the measurement for optical interference (like
hemoglobin) absorbing at the wavelength of
measurement.
 Ineffective in cases of turbidity (due to lipemia)
 Ultracentrifugation may be necessary to clear the serum
or plasma of chylomicrons

FLAME / ATOMIC EMISSION PHOTOMETRY

 It measures the light emitted by a single atom burned in a flame.

 Used for measurement of excited ions (sodium and potassium).
 Internal standard: Lithium/Cesium – corrects variation in flame
and atomizer characteristics
Principle

 Substances being exposed to high temperature results in

excited atoms which almost immediately return to ground state
giving off energy of a specific wavelength
 Excitation of electrons from lower to higher energy state.
 Concentration of the substance is directly proportional to the
amount of light produced
Components

1. Gas
 Source of flame
 Mixture of hydrogen and oxygen, natural gas, acetylene and
propane in conjunction with air and O2
 Flame temperature should be held constant
2. Atomizer / Burner
 Introduces heat to atoms
3. Light Source
 Each element produces an individually characteristic wavelength
 Sodium – yellow
 Potassium – violet
 Magnesium – blue

CLINICAL 4 of
  Flickering the light indicates changes in the fuel reading
of the instrument.
MLS109 Basic Principles of Clinical LE TRANS
 Not routinely used.

ATOMIC ABSORPTION SPECTROPHOTOMETRY

 It measures the light absorbed by atoms dissociated by heat.
 Used for measurement of unexcited trace minerals (calcium
and magnesium)
Principle
 Measures the amount of light absorbed by the element sample in
an unexcited state, unionized state, neutral atom ground state
and dissociated from its chemical bond
 Ground state atoms absorb light at defined wavelengths
 Measures light absorbed by ground state atoms
 Inverse of flame photometry
 After the flame dissociates the element from the chemical
bonds, the atom remain in the ground state and the amount if
light absorbed is measured rather than emitted.

More sensitive than EFP

 Used for detecting small amounts of metals (heavy metals,
trace elements)
 Reference method for calcium
 Phosphate would interfere so lanthanum or strontium are used
as diluents to bind the phosphorus.

Operation
 Sample is atomized in a flame.
 Atoms of the metals to be quantified are maintained at ground state
 Beam of light from Hollow Cathode Lamp (HCL) is passed
through a chopper to the flame.
 Ground state atoms in the flame absorb the same wavelength of
the from HCL.
 Light not absorbed by the atoms is measured as a decrease in
light intensity by the detector.
 Difference of light leaving the HCL and the amount of light
measured by the detector is indirectly proportional to the
concentration of the analyte.

Components
1. Hollow Cathode Lamp
 Source of radiation energy
 Filter gas such as neon or argon gas is placed inside the lamp
2. Nebulizer / Atomizer
 Sprays the sample into the flame
 Used to convert ions to atoms
3. Chopper
 Used to modulate the light source
4. Monochromator
 Isolates light by filtering out extraneous energy from the flame

CLINICAL 5 of
 MLS109 Basic Principles of Clinical LE TRANS
5. Slits
6. Detector  Migration of charged particles in an electric field.
 Measures the intensity of light energy produced from  Principle
the monochromator  The ionized ampotheric molecules will move either to the cathode
 Usually a photomultiplier (negative electrode) or the anode (positive electrode) depending
 Internal standard is not needed – changes in aspiration on the net charge on the molecule.
have little effect on the number of ground state atoms

Interferences

 Chemical
 Occurs when flame cannot dissociate the sample
 Ionization
 Occurs when sample in flame become excited
 Matrix
 Occurs when light absorption is enhanced by organic substance
 Occurs when sample is evaporated in the flame and produces a
solid particle

VOLUMETRIC / TITRIMETRIC

 The unknown sample is made to react with a known solution in

the presence of an indicator.
 Example
 Schales and Schales method = for chlride
 EDTA titration method = for calcium

TURBIDEMETRY

 Measures the amount of light reduced or blocked by

particle formation
 Determines the amount of light blocked (reduction of light) by a
particulate matter in a turbid solution.
 The measurement of reduction of light is due to particle formation.
 Specimen concentration is directly proportional to the amount of
light blocked which depends on the specimen concentration and
particle size.
 Solutions requiring quantitation by turbidimetry are measured
using visible photometers or visible spectrophotometers.

Uses

 Detection of bacterial growth in broth cultures

 Antibiotic sensitivity tests
 Coagulation studies
 Protein measurements in CSF and urine

NEPHELOMETRY

 Measures the amount of light scattered by a particular solution

 Amount of light scatter is directly proportional to the number and
size of particles in the solution
 Light scattered is measured at angles 15-90˚ of the cuvette
 Forward scatter - for large molecules
 Used for measurement of large particles like Ag-Ab complexes

Components

1. Light Source
 Mercury Arc Lamp
 Tungsten Filament Lamp
 LED
 Laser
2. Collimator
 A device that produces a beam of parallel rays
3. Monochromator
4. Cuvette
5. Stray Light Trap
6. Photodetectors

I. ELECTROPHORESIS
CLINICAL 6 of
 MLS109 Basic Principles of Clinical LE TRANS
 It is the movement of electrically charge compounds in a medium
resulting to their separation based on their electrical charges
when an electric current is applied. (kapag nilagyan ng kuryente
yun ang magseserve ng pang-pagalaw nung sample)
 Cation moves toward the cathode (Positive Charge)
 Anion moves toward the anode (Negative Charge)
 Remember: Cation ay para lang kay Cathode, Anion ay para
lang kay Anode. (SANA ALL!!!)
 A charged colloid particle or ion will migrate toward either anode
or cathode under the influence of an externally applied electrical
field.
 MIGRATION DEPENDS ON
 Electric charge of a molecule of a given pH
 Acid pH buffer = molecules are positive (cations) and
migrate toward the negatively charged electrode (cathode)
 Alkaline pH buffer = molecules are negative (anions) and
migrate toward the positively charged electrode (Anode)
 Electrophoretic mobility is proportional to the net charge
 A particle without a net charge will not migrate and remains
in the point of application.
 Particle size and shape
 Electrophoretic mobility is inversely proportional to the
molecular size and viscosity of the supporting medium.
(kapag viscous mabagal ang galaw ng particle)
 Electric field strength
 Higher voltage = faster migration
 Nature of the supporting medium (depende yung medium kung
ano yung pinaglagyan ng electrophoresis)
 Cellulose acetate and Agarose gel
 Most commonly used for protein electrophoresis
 Affected by electro-osmosis or endosmosis flow of ions
toward the negative electrode
 Polyacrylamide gel
 Not affected by endosmosis
 Temperature of operation
 Higher temperature = faster migration
 Buffer ionic strength
 Lower ionic strength = better separation = less resolution
= faster migration = longer pattern
 Higher ionic strength = better resolution = less separation
= slower migration = shorter pattern
 The ions carry the applied electric current and allow the
buffer to maintain a constant pH during electrophoresis.
 The more the pH of the buffer differs from the isoelectric
point, the greater is the magnitude of the net charge of that
protein and the faster it will move in the electric field.

A. Components
Electrical Power
 Voltage: 100-200 volts for about 25minutes

Support Medium
 Paper
 Oldest support medium
 No longer used

Cellulose acetate
 Replacement for paper
 Separates based on molecular size
 Advantages:
 Substances clears with alcohol and controlled heat or
acetic acid + alcohol
 Excess dye extracted rapidly from the background
NOTE
 Sensitivity depends on the absence of
background scatter from scratched cuvettes
and particulates in reagents.
USES
 Measurement of antigen-antibody complexes (proteins)
 Migration occurs in short time (20-60 minutes)

CLINICAL 7 of
 MLS109 Basic Principles of Clinical LE TRANS
 Can be used for isoenzymes and hemoglobin fractions  FraScreeningctionation of isoenzymes
 Tough when wet  Identification of abnormal hemoglobins

Agarose
 Separates by electrical charge
 It does not bind protein
 Advantages:
 Reproducibility (madaling siyang ulit-ulitin)
 7-8 distinct protein bands or improved resolution
 No clearing
 Increased sensitivity

Polyacrylamide Gel
 Separates on the basis of charge and molecular size
 Not affected by endosmosis
 Separates proteins into 20 fractions, used to study
isoenzymes. (isoenzyme may iba’t iba or hawig pero iba lang
yung chemical molecule arrangement)

Buffer
 Carries out the applied current. (galing sa ating kuryente)
 The pH of the buffer determines the charge on the protein
molecule and therefore its direction of migration.
 At pH 8.6, all proteins are negatively charged and migrate toward
the anode.
 Sodium barbital

Sampling and Detecting System

 After electrophoresis, the gel is treated with a mild fixative, such
as acetic acid, that precipitates the proteins at the positions to
which they have migrated. (after migration ifi-fix natin siya para
ma-hold doon yung sample tsaka i-stain)
 They are then stained, and the gel is dried and cleared of
excess stain.

II. STAINS FOR VISUALIZATION OF FRACTIONS

 Amido black
 Ponceau S
 Oil Red O
 Sudan Black
 Fat Red 7B
 Coomassie Blue
 Gold / Silver Stain
 Most sensitive
 Can detect nanogram quantities of proteins
 *****stains must bind all fractions

III. DENSITOMETRY

 Measures the absorbance of stain – concentration of the dye and

protein fraction. (after mai-stain, measure ime-measure sa
densitometry)
 It scans and quantitates the electrophoretic pattern.
 Measurements are reported in g/dL
Precautions
 If the electrodes are not properly aligned, the current may be denser
on one side of the gel than on the other; proteins will migrate farther
on the side with more current. (kapag hindi naka-alligned pwedeng
unbalance yung flow nung kuryente)
 If electrophoresis proceeds too long, the proteins may migrate off the
gel into the buffer.
 If there is a break in the electrical circuit and no current passes, the
proteins will not move from the point of application.
 “Smile” artifact
 Samples at the center of the gel migrate farther than those at the
edges

Uses

 For protein abnormalities

CLINICAL 8 of
 MLS109 Basic Principles of Clinical LE TRANS
 Phenotyping of lipoproteins
 Identifying the type and class of an abnormal protein by reacting it
with specific antibodies

ISOELECTRIC FOCUSING
 Separation of molecules by migration through a pH gradient
 Similar to electrophoresis

Principle
 pH gradient is created by adding acid to the anodic area of the
electrolyte cell and adding base to the cathode area.
 The separating molecules migrate into an area where pH is equal
to their isoelectric point
 Proteins move in the electrical field until they reach a pH equal to
their isoelectric point.
 Movement of molecules are based on pH
 Ideal for separating proteins of identical sizes but with different net
charges

Advantages
 Able to resolve mixture of proteins
 Detects isoenzymes
 Identify genetic variants of proteins such as alpha-1-antitrypsin
 Detect CSF oligoclonal banding

CAPILLARY ELECTROPHORESIS
 Sample molecules are separated by electro-osmotic flow
 Positively charged ions in the specimen emerge early at the
capillary outlet because the EOF and the ion movement are in
the same direction.
 Negatively charged ions in the specimen move towards the
capillary outlet but at a slower rate.

Advantage
 Uses nanoliter quantities of specimen

Uses
 Separation, quantitation and determination of molecular weights
of proteins and peptides
 Analysis of PCR products
 Analysis of organic and inorganic substances and drugs

CHROMATOGRAPHY
 A technique where solutes in a sample are separated for
identification based on physical differences that allow their
differential distribution between a mobile phase and a stationary
phase.

Mobile Phase
 Inert gas or a liquid

Stationary Phase
 may be silica gel bound to the surface of a glass plate or plastic
sheet
 may be silica or a polymer that is coated or bonded within a column
Involves separation of soluble components in a solution by specific differences in physical and chemical characteristics of the different constituents.

Principle
 Mixture can be separated into individual components on the basis
of specific differences of their physical characteristics
 Separation of chemical mixture into different components based
on their physical characteristics and the interactions of molecules
with the mobile and stationary phase through a support medium

CLINICAL 9 of
 MLS109 Basic Principles of Clinical LE TRANS
 Technique used to separate complex mixtures on the basis of  Silylation – most common
different physical interactions between the individual compounds and
the stationary phase of the system (a solid or a liquid - coated solid).
 The goal of this technique is to produce “fractions” for quantitation

Bases of Separation

 Rate of diffusion
 Solubility of the solute
 Nature of the solvent
 Sample volatility / solubility
 Distribution between 2 liquid phases
 Molecular size
 Hydrophobicity of the molecule
 Ionic attraction
 Differential distribution between two immiscible liquids
 Selective separation of substances
 Differences in adsorption and desorption of solutes

Forms of Chromatography

A. Planar
1. Paper Chromatography
 Used for fractionation of sugar and amino acid
 Separation is determined by the rate of diffusion, solubility of the
solute, and nature of the solvent
 Stationary Phase: Whatman paper
2. Thin Layer Chromatography
 The mobile phase moves through the stationary phase by
absorption and capillary action
 Principle
 Stationary phase is a thin layer of fine particles on a sheet of
glass or plastic
 Mobile phase is a solvent or a mixture of solvents
 Sample is applied on lower edge and is carried upward with
the solvent separated by differences between the solvent and
the sorbent
 Basic Steps
 Sample extraction using a liquid-liquid or column technique
 Concentration of the extracted sample
 Sample application by spotting onto the silica gel plate
 Development of the solute in the sample using stationary and
mobile phases
 Solute detection using chromogenic sprays, UV light,
fluorescence and heat
 Interpretation of chromatographic results utilizing Rf values of
solute in comparison to aqueous standards
 Rf value
 Retention factor value
 Relative distance of migration from the point of
application
 Used for semi-quantitative drug screening test
 Each drug has a characterisctic Rf value and it must match
the Rf value of the drug standard
 Sample components are identified by comparison with
standards on same plate
B. Column
1. Gas Chromatography
 Used for separation of compounds that are naturally volatile or
can be converted to gas.
 Has high resolution, accuracy, sensitivity and short TAT
 Components
 Gas tank
 Contains the source of gases for mobile phase (inert gases)
 Nitrogen, helium, argon, hydrogen, CO2, NH3, NO
2. Injector
 Introduces the sample to the machine
 Samples are heated and vaporized then introduced
 If volatilization cannot be carried out, derivation of a substance
into a more volatile form is done by
CLINICAL 10 of
 MLS109 Basic Principles of Clinical LE TRANS
 Alkylation
 Acylation
3. Column
 It is where interaction between molecules take place
 Types
 Packed column
 Large sample capacity
 Capillary column
 Has higher efficiency and better detection limits
4. Detectors
 Flame ionization detector
 Thermal conductivity detector
 Nitrogen-phosphorus detector
 Electron capture detector
 Flame photometric detector
 Mass spectrophotometric detector
5. Data System

Types of Chromatography
Gas Solid Chromatography
 Separation occurs based on differences in absorption at the solid
phase surfaces.

Gas Liquid Chromatography

 Separation occurs by differences in solute partitioning between
the gaseous mobile phase and the liquid stationary phase.

Mass Spectroscopy
 Based on the fragmentation and ionization of molecules using a
suitable source of energy
 It can also detect structural information and determination of
molecular weight.
 Before a compound can be detected and quantified by MS, it must
be first separated by GC
 Mass Spectroscopy
 Gas Chromatography – Mass Spectroscopy
 Gold standard for drug testing
 In this method, quantitative
 Tandem Mass Spectroscopy
 Can detect 20 inborn errors of metabolism for a single blood
spot.

Liquid Chromatography
 It is based on the distribution of solutes between a liquid mobile
phase and a stationary phase.
 Analyzes substances that are not readily volatile
 Uses low temperature, suitable for thermolabile compounds

Separation Mechanisms Used in Liquid Chromatography

1. Gel Filtration / Molecular Sieve Chromatography
 Separates based on size and shape
 As solutes travel through the gel, large molecules that remain
in the mobile phase are eluted rapidly from the column.
 Hydrophilic Gel (Gel Filtration)
 For separation of enzymes, antibodies and proteins
 Ex: dextran and agarose
 Hydrophobic Gel (Gel Permeation)
 For separation of TG and fatty acids
 Ex: sephadex
2. Ion Exchange Chromatography
 Separation depends on the molecular weight, size and charge
of the ions of the molecules
 Involves exchange of sample ions and mobile phase ions
with the charged group of stationary phase
 Stationary phase
 polymers with covalently bound ions
 Ions with the greatest densities are held most strongly on ion
exchange material
 Used in separation of

CLINICAL 11 of
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 Amino acids  Spectrophotometric measurement of light emitted by a substance
 Proteins that has been previously excited by a source of UV light
 Nucleic acid  After it is excited and driven into a higher energy state, a molecule
3. Partition Chromatography (Liquid – Liquid Chromatography) loses energy by fluorescing
 Separation is based on differences in solubility between 2 liquid  The amount of light emitted is proportional to the concentration of the
phases – one aqueous, one organic substance in solution.
 Types A. Components
 Normal phase LC  Light Source
 Uses polar stationary phase  Excitation/Primary Monochromator
 Reverse phase  Selects the wavelength that is best absorbed by the solution
 Uses non-polar stationary phase  Cuvette
 used for separation of the therapeutic drugs and their metabolites.  Emission/Secondary Monochromator
4. Affinity Chromatography  Prevents the incident light from striking the photodetector
 Separates solutes by using immobilized biochemical ligands as a Photodetector
stationary phase. B. Factors Affecting Fluorometry
 This type of separation uses the so-called lock-and-key binding  Light scattering
 occurs when the detector is positioned at the right angle to the
 Uses in separation of
incidence light
 Lipoproteins
 Self quenching
 Carbohydrates  as the absorbance in the sample with a high fluorophore
 Glycated hemoglobins concentration increases, more of the excitation light is absorbed
 Antibodies before it reaches the molecules near the center of the cuvette
 Adsorption Chromatography (Liquid – Solid  Self absorption
Chromatography)  occurs when excited light is absorbed before it gets out to the
 Separates based on the differences (competition) between the cuvette
adsorption and desorption of solutes at the surfaces of a solid  Temperature
particle.  pH
 The compounds are adsorbed to a solid support such as silica  1000x more sensitive than spectrophotometer
or alumina.  Used in measurement of
o Porphyrins
Types of Liquid Chromatography o Magnesium
1. High Performance Liquid Chromatography o Calcium
 Uses pressure for fast separations, controlled temperature, in-line o Cathecolamines
detectors and elution technique.
 Components CHEMILUMINESCENCE
 Solvent reservoir  Emission of light that occurs from excitation of chemical or
 Pumps electrochemical compounds
 Pushes / propels the solvent  Differs from fluorescence in that the emission of light is created from
a chemical or electrochemical reaction, and not from absorption of
 Injector
electromagnetic energy.
 For sample introduction
 The chemical reaction yields an electronically excited compound that
 Chromatographic column
emits light as it return to its ground state, or that transfers its energy
 Functions as stationary phase to another compound, which then produces emission
 Operates at room temperature  Involves oxidation of an organic compound to produce light – the
 Detector excited products formed in the oxidation reaction produce
 It is where the effluent from the column passes chemiluminescence on return to unexcited state.
 Solutes are introduced in the order that each was eluted  Organic compounds
 Computer o Dioxetane
 Process data and control the operation of the system o Luminol
o Acridinium ester
Uses o Oxidant
o Hydrogen peroide
 Fractionation of drugs, hormones, lipids, carbohydrates and proteins o Hypochlorite
 Separation and quantification of various hemoglobins associated o Oxygen
with specific diseases (HB A1c) o Catalysts
o Enzymes (ALP, HP, microperoxidase)
Liquid Chromatography – Mass Spectroscopy  Metal ions ( Cu2+, Fe3+)
 It requires interface methods to convert nonvolatile to volatile  Does not require light source and monochromators
compounds and is used for detecting nonvolatile substances in body  More sensitive than fluorometer
fluids.  Used in immunoassays.
 Utilized to confirm positive results from screening of illicit drugs OSMOMERTY
(complementary for GC-MS) Measures the amount of dissolved substances in a solution
 Based in measuring changes in the colligative properties of solutions
FLUOROMETRY that occur due to variations in particle concentration.
 Colligative properties
 It measures the amount of light intensity present over a zero  osmotic pressure
background.  increases with osmolarity
 Determines the amount of light emitted by a molecule after excitation  boiling point
by electromagnetic radiation.  ecreases
 Measures the amount of light emitted by a substance due to its with osmolarity
excitation from a source rendering a higher or equal energy from its  vapor pressure
original state

CLINICAL 12 of
 MLS109 Basic Principles of Clinical LE TRANS
 decreases with osmolarity
 Freezing point depression osmometry o Most commonly used
method for measuring the changes in colligative properties of a
solution.
ELECTROCHEMISTRY TECHNIQUES
A. Potentiometry
 Principle
 measurement of potential between 2 electrodes in a solution to
measure analyte concentration
 potentials are produced by interaction of metals and its ions when
ions of different concentration are separated by a semi-permeable
membrane
 technique used to determine the concentration of a substance
using an electrochemical cell that consists of 2 half cells, where
the potential difference between an indicator electrode and a
reference electrode is measured
 technique used to determine the concentration of a substance
using an electrochemical cell that consists of 2 half cells, where
the potential difference between an indicator electrode and a
reference electrode is measured
 Components that measures electrode potentials
 Reference Electrode
 constant voltage source
 Includes the ff :
o Standard Hydrogen Electrode
 international standard, seldom used in routine work
o Saturated Calomel Electrode
 consists of mercury covered layer by a layer of
mercurous chloride in contact with saturated solution
of KCl
 widely used as reference electroode
 unstable at 80˚C
o Silver-Silver Chloride Electrode
 more stable in high temperature than saturated
calomel electrode
 most common
 Indicator Electrode
 measuring electrode
 Ion Selective Electrode
 An electrochemical transducer capable of responding to one
given ion
 Measures the activity of one ion much more than other ions
present in the sample.
 Consists of a membrane separating a reference solution and
a reference electrode
 Its selectivity depends on the membrane / barrier composition
used.
o Glass aluminum silicate – sodium
o Valinomycin gel – potassium
o Organic liquid membrane ion exchangers – calcium and
lithium
 Types of ISE
 Direct ISE
o Without sample dilution
o Less prone to interference
 Indirect ISE
o With sample dilution
o Prone to interference due to lipemia
 Interferences
 Protein coating
 Defective ISE membrane

B. Coulometry

 An electrochemical titration in which the titrant is

electrochemically generated and the endpoint is detected by
amperometry.
 The number of equivalent weights of a reactant oxidized/reduced
is directly. proportional to the quantity of electricity used in the
reaction o Quantity is measured in coulomb
 Used in chloride test
C. Amperometry

CLINICAL 13 of
 MLS109 Basic Principles of Clinical LE TRANS
 Electrochemical technique that measures the amount of
current produced through the oxidation or reduction of the
substance to be measured at an electrode held at a fixed
potential
 Measurement of the electrical current when a constant
potential is applied
 Used in measurement of:
 pO2
 glucose
 chloride
 peroxidase
D. Voltammetry
 Measures solution composition based on the current-potential
relationship in an electrochemical cell when the potential is varied.
 The measurement of current after which a potential is applied to
an electrochemical cell
 It has the capacity for multi-element measurements.
 Allows sample to be preconcentrated, thus utilizing minimal analyte
 Used for lead and iron testing

REFERENCES
Instrumentation Powerpoint Presentation of Mr. William Christopher C.
Salazar, RMT
Chapter 5 -6 ebook: Clinical Chemistry 8th edition by Bishop.
REVIEW QUESTIONS
1. What type of spectrophotometer can splits the
monochromatic light into two components?
A. Single-beam spectrophotometer
B. Double-beam spectrophotometer
C. Double beam in space
D. Double beam in time
2. It is the one of the part of wave that has the high points of
the wave?
A. Through
B. Wavelength
C. Crest
D. Frequency
3. Mathematically it establishes the relationship between
concentration and absorbance?
A. Beer’s Law
B. Blanking Techniques
C. Percent Transmittance
D. Absorbance
4. It measures the amount of light reduced or blocked by
particle formation?
A. Volumetric
B. Titrimetric
C. Turbidimetry
D. Nephelometry
5. It is similar to electrophoresis and separation of molecules
by migration through a pH gradient?
A. Chromatography
B. Agarose
C. Capillary Electrophoresis
D. Isoelectric Focusing
6. It requires interface methods to convert nonvolatile to
volatile compounds and is used for detecting nonvolatile
substances in body fluids.
A. Mass Spectroscopy
B. Affinity Chromatography
C. Both
D. None of the above
7. Composed of selenium on a plate of iron covered
with transparent layer of silver.
A. Barrier Layer Cell
B. Photocell
C. Photovoltaic cell
D. All of the Above
8. Affected by electro-osmosis or endosmosis flow of ions
toward the negative electrode.
A. Cellulose acetate
B. Agarose gel

CLINICAL 14 of
 MLS109 Basic Principles of Clinical LE TRANS
C. Both B. The additional beam corrects for variation in light source
D. None of the Above intensity
9. Which of the following is INCORRECT? C. Uses only one photodetector and alternately passes the
A. Sodium – yellow monochromatic light through the sample cuvet and then
B. Potassium – violet reference cuvet using a chopper or rotating sector mirror.
C. Magnesium – green D. Uses 2 photodetectors, one each for the sample beam and
D. Lithium and Rubidium – red the reference beam.
10. Which is TRUE about Double Beam in Space
A. An instrument that splits the monochromatic light into two Answers: 1B, 2C, 3A, 4C, 5D, 6A, 7A, 8C, 9C, 10D
components one beam passes throughout the sample,
and the other through a reference solution or blank.

END OF TRANSCRIPTION

RAK NA ITU 2022!!! \m/

GOODLUCK FUTURE DOCTORS AND RMTs
RAVAN LANG NG RAVAN MGA KATUSOK

CLINICAL 15 of
 MLS109 Specimen Collection and Processing CLINICAL CHEMISTRY

WILLIAM SALAZAR, RMT SEPT 12, 2020

LE 1TRANS 3

OUTLINE
I. Pre-Analytical Variables
A.Exercise TRANS GROUP #1 Marquez, Datinggaling, Daya EDITORS

B. Fasting
C. Diet
D. Position
E. Tabacco Smoking
F. Alcohol Intake
G. Stress Anxiety
H. Drugs
II. Physiologic Variation
A. Affected by DIURNAL VARIATIONS
B. Affected by GENDER (increased levels)
C. Affected by FOOD INGESTION
III. Types of Blood Specimen
A. Venous blood
B. Arterial blood
IV. Specimen Collection
A. Venipuncture
B. Arterial Puncture
C. Skin Puncture
V. Major Causes of Sample interference
A. Hemolysis
B. Icterus
C. Lipemia
VI. Specimen Consideration transport and storage
A. Separate serum / plasma as soon as possible
B. Specimens that require chilling during transport and storage
VII. Specimen of Choice

LEGEND
Remember Lecturer Book Previous Presentation
Trans

I. Pre-Analytical Variables
 Factors that affects specimen before tests are performed
 Most common cause of error = mislabelling

A. Exercise

 Causes the following

 Volume shifting between intravascular and extravascular
compartment
 Volume loss through sweating
 Hormonal changes
 Increases the following
 Transient – LDH, fatty acid, ammonia
 Long term – Creatine kinase, Aspartate aminotransferase,
aldolase
 Protein in urine
 Vigorous hand exercise (fist clenching) – increases
potassium, lactate and phosphate

B. Fasting

 Required for some test like FBS, glucose tolerance tests, lipid profile,
gastrin and insulin
 Overfasting causes
 Increased serum bilirubin (48 hours)
 Increase plasma triglyceride (72 hours)
 Increased plasma glucose (72 hours)
 Basal state collection
 Blood is collected early in the morning, 12 hours after the last
ingestion of food

C. Diet

 High protein – increased urea

 Low carbohydrate – increased ketone in the urine
 Caffeine – increased glucose and cathecolamines
 Increased fat – increased potassium, triglycerides and 5-HIAA

D. Position
 Preferred: upright or supine
 Ideally, the patient must be supine/seated for at least 15-20
minutes before blood collection to prevent hemodilution or
hemoconcentration
 Supine  seating position = constriction of blood vessels 
reduction of plasma volume = hemoconcentration (albumin,
enzymes, and calcium)
 Sitting  supine = water and electrolytes shift into tissue =
hemoconcentration (proteins, lipids, BUN, iron and calcium
 Standing  supine = extravascular water to transfer to the
vascular system = hemodeodilution (cholesterol, triglycerides, and
lipoproteins

 Torniquet/ tourniquet application

 Must be applied no longer than 1 minute
 Prolonged tourniquet application results to
 Hemoconcentration
 Increased levels of Potassium, proteins (albumin),
enzymes, lactate, cholesterol, and ammonia
 Anaerobiasis
 It should not be applied for ammonia determination

E. Tabacco smoking (nicotine)

 Increases plasma cathecolamines and cortisol
 Increased in glucose, growth hormone, cholesterol, trigilcerides,
ammonia, urea, lactate, insulin, and urinary 5-HIAA, neutrophils
and monocytes
 For ammonia determination – patient and phlebotomist are not
allowed to smoke
 Decreased plasma levels of vitamin B12

F. Alcohol intake
 Increases in the following
 Urate
 Triglycerides
 Gamma Glutamyl Transferase (GGT)
 Causes hypoglycema (chronic alcoholism)

G. Stress (anxiety)
 Affects adrenal hormone secretion
 Increases the following
 Cathecolamines, cortisol, ACTH, prolactin, insulin, albumin,
glucose, and lactate
 Total cholesterol has been reported to increase with mild stress;
HDL cholesterol to decrease by as much as 15%
 It also result in hyperventilation which in turn affects acid-base
balance

H. Drugs
 Medications affecting plasma volume can affect protein, BUN, iron
and calcium concentrations
 Therapeutic Drug Monitoring (TDM) specimen collection should
be scheduled according to the time of the last dose
 Hepatotoxic drugs can elevate liver function enzymes
II. Physiologic Variation
 Refers to the changes that occur within the body such as cyclic
changes (diurnal or circadian) or those resulting from exercise,
diet, stress, gender, age, drugs, posture or underlying medical
conditions

A. Affected by DIURNAL VARIATIONS

 Increased in AM
 ACTH, aldosterone, cortisol, iron
 Increased in PM
 ACP, GH, PTH, TSH
 Affected by age (increased levels)
 Albumin, ALP, cholesterol, and phosphorus

1 of 3
 1.03 Specimen Collection and LE TRANS
Note: Diurnal ( day or night)  No traces of alcohol should remain – it may cause hemolysis and
contaminate
Circadian recurring naturally on a twenty-four-hour cycle, even in the absence of light the specimen
fluctuations.
Adrenocorticotropic hormone (ACTH)

B. Affected by GENDER (increased levels)

 Males – albumin, ALP, creatine, uric acid, cholesterol, BUN

 Females – HDL, iron, cholesterol
C. Affected by FOOD INGESTION

 Increased levels – glucose, triglycerides, gastrin, free Calcium

 Decreased levels – electrolytes (Chloride, Potassium, Phosphorus)
alkaline phosphatase, amylase

III. Types of Blood Specimen

A. Venous blood
 Obtained by venipuncture
 Deoxygenated blood
 Contains products of metabolic activities of different organs
 Specimen of choice for most chemistry tests
 Plasma
 Liquid portion of anticoagulated blood
 Used in medical emergencies
 Serum
 Liquid portion of a clotted blood
 Adequate time for clotting must be allowed to prevent latent fibrin
formation which may cause clogging in automated chemistry
analyzers
B. Arterial blood

 Obtained by arterial puncture

 Oxygenated blood
 Uniform in composition throughout the body
 Specimen of choice for blood gas analysis
Capillary blood
 Obtained by skin puncture.
 Mixture of venous and arterial blood
 May contain tissue fluids
 Suitable in
 Pediatric patients
 Obese individuals
 Patients with thrombotic tendencies
 Severe burns
IV. Specimen collection
A. Venipuncture
 Blood is obtained from a vein
 Median Cubital
 Preferred site for venipuncture
 Largest and best anchored
 Cephalic
 Second choice
 Basilic
 Third choice
 Close to the brachial artery
 PRECAUTION!!!
 Selection of site
 Sites adjacent to IV therapy must be avoided
 IV fluid contamination causes the ff
 Increase in glucose, chloride, potassium and sodium
o 10% contamination with 5% dextrose = glucose
increases up to 500 mg/dl
 Decrease in urea and creatinine
 Tourniquet application
 Pressure = 60mmHg
 Tourniquet too close to the site = vein collapses
 Must be applied only within 1 minute to avoid
hemoconcentration
 Disinfection

CLINICAL 2
 1.03 Specimen Collection and LE TRANS
 Ethanol testing = use benzalkonium chloride (Zephiran) for
skin disinfection
 Blood culture = 70% alcohol followed by iodophor
 If allergic to iodophor = use chlorhexidine gluconate
 Needle
 The gauge is inversely related to the size of the needle
 21-gauge = standard size, 1-1.5 in
 23 gauge = for children / small veins, 1-1.5 in
 25 gauge = premature infants, ½ - ¾ in, increases
chance of hemolysis
 Tubes for blood collection
 Plain tube = clotting time is about 60 minutes
 Tubes with silica clot activator = clotting time is within 15-
30 min
 Never transfer blood collected in an additive tube into
another additive tube
 Tubes containing gels are not used in immunologic testing
and TDM as gel may interfere with immunologic reactions
 Sites to be avoided
 IV lines
 Burned / scarred areas
 Hematoma
 Thrombosis
 Edematous limb
 Arms at the mastectomy side and AV shunt / fistula
 Fractured limb
 Complications of venipuncture
 Infection
 Thrombosis
 Thrombus develops within a blood vessel
 Thrombophlebitis
 Inflammation of the vein accompanied by a clot
 Caused by trauma to the vessel wall
 Hematoma
 Pooling of blood under the skin
B. Arterial puncture
 Blood is obtained from an artery
 Used in blood gas analysis and pH measurement
 Sample is collected without a tourniquet
 Preferred site
 Radial artery
 Brachial artery
 Femoral artery
 Scalp artery
 Umbilical artery
 Allen’s Test
 Test for patency of the ulnar and radial arteries
 Done before performing an arterial puncture
 COMPLICATIONS!!!
 Bleeding
 Harder to control compared to venous bleeding
 Apply pressure to the puncture site for 3-5 minutes
 Thrombosis
 Hemorrhage
 Infection
C. Skin puncture
 Done by FINGERSTICK TECHNIQUE
 Preferred for children
 Length of lancet : 1.75mm
 Length of incision : < 2 mm (children),
 Cut should be oriented across fingerprints to generate a large
drop of blood
V. MAJOR CAUSES OF SAMPLE INTERFERENCE
A. Hemolysis
 Destruction of RBC
 Causes
 In vitro
 Due to underlying disease
 Inherent to the patient

CLINICAL 3
 1.03 Specimen Collection and LE TRANS
 May be due to RBC fragility, inappropriate venipuncture  Gastrin
technique or specimen contamination.  Lactic acid
 Use of a small bore needle
 Renin
 Pulling the plunger too fast
 PTH
 Expressing the blood forcefully into the tube
 Pyruvate
 Vigorous shaking / mixing
 Alcohol contamination  Lactic acid
 Bubble formation C. Specimens that require protection from light
 Effects (photosensitive)
 Increased levels of intracellular analytes  Bilirubin
 Dilutional effect on the analytes present in the serum or plasma  Beta-carotene
 Interference in color reactions  Folate
 Inhibition of certain analytes  Porphyrins
 Vitamin A and B6
B. Icterus VII. SPECIMEN OF CHOICE
 Lactic acid and glucose
 Yellowish discoloration of the plasma / serum
 Sodium fluoride and potassium oxalate as anticoagulant
 Inherent to the patient
 Lactic acid must be kept on ice and separated from cells as soon
 Caused by increased bilirubin levels (>20 mg/dl)
as possible
 Causes optical interference by its high absorbance at 340 and
 Acid Phosphatase
500nm
 Serum must be separated as soon as possible and buffered with
C. Lipemia
citric acid.
 Caused by high triglyceride levels (>400 mg/dl)
 Ammonia
 Causes turbidity of the serum which eventually blocks transmission
 Heparinized plasma
of light
 Kept from ice
 Can be cleared from the plasma by ultracentrifugation.
 Separated from cells and perform immediately
 Inherent to the patient
 LD Isoenzymes
 Inhibits the following
 Serum
 Amylase
 Store at room temperature
 Urate
 Do not refrigerate
 Urea
 Ionized Calcium
 CK
 Heparinized whole blood
 Bilirubin
 Must be collected anaerobically and kept on ice
 Total protein
 Blood Gases
VI. SPECIMEN CONSIDERATIONS TRANSPORT AND STORAGE
 Heparinized whole blood
 Must be collected anaerobically and kept on ice
A. Separate serum / plasma as soon as possible
 Prevents movement of water into the cells resulting to
REVIEW QUESTIONS
hemoconcentration
1. It is the liquid portion of anticoagulated blood?
 Standing time
A. Plasma
 Plain tube = 60 minutes
B. Serum
 Tubes with silica particles = 30 minutes C. Platelet
 Tests unstable if not removed from clot after 30 min D. WBC
 Potassium 2. It is used for blood gas analysis and pH measurement?
 Phosphorus A. Venipuncture
 Glucose B. Arterial Puncture
 Tests unstable after 6 hours if serum was not separated from the clot C. Capillary puncture
 Albumin D. Allen’s Test
 Bicarbonate 3. It is the destruction of RBC?
 Chloride A. Icterus
 C-peptide B. Serum
 HDL and LDL C. Lipemia
 Iron D. Hemolysis
 Total protein 4. It is the inflammation of the vein accompanied by a clot and
 Centrifugation Caused by trauma to the vessel wall
 3000 RCF for 10 minutes A. Thrombosis
B. Hematoma
 Excessive centrifugation
C. Thrombophlebitis
 Causes red cell lysis
D. Edematous limb
 Elevates LD and K
5. T or F. To avoid hemoconcentration, the tourniquet must be
 Undercentrifugation
applied within 2 minutes?
 Incomplete barrier formation
 Failure of the cellular elements to settle Answers: 1A, 2B, 3D, 4C, 5F
END OF TRANSCRIPTION
Serum or plasma must be stored at 4-6C if analysis will be
delayed longer than 4 hours. Online Class be like:

B. Specimens that require chilling during transport and

storage
 Ammonia
 Blood gases
 Cathecolamines

CLINICAL 4
 MLS109 Quality Control
CLINICAL CHEMISTRY
William Christopher C. Salazar, RMT| SEPTEMBER 29, 2020
LE 1 TRANS 02

OUTLINE  Problem log books should be used to identify potential

I. QUALITY ASSURANCE C. Mode problems and course of action implemented.
II. TERMINOLOGIES D. Range  Incident reports are recorded reports to problems
III. QUALITY CONTROL E. Standard Deviation occurred inside the laboratory that is kept for future
A. Major Types of QC F. Coefficient of variation reference.
1. Internal QC / G. Variance  Indicators should be identified to monitor potential problems,
Intra- Laboratory H. T test thresholds set and data collected.
QC I. F test  A quality control program using control material that resembles
2. External QC / VI.QUALITY CONTROL patient samples must be run exactly as the patients
Inter- Laboratory CHARTS II. TERMINOLOGIES
QC A. Gaussian Curve  Sensitivity
B. Objectives of Quality (Bell- Shaped Curve)  The ability of an analytical method to measure the
Control B. Cumulative Sum smallest concentration of the analyte of interest.
C. Control Solutions / Graph (CUSUM)  High sensitivity
QC Materials C. Youden/Twin Plot  none or low number of false negatives
IV. ANALYTICAL ERRORS D. Shewhart Levey-  Specificity
A. Random Error Jennings Chart  The ability of an analytical method to measure only
B. Systematic Error E. Westgard Multirules the analyte of interest.
1. Trend F. Rules  High specificity
2. Shift 1. Random error  low number of false positives
V. Allowable 2. Systematic error  Accuracy
Error  The extent to which the mean measurement is close to
STATISTICS the true value.
A. Mean  Indicates how close to the true value the result is.
B. Medial  Counterchecked using the mean
 Estimated using 3 types of studies
LEGEND o Recovery
Remember Lecturer Book Previous Presentation  Determines how much of the analyte can be
Trans identified in the sample
o Interference
 Determines if specific compounds affect the
I. QUALITY ASSURANCE
laboratory tests like hemolysis, turbidity, etc.
 A complete system of creating and following procedures and o Patient sample comparison
policies to aim for providing the most reliable patient laboratory
 Used to assess presence of error in actual patient
results and to minimize errors in the pre analytical, analytical
sample
and post analytical phases.
 Precision
 Monitors quality performance starting from the ordering of a
 Indicates how close the values are to one another
laboratory determination to its reporting (MT), the interpretation
 Counterchecked using COV
of results (Doctors), and then application to patient care
 Terms related to precision o
(Doctors).
o Repeatability
 Involves total quality control which requires constant attention
 Closeness of agreement between results of
of all involved with the process or system.
successive instruments carried out under the same
 Principle
conditions
 Every laboratory section must have a plan to address o Reproducibility
preanalytical, analytical and postanalytical testing.  Closeness of agreement between results of
 Personnel must be trained in every procedure they perform measurements performed under changed
and they must be tested annually for competency. conditions of measurements.
 Pre-analytical o Reliability
 patient preparation, specimen collection, transport and  Ability of the analytical method to maintain accuracy
storage and precision over an extended period of time.
 Analytical o Practicability
 Testing of the sample  The degree to which a method is easily repeated.
 Condition of reagents/equipments, pipetting,
QC, personnel competency  Diagnostic sensitivity
 Post-analytical  Is the ability of the analytical method to detect the proportion of
 Reporting or results, timely releasing of results, record individuals with the disease.
keeping  It indicates the ability of the test to generate more true-positive
 TAT (Turn Around Time) minimum of 2 hrs results and few false-negative
 STAT – immediate results  Screening tests require high sensitivity so that no case is
QC is under QA which makes the level of QC lower than QA . missed
 Sensitivity
 Procedure manuals, formatted according to CLSI standards,
must be available for procedures and must be followed by The number of diseased individuals
personnel explicitly.
 SOP
 This is where we base the job and decisions MTs are
supposed to do in the laboratory  Maintenance must be TRA
NS
ing, Daya,
Marquez
 Instrument maintenance and calibration procedures and documented GRO
UP
documentation of the same must be in place. Dati
ngal
 with a positive result X100
Total number of diseased individuals tested

1
of
4
 MLS109 Quality LE TRANS
 Examples
 Diagnostic specificity  Variations in technique
 Is the ability of the analytical method to detect the proportion of  Pipetting error
individuals without the disease  Mislabeling of samples
 It reflect the ability of the method to detect true-negatives with  Improper aliquoting
very few false-positives  Environmental conditions
 Confirmatory test requires high specificity to be certain of  Temperature and voltage fluctuation
diagnosis B. Systematic Error
 Specificity =  Error that occurs predictably once a pattern of recognition
ut the disease with a negative test Total number of individuals tested without the disease is established.
 Problem is constant or predictable from day to day.
X100  Detected as either positive or negative bias
1. Trend
 Values increase or decrease for six consecutive days
(crosses the mean)
 Delta check
 Cause: deterioration of reagents
 The current test value is compared to a previous value or
values to detect large changes

III. QUALITY CONTROL

 QC is concerned with the analytic phase of QA
 It is a process of ensuring that analytical results are correct
by testing known samples that resembles patient samples.
 It involves the process of monitoring the characteristics of the
analytical process and detects analytical errors during testing,
and ultimately prevents the reporting of inaccurate patient test
results.
A. 2 Major Types of QC
1. Internal QC / Intra-Laboratory QC
 Involves analyses of control samples together with the
2. Shift
patient specimens.
 Primarily monitors day to day performance of laboratory tests.  Values distribute themselves on one side or either side of
 It is important for the daily monitoring of accuracy and precision the mean for six consecutive days (does not cross the mean)
of analytical methods.  Cause: improper calibration of the instrument
2. External QC / Inter-Laboratory QC
 Involves proficiency training programs that periodically provide
samples of unknown concentration to participating clinical
laboratories.
 It is important in maintaining long-term accuracy of the
analytical methods.
 Involves proficiency training programs that periodically provide
samples of unknown concentration to participating clinical
laboratories.
 It is important in maintaining long-term accuracy of the
analytical methods.
B. Objectives of Quality Control
1. To check the stability of the machine  Examples
2. To check the quality of reagents  Calibration problems
3. To check technical / operator errors  Deterioration of reagents and control materials
C. Control Solutions / QC Materials  Improper preparation of standards and controls
 Solutions that are used to check the precision and accuracy of  Contaminated solutions
an analytical method.  Unstable and inadequate reagent blanks
 It should resemble the patient specimen and be run with  Failing instrumentation
the patients.
 Poorly written procedures
 Characteristics of ideal control solution
C. Allowable Error
 Behaves like real sample
 The quantity of error that will not negatively affect clinical
 Available to last a minimum of one year
decisions.
 Should vary minimally in concentration and composition from  Total error must be less than the allowable error to be
vial to vial considered acceptable.
 No communicable diseases
V. STATISTICS
IV. ANALYTICAL ERRORS A. Mean
 Errors encountered in the collection, preparation, and
 Measure of central tendency associated with symmetrical or
measurement of specimens including transcription and
normal distribution
releasing of results
 Formula:
A. Random Error
 Error that occurs unpredictably
 Affects precision and is the basis for varying differences
between repeated measurements

CLINICAL 2
 MLS109 Quality LE TRANS
 Occurs when the data elements are centered around the
mean with most elements close to the mean
 Focuses on the distribution of errors from the analytical
method rather than the values from a healthy or patient
population
 Where:  The total area under the curve is 1.0 or 100%
 mean
 ∑𝑥 summation of x
 𝑛 total number of samples
B.Medial
 Midpoint, 50th percentile
 The value that divides the observations into two groups,
each containing equal numbers of observation.
C. Mode
 The most frequently occurring value
 Used to describe data with two centers (bimodal)
D. Range
 The simplest expression of distribution
 Difference between the highest and the lowest score in a data 2. Cumulative Sum Graph (CUSUM)
E. Standard Deviation  Calculates the difference between QC results and the
 Measure of dispersion of the values from the mean target means
 A measure of distribution range  Common method: V-mask
 Most frequently used measure of variation  Identifies consistent bias problems; it requires
 Formula: computer implementation
 This plot will give the earliest indication of systematic
errors (trend) and can be used with the 13s rule
 Very sensitive to small, persistent errors that commonly
occur in the modern, low calibration-frequency analyzer
 Rules are out of control when the slope exceeds 45° or
a decision (+- 2.7 SD) is exceeded.

 In a normal distribution
 1 SD = 68% of the total population
 2 SD = 95% of the total population (maximum allowable SD)
 3 SD = 99.7% of the total population
F. Coefficient of variation
 Expresses SD as a percentage of mean value
 Used to determine precision
 Formula:

3. Youden/Twin Plot
 Interlab
 Used to compare results obtained on a high and low
control serum from different laboratories
G. Variance  It displays the result of the analyses by plotting the mean
values for one specimen on the ordinate (y-axis) and the
 Standard deviation squared
other specimen on the abscissa (x-axis)
 Measure of variability
 Difference between each value and the average of the data
 Formula:
Variance = (SD)2
H. T test
 Used to determine whether there is a statistically
significant difference between the means of two groups of
data.
I. F test
 Used to determine whether there is a statistically significant
difference between the standard deviations of two groups of 4. Shewhart Levey-Jennings Chart
data  Most widely used QC chart in the clinical laboratory
VI. QUALITY CONTROL CHARTS
 It allows the laboratorians to apply multiple rules without the
1. Gaussian Curve (Bell-Shaped Curve) aid of a computer
 Occurs when the data set can be accurately described by  It is a graphic representation of the acceptable limits
the SD and the Mean. of variations in the results of an analytical method
 Obtained by plotting the values from multiple analyses of  Easily identifies random and systematic errors
a sample
 Population probability distribution, that is symmetric about
the mean

CLINICAL 3
 MLS109 Quality
5. Westgard Multirules
 Establishes a criteria whether an analytic process is out
of control
 According to Westgard, the use of simple upper and
lower control limits is not enough to identify analytical
LE TRANS
4. Is the ability of the analytical method to detect the proportion
of individuals with the disease.
5. A complete system of creating and following procedures and
policies to aim for providing the most reliable patient laboratory
results and to minimize errors in the pre analytical, analytical
and post analytical phases.
6. The ability of an analytical method to measure the
smallest concentration of the analyte of interest.
7. Expresses SD as a percentage of mean value
8. Solutions that are used to check the precision and accuracy of
an analytical method.
9. Used to compare results obtained on a high and low
control serum from different laboratories
10. Measure of central tendency associated with symmetrical or
normal distribution
1J2I3H4G5F6E7D8C9B10A

problems.
END OF TRANS
 The combination of the control values used in conjunction with
a control chart has been called the Multirule Shewhart Plot
“Let this journey be the start of change towards a better Healthcare
6. Rules
System. Because our generation is different, our generation wants
 Random error change and we should hold to that idea of change. Laban future
 12s RMTs kaya natin „to!”
 1 control value exceeds 2 SD -018088
 Warning rule – accept the run
 13s
 1 control value exceeds 3 SD
 R4s
 The difference between 2 control values are equal
to or greater than 4 SD
 Systematic error
 22s
 2 consecutive control values are greater than +/- 2SD
 41s
 4 consecutive control values on one side of the mean
 10x
 10 consecutive control values on one side of the mean

REFERENCE
Module 5 / Quality Control Powerpoint
REVIEW QUESTIONS
A. Youden/Twin Plot
B. Mean
C. Control Solutions / QC Materials
D. Coefficient of variation
E. Sensitivity
F. Quality assurance
G. Diagnostic sensitivity
H. Trend
I. Variance
J. Gaussian Curve

1. Occurs when the data set can be accurately described by the

SD and the Mean.
2. Standard deviation squared
3. Values increase or decrease for six consecutive days (crosses
the mean)

CLINICAL 4
 MLS109 Carbohydrates CLINICAL CHEMISTRY

WILLIAM SALAZAR, RMT OCTOBER 15, 2020

LE 2TRANS 3

OUTLINE
I. CARBOHYDRATES
A.Monosaccharide
B. Disaccharide
C. Oligosaccharides and Polysaccharides
II. CHEMICAL PROPERTIES
III. DIGESTION AND ABSORPTION
IV. INTERMEDIATE METABOLISM
V. SPECIMEN COLLECTION AND STORAGE
VI. SAMPLES FOR GLUCOSE MEASUREMENTS
VII. KINDS OF GLUCOSE TOLERANCE TEST
VIII. INTRAVENOUS GLUCOSE TOLERANCE TEST
IX. GLYCOYLATED HEMOGLOBIN (HEMOGLOBIN A1C)
X. FRUCTOSAMINE
XI. DIABETES MELLITUS
XII. GALACTOSEMIA
XIII. ESSENTIAL FRUCTOSURIA Figure 3
XIV. HEREDITARY FRUCTOSE INTOLERANCE C. Oligosaccharides and Polysaccharides
XV. GLYCOGEN STORAGE DISEASE Oligosaccharides
 Contains 2-10 monosaccharides

LEGEND Polysaccharides
Remember Lecturer Book Previous Presentation  Contains more than 10 monosaccharides
Trans
II. Chemical Properties
 Some are reducing substances
I. CARBOHYDRATES  Glucose
 Maltose
 Compounds containing carbon, hydrogen and oxygen  Fructose
 Hydrates of aldehyde or ketone derivatives  Lactose
 Aldose – terminal carbonyl group (aldehyde)  Galactose
 Ketose – carbonyl group  Can form glycosidic bonds with other carbohydrates and
noncarbohydrates

Figure 1. Ketone (Left) Aldehyde (Right)

A. Monosaccharides
Figure 4
 Single polyhydroxy aldehyde or ketone unit and cannot be
hydrolyzed to a simpler form III. DIGESTION AND ABSORPTION
 Most common include glucose, fructose and galactose  Starch and glycogen are partially digested by salivary amylase to
form intermediate dextrin and maltose
 Acid pH of the stomach inhibits amylase activity
 Alkaline pancreatic secretions allows pancreatic amylase to complete
digestion to oligosaccharides, predominantly maltose
 Disaccharides are hydrolyzed by appropriate dissacharidase
(maltase, lactase and sucrase)
 Monosaccharides are absorbed across the wall of the duodenum
Figure 2 and ileum by an active, energy-requiring, carrier mediated transfer
B. Disaccharides process
 The rate of absorption for glucose and galactose is greater than for
 Formed when two monosaccharide units are joined by glycosidic other similar molecules
linkage  Conversion of glucose to fructose may occur during the process of
 Yields 2 monosaccharides on hydrolysis absorption
 Includes the following:  Monosaccharides are transported by the portal vein to the liver
 Maltose = glucose + glucose
 Lactose = glucose + galactose IV. INTERMEDIARY METABOLISM
 Sucrose = glucose + fructose (non-reducing sugar)  Metabolism of hexoses proceeds and results in:
 Energy production by conversion to carbon dioxide and water
 Storage
 Glycogen in the liver
 Triglycerides in adipose tissue
 Conversion to keto acids, amino acids or proteins

TRANS GROUP #1 Marquez, Datinggaling, Daya EDITORS

1 of 4
 2.03
LE TRANS

 Addition of peroxidase and a chromogenic oxygen acceptor, such as o-

dianisidine, results in the formation of a colored compound that is
measured.
 End color: yellow (slightly acidified) to pink (strong acid)

Figure 5
A. Terms Related to Glucose Metabolism

 Glycogenesis
 Conversion of glucose to glycogen
 Glycogenolysis
 Breakdown of glycogen to glucose and other intermediate
products
 Gluconeogenesis
 Formation of glucose from noncarbohydrate sources like amino
acids, glycerol, or lactate
 Glycolysis
 Conversion of glucose or other heoxese into pyruvate and lactate

V. SPECIMEN COLLECTION AND STORAGE

A. Plasma
 Whole blood glucose is approximately 10-12% lower than plasma
glucose
 Water content of plasma (93%) is approximately 11% higher
than that of the whole blood
 Venous plasma is recommended for diagnosis of diabetes
 Anticoagulants
 EDTA, Citrate, Oxalate + NaF – inhibits glycolysis
 Can be readily separated from the RBC
B. Serum

 Most commonly used specimen

 Glycolysis decreases serum glucose by 7% per hour
 Higher in the presence of leukocytosis or bacterial
contamination
 May be inhibited by NaF (inhibits enolase)
 Inhibitor of glycolysis are required in patients with greatly
increased leukocyte counts
 RBC must be separated within 30 minutes of collection
 Stability
 8 hours at 25 C
 Up to 72 hours at 25
C. Whole Blood

 Obtained by capillary puncture = higher than venous blood

D. CSF

 Must be analyzed immediately

 If cannot be analyzed immediately, centrifuge and refrigerate

E. Urine

 For 24 hour collection, should be stored at 4 C during collection

 Appropriate preservatives
 Glacial acetic acid
 5g sodium benzoate
 Chlorhexidine
 0.1% sodium nitrate with 0.01% benzethonium chloride
V. SPECIMEN COLLECTION AND STORAGE
A. ANALYTICAL METHODS – GLUCOSE OXIDASE
 Glucose oxidase catalyzes the oxidation of glucose to gluconic acid
and hydrogen peroxide

CLINICAL 2
 2.03
LE TRANS
Figure
6
Figure 6
 Highly specific for B-D-glucose
 Mutarotase accelerates the conversion of alpha to beta-D-glucose
 The second step involving peroxidase is not specific
 False decrease
 Substances that compete with the chromogen for H202
 Uric acid, ascorbic acid, bilirubin, haemoglobin, tetracycline,
glutathione
 Potassium ferricyanide decreases interference with bilirubin
 Most interference can be eliminated by the use of Somogyi
filtrate

B. Analytical Methods – Glucose Dehydrogenase

 Reaction
 Glucose dehydrogenase catalyzes the oxidation of glucose to
gluconolactone and NADH.
 The amount of NADH generated is proportional to the glucose
concentration.
 Mutarotase is added to shorten the time necessary to reach
equilibrium
 glucose + NAD > GLDH = D-gluconolactone + NADH
 chromogen(MTT) + NADH > diaphorase = reduced chromogen
(MTT = blue) + NAD
 Advantages
 Highly specific for glucose
 No interferences from anticoagulants and substances found in
serum
 Close agreement with the hexokinase procedure
 Disadvantages
 Positive interference from maltose, icodextrin, or galactose

C. Analytical Methods – Hexokinase

 Glucose is phosphorylated by ATP in the presence of hexokinase
and Mg.
 The glucose-6-phosphate formed is oxidized by glucose-6-
phosphate dehydrogenase to 6-phosphogluconate in the
presence of NADPH.
 The amount of NADPH is proportional to the amount of glucose in
the sample and is measured at 340nm.

Figure 7
 Generally accepted reference method for glucose
 Interference
 Mannitol and fructose - increased
 Hemolysis
 Lipemia (>500 mg/dl triglyceride) = increased

 Based on the ability of glucose to reduce cupric ions to cuprous D. Analytical Methods – Alkaline Copper Reduction (Oldest
ions in an alkaline solution Method)
 Benedict’s and Fehling’s reagents
 END PRODUCT : MOLYBDENUM BLUE

CLINICAL 3
 2.03
LE TRANS
 Folin-Wu = cuprous ions + phosphomolybdate =
phosphomolybdenum blue  The patient should avoid exercise, eating, drinking (except water)
 Somogyi-Nelson = cuprous ions + arsenomolybdate = and smoking during the test
arsenomolybdenum blue  For nonpregnant and adults, only the fasting and the 2nd hour sample
may be measured
E. Analytical Methods – Ortho-Toluidine  Instruct the patient to drink the glucose load within 5 minutes
 Collect blood samples before intake of glucose solution (fasting), 1
 o-Toluidine reacts with glucose in acetic acid to form a colored hour, 2 hours and 3 hours
product
 Colorimetric Reaction
 Aldehyde group of glucose + o-toluidine > acetic acid = green
color
 Measures any aldohexose
 Interference
 mannose and galactose = increased
 bilirubin = decreased

F. Analytical Methods – Seliwanhoff’s Test

 For fructose
 Specimen must be glucose free
 reaction
 HCl + resorcinol + fructose > heat > red color within 30 seconds

VI. SAMPLES FOR GLUCOSE MEASUREMENT

A. RBS
 Requested in blood sugar monitoring, insulin shock and
hyperglycemic ketonic coma
B. FBS

 Better indicator of overall glucose homeostasis than RBS

C. 2-hour postprandial blood sugar

 Taken 2 hours after meal

D. Glucose tolerance test

 Used to determine how well the body metabolizes glucose over a

given time
 Aids in the diagnosis of DM
 Involves multiple blood extraction

VII. KINDS OF GLUCOSE TOLERANCE TEST

 Janey-Isaacson Method
 Single dose, most common
 Exton Rose Method
 Divided oral dose or double dose method
 Glucose elevation after OGTT
 30min = 30-60 mg/dl
 1 hour = 20-50 mg/dl
 2 hour = 5-15 mg/dl
 3 hour = fasting level or below
VIII. INTRAVENOUS GLUCOSE TOLERANCE TEST

 Used for diabetes patient with gastrointestinal disorders

 0.5 g of glucose per kg body weight (given within 3 minutes)
administered intravenously
 Fasting sample is required
 First blood collection is after 5 minutes of IV glucose
A. Requirement for OGTT

 Patient should be ambulatory

 CHO depletion and inactivity or bed rest impair glucose tolerance
 Fasting of 8-10 hours
 Unrestricted diet of 150g/carbohydrate for 3 days prior to testing
 The patient should not smoke and drink alcohol prior to testing
 Glucose load
 75 grams – standard glucose load
 100 grams
 1.75g/kg body weight for children
B. Procedure for OGTT
CLINICAL 4
 2.03
LE TRANS
 Criteria for fasting plasma glucose
 Non-diabetic = <100 mg/dl
 Impaired glucose tolerance = 100-125 mg/dl
 Diabetes mellitus = equal to or greater than 126 mg/dl

C. DIAGNOSTIC CRITERIA FOR DM

 RBS = >200 mg/dl with symptoms of DM
 FBS = equal to or >126 mg/dl
 2-hour post-prandial glucose = equal to or >200 mg/dl

IX. GLYCOYLATED HEMOGLOBIN (HEMOGLOBIN A1C)

 Glucose molecule attached to one or both N-terminal valines of
beta chains of hemoglobin A1
 The largest subfraction of normal hemoglobin A in both diabetic
and non-diabetic individuals
 Reliable method in the monitoring of long term glucose control
 Reflects the average blood glucose level over the previous 2-3
months
 For every 1% change in the HBA1C value, 35 mg/dl is added to
plasma glucose
 Limitation
 Older RBC and iron deficiency anemia = falsely elevated
 Hemolytic disease / shortened RBC survival = falsely decreased
 Specimen
 EDTA whole blood
 Methods
 Electrophoresis
 Immunoassay
 HPLC – gold standard
 Chromatography

X. FRUCTOSAMINE
 Also called glycated albumin / plasma protein ketoamine
 Reflects short-term glucose control (2-3 weeks)
 Useful for monitoring diabetic individuals with chronic hemolytic
anemias and hemoglobin variants
 Should not be measured in cases of low plasma albumin (<30
g/L) = falsely decreased results

XI. DIABETES MELLITUS

Figure 8
XII. GALACTOSEMIA
 Cause of “failure to thrive syndrome”
 Excessive levels of galactose in the blood and excretion in the
urine caused by congenital deficiency of the following enzymes
 Galactose-1-phosphate uridyl transferase = most common
 Galactokinase
 Uridine-diphosphate galactose-4-epimerase
CLINICAL FEATURES
 Jaundice, hepatomegaly, easy bruising, galactosuria, E. coli
sepsis, cataract, hypotonia and sensory neural deafness
DIAGNOSTIC TEST
 Erythrocyte galactose-1-PO4 uridyl transferase activity

XIII. ESSENTIAL FRUCTOSURIA

 Autosomal recessive disorder characterized by
fructokinase deficiency
 Fructokinase catalyzes the conversion of fructose to
fructose-1- phosphate

CLINICAL 5
 2.03
LE TRANS
 Diagnostic indicator = presence of fructose in the urine

XIV. HEREDITARY FRUCTOSE INTOLERANCE

 Defect of fructose-1-biphosphate aldolase B activity in the liver,
kidney and intestine
 Inability to convert fructose-1-phosphate and fructose-1,6
biphosphate into dihydroxyacetone phosphate, glyceraldehydes-3-
phosphate and fructose-1,6 biphosphate and glyceraldehydes
CLINICAL FEATURES
 Irritability
 Lethargy
 Seizures
 Hepatomegaly

XV. GLYCOGEN STORAGE DISEASE

 Deficiency of specific enzyme involved in the metabolism of glycogen
 Inherited as autosomal recessive trait
 Serial blood specimens are collected for 2 hours at 15 minutes
interval for testing
 Von Gierke
 Most common type, associated with hyperlipidemia
 Test: intravenous galactose tolerance test (decreased glucose
levels)
 Liver damage – types I, III, IV, VI, IX
 Muscular defect – types V, VII
A. Types of Glycogen Storage Disease – Name – Enzyme
Deficiency
Type I Von Gierke Glucose-6-
phosphatase
Type II Pompe 1,4 glucosidase
Type III Cori-Forbes Amylo-1,6-
glucosidase
Type IV Andersen Brancher enzyme
Type V McArdle Muscle
phosphorylase
Type VI Hers Liver phosphorylase
Type VII Tarui phosphofructokinase
Type VIII Adenyl kinase
deficiency
END OF TRANSCRIPTION
Type IX Phosphorylase
kinase deficiency WHAT DO YOU GIVE A SICK BIRD?
Type X Cyclic AMP-
dependent kinase
EDI “TWEETMENT!”
deficiency
Type XI Fanconi-Bickel Glucose transporter-
2

CLINICAL 6
Nonprotein Nitrogenous Compounds
 Refers to the analytes that required removal of protein from the sample before analysis
 Majority arise from the catabolism of proteins and nucleic acids

Urea
 Major excretory product of protein metabolism
o NPN present in highest concentration in the blood
 Formed in the liver
 Came from amino groups and free ammonia generated during protein catabolism
 Excretion
o Kidneys
 Majority (>90%) are excreted by glomerular filtration
 Concentration in the plasma is determined by renal function and perfusion, protein content
and rate of protein catabolism
o Gastrointestinal tract
o Skin
 Pathophysiology
o Increased in
 Azotemia
 Elevated concentrations of urea in the blood
 Prerenal Azotemia
o Reduced renal blood flow
 Less blood is delivered, less urea is filtered
 Congestive heart failure
 Shock
 Hemorrhage
 Dehydration
o Increased protein catabolism
 High protein diet
 Stress
 Fever
 Corticosteroid therapy
 Gastrointestinal hemorrhage
 Renal Azotemia
o Caused by decreased renal function
o Results to compromised urea excretion
 Acute renal failure
 Glomerular nephritis
 Tubular nectosis
 Intrinsic renal disease
 Postrenal Azotemia
o Due to obstruction of urine flow anywhere in the urinary tract
 Renal calculi
 Tumors of the bladder and prostate
 Severe infection
 Uremia

Prepared by:
John Gabriel B. Abcede, RN, RMT, MSMLS©
Professor – Clinical Chemistry, Calayan Educational Foundation
Incorporated Section Head – Clinical Chemistry, Mount Carmel Diocesan
  Very high plasma urea concentration accompanied by renal failure
 Fatal if not treated
o Decreased in
 Late pregnancy
 Infancy
 Low protein intake
 Severe vomiting and diarrhea
 Liver disease
 Clinical use
o Evaluation of renal function
o Verify adequacy of dialysis
o Differentiation of the cause of azotemia
 Urea Nitrogen/Creatinine Ratio
 Normal : 10:1 – 20:1
 Prerenal
o Elevated urea, normal creatinine
o High BUN/Crea ratio
 Postrenal
o Elevated urea, elevated creatinine
o High BUN/Crea ratio
o Assessment of hydration status
o Determine nitrogen balance
 Specimen requirements and interfering substances
o May be measured in plasma, serum or urine
 Plasma
 Use heparin
 Citrate and fluoride inhibits urease
 Urine
 Timed collection should be refrigerated during the collection period
o Avoid hemolysis
o Refrigerate if cannot be analyzed in a few hours
 Analytical Methods
1. UREASE
 indirect method, enzymatic reaction
 urea is hydrolyzed to yield ammonium and bicarbonate ions
 ammonium is quantitated by either spectrophotometric or electrochemical process
 inhibited by NaF
 UREASE / GLUTAMATE DEHYDROGENASE
o urease hydrolyzes urea into NH3 and CO2
o glutamate dehydrogenase converts the products into glutamate and NADP
o disappearance of NADP is measured at 340nm
o indirect method
 COUPLED ENZYMATIC
o ammonium ions produced from urease hydrolysis is measured by a series of coupled
reactions that produce hydrogen peroxide
o hydrogen peroxide is quantitated by the formation of quinone-amine dye in the presence
of phenol and 4-aminophenazone

Prepared by:
John Gabriel B. Abcede, RN, RMT, MSMLS©
Professor – Clinical Chemistry, Calayan Educational Foundation
Incorporated Section Head – Clinical Chemistry, Mount Carmel Diocesan
  INDICATOR DYE
o urease is added to the test and ammonia is liberated from the specimen
o ammonia + pH indicator = color change
o used on many automated instruments
o best used as kinetic measurement
 CONDUCTIMETRIC
o urease is added to the test and ammonia is liberated from the specimen
o conversion of unionized urea to ammonia result in increased conductivity
o specific and rapid
 BERTHELOT REACTION
o urease is added to the test and ammonia is liberated from the specimen
o reaction
 ammonia + phenolhypochlorite > sodium nitroprusside = indophenol
o ammonia is measured by addition of phenolhypochlorite
o uses nitroprusside as catalyst to convert ammonium to indophenol
o forms blue colored end product (indophenol) and is measured by spectrophotometer
o yields satisfactory result, inexpensive
o prone to ammonia contamination
 NESSLER REACTION
o ammonia + Nessler's Rgt > yellow orange color
 Nessler Reagent
 alkaline double iodide of Hg & K)
 End Product
 ammonium dimercuric iodide
2. DIACETYL MONOXIME
 direct method, colorimetric reaction
 urea + diacetyl monoxime [strong acid] > yellow diazine derivative
 measured at 540nm
 based on Fearon Reaction
 shows no interference from ammonia or protein
 disadvantages:
o lacks specificity
o limited linearity, uses caustic chemicals
3. ISOTOPE DILUTION MASS SPECTROMETRY
 detection of characteristic fragments following ionization
 quantification using isotopically labeled compounds
 reference method

Uric Acid
 The product of catabolism of the purine nucleic acids
o Purines from the breakdown of ingested nucleic acids or from tissue destruction are converted into
uric acid, primarily by the liver.
o Present in the plasma as monosodium urate
 Insoluble at pH 7
 Plasma is saturated at 6.8 mg/dl, may precipitate in the tissues
 Precipitates in urine as uric acid crystals
o Elimination
 Filtered by the glomerulus (70%)
 Gastrointestinal tract (degraded by bacterial enzymes)
Prepared by:
John Gabriel B. Abcede, RN, RMT, MSMLS©
Professor – Clinical Chemistry, Calayan Educational Foundation
Incorporated Section Head – Clinical Chemistry, Mount Carmel Diocesan
  Clinical significance
o Assessment of inherited disorders of purine metabolism
 Lesch-Nyhan Syndrome
 X linked genetic disorder caused by the complete deficiency of hypoxanthine
guanine phosphoribosyltransferase
o Enzyme required in the biosynthesis of purines
o Lack of this enzyme prevents reutilization of purine bases and increases
synthesis of purine nucleotides and high plasma and urine
concentrations of uric acid.
 Manifestations
o Neurologic symptoms
o Mental retardation
o Self-mutilation
 Carbohydrate metabolism defect
 Glycogen storage diseases (G-6-PD Deficiency), Fructose intolerance
 Lactate and triglycerides are produced in excess and compete with urate for
renal excretion.
o Diagnosis and treatment of gout
 Gout
 Pain and inflammation of the joints caused by precipitation of sodium urates
 Hyperuricemia is caused by overproduction of uric acid
o Exacerbated by a purine rich diet, drugs and alcohol
o UA levels are >6.0 mg/dl
 Prone to development of renal calculi
 Tophi
 Deposits of crystalline uric acid and urates in tissues, causing deformity
o Prevent uric acid nephropathy
 In chemotherapy, there is increased metabolism of cell nuclei
 Monitoring uric acid is important to avoid nephrotoxicity
o Detect kidney dysfunction
 Chronic renal disease can increase uric acid levels because secretion and filtration are
impaired.
 Not useful as an indicator of renal function because many other factors affect its plasma
concentration.
o Clinical correlation
 Increased in
 Hemolytic and megaloblastic anemia
 Toxemia of pregnancy
 Lactic acidosis
o Competition for binding sites in the renal tubules
 High purine diet
o Liver, kidney, sweetbreads, shellfish
 Starvation
o Increased tissue catabolism
 Decreased in
 Fanconi’s Syndrome

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Professor – Clinical Chemistry, Calayan Educational Foundation
Incorporated Section Head – Clinical Chemistry, Mount Carmel Diocesan
 o
Any of a group of diseases marked by dysfunction of proximal renal
tubules
o Characterized by
 Hyperaminoaciduria
 Renal glycosuria
 Hyperphosphaturia
 Bicarbonate and water loss
o Due to defective tubular reabsorption
 Severe liver disease
 Specimen requirements and interference
o May be measured in plasma, serum or urine
 Plasma
 Must be heparinized
 Avoid EDTA or fluoride additives (affects uricase method)
 Serum
 Separated asap to prevent dilution by intracellular contents
 Urine
 Must be alkaline (pH 8)
o Storage
 Refrigerator
o Diet
 Recent meal has no significant effect and a fasting specimen is not necessary
o Lipemia
 Gross lipemia should be avoided
o Icterus
 May falsely decrease results obtained by peroxidase methods
o Hemolysis
 May decrease results due to glutathione release
o Thiazides and salicylates
 Increases uric acid values
 Analysis
1. HENRY/ CARAWAY / PHOSPHOTUNGSTIC ACID
 Based on the oxidation of uric acid in a protein free filtrate, with subsequent reduction of
phosphotungstic acid in alkaline solution to tungsten blue
 PTA is reduced by urate in an alkaline medium at a protein free solution
 uses PFF
 measured at 700nm
 colorimetric method = blue
 NaCN - used to increase color and inhibit fading
 disadvantages:
o lacks specificity
 colorimetric method gives higher results than enzymatic method
o less desirable than the enzymatic, prone to interference
o uric acid co-precipitates with proteins, turbidity is formed during color development
 interference
o aspirin
o acetaminophen

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John Gabriel B. Abcede, RN, RMT, MSMLS©
Professor – Clinical Chemistry, Calayan Educational Foundation
Incorporated Section Head – Clinical Chemistry, Mount Carmel Diocesan
 o caffeine
o theophylline
2. URICASE
 enzyme that catalyzes the oxidation of uric acid to allantoin
 URICASE / UV ABSORBANCE
o uricase oxidizes urate to allantoin and CO2 and H2O2
o urate absorbs light at 292nm, allantoin does not
o the concentration is proportional to the differences in the absorbance before and after
treatment of the sample with uricase
 measures allantoin
o read with a spectrophotometer
 must use special QUARTZ CUVETTES
 glass does not transmit light below 340nm
 URICASE / H2O2 COUPLED REACTIONS
o uricase oxidizes urate to allantoin and CO2 and H2O2
o in the presence of peroxidase, H2O2 reacts oxidatively with 3,5-dichloro-2-
hydroxybenzenesulfonic acid and 4-aminophenazone to form a red dye
o interference from ascorbic acid
 eliminated by
 Potassium ferricyanide
 Sodium hydroxide
 advantages:
o avoids protein precipitation
o superior sensitivity and specificity
o adopted for most automated analyzers
 interference
o reducing substances
3. Isotope Dilution Mass Spectrometry
 detection of characteristic fragments following ionization
 quantification using isotopically labeled compound
 proposed reference method

Creatinine
 A nitrogenous compound formed as the end product of creatine metabolism
o Creatine phosphate loses phosphoric acid and creatine loses water to form creatinine
 Creatine
 Substance transported to muscles where it is converted to creatine phosphate
which serves as high energy source
o Released in the circulation at a constant rate proportional to individual’s muscle mass
 Removed by glomerular filtration
 Clinical Use
o Determine the sufficiency of kidney function and severity of kidney damage and to monitor the
progression of kidney disease
 Creatinine clearance
 Measure of the amount of creatinine eliminated from the body by the kidneys
 Measures GFR – the volume of plasma filtered by the glomerulus per unit of time
o Used to gauge renal function
o Reported in ml/min

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Professor – Clinical Chemistry, Calayan Educational Foundation
Incorporated Section Head – Clinical Chemistry, Mount Carmel Diocesan
 o

o Used as a measure of completeness of 24-hour urine collections
 Specimen considerations
o May be measured in plasma serum or urine
 Serum and plasma
 Avoid hemolysis
o Causes a negative bias
 Lipemia may lead to erroneous results
 Icterus may cause interference
o Causes a negative bias in both jaffe and enzymatic methods
 Urine
 Must be refrigerated for 24 hour collection
 Must be frozen if longer storage (4 days) is required
o Diet
 Protein content of the diet does not influence plasma concentration
 High protein ingestion may transiently elevate serum concentrations
o Other interfering substances
 Ascorbate
 Causes a positive bias in the Jaffe reaction
 Interferes in the enzymatic method that use peroxidase as reagent
 Glucose
 Alpha ketoacids
 Uric acid
 Cephalosporin
 Causes a falsely increased results when the Jaffe reaction is used
 Dopamine
 Affects both Jaffe and enzymatic methods
 Lidocaine
 Causes a positive bias in some enzymatic methods
 Analytical Methods
o Jaffe Reaction
 Creatinine reacts with picric acid in alkaline solution to form a red-orange chromogen
 Reagents
o Saturated picric acid
o 10% NaOH
 The reaction is nonspecific and is affected by interference from
 Acetoacetate
 Acetone
 Ascorbate
 Glucose
 Pyruvate
 Adsorbent
 Removes some interference present in the specimen
 More accurate results are obtained when creatinine is absorbed onto
o Fuller’s Earth – aluminum magnesium silicate
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John Gabriel B. Abcede, RN, RMT, MSMLS©
Professor – Clinical Chemistry, Calayan Educational Foundation
Incorporated Section Head – Clinical Chemistry, Mount Carmel Diocesan
 o Lloyd’s Reagent – sodium aluminum silicate
 Elution techniques are then utilized to separate the creatinine from the adsorbent
which is then made to react with the Jaffe reagent
 Modification
 Kinetic Jaffe
o Serum is mixed with alkaline picrate and the rate of change in
absorbance is measured
o Eliminates some nonspecific reactants
o Affected by
 Alpha ketoacids
 Cephalosporins
 Bilirubin
 Hemoglobin
o Rapid and inexpensive
 Coupled enzymatic methods
o Uses different enzymes
 Creatininase
 Creatinase
 Sarcosine oxidase
 Peroxidase
o Used on a dry slide chemistry analyzer
o Enzymatic Method
 Used to eliminate Jaffe chromogens
 More specific
 1. Creatinine Aminohydrolase – CK Method
 Requires large sample
 Not widely used
 2. Creatinase-Hydrogen Peroxide Method
 Adapted for dry slide method
 Potential to replace Jaffe
 No interference from acetoacetate or cephalosporins
 Positive bias due to lidocaine
o Isotope Dilution Mass Spectrometry
 Reference method
 Assays on automated analyzers are calibrated to an IDMS method

Ammonia
 Produced in the
o catabolism of amino acids
 deamination during protein metabolism
o bacterial metabolism in the lumen of the intestine
o anaerobic metabolic reactions in skeletal muscles during exercises
 Highly toxic
 Exists as ammonium ion in the blood
 Elimination
o Converted to urea in the liver

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John Gabriel B. Abcede, RN, RMT, MSMLS©
Professor – Clinical Chemistry, Calayan Educational Foundation
Incorporated Section Head – Clinical Chemistry, Mount Carmel Diocesan
 o Excreted as ammonium by the kidney
 Acts as buffer to the urine
 Clinical Significance
o Assessment of hepatic detoxification function
 Severe liver disease is the most common cause of disturbed ammonia metabolism
o Hepatic Coma
 Ammonia is converted to glutamine in the brain causing lack of ATP and increases CNS
pH
 Ammonia also lowers GABA, an important neurotransmitter in the brain
o Reye’s Syndrome
 A disease preceded by a viral infection and administration of aspirin
 Acute metabolic disorder of the liver
 In autopsy, it shows severe fatty infiltration of the organ
 Specimen Considerations
o Specimen : Blood (venous or arterial)
 Patient and the phlebotomist must smoke
 Heparin and EDTA may be used
 Extracted without trauma and placed on ice immediately, avoid hemolysis
 Minimal use of tourniquet
 Avoid fist clenching and relaxing
 Must be centrifuged at 0 to 4 degree Celsius within 20 minutes of collection and the
plasma or serum
 Assayed ASAP
 Ammonia increases rapidly following specimen collection because of in vitro
amino acid deamination
 Frozen plasma is stable for several days at -20 degree celsius
 Methods (refer to the methods used in urea nitrogen measurement)
o Nesslerization reaction
o Berthelot’s reaction
o Glutamate dehydrogenase
 Most commonly used method
 Decrease in absorbance at 340nm as NADPH is consumed
o Ion Selective Electrode
 The electrode measures the change in pH of a solution of ammonium chloride as
ammonia diffuses across a semipermeable membrane

References:
Clinical Chemistry – Techniques, Principles, Correlations, 7 th Edition by Bishop Et.al (2014)
Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 5th Edition by Burtis Et.al (2012)
Clinical Chemistry Review Handbook for Medical Technologists, Revised 2014 by Rodriguez (2014)
Clinical Chemistry, 7th Edition by Marshall Et. al

Prepared by:
John Gabriel B. Abcede, RN, RMT, MSMLS©
Professor – Clinical Chemistry, Calayan Educational Foundation
Incorporated Section Head – Clinical Chemistry, Mount Carmel Diocesan
 MLS109 AMINO ACIDS, PEPTIDES AND PROTEINS
WILLIAM SALAZAR, RMT | AUGUST 8, 2018
OUTLINE
I. Amino Acids
A. Biochemistry
B. Metabolism
C. Specimen Consideration
D. Clinical Significance
1. Aminoacidopathies
2. Alkaptonuria
3. Homocysteinuria
4. Maple-Syrup Urine
Disease
5. Phenylketonuria
6. Tyrosinemia
7. Cystinuria
II. Peptides
III. Protein
A. Functions
B. Elements
1. Primary Structure
2. Secondary Structure
3. Tertiary Structure
4. Quaternary Structure
C. Classification
1. Simple Protein
2. Conjugated Proteins
D. Physical Properties
E. Differences Between
Plasma And Serum
Proteins
F. Specimen
Considerations
1. Analytical Methods For
Total Protein
2. Kjeldahl Method
3. Biuret Method
4. Direct Optical Method
5. Dye-Binding Methods
6. Lowry (Felin-
Ciocalteu) Method
7. Refractometry
8. Turbidimetric And
Nephelometric Methods
G. Reference Range For
Total Protein
H. Disease Correlation
1. Increased
2. Decreased
IV. Clinically Significant
Proteins
A. Prealbumin
1.Clinical Significance
2.Laboratory
Considerations
3.Reference Intervals
LEGEND
Remember Lecturer Book
OBJECTIVES:
At the end of the lecture, the student should be able to:
TRANS GROUP #1 Marquez, Datinggaling, Daya
B. Albumin
1. Biochemistry
2. Functions
3. Clinical Significance
a)Increased
b)Nonpathologic Cause
of Increase
c)Decreased
4. Measurement
5. Dye-Binding Methods
6. Salt Fractionation
C.Alpha-1-Acid Glycoprotein
1. Clinical Significance
2. Increased
3. Decreased
4. Laboratory
Considerations
5. Measurement
Techniques
D. Alpha-1-Antitripsin
1. Reference Intervals
2. Clinical Significance
3. Laboratory
Considerations
4. Reference Values
E. Alpha-2-Macroglobulin
1. Functions
2. Biochemistry
3. Clinical Significance
4. Laboratory
Consideration
5. Reference Intervals
F. GC Globulin
G.Ceruloplasmin
1. Functions
2. Clinical Significance
3. Laboratory
Consideration
H.Haptoglobin
1. Clinical Uses
2. Analytical Methods
3. Reference Range
I. Beta-2-Microglobulin
1. Biochemistry
2. Clinical Significance
J. Beta-2-Microglobulin
K. Transferrin
1. Clinical Significance
L. Hemopexin
M. Fibrinogen
1. Clinical Use
2. Reference Values
N.Complement
1. Clinical Significance
2. Analytical Methods
O.Immunoglobulin
1. 5 Subtypes
2. Increased In
3. Analytical Methods
Previous Presentation
Trans
EDITORS
CLINICAL CHEMISTRY
LE 2TRANS 02
I. AMINO ACID
 Major metabolic intermediates and the basic structural units of
proteins
A. Biochemistry
 “Organic compounds containing both an amino group (-NH2) and a
carboxyl group (-COOH)
 Acid-base properties
 At neutral pH, they have both positively and negatively charged
groups and occurs as zwitterions.
 Isoelectric point (pI)
 The pH where an amino acid or other molecule has a net charge of
0
B. Metabolism
 Serves as substrate for protein synthesis
 Sources
 Diet
 Endogenous turnover
 Daily requirement: 0.8 g/kg body weight
 Increased during growth and many disease state
 Essential amino acids
 Amino acids used for protein synthesis that are not synthesized
by humans
 Isoleucine
 Leucine
 Lysin
 Methionine
 Phenylalanine
 Threonine
 Tryptophan
 Valine
 Amino acids are released from dietary protein by degradation with
pepsin in the acidic environment of stomach and with pancreatic
hydrolases
 Digestion of protein may be impaired by pancreatic or gastric
disorders
 Amino acids are actively transported by GGT
 Maintains higher concentration of amino acids inside cells than
outside
 They also act as intermediates in many metabolic pathways.
 Urea cycle
 Converts ammonia to urea in the liver
 Alanine cycle
 Metabolic pathway that allows muscle cells to use amino acids
as fuel source and export excess nitrogen in the form of
alanine.
 Undergo efficient glomerular filtration but are reabsorbed in the
proximal tubules in the kidneys
 Aminoaciduria
 Increased renal excretion of amino acids
 May be due to overload or from tubular impairment
 Premature neonates have a generalized aminoaciduria
C. Specimen Consideration
 Concentrations are high during the first days of life
 Blood specimen should be collected the same time each day
 Values are highest in midafternoon and lowest in early morning
 General rules for amino acid analysis
 In plasma
 Requires fasting of 6-8 hours to avoid the effect of dietary
proteins
 Collect in heparinized tubes
 Separate cells from the plasma because cells contain 100 fold
increase in aspartic and glutamic acid
 Hemolysis should be avoided
 De-proteinization must be carried out within 30 minutes of
sample collection
 In urine
1 of 8
 2.02 AMINO ACIDS, PEPTIDES AND LE 2 TRANS
 Random specimen is suitable  Spores of B. subtilis are incorporated into an agar plate
 For quantification purposes, 24 hour urine preserved containing an inhibitor (B-2-thienylalanine)
with thymol or organic solvent is required  A paper disk impregnated with dried blood sample is placed
D. Clinical significance onto the agar
 Amino acids are generally used to monitor inborn errors of  If the blood phenylalanine level is high, it counteracts the
metabolism antagonist, and the bacteria grows
 Aminoacidopathies  Affected by the presence of antibiotics
 Inherited disorders of amino acid metabolism  Microfluorometric Assay
 Alkaptonuria  Filter paper disk containing the sample is treated with
 Absence of homogentisate oxidase in the tyrosine pathway which trichloroacetic acid
leads to accumulation of homogentisic acid  The extract is then reacted with a mixture of ninhydrin,
 Clinical features succinate and leucylalanine in the the presence of copper
 Onorchosis – tissue pigmentation tartrate
 Darkening of urine upon standing at room temperature  Fluorescence of the complex is measured using excitation /
 Homocysteinuria emission wavelength of 360nm and 530nm respectively
 Impaired activity of cystathionine-B-synthethase  Direct and quantitative measurement of phenylalanine in
 Results to eleveated levels of homocysteine and methionine in blood stained filter disks
blood and urine  Not affected by the presence of antibiotics
 Clinical features  Urine testing
 Physical defects  Used for monitoring dietary therapy
 Thrombosis  Urine is reacted with ferric chloride > if + for phenylpyruvic
 Osteoporosis acid = green color
 Lens abnormality  Tyrosinemia
 Mental retardation  Characterized by the deficiency of the following enzymes
 Screening tests  Fumarylacetoacetate hydrolase – Tyrosinemia I
 Modified Guthrie Test  Tyrosine aminotransferase – Tyrosinemia II
 Used for blood  4-hydroxyphenylpyruvic acid oxidase – Tyrosinemia III
 Procedure  Elevated tyrosine and tyrosine catabolytes (methionine and
o Blood specimen is placed on filter paper which is then phydroxyphenylpyruvic acid) in blood
placed on agar plates with a strain of Bacillus subtilis  Leads to liver damage
 L-methionine sulfoximine as antagonist  Cystinuria
o Increased plasma levels of methionine will allow bacterial  Defect in amino acid transport system rather than metabolic
growth by suppressing the antagonistic effect of L- pathway affecting renal system and other vital organs
methionine sulfoximine  Genetic defect in renal reabsorption mechanism
 Cyanide-Nitroprusside Test
 20-30fold increase in urinary excretion of cysteine
 Cyanide-nitroprusside is added to urine
 Cysteine is insoluble and precipitates in kidney tubules
 High levels of homocysteine will be detected by the
development of purple red color
 Has positive interference with cysteine II. Peptides
o Eliminated by adding silver
 Polymers of several amino acids joined by peptide bonds
 Reduces homocysteine but not cysteine
III. Protein
 Homocysteine then reacts with nitroprusside to
 Any large compound made from one or more polypeptides joined by
produce reddish color
peptide linkages between amino group of one and the carboxylic
 Maple-Syrup Urine Disease
acid group of the next
 Complete absence or reduction in activity of branched-chain A. Functions
ketoacid decarboxylase enzyme, hence, blocking the normal
 Forms intracellular and extracellular structures
metabolism of leucine, isoleucine and valine which results in their
 Generate energy through catalysis and electron transfer
accumulation in the blood, urine and CSF
 Produce motility through contractile elements
 Clinical features
 Characteristic maple-syrup or burnt sugar type odor of urine,  Assemble molecule
breath and skin  Serve as ion channels and pumps
 Failure to thrive  Acts as carriers
 Muscular rigidity  Perform immune defense
 Mental retardation  Serves as receptors, hormones and cytokines
 Hypoglycemia B. Elements
 Screening test 1. Primary Structure
 Modified Guthrie Test  Refers to the sequence of amino acids in a peptide or protein
 4-azaleucine as antagonist 2. Secondary Structure
 Confirmatory test  Involves winding of the polypeptide chain
 Amino acid analysis by HPLC  Refers to the specific 3-dimensional conformations (alpha, helix, beta
o 4 mg/dl of leucine is indicative of MSUD pleated and random structure)
 Phenylketonuria 3. Tertiary Structure
 Deficiency of phenylalanine hydroxylase that catalyzes the  Actual 3-dimensional configuration
conversion of phenylalanine into tyrosine  The folding of the polypeptide chains and elements of secondary
 Phenylpyruvic acid structure into a compact 3-dimensional shape
 Substance accumulated in phenylketonuria  Responsible for the physical and chemical properties of the protein
 Product of phenylalanine deamination 4. Quaternary Structure
 Neurotoxic – excessive amounts may cause mental retardation
 Gives a characteristic musty odor to urine  Incorporation of two or more polypeptide chains or subunits into a
 Screening Test larger unit
 Guthrie Bacterial Inhibition Test C. Classification
1. Simple Protein

CLINICAL 2
 2.02 AMINO ACIDS, PEPTIDES AND LE 2 TRANS
 Contains peptide chains which on hydrolysis yield only amino acids 1. Kjeldahl Method
 Types  Reference method for total protein measurement
 Fibrous  Principle:
o Fibrinogen  Protein nitrogen is converted to ammonium ion by heating with
o Troponin sulfuric acid in the presence of a catalyst
o Collagen  Ammonium ion is measured by
 Globular
o distillation into acid and titration (recommended)
o Hemoglobin
o berthelot or nessler reaction
o Plasma proteins  Total serum protein is obtained by multiplying TPN by 6.25
o Enzymes
o Based on the estimation that proteins contain 16%
o Peptide hormones nitrogen
2. Conjugated Proteins
 Disadvantages
 Composed of a protein (apoprotein) and a non protein compound  Time-consuming and impractical for routine use
(prosthetic group)  Inaccurate if the amino acid composition is unusual
 Metalloproteins  Interference from nonprotein nitrogenous compounds and
 Ferritin, ceruloplasmin, hemoglobin, flavoproteins amino acids
 Lipoproteins o Protein precipitation step is required
 HDL, LDL, VLDL 2. Biuret Method
 Glycoproteins  Most widely used and IFCC recommended method
 Haptoglobin, alpha-1-antitrypsin  Principle:
 Mucoproteins  Under strongly alkaline conditions, copper ions form multivalent
 Higher carbohydrate content than protein (mucin) complexes with peptide bonds in proteins.
 Nucleoproteins  Binding shifts the color from blue to violet
 Chromatin  Color change depends on the presence of 2 or more peptide
D. Physical Properties bonds
 Have peak absorbance at 220-280nm  Intensity depends on the number of peptide bonds present
 Contains 16% nitrogen  Absorbance is measured spectrophotometrically at 540nm
 Amphoteric  Reagents:
 Can bear both positive and negative charge  alkaline copper sulfate
 Buffer  rochelle salt (NaK Tartrate)
 Proteins can act both as a weak acid and weak base  NaOH and potassium iodide
 Advantages
 Buffering capacity is primarily in a range within -/+1 of the pK of
 More specific
the respective group
 Complex in structure  Biuret reaction has no interference from amino acids and
dipeptides
 Can act as a good antigen or immunogen
 More practical to use
E. Differences Between Plasma And Serum Proteins
 Linear up to 16 g/dl of proteins
 Effects of anticoagulant
 Interference
 Dry additives  Hemoglobin
 slight osmotic effect on plasma volume  Bilirubin
 Citrate solutions 3. Direct Optical Method
 dilute specimens
 Principle:
 Platelet activation
 Proteins absorb light optimally at UV range
 Causes release of platelet granules that are protein in nature
 200-225nm and 270-290nm are the most commonly used
 Preparation of platelet-poor plasma requires spinning of the
wavelength
specimen twice
o 280nm – tryptophan and tyrosine
 Serum
o 200-225nm – peptide bonds
 Has 4% decrease in total protein content – less viscous than
 Disadvantages
plasma
 Many low molecular weight compounds have absorbance
 The decrease is due to removal of fibrinogen during
below 220nm
coagulation
 Requires removal of the interfering substances before
 More advantageous to use for analysis of abundant
analysis
components
 Ultraviolet radiation
 Improves specimen delivery
4. Dye-Binding Methods
 Decreases precipitates formed during freezing and thawing
 Decreases the opportunity for fibrin formation in specimens  Principle:
 Based on shifts in the absorbance spectra of dyes when they
 Preferred for protein electrophoresis – fibrinogen yields a
bind to proteins
prominent band in the beta-gamma border
 Pyrogallol red
 Plasma
 most commonly used
 Has all the coagulation proteins lacking in the serum
 Advantage:
 Represents a protein and peptide composition more similar to that
 Good sensitivity, may be used for fluids with lower protein
seen in in-vivo conditions
concentrations such as urine and CSF
 Recommended for proteometric analysis 5. Lowry (Felin-Ciocalteu) Method
F. Specimen Considerations
 Principle:
 Total protein concentrations are higher for ambulatory than patients
 Specimens are mixed with an alkaline copper solution followed
under bed rest or in recumbent position
by addition of Folin-Ciocalteu reagent
 Due to redistribution of extracellular fluid to the intravascular
 Phosphotungstic acid and phosphomolybdic acid are
space – resulting to hemodilution
reduced to tungsten blue and molybdenum blue by copper
 Higher levels are expected with prolonged standing due to reduced complexed with peptide and by tyrosine and tryptophan
intravascular volume (hemoconcentration residues
G. Analytical Methods For Total Protein

CLINICAL 3
 2.02 AMINO ACIDS, PEPTIDES AND LE 2 TRANS
 Involves oxidation of phenolic compounds such as tyrosine,  Transport protein for all trans-retinol, the active form of
tryptophan and histidine to give a deep blue color vitamin A
 Absorbance of products are measured at 650-750nm  Synthesized in the liver and in adipose tissue
 Disadvantage o Requires zinc for synthesis
 Reactivity varies with the content of tyrosine and tryptophan  CLINICAL SIGNIFICANCE
 Positive interference  Increased
o Tyrosine and tryptophan  Chronic renal disease
o Salicylates  Corticosteroid or NSAID theraphy
o Chlorpromazine  Obesity
o Tetracycline  Decreased
o Sulfa drugs  Liver disease
6. Refractometry
 Protein malnutrition
 Measurement of the light bending capacity of solutions o Useful indicator of adequacy of protein nutrition because
 Principle:  It has relatively short half life
 Estimation of protein concentration based on measurement of  High proportion of essential amino acids
refractive index due to solutes in serum  Small pool size
 Used to rapidly estimate protein at high concentrations o Should be assayed along with CRP
 Accuracy decreases at <35 g/L  Acute phase response
 Salts, glucose and other low molecular weight compounds  Negative APR
have a larger proportional effect on refractive index  Protein-losing diseases
7. Turbidimetric And Nephelometric Methods  In the gut and in kidneys
 Principle  LABORATORY CONSIDERATIONS
 Proteins are aggregated before analysis by  Migrates as minor component anodal to albumin
o Trichloroacetic acid  Not routinely observed in serum by most methods
o Sulfosalicylic acid  Measured by immunonephelometric or immunoturbidimetric
o SSA with Sodium sulfate methods
o Benzethonium chloride  REFERENCE INTERVALS
o Benzalkonium salts under alkaline conditions  Adult: 30-40mg/dl
H. Reference Range For Total Protein  In neonates, concentrations are approximately half of the
normal adult values.
 Increases in puberty
 Decreases after age 50
B. Albumin
 Most abundant plasma protein
 BIOCHEMISTRY
 MW: 66,438 Da
 Has a high abundance of charged amino acids
 High solubility
 Net negative charge of -12 at neutral pH
I. Disease Correlation
 Contributes about 10mmol/L to the anion gap at normal
1. Increased
concentrations
 Dehydration  Synthesized by the hepatocytes
 Decreased water intake  Rate is controlled by
 Increased water loss  Colloidal osmotic pressure
o Diarrhea  Protein intake
o Severe vomiting  Half-life: 15-19 days
o Addison’s disease  Lost through the following routes
o Diabetic acidosis
 Catabolism
 Causes relative increase - hemoconcentration
2. Decreased  Gastrointestinal tract
 Glomerular filtration
 Overhydration
 FUNCTIONS
 Water intoxication
 Serves as storage form of amino acids
 Salt retention syndromes
 Massive intravenous infusions  Major component of colloid osmotic pressure
 Causes relative decrease  Maintains intravascular volume
 Hypoalbuminemic states are prone to develop edema
 hemodilution
 Transporter for a diverse range of substances (especially
 Inflammation
hydrophobic metabolites and drugs, it has 6 binding sites on its
 Inflammation decreases albumin – the largest fraction of serum
molecule)
protein
 Fatty acids
IV. Clinically Significant Proteins
 Lipids
A. Prealbumin  Bilirubin
 An alpha globulin that transports retinol binding protein and throxine  Drugs
in the blood.  Minerals
 Biochemistry  CLINICAL SIGNIFICANCE
 MW: 35kDa  Increased in
 Composed of 4 identical, noncovalently bound subunits, forming  Dehydration
T3 and T4 binding sites  High concentrations suggest problem with hydration
 Synthesized in the liver and in the choroid plexus of the brain  Artifactual
 Has a high concentration in the CSF  Prolonged tourniquet application
 Retinol-binding protein  Nonpathologic cause of Increase
 Postural changes

CLINICAL 4
 2.02 AMINO ACIDS, PEPTIDES AND LE 2 TRANS
 More pronounced in standing positon  Disadvantages
 Decreased by collecting the first or second voided specimen  Heparin interference
 Strenuous exercise
 Less accurate when the serum or plasma protein composition is
 Fever abnormal
 Decreased in  Decreased accuracy for patients with cirrhosis due to modified
 Acute phase response forms of albumin
 Negative acute phase reactant
 Lowers albumin levels by 2. Salt Fractionation
 Increasing capillary permeability – increased distribution to
 Globulins are separated from albumin using salting-out procedures
the extravascular space
 Sodium sulfate
 Decrease synthesis in response to inflammatory cytokines
 The albumin that remains in the solution can be measured by any of
 Increased quantities of acute phase reactants contribute to
the routine total protein methods.
oncotic pressure
 Increasing the catabolism of albumin by cells.
ALBUMIN – REFERENCE INTERVALS
 Kidney Disease
 ADULTS: (20-60): 35-52 g/L
 Loss from glomerular filtration
 Concentrations are posture dependent due to fluid shifts
 Normally, only 1-2 g/day passes through the glomerular
 10-15% increase if an individual is standing vs recumbent
barrier and 99% are taken up by the proximal tubules and
degraded
C. Alpha-1-Acid Glycoprotein
o Only about 10 mg/day excreted in the urine as
microalbuminuria  Glycoprotein
 Marker for prognosis  Major constituent of the seromucoid fraction of the plasma that
o Low concentrations may indicate poor outcome consist of a group of proteins that are slimy due to their high
 Hepatic Disease carbohydrate content.
 Not decreased until parenchymal damage or loss is severe  Also called orosomucoid
(>50%)  Synthesized mainly by the hepatocytes
 May be due to  Granulocytes and monocytes contribute to its concentrations in
 Nutritional deficiencies sepsis.
 Direct inhibition of synthesis by toxins FUNCTIONS
 Increased distribution in extravascular spaces  Binds basic and lipophilic substances
 Gastointestinal Loss  Drugs – propranol, quinidine, chlorpromazine, cocaine and
 Due to protein-losing enteropathy benzodiazepines
 Menetrier’s Disease  Hormones – progesterone
 Excessive proliferation of the gastric mucosa, producing  Down regulation of the immune response
diffuse thickening of the wall  Depression of phagocytosis by neutrophils
 Also called hypertrophic gastritis  Inhibition of platelet aggregation
 Inflammatory Bowel Disease  Inhibition of mitosis
 Crohn’s Disease  Inhibition of viruses and parasite
o Inflammation of the GIT, usually in the terminal ileum  Cofactor of lipoprotein lipase
o Chronic, relapsing disease that produces bouts of  Contributor to capillary selectivity
CLINICAL SIGNIFICANCE
diarrhea cramping in the abdomen and fever.
 Ulcerative Colitis  Increased in
o A recurrent acute and chronic disorder characterized by  Acute phase response
extensive inflammatory ulceration in the mucous  Up to fourfold increase
membranes and the submucosa of the colon  Noted around 3-5 days after the injury
o Characterized by bloody, mucoid diarrhea brought on by  Hormonal effects
physical or emotional stress.  Increased by glucocorticoid hormones
 Protein-Calorie Malnutrition  Decreased in
 Albumin concentrations vary directly with adequacy of intake  Hormonal effects
 Effects of inflammation decrease the correlation of albumin  Decreased by estrogens
with nutrition  Sieving protein loss
 Burn Injury  Slightly smaller than albumin
 Severe loss of albumin from wounds  Lost in the urine and gastrointestinal secretions in protein-
 Combined effects of losing enteropathy.
 Epithelial losses LABORATORY CONSIDERATIONS
 Accelerated catabolism  Monitoring APR
 Stimulation of acute phase response  Similar pattern to haptoglobin
 Edema and Ascites  Assess effect of drug binding
 Occurs secondary to increased vascular permeability which MEASUREMENT TECHNIQUES
permits the loss of albumin
 Immunotubidimetry, immunonephelometry or radial immunodiffusion
1. Dye Binding Methods (RID).
 It can be found on the alpha-1 globulin region in electrophoresis but
 Albumin binds the dye causing a shift in its absorption spectrum. does not stain well due to its high carbohydrate content
 The affinity is higher for albumin than for other proteins  Can be visualized using PAS and other carbohydrate stains.
 Dyes REFERENCE INTERVALS
 Bromcresol green (BCG)
 Adults: 0.5-1.2 g/L
 Bromcresol purple (BCP)
 More specific for albumin
D. Alpha-1-Antitripsin
 Yields lower values than BCG
 Inhibitor of a variety of serine proteases
 Ligands of albumins do not affect unless the considerations are very
high  Covalently binds to the active sites of serine proteases, thus
blocking their enzymatic activity.

CLINICAL 5
 2.02 AMINO ACIDS, PEPTIDES AND LE 2 TRANS
 Inhibits elastase  Increased hepatic synthesis in attempt to compensate for the
 Deficiency is associated with emphysema related to elastin decrease oncotic pressure
degradation in the lung by neutrophil elastase  Decreased in
 Complicated by smoking (promotes inactivation of AAT)  Pancreatitis
 Synthesized by the hepatocytes  Due to increase in protease-antiprotease complexes
CLINICAL SIGNIFICANCE  Prostatic carcinoma
 Increased in  Decreased to normal before treatment and returns to normal
 Acute phase response with successful treatment
 Rise after 24 hours and peak at 3-4days LABORATORY CONSIDERATION
 Synthesis is stimulated by cytokines (IL-6)  Found on alpha-2-globulin region on routine serum electrophoresis
 Estrogen
 Synthesis is also stimulated by estrogen REFERENCE INTERVALS
 Elevated during late pregnancy and during estrogen therapy
 Adults: 1.3-3.0 g/L
 Decreased in
 Twice in children 2-4 yrs old
 Genetic deficiency
 Women are 20-30% higher than in men after age 40
 Increases risk for pulmonary emphysema
 Liver disease
F. GC Globulin
 Increased utilization
 Group specific globulin
 Pancreatitis due to increased concentration of trypsin
 Exhibits activity with vitamin D (Vitamin D binding protein) and actin
 Urinary / Gastrointestinal Loss
 Migrates in the alpha-1 and alpha-2 interzone during electrophoresis
 Similar in size to albumin
 Present in excreted stool mostly complex with trypsin and  Analytical method: radial immunodiffusion
elastase  Reference value: 20-55 mg/dL
LABORATORY CONSIDERATIONS
G. Ceruloplasmin
 Quantified by
 Alpha-2 globulin in the plasma, being the form in which most of the
 Immunoturbidimetry
plasma copper is transported
 Immunonephelometry
 In electrophoresis it is the major serum component stained in the FUNCTIONS
alpha-1-globulin region
 Considerations  Catalyst for redox reactions in plasma
 Leukocytes proteases may be released into serum stored on the  Cp oxidizes Fe2+ to Fe3+
clot after blood drawing and may bind with AAT  Controls membrane lipid oxidation
 Bacterial contamination will cause the same effects  In the presence of superoxide, promotes LDL oxidation, which
 Serum must be removed from the clot aseptically and stored at may contribute to atherosclerosis
 4’C – up to 3 – 4 days  Limited role in plasma transport to tissue
 -70’C for long term storage CLINICAL SIGNIFICANCE
REFERENCE VALUE  Increased in
 Reference value 0.9-2.0 g/L  Acute phase response
 Slightly higher in women in childbearing years and in elderly  Peaks at 4-20days after injury
 Higher in  Hormonal effect
 Inflammatory disorders  Increased by estrogen theraphy
 Malignancy  Decreased in
 Trauma  Genetic Deficiency
 Pregnancy  Inherited aceruloplasminemia
 Estrogen theraphy and oral contraceptives  Neurodegeneration occurs with iron deposition in the brain
 Neonates – due to maternal estrogen  Dietary deficiency and malabsorption
 Lack of incorporation of copper into the molecule during
E. Alpha-2-Macroglobulin synthesis
 Largest major nonimmunoglobulin protein in plasma  Nutritional copper deficiency
FUNCTIONS  Menke’s Disease
 Versatile inhibitor of many proteases including enzymes in the kinin,  X-linked inherited disorder in which dietary copper is absorbed
complement, coagulation and fibrinolytic pathways and proteins not by the gastrointestinal cells that lack an ATPase for copper
inhibited by serpins transfer to plasma, hence, copper is not available to the liver
 Serves as a transport molecule for cytokines, growth factor and zinc for incorporation in to Cp.
BIOCHEMISTRY  Manifested by
 Sparse, brittle and kinky hair
 Synthesized by the hepatocytes
 Growth restriction
 Found principally in the intravascular spaces – does not diffuse from  Neurologic degeneration
the plasma space.
 Death during the first few years of life if untreated
 Forms complex with PSA
 Wilson’s Disease
CLINICAL SIGNIFICANCE
 Hepatolenticular degeneration
 Increased in  Body copper is markedly increased and is deposited in tissue,
 Hormonal effects including in the hepatic parenchymal cells, the brain, and the
 Estrogen periphery of the iris.
 Women of childbearing age have higher concentration than  Characteristic Kayser-Fleischer rings
men of the same age  the absence of P type of ATPase prevents incorporation of
 Age copper into Cp
 Synthesis in infants and children are up to 3x the adult  manifestations (begin during the 2nd or 3rd decade of life)
concentrations  chronic active hepatitis
 Nephrotic syndrome  neurologic signs (clumsiness, dysarthria, ataxia, tremors)
 renal signs (renal tubular acidosis with aminoaciduria

CLINICAL 6
 2.02 AMINO ACIDS, PEPTIDES AND LE 2 TRANS
 hemolysis  Deficiency may result in the accumulation of iron into tissues
LABORATORY CONSIDERATION producing hemosiderin
 Measured by  Major component of the beta-2 globulin fraction
 Immunoturbidimetry
 Immunonephelometry CLINICAL SIGNIFICANCE
 Subject to proteolytic degradation during storage  Used to determine the iron carrying capacity of the blood
 Serum or plasma should be separated as soon as possible after  Increased in
collection  Iron Deficiency Anemia
 Proper storage  Low plasma transferrin levels can impair hemoglobin
 Up to 3 days at 4C production and lead to anemia
 Longer storage at -70 C  Used to determine the cause of anemia
REFERENCE VALUE  Decreased in
 Adults: 0.2-0.6 g/L  Acute phase response
 25-40% of adult values in neonates  Liver disease
 Normal adult concentrations at 6months of age  Malnutrition
 Levels are maximum at 2-3 years of age  Nephrotic syndrome
 Higher in women during their premenstrual years
ANALYTICAL METHOD
H. Haptoglobin  Immunodiffusion
 Alpha – 2 glycoprotein that binds free hemoglobin  Immunonephelometry
 Prevents loss of hemoglobin into the urine
REFERENCE VALUES
CLINICAL USES  Male: 215-365 mg/dl
 Evaluating patients with slow but steady rate of red cell breakdown  Female: 250-380 mg/dl
such as by mechanical heart valves, hemoglobinopathies or
exercise-associated trauma K. Hemopexin
 Evaluates degree of hemolysis  Binds heme released by degradation of hemoglobin
 Evaluation of rheumatic diseases  Has the strongest affinity for heme
 Increased in  Helps in the diagnosis of early hemolysis
 Acute phase response  Migrates in the beta region during electrophoresis
 Stress
 Myoglobinuria REFERENCE VALUE: 50-115 mg/dl
 Decreased in
 Intravascular disease L. Fibrinogen
 Hemoglobinuria  A coagulation factor that is converted to fibrin through the action of
thrombin
ANALYTICAL METHODS  Forms fibrin clot when activated
 Radial immunodiffusion  Most abundant coagulation factor and one of the largest protein in
 Immunonephelometry the blood
 Appears as a distinct band between the beta and gamma region
REFERENCE RANGE during electrophoresis
CLINICAL USE
 26-185 Mg/dl
 Marker for long term prognosis of cardiovascular disease
I. Beta-2-Microglobulin  Increased in
 it is the light chain component of the major histocompatibility  Acute phase response
complex  Increased causes elevation of ESR as fibrinogen coats RBC
 found in the surface of most nucleated cells and allowing them to sediment faster in clumps
 Pregnancy
BIOCHEMISTRY  Use of birth control pills
 Needed in the production of CD8+ cells  Decreased in
 Freely filtered in the glomerulus and then reabsorbed by the proximal  Extensive coagulation
tubule
REFERENCE VALUES: 200-400 mg/dl
CLINICAL SIGNIFICANCE
M. Complement
 Elevated in
 One of the natural defense mechanisms that protects the human
 Renal failure
body from infection
 Due to impaired renal clearance
 heat labile factor that cause immune cytolysis
 Multiple myeloma
 circulated as nonfunctional precursors
 Rheumatoid arthritis
 C3 is the most abundant form in serum
 SLE
 HIV CLINICAL SIGNIFICANCE
ANALYTICAL METHOD: Immunoassay  Increased in
REFERENCE VALUE: 0.2-28 ug/dl  Inflammatory conditions
 Decreased in
J. Transferrin  Genetic deficiency
 A glycoprotein synthesized by the liver that transports iron to its  DIC
storage sites  Hemolytic anemia
 Prevents iron loss through the kidneys  Malnutrition

CLINICAL 7
 2.02 AMINO ACIDS, PEPTIDES AND LE 2 TRANS
ANALYTICAL METHODS  Toxoplasmosis
 Immunonephelometry  Cytomegalovirus infections
 Turbidimetry  Rubella
 Herpes
 Syphilis
 Allergic reactions
N. Immunoglobulin  Multiple myeloma
 A protein with known antibody activity and are the major participants IMMUNOGLOBULIN – ANALYTICAL METHODS
in humoral immunity
 Nephelometry
 Produced by plasma cells
 Turbidimetry
 Also termed as gamma globulins because they migrate to the
 Radial immunodiffusion
gamma region during electrophoresis
 Immunoassay
5 SUBTYPES
 IgG – most abundant in plasma
 IgA – main antibody found in secretions
END OF TRANSCRIPTION
 IgM – first antibody to increase in response to stimulation
 IgD – found mostly on the surface B cells
“Believe in yourself and all that you are. Know that there is something
 IgE – associated with anaphylactic reactions
inside you that is greater than any obstacles.”
Dati pangarap mo lang makapasok sa 3rd year pero ses eto na
IMMUNOGLOBULIN – INCREASED IN
yun oo eto na nga yun 
 Hepatic disease
 Infections

V. APPENDIX
 1 column
 may be started on a new page
 follow Tables and Figures formatting

CLINICAL 8
Tumor markers

Primary use

1. Establish prognosis
2. Monitor results of therapy
3. Detect recurrence
*not a major tool for diagnosis

TUMOR MARKER
AFP Liver cancer, germ cell tumor/ testicular cancer
CEA Colorectal cancer, others (breast, gastric, prostate, ovarian, bladder cancer
CA 125 Ovarian cancer, others (pregnancy, benign cyst)
CA 15.3 Breast cancer, others (lung, pancreatic, colorectal, liver cancer)
CA 19.9 Pancreatic Cancer, others (hepatobillary, gastric, colorectal)
CA 50 Gastric and pancreatic cancer
CA 549 Malignant breast, lung, prostate, colon cancer
CA 27.29 Breast Cancer
5-HIAA Argenaffinoma
Calcitonin Medullary thydoid cancer
Gastrin Zollinger Ellison syndrome
HUA/VMA Pheochromocytoma
Chromogranin A Pheochromocytoma
FOBT Colorectal cancer
HCG Choriocarcinoma, hydatidiform mole, teratocarcinoma, seminoma
CYFRA 21-1 Squamous cell lung cancer
NMP 22 (nuclear matrix protein) Urinary bladder cancer
ER/PR (estrogen receptor/progesterone receptor) determines if breast tumor is
amenable to hormone therapy
HER 2/ NEU Breast cancer
TDT Lymphoblastic leukemia
CA= Cancer Antigen

Enzyme Tumor Markers

Alkaline phosphatase Liver, bone; nonspecific elevation in many bone-related and liver
cancers
Lactate dehydrogenase Prognostic indicator; elevated non-specifically in numerous cancers
Neuron-specific enolase Neuroendocrine tumors
Prostate-specific Prostate cancer
antigen
Clinical Chemistry 2- MODULE 4
Blood Gases
TERMINOLOGIES
 Acid is a substance that can yield H+ (hydronium
ions) when dissolved in water
 Base is a substance that can yield OH- or hydroxyl
ions
 𝒑𝑲𝒂 is a constant.
- pH in which protonated and unprotonated forms are
equal in concentration
- Example. Bicarbonate-carbonic acid system – 6.1
 Buffer
- Combination of weak acid or weak base and its salt
- H2CO3 weak acid
- HCO3 weak/conjugate base
+ Buffers are used to control the pH level of
substances or solutions. We can adjust the pH level
depending on the required pH level using these
buffers.
 pH
- pH means power of hydrogen
- It is the negative logarithm of H or Hydrogen
concentration.
- It is inversely proportional to the H or Hydrogen
concentration.
- Venous blood pH= 7.35
- Arterial blood pH= 7.45
ACID BASE BALANCE
A. Maintenance of Hydrogen
 Hydrogen concentration: 36-44 nmol/L
 pH: 7.35-7.45
 Hydrogen excess can lead to alterations in
consciousness, tetany, coma, and death.
 Acidosis pH of <7.5 and alkalosis pH of >7.45
 Our pH is maintained by the lungs and the kidneys.
B. Buffer Systems: Regulation of Hydrogen
1. The bicarbonate-carbonic acid system
 Weak acid: carbonic acid (H2CO3)
 Conjugate base: bicarbonate (HCO3)
 Carbonic acid (H2CO3) dissociates into carbon
dioxide (CO2) and water (H2O)
 Carbon dioxide (CO2) can be modified by the
ventilation rate.
- Carbon dioxide can be controlled through
ventilation rate (frequency of breathing)
- Hypo and hyperventilation give an impact in
our carbon dioxide levels.
 Bicarbonate (HCO3) can be altered by the
kidneys.
- Bicarbonate is controlled by our kidneys.
 Major extracellular buffer
2. Hemoglobin buffer system
 Used hemoglobin in red blood cells to minimize
pH changes in the blood
 Most important intracellular buffer
3. Plasma protein buffer system
 Uses plasma proteins to minimize pH changes in
the blood
4. Phosphate buffer system
 Uses hydrogen phosphate (HPO4) and
phosphoric acid (H2PO4) to minimize changes
in the plasma and erythrocytes.
C. Regulation of Acid-Base Balance: Lungs and
Kidneys
ASSESMENT OF ACID-BASE HOMEOSTASIS
A. The bicarbonate buffering system and Henderson-
Hasslebalch equation
Reference ranges at 37-degree Celcius
 pH 7.35 – 7.45
 PCO2 (mmhg) 35 – 45
 HCO3 (mmol/L) 22 – 26
B. Organs in Acid-Base balance
1. Lungs (Respiratory Mechanism)
 Hyperventilation: carbon dioxide is released
quickly.
 Hypoventilation: slow respiration
 Once we intake air, oxygen will be converted
into carbon dioxide. The release of carbon
dioxide depends on our respiration.
2. Kidney (Renal Mechanism)
 Excretion of Hydrogen
 Reabsorption of filtered H2O
C. Acid-Base Disorders
1. Respiratory Origin (lungs)
= alterations in H2CO3 or CO2
A. Acidosis
= excess H2CO3 or CO2
= high acid concentration low pH level
  Diuretics, Excessive gastric suctioning
Caused by:  Internal Obstructions
 Asthma, Emphysema Compensation
 Bronchoneumia  Hypoventilation
 Depression of Respiratory Center
 Hypoventilation due to drugs (Barbiturates) COMPENSATION
 Congestive Heart Failure  Goal: To maintain normal pH
 OPD (Obstructive Pulmonary Disease)  Organs: either lungs or kidneys
Compensation  Henderson-Hasselbalch Equation
 Compensation is the mechanism we use to
counteract the change that is happening.
 Coping mechanism or compensation is done
through the opposite organ. (Example.
Respiratory, sa lungs. Ang tutulong
magnormalize ng pH levels are the kidneys.
Metabolic ang tutulong)
 If the patient is acidic, the Sodium/Hydrogen
exchange will increase and there will be high
NH3 production, reabsorption of bicarbonate
(HCO3) and excretion of acidic hydrogen.
B. Alkalosis
= excessive reduction of H2CO3 or CO2
= release of acids in the body is quick
Caused by: DO NOT FORGET! ROME, Respiratory Opposite,
 High fever Metabolic Equal
 Hysteria (Hyperventilation)
Compensation Mechanisms
 Pulmonary emboli and fibrosis
1. Uncompensated
 Drugs (salicylates)
 Abnormal pH
Compensation
- either acidic or alkaline
 Opposite of the compensation for alkalosis
 No action of compensating organ
 Decrease of Sodium/Hydrogen exchange, NH3
- the body is not yet counteracting the imbalance
production, reabsorption of HCO3, and
2. Partially Compensated
excretion of acid hydrogens
 Abnormal pH
2. Metabolic Origin (kidneys)
= alterations in HCO3  Compensation takes place
A. Acidosis 3. Fully Compensated
= primarily bicarbonate deficient <24 mmol/L  Normal pH
Caused by:
 Addition acidosis (diabetic ketoacidosis) OXYGEN TRANSPORT
 Subtraction acidosis (diarrhea)  Oxygen is transported to the tissues by
hemoglobin.
 Renal Tubular Acidosis
 Starvation, Lactic acidosis
 Toxins
 Excessive loss of electrolytes and intestinal
fluids Compensation
 Hyperventilation
- If the patient is acidic, kailangan bawasan ang
acid through hyperventilation.
- Coping mechanism or compensation is done
through the opposite organ.
B. Alkalosis
= bicarbonate excess
Caused by:
 Additional alkalosis (ingestion of alkali)
 Subtraction alkalosis (vomiting)
  When inhaling, oxygen from the external Oxygen content
environment will go inside the alveoli of the
lungs, then go to the capillaries, then to the
blood.
 When exhaling, CO2 is from the blood, it will
go through the capillaries, going to the alveoli of BLOOD GAS ANALYSIS
the lungs, and to the external environment for
photosynthesis.
Variations in the hemoglobin
 Oxyhemoglobin
- regular hemoglobin
- oxygen reversibly bound to hemoglobin
- color is bright red
- normal
 Deoxyhemoglobin
- reduced hemoglobin
- hemoglobin not bound to oxygen
- color is dark red
- normal
 Carboxyhemoglobin
- hemoglobin bound to carbon monoxide
- 200 x stringer affinity for hemoglobin than
oxygen
- cherry red appearance
- abnormal
 Methemoglobin
- hemoglobin unable to bind oxygen because
iron is oxidized
- chocolate brown in appearance
- methyl is attached to the blood
- abnormal
MEASUREMENT OF OXYGEN SATURATION
Dedicated spectrophotometer (cooximeter)
 Used to determine the relative concentration of
each of the forms of hemoglobin
4 parameters used to asses a patient’s oxygen status
1. Oxygen Saturation (SO2)
 Represents the ratio of oxygen that is bound to
its carrier protein, hemoglobin
2. Fractional (percent) oxyhemoglobin (FO2Hb) NOTE!
 Ratio of the concentration of oxyhemoglobin to
the concentration of total hemoglobin
3. Partial pressure of oxygen dissolved in plasma (PO2)
 Counts for the little of the body’s oxygen stores
4. Pulse oximetry (SO2)
 Uses a device to pass light to two or more
wavelength through the tissue in the capillary
bed of toe, finger, or ear.
Hemoglobin oxygen binding capacity
 Maximum amount of oxygen that can be carried
by hemoglobin in a given quantity of blood.
 Total oxygen in blood and is the sum of the
oxygen bound to hemoglobin (O2Hb) and the
amount dissolved in plasma (pO2)
 pH, pCO2, pO2
- Blood gas analyzers use electrodes
(microelectrochemical or microelectrochemical)
as sensicing device to pressure pO2, pCO2, and
pH.
- pO2 measurement is amperometric
- pCO2 and pH measurement are potentiometric.
- similar technique through direct titration of a
redox reaction
 Cathode
- negative electrode
- site to which cations tent to travel
- site at which reduction occurs (reduction- gain
of electron by particle)
 Anode
- positive electrode
- site to which anions tend to travel
- site at which oxidation occurs (oxidation- loss
of electron by particle)
 pO2 electrodes (clark electrode)
- Measure amounts of current flow in a circuit
that is release to the amount of oxygen being
reduced at the cathode.
 Continuous measurement of pO2
- Made possibly by using transcutaneous
electrode (TC) placed directly in the skin.
 pH
- Glass membrane coated electrode placed
around an internal Ag-AgCl electrode
(measuring electrode)
 pCO2
- Severinghaus electrode: an outer
semipermeable membrane that allow carbon
dioxide to diffuse into a layer of electrode.
 H2CO3- carbonic acid
 HCO3- bicarbonate
 CO2- carbon dioxide
 O2- oxygen
 H2O- water
 PCO2- partial pressure of carbon dioxide
 HPO4- hydrogen phosphate
 H2PO4- phosphoric acid
 CLINICAL ENDOCRINOLOGY: OTHER
Professor: William Christopher C. Salazar, RMT Happy Reviewing, RMT.
PARATHYROID GLAND  Lab results: increase PTH, decrease
 It is located on or near the thyroid capsule ionized calcium
(region of the thyroid gland); sometimes within
the thyroid gland c) Tertiary hyperparathyroidism
 It may also be found outside their normal  It occurs with secondary
anatomical site – between the hyoid bone in the hyperparathyroidism
neck and mediastinum  The phosphate levels are normal to
high, calcium phosphates precipitate in
 Most people have 4 parathyroid glands but some
soft tissues
have 8 or as few as 2
 It is the smallest endocrine gland in the body
 It secretes parathyroid hormone (PTH) – HYPOTHYROIDISM
hypercalcemic hormone  Is due to accidental injury to the parathyroid
glands (neck) during surgery – postsurgical
cause
ROLE OF PARATHYROID GLAND:
 Other causes: autoimmune parathyroid
 Primary role: to prevent hypocalcemia destruction
(regulates blood calcium)  Individuals are unable to maintain calcium
 It preserves calcium and phosphate within concentration in blood without calcium
normal range supplementation
 It promotes bone resorption – release calcium
into the blood stream ADRENAL GLAND
 It has a pyramid-like shape (adult gland) located
 It increases renal reabsorption of calcium
above the kidneys
 It stimulates conversion of inactive Vitamin D to
 It is composed of distinct but conjoined glands,
activated Vitamin D3.
the outer adrenal cortex (yellow) and inner
 Indirectly stimulates intestinal absorption of adrenal medulla (dark mahogany)
calcium
 As calcium level increases, PTH secretion is
suppressed allowing urinary loss of calcium and
calcium to remain in bone
 If calcium level decreases, PTH is released

CLINICAL DISORDERS
HYPERPARATHYROIDISM
a) Primary Hyperparathyroidism
 Physiologic defect lies with the
parathyroid gland
 Is the most common cause of
hypercalcemia
 Is due to the presence of a functioning
parathyroid adenoma
 It is accompanied with phosphouria
 If it goes undetected, severe
demineralization may occur (osteitis ADRENAL CORTEX
fibrosa cystica)  It is the outer region of the adrenal gland
 Lab results: increased PTH or high secreting the steroid hormone
normal range, increased ionized  Is the major site of steroid hormone production
calcium, hypercalciuria,
hypophosphatemia (fasting state) 3 ZONAL LAYERS OF ADRENAL CORTEX
1. G – zone – Zona Glomerulosa (10%)
b) Secondary hyperparathyroidism  Synthesize mineralocorticoid –
 It develops in response to decrease Aldosterone
serum calcium  Aldosterone is responsible for Na, K and
 There is diffuse hyperplasia of all 4 acid-base homeostasis
glands 2. F – Zone – Zona Fasciculata (75%)
 The patient develops severe bone  Synthesize glucocorticoids – Cortisol
disease  Cortisol is responsible for blood glucose
 Causes: vitamin D deficiency and homeostasis
chronic renal failure 3. R – Zone – Zona Reticularis (15%)
 CLINICAL ENDOCRINOLOGY: OTHER
Professor: William Christopher C. Salazar, RMT Happy Reviewing, RMT.
 Sulfates DHEA to  Includes: dopamine, epinephrine (increase BP)
dehydroepiandrosterone – DHEAS, a and norepinephrine (vasoconstrictor)
precursor for adrenal sex hormones  Possess α and β adrenergic effects with a net
effect on increasing the blood pressure
CORTISOL  Stimulates cardiac muscle (major contributor is
 Is the principal glucocorticoid epinephrine)
 Its synthesis is regulated by ACTH; it is mostly  Synthesis occurs in the Chromaffin cells
bound to glycoprotein, transcortin (pheochromocytes)
 It stimulates gluconeogenesis in the liver  Storage occurs in the Chromaffin granules
resulting in hyperglycemia (anti-insulin effect)  Major catecholamine in blood: Epinephrine
 It is the only adrenal hormone that inhibits the  Major catecholamine in neurons:
secretion of ACTH (when plasma level of cortisol Norepinephrine
is elevated)
 It has anti-inflammatory and immunosuppressive NOREPINEPHRINE
actions – a valuable therapeutic agent for  Primary amine
rheumatoid arthritis, SLE and multiple sclerosis  It is produced by the sympathetic ganglia
 Its secretion is diurnal and is associated with a  The highest concentration is found in the brain
person’s sleep-wake cycle (CNS)
 High levels in the morning (8:00am to 10:00am)  It acts as a neurotransmitter in both CNS and
and lowest at night (10:00pm to 12:00mn) sympathetic nervous system (SNS)
 Specimen: serum (red top), urine; blood sample  MHPG is the major metabolite in CNS
should be drawn at 8:00am  Major metabolites: 3-methoxy-4-
 Urine free cortisol levels are sensitive indicators hydroxyphenylglycol (MHPG) – CSF and urine
of adrenal hyperfunction (endogenous vanillylmandelic acid (VMA)
corticolism)
– 24-hour urine collection EPINEPHRINE
 Reference value: 5-25 ug/dL (140-690 nmol/L)  Adrenaline/ secondary amine
at 8am to 10am  Is the most abundant medullary hormone
 It is produced from norepinephrine and comes
ALDOSTERONE (Aldo) from the adrenal
 Is the most potent mineralocorticoid (electro-  It is called the “flight or flight hormone”,
regulating hormone) because it is released in response to physiologic
 Is a steroid hormone that helps regulate water (injuries) or psychological (stress, anxiety)
and electrolytes (sodium, chloride and threats
potassium) and blood pressure.  It increases glucose concentration
 Is the main determinant of renal excretion of (glycogenolysis).
potassium  Any form of stress that increases cortisol levels
 It acts on renal tubular epithelium to increase stimulates its production
retention of Na and Cl, and excretion of K and H  Major metabolite: vanillylmandelic acid (VMA)
 It promotes the 1:1 exchange of sodium for  Minor metabolites: metanephrines,
potassium or hydrogen ions normetanephrines homovanillic acid
 The synthesis of this hormone is primarily  VMA is the major (60%) catecholamine
controlled by the renin-angiotensin system metabolite in urine derived largely from
 18-hydroxysteroid dehydrogenase is an enzyme norepinephrine
needed for the synthesis of aldosterone
 Method: RIA and chromatography DOPAMINE
 Primary amine
ADRENAL MEDULLA  Is a catecholamine produced in the body by the
 Is composed primarily of chromaffin cells that decarboxylation of 3,4-dihydroxyphenylalanine
secrete catecholamines (DOPA)
 L-tyrosine is the precursor of the catecholamines  It is present in highest concentration in the
 Cathecolamines are the “First Responders” to regions of the brain
stress by acting within seconds to promote fight  Dopamine is the major intact catecholamines
or flight response (cortisol takes 20 minutes) present in urine
 Major metabolite: homovanillic acid (HVA)
CATHECOLAMINES
 Produced by adrenal medulla and neuron of CNS
 Derived from amino acid tyrosine
 CLINICAL
Professor: William Christopher C. Salazar, RMT
CLINICAL DISORDER - Pheochromocytoma
 Is tumor of the adrenal medulla or sympathetic
ganglia
 It is due to overproduction of catecholamines
 Is commonly seen in 3rd to 5th decades of life
 Classic “spells”: hypertension, tachycardia,
headache, tightness of chest and sweating
 Screening test: plasma metanephrines and
normetanephrine by HPLC (four-fold increased)
 Diagnostic test: 24-hour urinary excretion of
metanephrines and normetanephrine (increased
levels)
 Patient preparation: avoid caffeine, nicotine,
alcohol and acetaminophen, monoamine
oxidase inhibitors, and tricyclic anti-drepressants
for at least 5 days before testing
CLINICAL DISORDER - Neuroblastoma
 Is a fatal malignant condition in children resulting
to excessing production of norepinephrine
 (+) high urinary excretion of HVA or VMA or
both, and dopamine
REPRODUCTIVE HORMONES
Testosterone
 Is the principal androgen hormone in the blood –
most potent male androgen
 Is synthesized by the Leydig cells of the testis of
the male; also derived from progesterone
 Controlled primarily by FSH and LH
 Functions: growth and development of the
reproductive system, prostate and external
genitalia
 Levels demonstrate a circadian pattern and peak
at the time of awakening (8am); fall to their
lowest level at 8 pm
 There is a gradual reduction in testosterone after
age 30, with an average decline of about 110
ng/dL every decade
 Test for male infertility: semen analysis,
testosterone, FSH and LH
 Reference values: 3.9-7.9 ng/mL (serum)
DEHYDROEPIANDROSTERONE (DHEA)
 Is the principal androgen formed by adrenal
cortex, weak androgen.
 This androgen is primarily derived from the
adrenal gland
 It is valuable in the assessment of adrenal cortex
function
ESTROGEN
 Is a carbon-18 steroid hormone that have a
phenol A ring
 It arises through structural alteration of the
testosterone molecule
 It is not produced by the ovaries after
menopause
ENDOCRINOLOGY: OTHER
Happy Reviewing, RMT.
 Functions: promote breast development,
maturation of the external genitalia, deposition of
body fat and termination of linear growth.
 In conjunction with progesterone, they function
in uterine growth and regulation of menstrual
cycle, and maintenance of pregnancy
 Deficiency: irregular and incomplete
development of endometrium
 3 forms: Estrone, Estradiol, Estriol
 Estrone and estriol are metabolites of intra
ovatian and extra glandular conversion
ESTROGEN – ESTRONE (E1)
 Is the most abundant estrogen in post
menopausal women
ESTROGEN – ESTRADIOL (E2)
 Most potent estrogen secreted by the ovary;
major estrogen
 Most abundant estrogen in pre-menopausal
women; low in the menopausal stage
 Synthesized from the testosterone, then diffuses
out of the thecal cells of the ovaries in the
female
 It is the precursor of both E1 and E3
 Used to assess ovarian function; serves as
negative feedback for FSH
 Transport protein: albumin (60%) and SHGB
(38%)
 The free form of E2 is approximately 2%
ESTROGEN – ESTRIOL (E3)
 Metabolite of estradiol
 Estrogen found in maternal urine
 Major estrogen secreted by the placenta during
pregnancy
 used to assess the fetoplacental unit
(fetoplacental viability), postdate gestations and
intrauterine retardation)
 used as a marker for down syndrome (together
with AFP and HCG)
 Preferred specimen: Plasma
PROGESTERONE
 Produced mainly by the granulose (lutein) cells of
the corpus luteum in the female
 It is the prime secretory product of the ovary
 It is a dominant hormone responsible for the
luteal phase cycle among females
 It is the single best hormone to determine
whether ovulation has occurred
 It is used primarily for the evaluation of fertility in
female
 It serves to prepare the uterus for pregnancy
and the tubules of the breast for lactation
 It is intermediate in the synthesis of adrenal
steroids and androstenedione
 Deficiency: failure of the implantation of embryo.
 CLINICAL ENDOCRINOLOG
Professor: William Christopher Salazar Happy Reviewing, RMT.

ENDOCRINE SYSTEM
 A network of ductless glands that secrete
hormones directly into the blood.
 It is considered to be the regulatory hormone
of the body.
 It is regulated by means of control of
hormone synthesis rather than by
degradation.

Note: ‘Yun ibang images na involve sa PPT ay nasa dulong part ng

trans. Larger images para readable.

HYPOTHALAMIC-HYPOPHYSIAL UNIT
 Response pattern are characterized by:
o Open-loop negative feedback
mechanism
 Feedback of a
specific hormone to
pituitary
= short feedback loop
 Feedback of a
HORMONES
specific hormone to
 Are chemical signals produced by
hypothalamus = long
specialized cells secreted into the blood
feedback loop
stream and carried to a target issue.
 Feedback between
 They play an important role in the growth
pituitary and
and development of an individual.
hypothalamus =
 They are regulated by metabolic activity
ultrashort feedback
either positive or negative feedback
loop.
mechanism.
 Major Function: to maintain the constancy TYPES OF HORMONES
of chemical composition of extracellular and 1. Endocrine
intracellular fluids, and control metabolism, o is secreted in one location and release
growth, fertility, and responses to stress. into blood circulation, binds to
specific receptor to elicit physiological
FEEDBACK MECHANISM response.
 Positive Feedback Mechanism 2. Paracine
o Is a system in which an increase in o Is secreted in endocrine cells and
the product results to elevation of released into interstitial space; binds to
the activity of the system and the specific receptor in adjacent cells and
production rate (example; gonadal, affects its function.
thyroidal, adrenocortical hormones) 3. Autocrine
 Negative Feedback Mechanism o Is secreted in endocrine cells and
o Is a system in which an increased in sometimes released into interstitial
the product results to decreased space’ binds to specific receptor on cell
activity of the system and the of origin resulting to self-regulation of
production rate (example: lutenizing its function.
hormones) 4. Juxtacrine
o Is secreted in endocrine cells and
remains in relation to plasma
membrane; acts on immediately
adjacent cell by direct cell to cell
contact.
5. Inracrine
o Is secreted in endocrine cells and
remained as well as function inside the
1| Clinical Chemist
CLINICAL ENDOCRINOLOG
synthesis of origin.

2| Clinical Chemist
 CLINICAL ENDOCRINOLOG
Professor: William Christopher Salazar Happy Reviewing, RMT.

6. Exocrine HYPOTHALAMUS
o Is secreted in endocrine cells and  It is the portion of the brain located in the
released into lumen of gut; it affects walls and floor of the third ventricle.
their function.
 It is above the pituitary gland, and is
7. Neurocine
connected to the posterior pituitary by the
o Is secreted in neurons and released
infundibulum (pituitary stalk).
intro extracellular space; bind to receptor
 It is the link between the nervous system
in nearby cell and affects its function.
and the endocrine system.
8. Neuroendocrine
 The supraoptic and paraventricular nuclei
o Is secreted in neurons and released
produce vasopressin and oxytocin.
from nerve endings, interacts with
 The neurons in the anterior portion release
receptors at a distant site.
the following hormones (hypophysial
hormones)
o Thyrotropin-releasing hormone (TRH)
CLASSIFICATION OF HORMONES BY o Gonadotropin-releasing hormone
STRUCTURE (Gn-RH)
o Somatostatin (Growth Hormone
A. PEPTIDES AND PROTEINS inhibiting hormone (GH-IH))
TYPES OF PEPTIDE AND PROTEIN HORMONES o Growth Hormone releasing hormone
1. GLYCOPROTEIN (GH-RH)
a. FSH (Follicle Stimulating Hormone), o Prolactin-inhibiting factor (PIF)
HCG (Hyman Chorionic Gonadotropin
ENDOCRINE GLANDS
Hormone), TSH (Thyroid Stimulating A. Pineal Gland
Hormone, and EPO (Erythropoietin) B. Pituitary Gland
C. Thyroid Gland
2. POLYPEPTIDES D. Parathyroid Gland
a. ACTH (Adrenocorticotropic Hormone) E. Adrenal Gland
b. ADH (Antidiuretic Hormone)
c. GH (Growth Hormone) PINEAL GLAND
d. Angiotensin, calcitonin, cholecystokinin,  Is attached to the midbrain
gastrin, glucagon, insulin, melanocyte  Secretes melatonin that decreases the
stimulating hormone (MSH), oxytocin, pigmentation of the skin
PTH, prolactin, somatostatin  Secretions are controlled by nervus stimuli

3. STEROIDS PITUITARY GLAND (HYPOPHYSIS)

 Are lipid molecules that have cholesterol as  It is known as the Master Gland.
a common precursor  It is located in a small cavity in the sphenoid
 They are produced by adrenal glands, bone of the skull called the sella turcica or
ovaries, testes, and placenta. Turkish saddle.
 They are water insoluble, (hydrophobic), and  It is connected by the infundibular stalk to
circulate bound to a carrier protein. the median eminence of the hypothalamus.
 Examples: aldosterone, cortisol, estradiol,  All pituitary hormones have circadian rhythm.
progesterone, testosterone, and activated  3 Distinct Parts:
vitamin D. o Anterior Pituitary
(adenohypophysis) – largest
4. AMINES oprtion of the gland.
 They are derived from an amino acid and o Intermedeiate lobe – little
they are intermediary between steroid and functional capacity
protein hormones. o Posterior pituitary
 Examples: epinephrine, norepinephrine, (neurohypophysis) – for storage and
triiodothyronine and thyroxine release of oxytocin and vasopressin.

3| Clinical Chemist
 CLINICAL
Professor: William Christopher Salazar
PITUITARY GLAND
ANTERIOR PITUITARY (Adenohypophysis)
 Is the “true endocrine gland”
 It regulates the released and production of
hormones such as prolactin, grown
hormone, gonadotropins (FSH and LH), TSH
and ACTH
 The hormones secreted by the anterior lobe
are either peptides or glycoproteins.
5 TYPES OF CELLS BY IMMUNOCHEMICAL TEST
1. Somatotrophs – secrete growth hormone
2. Lactotrophs or mammotrophs – secrete
prolactin
3. Thyrotrops – secrete TSH
4. Gonadotrophs - secrete LH and FSH
5. Corticotrophs – secretes proopiomelanocortin
(POMC is cleaved within the pituitary to produce
ACTH, β-endorphin, and β-lipotropin)
GROWTH HORMONE | SOMATOTROPIN
 The most abundant of all pituitary hormone
 Controlled by GH-RH (the amount of
release) and somatostatin (governs the
frequency and duration of secretory pulse)
 It is structurally similar to prolactin and
human placental lactogen
 Its secretion is erratic and occurs in short
bursts.
 Its overall metabolic effect is to metabolize
fat stores while conserving glucose.
 Major stimulus: deep sleep (markedly
increased GH)
 Major inhibitor: somatostatin (also insulin
and gastrin)
 Physiologic Stimuli (increased GH): stress,
fasting, and high protein diet.
 Pharmacologic stimuli (increased GH):
sex steroids, apomorphine and levodopa.
 GH suppressors: glucocorticoids and
elevated fatty acids.
 Increased: acromegaly, gigantism, chronic
malnutrition, renal disease, cirrhosis, and
sepsis.
 Decreased: hyperglycemia, obesity, and
hypothyroidism
 Method: chemiluminescent immunoassay
 Reference value (fasting): <7ng/ml
ENDOCRINOLOG
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GROWTH HORMONE | SOMATOTROPIN
DISORDERS
1. GH Deficiency (GHD)
a. Idiopathic Growth Hormone Deficiency
 Is the most common cause of GH deficiency
in children
 In children with pituitary dwarfism, normal
proportions are retained and show no
intellectual abnormalities
b. Pituitary adenoma
 Is the most common etiology in adult-onset
GH deficiency
2. Acromegaly
 Is due to overproduction of GH (>50ng/ml or
2210 pmol/L)
GROWTH HORMONE | SOMATOTROPIN
DIAGNOSTIC TEST
 Patient preparation: complete rest 30
minutes before blood collection
 Specimen requirement: preferably fasting
serum.
1. For GH Deficiency:
a. Screening Test: Physical Activity Test
(Exercise Test)
 Result of the test: elevated serum GH
 If GH fails to increase, confirmatory must be
made.
b. Confirmatory Test:
 Insulin tolerance test – gold standard
 Arginine Stimulation Test – 2nd confirmatory
test
 Procedure: 24 hour or night time
monitoring of GH
 Interpretation: Failure of GH to rise >5ng/ml
(adults) and >10 ng/ml (child) in all the test
is confirmed of GH deficiency
 GHD in childhood is defined by failure of
serum GH to reach defined levels when at
least two different pharmacologic stimuli are
used.
2. For Acromegaly:
a. Screening Test: somatomedin C or
insulin-like growth factor 1(IGF1)
 IGF-1 is produced in the liver.
 IGF-1is increased in patients with
acromegaly.
 IGF-1 is low in GHD.
4| Clinical Chemist
 CLINICAL
Professor: William Christopher Salazar
b. Confirmatory Test: Glucose
Suppression test – OGTT (75g Glucose)
 Blood is collected every 30 minutes for 2
hours; fasting sample is required.
INTERPRETATION OF RESULT FOR ACROMEGALY:
 A normal response for this test is a
suppression of GH less than 1 ng/ml.
 If GH fails to decline less than 1 ng/ml, its
acromegaly
 Failure of GH to be suppressed below 0.3
ug/l, accompanied by elevated by IGF-1, is
diagnostic of acromegaly
 Suppression of GH below 0.3 ug/l with
normal IGF-1 excludes acromegaly
 Suppression of GH but increased IGF-1,
requires follow up and monitoring.
GONADOTROPIN – FSH AND LH
 Is an important marker in diagnosing fertility
and menstrual cycle disorders
 Is present in the blood of both male and
female at all ages
 FSH aids in spermatogenesis (male)
 LH helps Leydig cells to produce
testosterone (male) and for female, it is
necessary for ovulation and the final
follicular growth.
 LH acts on theca cells to cause the
synthesis of androgens, estrogens (estradiol
and estrone) and progesterone.
 Elevation of FSH is a clue in the diagnosis of
premature menopause.
 Increased FSH and LH after menopause is
due to lack of estrogen.
THYROID STIMULATING HORMONE (TSH)
 Is also known as thyrotropin
 Is the main stimulus for the uptake of iodide
by the thyroid gland
 Is acts to increase the number and size of
follicular cells; it stimulates thyroid hormone
synthesis
 It is composed of 2 mono-covalently linked
to α and β subunits; α subunit has the same
amino acid sequence of LH, FSH, and HCG
 The β subunit carries the specific
information to the binding receptors for
expression of hormonal activities
 Blood levels may contribute in the evaluation
of infertility.
ENDOCRINOLOG
Happy Reviewing, RMT.
ADRENOCORTICOTROPHIC HORMONE (ACTH)
• It is a single-chain peptide without sulfide bonds
• It is produced in response to low serum
cortisol; regulator of adrenal androgen
synthesis
• Deficiency of ACTH will lead to atrophy of the
zona glomerulosa and zona reticularis (layers of
the adrenal cortex)
• Highest level is between 6:00am to 8:00am;
lowest level is between 6:00pm to 11:00pm
• Increased: Addison’s disease, ectopic tumors,
after protein-rich meals.
• Specimen requirement
o Blood should be collected into
prechilled polystyrene (plastic) EDTA
tubes to prevent degradation of ACTH
o Specimen for test should not be allowed
to have contact with glass because
ACTH adheres to glass surface resulting
to decreased the levels
o Best time for collecting specimen:
8:00am to 10:00am
PROLACTIN (PRL)
 It is a pituitary lactogenic hormone; a stress
hormone; a direct effector hormone
 Its amino acid structure is similar to GH
 It functions in the initiation and
maintenance of lactation
 It also acts in conjunction with estrogen and
progesterone to promote breast tissue
development
 Major inhibitor: dopamine (secreted by the
hypothalamus)
 Consequence of prolactin excess:
hypogonadism
 Increased: pituitary adenoma, infertility,
amenorrhea, galactorrhea acromegaly, renal
failure, polycystic ovary syndrome, cirrhosis,
and primary and secondary hypothyroidism
 Prolactin serum level: >200 mg/dl
 Specimen requirement: blood should be
collected 3 to 4 hours after the individual
has awakened: fasting sample
 Highest serum level (during sleep):
4:00am to 8:00am; 8:00pm to 10:00pm
 Method: immunometric
 Physiologic stimuli (increased): exercise,
sleep, stress, postprandially, pain, coitus,
pregnancy, nipple stimulation or nursing
5| Clinical Chemist
 CLINICAL ENDOCRINOLOG
Professor: William Christopher Salazar Happy Reviewing, RMT.

 Pharmacologic stimuli (increase): intake of  Synthetic preparations; to increase weak uterine

verapamil, phenothiazines, olanzapine,
Prozac, cimetidine and opiates
 Reference values:
o male 1-20 ng/ml (1-20 ug/L)
o Female 1-25 ng/ml (1-25 ug/L)
NOTES TO REMEMBER: Prolactin
 It is essential to obtain TSH a free T4 (or
total thyroxine and T3 resin uptake) to
eliminate primary hypothyroidism as a cause
for the elevated prolactin.
 Thyroid hormone replacement therapy will
usually return the PRL to normal plasma
level.
 Three specimens should be obtained at 20-
to 30- minute intervals because of
physiologic stimuli, these samples can be
measured separately and their results
averaged, or, equal aliquot from each
sample can be pooled into one final sample
and then it is analyzed.
 Elevations in PRL due to physiologic and
pharmacologic stimuli rarely exceed 200
ng/mL.
contractions during labor and to aid in lactation.
ANTI-DIURETIC HORMONE (ADH) ARGININE
VASOPRESSIN (AVP)
 Acts on the distal convoluted and
collecting tubules of the kidneys
 Major function: to maintain osmotic
homeostasis by regulating water balance
 It decreases the production of urine by
promoting reabsorption of water by the renal
tubules maintains water homeostasis
 It increases blood pressure – a decrease
in blood volume or blood pressure will
likewise stimulate ADH release
 It is a potent pressor agent and affects
blood clotting by promoting factor VII
release from hepatocytes and Factor VIII
(von willebrands factor) release from the
endothelium
 Reference value: 0.5 -2 pg/uL
END OF LESSON. GOOD LUCK.

POSTERIOR PITUITARY (Neurohypophysis)

 Is capable of releasing the hormones oxytocin
and vasopressin but not capable of producing
it.

 The hormones released by neurohypophysis are

synthesized in the magnicellular neurons of
the supraoptic (ADH) and paraventricular
nuclei (oxytocin) of the hypothalamus, and
stored in the nerve terminals that end in the
posterior pituitary.

 The release of the hormones occurs in response

to serum osmolality or by suckling.

 Hormones produced by the neurohypophysis are

controlled by the central nervous system.

OXYTOXIN
 Very similar in composition to ADH

 It stimulates contraction of gravid uterus at term

– “Ferguson reflex”

 Stimulates contraction of the uterus and also

milk ejection.

 Oxytocin secretion is also inhibited by alcohol,

which has been used clinically intravenous
administration for stopping premature labor.

6| Clinical Chemist
MLS 115: CLINICAL CHEMISTRY 2 SELENIUM
LESSON 7 | TRACE ELEMENTS
 Function: prevents oxidative damage of lipids
TRACE ELEMENTS  Deficiency: keshan disease
• Consists of metals, halogens, fluoride, and iodine  Toxicity: hair and nail loss, liver failure
• Essential trace elements are important for the ZINC
maintenance of normal health, and tissue and organ
functions.  Function: protein synthesis

• Trace elements have specific in vivo metabolic functions  Deficiency: acrodermatitis, enteropathica, growth
that cannot be effectively performed by other similar retardation, immune deficiency, infertility, delayed wound
elements healing, osteoporosis
• Concentration in tissue: <1 µg/g of wet tissue and  Toxicity: gastrointestinal irritation
<0.01% of dry body weight
End of PPT for Trace Elements
CHROMIUM
REFERENCE
 Function: enhances insulin action; for glucose and lipid
metabolism Bishop, Michael L. et.al. CLINICAL CHEMISTRY:
PRINCIPLES, PROCEDURES, CORRELATIONS 8th ed.
 Deficiency: insulin resistance, impaired glucose Philadelphia: Lippincott Williams,
tolerance (type 2 DM), hyperlipidemia
Philadelphia, 2018
 Toxicity: skin ulcers, renal and hepatic necrosis
Clinical Chemistry Review Handbook for Medical
COBALT Technologists by Maria Teresa T. Rodriguez,
 Function: hemoglobin synthesis; component of vitamin
Legend[s]:
B12
Professor | instructor
 Deficiency: anemia, growth depression
Ajiuaifngtepqok – good to know | must remember
 Toxicity: heart failure, hypothyroidism either of the two. Can be both.
COPPER Note:
 Function: cellular respiration, collagen synthesis A4 size
 Deficiency: menkes’ kinky hair syndrome, muscle
weakness
 Toxicity: interferes with absorption of iron and zinc
FLUORIDE
 Function: prevents dental caries
 Deficiency: dental caries
 Toxicity:
IODINE
 Function: thyroid hormone synthesis
 Deficiency: goiter, cretinism, myxedema
 Toxicity: thyrotoxicosis
IRON
 Function: oxygen transport, component of hemoglobin
 Deficiency: anemia
 Toxicity: Hemachromatosis
MANGANESE
 Function: bone and connective tissue functions
 Deficiency: skeletal defects
 Toxicity: psychiatric disorders, Parkinson’s disease
MOLYBDENUM
 Function: DNA metabolism
 Deficiency: Growth depression, cretinism, goiter
 Toxicity: anemia, thyrotoxicosis

1 | LE 7. T r a c e E l e m e n
MLS 115: CLINICAL CHEMISTRY 2  Deficiency: angular stomatitis, dermatitis, photophobia
LESSON 6 | NUTRITIONAL MARKERS
Vitamin B3
VITAMINS  Chemical name: pantothenic acid
 Essential organic substances that the body cannot  Function: enzyme cofactor
synthesize, or does bit sufficiently synthesize
 Present in almost all foods, but no single food group is  Deficiency: depressed immune system, muscle
the source of all vitamins. weakness

VITAMINS – FUNCTIONS Vitamin B6

• Antioxidants  Chemical name: Pyridoxine, pyridoxal
• Enzyme cofactor  Function: enzyme cofactor
• Hormones and important in blood cell maturation  Deficiency:
• Bone formation and active in energy metabolism o Infants: irritability, seizures, anemia
FAT SOLUBLE VITAMINS o Adult: facial seborrhea
VITAMIN A
Vitamin B12

 Chemical name: Retinol  Chemical name: Cyanocobalamin

 Function: maintenance of good vision, resistance to  Function: synthesis of DNA and Folate
infection
 Deficiency:
 Deficiency: night blindness, growth retardation,
abnormal taste response, dermatitis o megaloblastic anemia, neurologic abnormalities
Vitamin C
VITAMIN E
 Chemical name: Tocopherols (α, β, γ, δ)  Chemical name: ascorbic acid
 Function: antioxidant; for cellular respiration  Function: hydroxylation of collagen, participates in
 Deficiency: mild hemolytic anemia (newborn), RBC redox reaction
fragility, ataxia
o Ataxia is a degenerative disease of the nervous  Deficiency: scurvy
system Biotin
VITAMIN D2  Chemical name:
 Chemical name: ergocalciferol, cholecalciferol  Function: enzyme cofactor
 Function: helps body absorb calcium and phosphorus  Deficiency: dermatitis, hair loss, depression
 Deficiency: rickets (young), osteomalacia (adult) Carnitine
VITAMIN D3  Chemical name:
 Chemical name: Activated sterol-1,25  Function: fat catabolism
dihydroxycholecalciferol
 Deficiency: muscle weakness, fatigue
 Function: absorption of dietary calcium
Folic acid
 Deficiency: hypocalcemia  Chemical name: pteroylglutamic acid
VITAMIN K  Function: synthesis of amino acids and DNA
 Chemical name: phytomenadione
 Deficiency: megaloblastic anemia
 Function: cofactor of procoagulants like prothrombin
Niacin/Niacinamide
 Deficiency: bleeding disorder, hemorrhage  Chemical name: nicotinic acid/nicotinamide
WATER SOLUBLE VITAMINS  Function: enzyme cofactor
VITAMIN B1
 Deficiency: pellagra, dermatitis, disorientation, weight
 Chemical name: thiamine loss
 Function: enzyme cofactor Thank you!
 Deficiency: REFERENCE
o Infants: dyspnea, cyanosis, diarrhea and vomiting Bishop, Michael L. et.al. CLINICAL CHEMISTRY:
o Adults: Beri-beri, Wernicke-Korsakoff syndrome, PRINCIPLES, PROCEDURES, CORRELATIONS 8th ed.
apathy, ataxia, and visual problems Philadelphia: Lippincott Williams,
Vitamin B2 Philadelphia, 2018
 Chemical name: riboflavin
Clinical Chemistry Review Handbook for Medical
 Function: enzyme cofactor Technologists by Maria Teresa T. Rodriguez,

1 | LE 6.N u t r i t i o n a l M a r k e
Legend[s]:
Professor | instructor
Ajiuaifngtepqok – good to know | must remember
either of the two. Can be both.
Note:
A4 size

2 | LE 6.N u t r i t i o n a l M a r k e
CLINICAL CHEMISTRY
Professor: William Christopher
THYROID GLAND
• Is also known as the butterfly shaped gland.
• It consists of two lobes (one on either side of
the trachea) located in the lower part of the neck
just below the voice box.
• The lobes are connected by a narrow band
called the isthmus.
• By 11 weeks of gestation, the gland begins to
produce measurable amount of hormone.
• Follicle is the fundamental structural unit of the
thyroid gland
• 2 types of cells:
o Follicular cells
• Secretory and produce
thyroxine – T4 and
triiodothyronine - T3
• Each follicle is in the shape of a
sphere that surrounds a viscous
substance called colloid. The
major component of colloid is
the Thyroglobulin, which is rich
in tyrosine
o Parafollicular Cells or C-cells
• Situated in clusters along the
interfollicular spaces
•
They produce the polypeptide
calcitonin, for calcium
regulation.
THYROID HORMONES
• There is more T4 than T3 in serum, but T3 is
more potent and significant physiologically
• About 80% of circulating T3 is formed following
monodeiodination of T4 in peripheral tissues
• Thyroid hormones are almost completely protein
bound
• T4 = 99.97% bound = only 0.03%
FREE
• T3 = 99.70% bound = only 0.30%
FREE
TRIIODOTHYRONINE (T3) / 3,5,3’
TRIIODOTHYRONINE
• It has the most active thyroid hormonal activity
• Almost 75-80% is produced from the tissue
deiodination of T4 conversion of T4 to T3 takes
place in many tissues, particularly in the liver
THYROID GLAND
Happy Reviewing,
• The principal application of this hormone is in
diagnosing T3 thyrotoxicosis.
• Is a better indicator of recovery from
hyperthyroidism as well as the recognition of
recurrence of recurrence of hyperthyroidism – it
is helpful in confirming the diagnosis of
hyperthyroidism, especially in patients with no or
minimally elevated T4
• An increase in the plasma level of T3 is the first
abnormality seen in cases of hyperthyroidism
• Reference values:
o 60-160 ug/dl or 0.9-2.46 nmol/L (adult)
o 105-245 ug/dl or 1.8-3.8 nmol/L (children
1-14 years old)
Tetraiodothyronine (T4)/ 3,5,3’,5’
tetraiodothyronine
• Is the principal secretory product
• It has the major fraction of organic iodine in the
circulation
• Is a prohormone for T3 production
• All circulating T4 originates in the thyroid gland –
it is secreted 100% in the thyroid gland
• The amount of serum T4 is a good indicator of
the thyroid secretory rate
• Elevated thyroxine causes inhibition of TSH
secretion and vice versa
• Reference values:
o 5.5-12.5 ug/dL or 71-161 nmol/L (adult)
o 11.8-22.6 ug/dL or 152-292 nmol/L
(neonate)
THYROID HORMONE BINDING PROTEINS
1. Thyroxine-Binding Globulin (TBG)
 It transports majority of T3 (affinity for T3
is lower than T4)
 It transports 70-75% of Total T4
2. Thyroxine-Binding Prealbumin (transthyretin)
 It transports 15-20% of Total T4
 T3 has no affinity for prealbumin
3. Thyroxine-Binding Albumin
 Transports T3 and 10% of T4
THYROID HORMONE RESPONSIBLE FOR
AUTOIMMUNE DISORDERS
and the kidneys
1| Clinical Chemist
CLINICAL CHEMISTRY THYROID GLAND
1. Thyroperoxidase
Professor: William Christopher (TPO)  Thyroid cancer Happy Reviewing,
Involved in the tissue destructive 3. TSH receptor (TR)
process (hashimoto’s disease)  Involved in Grave’s Disease
2. Thyroglobulin (Tg)

2| Clinical Chemist
 CLINICAL CHEMISTRY THYROID GLAND
Professor: William Christopher Happy Reviewing,

CLINICAL DISORDERS
HYPERTHYROIDISM – RIEDEL’S THYROIDITIS
• The thyroid turns into a
 Screening of thyroid disorders is recommended woody or stony-hard mass
when a person’s reaches 35 years old and every
year thereafter

HYPERTHYROIDISM
 It refers to an excess of circulating thyroid
hormones
 Signs and symptoms Hyperthyroidism – SUBCLINICAL
o Tachycardia, tremors, weight loss, heat HYPERTHYROIDISM
intolerance, emotional lability and menstrual  Shows no clinical symptoms but TSH level is
cycle changes low, and TF3 and FT4 are normal
 Primary hyperthyroidism
o Elevated T3 and T4, decreased TSH HYPERTHYROIDISM – SUBACUTE
 Secondary hyperthyroidism GRANULOMATOUS
o Increased FT4 and TSH (due to primary  Subacute nonsuppurative thyroiditis, De
Quevain’ thyroiditis (painful thyroiditis)
lesion in the pituitary gland)
 It is associated with neck pain, low-grade fever
and swings in thyroid function test
HYPERTHYROIDISM - THYROTOXICOSIS
 The thyroidal pexidase (TPO) antibodies are
 Is applied to a group of syndromes caused by
absent; ESR and thyroglobulin levels are
high levels of Free Thyroid hormones in the
elevated
circulation
 T3 thyrotoxicosis or plummer’s disease: FT3 is
HYPOTHYROIDISM
increased but FT4 is normal with low TSH
 T4 thyrotoxicosis: T3 is normal of low but T4 is  It develops whenever insufficient amounts of
increased with low TSH thyroid hormone are available to tissues.

 It is treated with thyroid hormone replacement

therapy (levothyroxine)

 Signs and symptoms: bradycardia, weight gain,

coarsened skin, cold intolerance and mental
dullness

HYPOTHYROIDISM –
PRIMARY HYPOTHYROIDISM
 Is primarily due to deficiency of elemental Iodine
HYPERTHYROIDISM – GRAVE’S DISEASE  T3 and T4 are decreased while TSH is
 Diffuse toxic goiter increased
 It is the most common cause of thyrotoxicosis  It is also caused by destruction or ablation of
the thyroid gland
 It is an autoimmune disease in which
antibodies are produced that active the TSH  Other causes: surgical removal of the gland,
receptor use of radioactive iodine for hyperthyroidism
 It occurs 6x more commonly in women than in treatment, radiation exposure; drugs such as
men lithium
 Features: exophthalmos (bulging eyes) and PRIMARY HYPOTHYROIDISM
pritibial myxedema HASHIMOTO’S DISEASE
Chronic autoimmune
Diagnostic test: thyroiditis
TSH receptor antibody test
It is the most common cause of primary hypothyroidism
It is characterized by a thyroid replaced by a nest of lymphoid tissue – sensitized T lymphocytes/autoantibodies bind to cell

3| Clinical Chemist
 CLINICAL CHEMISTRY THYROID GLAND
Professor: William Christopher Happy Reviewing,

membrane causing cell lysis and inflammatory
reaction
 It is associated with enlargement of the thyroid
gland (goiter)
 Lab result: high TSH and positive TPO antibody
o TSH value >20 mIU/L – for endocrinologic
evaluation to diagnose hypothyroidism.
HYPOTHYROIDISM – SUBCLINICAL
HYPOTHYROIDISM

 Lab result: T3 and T4 are normal but TSH is

slightly increased

THYROID FUNCTION TEST

TRH Stimulation Test (Thyrotropin Releasing Hormone)

 It measures the relationship between the TRH

and TSH secretions
 It is used to differentiate euthyroid and
hyperthyroid patients
 It may also be helpful in the detection of thyroid
hormone resistance syndromes
 It is used to confirm borderline cases and
PRIMARY HYPOTHYROIDISM euthyroid Grave’s disease.
MYXEDEMA  Dose needed: 500 ug TRH by IV
 It describes the peculiar nonpitting swelling of  Increased: primary hypothyroidism
the skin  Decreased: hyperthyroidism
 The skin becomes infiltrated by
mucopolysaccharides TSH Test
 Clinical features: “puffy” face, weight gain,
slow speech, eyebrows thinned, dry and yellow  Is the most important thyroid function test
skin, and anemia  The best method for detecting clinically
 Myxedema coma is the severe form of primary significant thyroid disfunction
hypothyroidism  It is the most clinically sensitive assay for the
detection of primary thyroid disorders
HYPOTHYROIDISM –  It helps in the early detection of hypothyroidism
SECONDARY HYPOTHYROIDISM  It is used to differentiate primary hypothyroidism
 Due to pituitary destruction or pituitary adenoma from secondary hypothyroidism.
 Lab result: T3 and T4 levels are low, TSH is  It is used to monitor and adjust thyroid hormone
also decreased replacement therapy
 the sensitivity of the third generation TSH assay
HYPOTHYROIDISM – has led to the ability to detect what is termed as
TERTIARY HYPOTHYROIDISM subclinical disease- or a mild degree of thyroid
 Due to hypothalamic disease dysfunction (due to large reciprocal change in
 Lab results: T3 and T4 levels are low, TSH is TSH levels seen for even small changes in FT4)
also decreased  Reference value: 0.5 – 5 uU/mL
HYPOTHYROIDISM
 Increased TSH
CONGENITAL HYPOTHYROIDISM
o Primary hypothyroidism
 Also known as cretinism
o Hashimoto’s thyroiditis
 It is a defect in the development or function of o Thyrotoxicosis due to pituitary tumor
the gland o TSH antibodies
 Symptoms: physical and mental development o Thyroid hormone resistance
of the child are retarded
 Decreased TSH
 Screening test: T4 (decreased) o Primary hyperthyroidism
 Confirmatory test: TSH (increased) o Secondary and Tertiary hypothyroidism
 Interpretation: o Treated Grave’s Disease
o TSH value <10 mIU/L – no further test o Euthyroid sick disease
o TSH value 10 – 20 mIU/L – repeat test in 2-
6 weeks

3| Clinical Chemist
CLINICAL CHEMISTRY THYROID GLAND
Professor: William Christopher Happy Reviewing,

o Over replacement of thyroid hormone in REVERSE T3 (rT3)

hypothyroidism
RADIOACTIVE IODINE UPTAKE (RAIU)
 It is used to measure the ability of the thyroid
gland to trap iodine
 It is helpful in establishing the cause of
hyperthyroidism
 Radioactive iodine is ingested by mouth and
measured after 4-6 hours and 24 hours
 Rt3 is formed by removal of one iodine from the
inner ring of T4
 It is an end product of T4 metabolism; the 3rd
major circulating thyroid hormone
 It identifies patients with euthyroid sick
syndrome (elevated rT3)
 It is used to assess the borderline or conflicting
laboratory results
 Reference value: 38 – 44 ng/dL

FREE THYROXINE INDEX (FTI or T7)

THYROGLOBULIN (Tg) Assay
 It is normally used as a postoperative marker of  It indirectly assesses the level of Free T4 in the
thyroid cancer blood
 It is based on equilibrium relationship of bound
 It is used in monitoring the course of metastatic T4 or FT4
or recurrence of thyroid cancer (a well  It is important in correcting euthyroid individuals
differentiated tumor typically displays a 10-fold  It is elevated in hyperthyroidism and decreased
increase in Tg in response to a high TSH) in hypothyroidism
 When measuring Tg as a tumor marker for  Reference value: 5.4 – 9.7
𝑇𝑇4 𝑥 𝑇3𝑈(%)
thyroid cancer, always check simultaneous  𝐹𝑇𝐼 = 𝑜𝑟 𝑇𝑇4 𝑥 𝑇𝐻𝐵𝑅
100
sample for thyroglobulin antibodies

 It differentiates subacute thyroiditis (increased TOTAL T3 (TT3), Free T3 (FT3), and Free T4 (FT4)
Tg) from thyrotoxicosis factitia (decreased Tg)  FT4 test is used to differentiate drug induced
TSH elevation and hypothyroidism
 Increased: untreated and metastatic
 The value of TT3 or FT3 is in confirming
differentiated thyroid cancer, nodular goiter and
hyperthyroidism
hyperthyroidism
 Direct/reference method: Equilibrium dialysis
 Decreased: infants with goitorous (FT4)
hypothyroidism and thyrotoxicosis factitial
(decreased Tg) T3 UPTAKE
 It measures the number of available binding
 Reference value: sites of the thyroxine binding proteins, most
o 3-42 ng/ml or ug/ml (adult) notably TBG; a test for TBG
o 38-48 ng/ml or ug/ml (infant)  Increased: hyperthyroidism, euthyroid patients,
chronic liver disease
 Methods for testing: double-antibody RIA,  Decreased: hypothyroidism, oral contraceptives,
ELISA, IRMA, Immunochemiluminescent assay pregnancy, acute hepatitis
(ICMA)  Reference value: 25-35%
 It differentiates subacute thyroiditis (increased
Tg) from thyrotoxicosis factitia (decreased Tg) FINE NEEDLE ASPIRATE (FNA)

 Increased: untreated and metastatic  The most accurate tool in the evaluation of
differentiated thyroid cancer, nodular goiter and thyroid nodules
hyperthyroidism

 Decreased: infants with goitorous END OF LESSON. GOOD LUCK!

hypothyroidism and thyrotoxicosis factitial
(decreased Tg).

4| Clinical Chemist
 MLS115 Enzymes CLINICAL CHEM 2

WILLIAM CHRISTOPHER C. SALAZAR, RMT | JANUARY, 2021 LE 1 TRANS 01

OUTLINE
I. What are Enzymes?
II. Function of Enzymes
III. General Properties of Enzymes
IV. Measurement of Enzyme Activity
V. Clinical Significance of Enzymes
LEGEND
Remember Lecturer Book
I. WHAT ARE ENZYMES?
 biological proteins that catalyze biochemical reactions
 not consumed or changed in composition
 found in all body tissues
 increased in serum after cell injury
II. FUNCTION OF ENZYMES
 Hydration of Carbon dioxide (respiration)
 Nerve induction
 Muscle contraction
 Nutrient degradation
 Growth and reproduction
 Energy Storage and use
III. GENERAL PROPERTIES OF ENZYMES
A. Components of Enzymes
 Active site – a cavity of an enzyme where the substrate binds and
undergo chemical reaction
 Holoenyme – active enzyme/substrate formed by combination of a
coenzyme and an apoenzyme
 Zymogen/Proenzyme – inactive enzyme precursor
(coagulation cofactors, digestive enzymes)
B. Terms Associated with Enzymes
 Substrate – substance acted upon by enzymes specific for each of
Previous Presentation their particulate enzyme
Trans  General Form of Enzyme Reaction
 --
 Cofactor – non protein substance added in enzyme substance
complex to manifest the enzyme activity
 Two types of cofactors
1. Coenzyme/Prosthetic Group
Organic cofactor
Nucleotide (NAD, NADP, Vitamins)
2. Activator
Inorganic cofactor
Metal in (Cl, Mg, Cu, Zn)
C. Enzyme Classification
 Oxidoreductase
 Catalyzes the REDOX reaction between two substances
 Lactate dehydrogenase, Glucose-6-phospate dehydrogenase
 Transferase
 Catalyzes the transfer of a group other than hydrogen ion
 ALT, AST, GGT, CK
 Hydrolase
 Catalyze hydrolysis of bonds
 Esterase – ALP , ACP, Cholinesterase, Lipase
 Peptidase – trypsin, pepsin, leucine amino peptidase
 Glycosidase – amylase, galactosidase
 Lyases
 Catalyzes removal of groups of substances without hydrolysis
 Aldolase, pyruvate decarboxylase, glutamate decarboxylase,
tryptophan decarboxylase.

Hydrolysis : splitting of substances involving H2O

Esterase : splits esters into acid and an alcohol
Peptidase : breaks down peptides into amino acids
Glycosidase : catalyze the hydrolysis of glycosidic bonds in
complex sugars

 Isomerase
 Catalyzes the interconversion of geometric, optical, or positional
isomers
Figure 1. General Properties of Enzymes
 Triphosphate isomerase, ribose phosphate isomerase, glucose
 Allosteric site – a cavity other than the active site that binds the phosphate isomerase.
regulatory molecule (effector)  Ligase
 Catalyzes the joining of two substrate molecules
 Glutathione synthase

D. Enzyme kinetics
 Factors that influence Enzymatic Reaction
 Substrate concentration
 First Order Kinetics
 Reaction rate is directly proportional to Substrate
Concentration
 Enzyme excess
First “S” for SUBSTRATE
 Enzyme concentration
Figure 2. Enzyme Activity: Allosteric site
 Zero Order Kinetics
 Isoenzyme – enzymes of similar enzymatic activity but differs in
 Reaction rate depends on Enzyme Concentration
physical, biochemical and immunologic characteristics
 Substrate excess
 Apoenzyme – protein portion of the enzyme subject to denaturation
Zero “Z” for ENZYME
in which it loses its activity.
 pH

MICROBIOLO
1
1.01 LE TRANS
 Most physiologic reactions occur in the pH range of 7 – 8 Figure 5. Uncompetitive Inhibitor
 Controlled by a buffer
 Temperature IV. MEASUREMENT OF ENZYME ACTIVITY
 Increase Temp = increased rate of chemical reaction A. Enzyme Quantitation
 For every 10 degrees of Celsius increase, the reaction rate  Measurement of catalytic enzymes
will be DOUBLE  Activity is related to the concentration
 Enzyme is active at 25, 30, and 37 degrees of Celsius  Photometric
 40 – 50 degrees of Celsius = significant denaturation  Increase in product concentration
of enzyme.  Decrease concentration
 Cofactor  Increase in concentration of altered coenzyme
 Activator
 Metallic (Ca) B. General Methods of Measuring Enzymatic Reactions
 Non-metallic (Cl)  Fixed time
 Coenzymes (prosthetic group)  End point
 Secondary substrate (NAD)
 Reagents are combined and the amount of reaction is measured
 Inhibitor
Measures the concentration after the endpoint has been
 Competitive Inhibitor
reached
 Binds to active site (reversible)
 Continuous time
 Non-Competitive Inhibitor
 Kinetic
 Does not bind to the active site but to the allosteric site
 Uncompetitive Inhibitor  Measurement at specific time intervals
 Binds to the Enzyme-Substrate Complex  Continuous measurement
 Competitive Inhibitor
 Binds to active site (reversible) V. CLINICAL SIGNIFICANCE OF ENZYMES
A. MI (Myocardial Infarction) Profile
 Creatine Kinase (CK)
 Aspartate aminotransferase (AST)
 Lactate Dehydrogenase (LDH)

1. Creatine Kinase
 Storage of high energy creatine phosphate in muscle cells
 Highest activity is in skeletal muscle, heart and brain tissue
 Creatine kinase is used to assess Myocardial Infarction
 Isoenzymes
 CK – 1 (CK-BB) Brain type
- Serum rarely contains CK-BB of brain because of its
molecular size (80,000); its passage across the BBB
is Hindered
- Increased in
o CNS Shock, Seizures
Figure 3. Competitive Inhibitor o Lung and gastrointestinal diseases
 CK – 2 (CK-MB) Hybrid type
 Non-Competitive Inhibitor - Values for MB range should be <6% of Total CK
 Does not bind to the active site but to the allosteric site - >6% of total CK = AMI
o Rise: 4-8 hours
o Peak 12-24 hours
o Return to normal 48-72 hours
- Other increased levels are seen in other cardiac
disorders, probably reflect ischemic heart
damage.
 CK -3 (CK-MM) Muscle type

Figure 4. Non-Competitive Inhibitor

 Uncompetitive Inhibitor
 Binds to the Enzyme-Substrate Complex

Table 1. Isoenzyme CK
 Macro-CK
 Largely comprises CK-BB complexed with an
immunoglobulin
 Mitochondrial-CK
 Not present in normal serum and NOT present following AMI

CLINICAL
2
 1.01 LE TRANS
 Extensive tissue damage must occur first (breakdown Lactate Dehydrogenase
of mitochondrion and cell wall)  Electrophoresis
 Appears to be an indicator of SEVERE ILLNESS  LD-1 FASTEST; MOST ANODAL
 LD-2
2. Aspartate Aminotransferase  LD-3
 Not absolute  LD-4
 Not used alone because it is not specific and it has a wide  LD-5 SLOWEST; MOST CATHODAL
distribution to various organs  Normal isoenzyme concentration:
 Involved in synthesis and degradation of amino acids  LD2>LD1>LD3>LD4>LD5
 Formerly SGOT (Serum Glutamic-Oxaloacetic transaminase  “LD Flipped Pattern”
 Highest activity in cardiac, liver, and skeletal muscles  LD1>LD2>LD3>LD4>LD5
Occurs in:
 AMI
 Major clinical uses:  Intravascular Hemolysis
 Myocardial Infarction  Hemolyzed Specimen
 Hepatocellular Disorders
 Viral Hepatitis (100x ULN)
 Cirrhosis (4x ULN)
- Skeletal muscle disease (4x ULN)
- Also a marker for AMI
o Rise: 8-12 hour
o Peak: 24 hours
o Return to normal: 5
days ULN : Upper limits of normal
De ritis ratio?
AST/ALT Table 3. MI Profile
<1 = acute disorders of the liver, acute viral hepatitis,
infectious mononucleosis B. Liver Enzymes
>1 = chronic disorders of the liver, alcoholic liver disease,  Alanine aminotransferase (ALT)
post-necrotic cirrhosis, chronic active hepatitis  Alkaline phosphatase (ALP)
 Gamma-glutamyltransferase (GGT)
3. Lactate Dehydrogenase
 Lactate dehydrogenase (LD)
 Widely distributed
 Aspartate aminotransferase (AST)
 Most Non-Specific enzyme
 Highest activity in heart, hepatic, skeletal muscle, and RBC 1. Alanine Aminotransferase
 Increased levels are seen in:  Formerly called – Serum glutamic-pyruvic transaminase (SGPT)
 Cardiac diseases
 Similar activity to AST, catalyzes transfer of an amino group from
 Hepatic diseases
alanine to a-ketoglutarate with the formation of glutamate and
 Skeletal diseases
pyruvate
 Renal diseases
 Distributed in many tissues, highest concentration in the liver
 5x increase in megaloblastic anemia
 It is the more liver specific enzyme among the transferases
 Highest levels are seen in Pernicious anemia and hemolytic
 Used to evaluate hepatic disorders (ALT elevations are higher
disorders
than AST)
 Marked elevations are also seen in Acute lymphoblastic
 Cardiac tissue contains small amount of ALT activity.
Leukemia
 Also a marker for AMI:
2. Alkaline phosphatase
 Rise: 12-24 hours
 Catalyze hydrolysis of phosphoesters
 Peak: 48-72 hours
 Cleaves phosphate group of the phosphomonoesters producing
 Return to Normal:10 days (best indicator for delayed check-ups
phosphate ion
due to chest pain)
 Not specific, can react with many different substrate
 More active in an alkaline pH
 Requires Mg activator
 Used in the evaluation of heptobillary and bone disorder
 Isoenzymes
 Liver ALP isoenzymes
 Increased in liver diseases
 Bone ALP isoenzymes
 Increased in bone disease, healing of bone fractures, and
physiologic bone growth
 Placental ALP isoenzyme
 Increased in pregnancy
 Intestinal ALP isoenzyme
 Increased in GIT disorders

Table 2. Isoenzyme LD

CLINICAL
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 1.01 LE TRANS
Table 5. Amylase
Lipase
 Hydrolyzes ester linkages of fats to produce alcohol and fatty acids
 Most specific marker for acute pancreatitis
 Large molecule remains in the circulation for up to 7 days
 During acute pancreatitis
Rise: 8 hours
Peak 24 hours
Normalize: 8-14 days

Table 4. Alkaline Phosphatase

3. 
Gamma-glutamyltransferase
Catalyze transfer of the gamma-glutamyl residue from peptides to
Table 6. Lipase
amino acids or water molecules
 Present in cells in bile and hepatic duct D. Other Enzymes
 Increased in: Acid Phophatase
 Hepatobillay disorders (billary tract obstruction)  Prostate enzyme
 Chronic alcoholism
 Catalyze hydrolysis of phosphomonoesters
 Enzyme inducing medications: warfarin,
 Evaluation of metastatic carcinoma of prostate
phenobarbital, phenytoin
 smoking; 10% increase for moderate smokers, 20% increase  Forensic investigation of rape cases
for heavy smokers  12 hours up to 4 days
 Useful in differentiating the source of an elevated ALP  Non specific
 GGT levels are increased in liver disorders  Found in
 GGT levels are normal in skeletal disorders and pregnancy  Liver, spleen, kidney
 Bone, RBCs, platelets
C. Pancreatic Enzymes  Prostate – Richest source (prostatic carcinoma)

 Amylase
V. METHODS OF DETERMINATION
A. Creatine Kinase
 Lipase
 Tanzer-Gilvarg
 Forward reaction
1. Amylase
 pH = 9.0
 Increased in acute pancreatitis (non-specific)
 Measures decrease in absorbance @340 nm
 Requires calcium and chloride for activation
CK
 Smallest enzyme (mol wt. 50,000)
 Creatine + ATP  Creatine phosphate + ADP
 Only protein that can be cleared by the kidneys
 Amylase levels during acute pancreatitis
 Oliver Rosalki
 Rise: 2-12 hours
 Reverse reaction
 Peak 24 hours
 pH = 6.8
 Normalize 3-5 days
 Other elevations are seen in:  6x faster than the forward reaction
 Mumps and parotitis (salivary gland lesions)  Measures increase in absorbance @340nm
 Diabetic ketoacidosis CK
 Renal insufficiency  Creatine phosphate + ADP  Creatine + ATP
 Intraabdominal diseases
 Peptic ulcer, intestinal obstruction B. Aspartate aminotransferase
 Cholecystitis, acute appendicitis  Karmen method
 pH = 7.3 – 7.8
Macroamylasemia  Uses malate dehydrogenase as the indicator enzyme
 Results when AMS molecules combine with immunoglobulins to  Measures decrease in absorbance @340nm
form a complex that is too large to be filtered across the  Used to diagnose hepatocellular disorders
glomerulus AST
 Serum AMS levels increase because of the reduction in renal  Aspartate + a-ketogluterate  glutamate + oxaloacetate
clearance
C. Lactate Dehydrogenase
 Wacker method
 Forward reaction

 pH = 8.3-8.9
 Measures increase in absorbance @340nm
 Source of error
 Hemolysis – RBCs contain 100x LD concentration than
that found in serum
 Cold – loss of LD-5 activity

 Wrobleuski-Ladue
 Reverse reaction

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 1.01 LE TRANS
 3. Chromogenic
 pH = 7.2-7.4  Measures the increasing odor from products
 Measures decrease in absorbance @340nm 4. Continuous coupled
 Utilized by dry-slide methods

D. Alanine aminotransferase

 Coupled enzymatic reaction

 Uses lactate dehydrogenase as indicator enzyme
 pH = 7.3–7.8
 Measures change in absorbance @340nm
ALT
 Alanine + a-ketogluterate  pyruvate + glutamate

E. Alkaline phosphatase

 Bower’s and McComb

 Uses p-nitrophenylphosphate (colorless) as substrate
 Based on molar absorptivity of p-nitrophenol (yellow color)
 Optimal pH = 10
 Measures increase in absorbance @405nm
ALP
 P-nitrophenyl-phosphate  p-nitro-phenol + phosphate ion

 Alkaline Phosphatase
 ALP isoenzymes
 Methods to determine ALP isoenzymes
1. Heat stability – heating serum at 56C for 10mins
 Placenta – heat stable (resist heat denaturation at 65C
for 30mins)
 Intestine
 Liver
 Bone – heat labile

 ALP activity is measured before and after heating serum at 56

degrees celcius for 10 mins.
 If the residual activity after heating is less than 20% of the
total activity before heating, then ALP elevation is assumed
to be a result of bone phosphatase.
 If greater than 20% of the activity remains, the elevation
is probably a result of liver phosphatase.
 Imprecise method due to
 Correct temperature control
 Timing
2. Electrophoresis – mobility
 Liver: most anodal
 Bone
 Placenta
 Intestine: least anodal
3. Chemical inhibition
 Phenylalanine – inhibits placenta and intestine
 L-leucine – inhibits the abnormal nagao isoenzyme
 Levamisol – inhibits liver and bone
 3M urea (synthetic urea) – inhibits bone

F. Gamma-glutamyltransferase

 SZASZ assay
 Uses y-glutamyl-p-nitroanilide as substrate
 Measures absorbance @405-420nm
 GGT activity is stable with no loss of activity for 1 week at 4C
 Not affected by hemolysis

G. Amylase

1 .Amyloclastic (Iodometric)
 Measures disappearance of starch
 Substrate: starch molecule with iodine
 Decrease in color is proportional to AMS concentration
2. Saccharogenic (Reference method)
 Measures appearance of product
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 1.01 LE TRANS
 Coupling of several enzymes to measure AMS activity

Table 7.
Lipase

H. Acid Phosphatase

 Same as ALP but done in an acid pH

 Inhibitors
 L-tartrate ions
 Formaldehyde and cupric ions
 Quantitative
 Thymolpthalein monophosphate
 Continuous monitoring
 Alpha-napthyl-phosphate
 pH 5.0

CLINICAL
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IMMUNOLOGY & SEROLOGICAL
SEROLOGY APPLICATION

SEROLOGICAL APPLICATION  Identify serological and molecular techniques

LEARNING OBJECTIVES: to detect rickettsial infections.
 Discuss the advantages of direct fluorescent
staining for T. pallidum over darkfield  Explain the antibody testing that is performed
examination without staining. to detect infections to Mycoplasma
pneumoniae.
 Define reagin.
 State important diagnostic information
 Distinguish treponemal from nontreponemal provided by serological testing for Toxoplasma
tests for syphilis. gondii.

 Describe the principle of the following tests for SYPHILIS

syphilis: Venereal Disease Research
Laboratory (VDRL), rapid plasma reagin
(RPR), fluoresecent treponemal antibody
absorption (FTA-ABS), and agglutination
assays.
 Give reasons for false-positive reagin test
results.
 Compare and contrast sensitivity and
specificity of treponemal and nontreponemal
testing for the various stages of syphilis.
 Discuss the advantages and disadvantages of
polymerase chain reaction (PCR) and enzyme
immunoassay (EIA) testing for syphilis.
 Spirochetes are long, slender, helically coiled
bacteria containing axial filaments, or
periplasmic flagella, which wind around the
bacterial cell wall and are enclosed by an outer
sheath. These gram-negative, microaerophilic
bacteria exhibit a characteristic corkscrew
flexion or motility.
 Diseases caused by these organisms have
many similarities, including a localized skin
infection that disseminates to numerous
organs as the disease progresses, a latent
stage, and cardiac and neurological
involvement if the disease remains untreated.

 Discuss limitations of cerebrospinal fluid (CSF)

testing for neurosyphilis and testing for
congenital syphilis.

 Discuss reasons for performing antibody

rather than antigen testing for sequelae of
streptococcal infections.
 State the principle of the antistreptolysin O
(ASO) titer test.
 Explain how the presence of anti-
deoxyribonuclease B (anti-DNase B) is
detected.
 Compare the sensitivity of the streptozyme test
to other tests for streptococcal antibodies.
 Interpret laboratory data to diagnose sequelae
of streptococcal infections.
 Discuss the various types of testing that may
be done to detect H. pylori infection.
 Explain the testing that may be done to detect
antibodies to H. pylori.
[Yun malalaking table images, nasa dulong page.
Hindi ko isasama dito kasi hindi readable.]
LABORATORY DIAGNOSIS
 The first diagnostic blood test for syphilis was
the Wassermann test, a complement fixation
test developed in 1906.
 Serological testing plays a key role in
diagnosis of these diseases, because isolation
of the organism itself is difficult to accomplish
in the laboratory, and clinical symptoms are
not always apparent.
 Traditional laboratory tests for syphilis can be
classified into three main types: direct
detection of spirochetes, nontreponemal
serological tests, and treponemal
serological tests.
DIRECT DETECTION OF SPIROCHETES
 Direct detection of spirochetes can be
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accomplished by darkfield
microscopy or

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fluorescent antibody testing. The
performance of either test requires that the
patient have active lesions.
DARK-FIELD MICROSCOPY
 Primary and secondary syphilis can be
diagnosed by demonstrating the presence of
T. pallidum in exudates from skin lesions.
 In dark-field microscopy, a dark-field
condenser is used to keep all incident light out
of the field except for that captured by the
organisms themselves.
 It is essential to have a good specimen in the
form of serous fluid from a lesion.
 Pathogenic treponemes are identified on the
basis of characteristic corkscrew morphology
and flexing motility.
 False-negative results can occur due to delay
in evaluating the slides, an insufficient
specimen, or pretreatment of the patient with
antibiotics. Thus, a negative test does not
exclude a diagnosis of syphilis.
 In addition, an experienced microscopist
should perform testing. If a specimen is
obtained from the mouth or the rectal area,
morphologically identical nonpathogens can be
found, so these must be differentiated from the
true pathogens.
FLUORESCENT ANTIBODY
TESTING OF A SPECIMEN
 The use of a fluorescent-labeled antibody is a
sensitive and highly specific alternative to
dark- field microscopy.
 This can be performed by either a direct
method, which uses a fluorescent-labeled
antibody conjugate to T. pallidum, or an indirect
method using antibody specific for T. pallidum
and a second labeled anti-immunoglobulin
antibody.
 An advantage of this method is that live
specimens are not required.
SEROLOGICAL TESTS
 Serological tests can be classified as
nontreponemal or treponemal, depending on
the reactivity of the antibody that is detected.
SEROLOGICAL
APPLICATION
 Nontreponemal tests, which detect antibody
to cardiolipin, have traditionally been used to
screen for syphilis because of their high
sensitivity and ease of performance.
 However, false-positive results are common
because of the nonspecific nature of the
antigen. Therefore, any positive results must
be confirmed by a more specific treponemal
test, which detects antibodies to T. pallidum.
NONTREPONEMAL TESTS
 Nontreponemal tests determine the presence
of reagin, an antibody that forms against
cardiolipin, a lipid material from damaged cells.
This is found in the sera of patients with
syphilis and several other disease states.
 An antigen that is a combination of cholesterol,
lecithin, and cardiolipin is used in the reaction
to detect the nontreponemal reaginic
antibodies, which are either of the IgG or IgM
class.
 The traditional nontreponemal tests are based
on flocculation reactions in which patient
antibody complexes with the cardiolipin
antigen.
 Flocculation is a specific type of precipitation
that occurs over a narrow range of antigen
concentrations. The antigen consists of very
fine particles.
 In general, nontreponemal tests are positive
within 1 to 4 weeks after the appearance of
the primary chance. Titers usually peak during
the secondary or early latent stages.
 In primary disease, between 13 and 41
percent of individuals appear nonreactive,
but by the secondary stage, almost all patients
have reactive test results.
 However, testing of sera from patients in the
secondary stage is subject to false negatives
because of the prozone phenomenon
(antibody excess).
 Reagin titers tend to decline in later stages of
the disease even if the patient remains
untreated.
 A first-time infection, if in the primary or
secondary stage, should show a fourfold
decrease in titer by the third month following
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SEROLOGY APPLICATION

treatment and an eightfold decrease by 6 to 8 to large clumps), weakly reactive (small

months.

 Following successful treatment, tests typically

become completely nonreactive within 1 to 2
years.

 Examples of nontreponemal tests include

Venereal Disease Research Laboratory
(VDRL), rapid plasma reagin (RPR),
toluidine red unheated serum test (TRUST),
unheated serum reagin (USR), and reagin
screen test (RST).

 The RPR and the VDRL are the most widely

used of these tests.

VENEREAL DISEASE RESEARCH

LABORATORY TEST
 The VDRL TEST, which was designed by the
Venereal Disease Research Laboratories, is
both a qualitative and quantitative slide
flocculation test for serum, and there is a
modification for use on spinal fluid.

 The antigen is an alcoholic solution of 0.03

percent cardiolipin, 0.9 percent cholesterol,
and
0.21 percent lecithin.

 Basically, the antigen suspension is prepared

by adding the VDRL antigen via a dropper to a
buffered saline solution while continuously
rotating on a flat surface; attention must be
paid to required rotation speed and timing. A
daily calibrated Hamilton syringe is used to
deliver one drop of antigen for the slide test.

 The serum specimens to be tested are heated

56oC for 30 minutes to inactive complement,
and 0.05 mL is pipetted into a ceramic ring of a
glass slide.

 Three control sera – nonreactive, minimally

reactive, and reactive – are pipetted into rings
on the glass slide in the same manner. Sera
and patient samples are spread out to fill the
entire ring. One drop (1/60 mL) of the VDRL
antigen is then added to each ring. The slide is
rotated for 4 minutes on a rotator at 180
rpm.

 It is read microscopically to determine the

presence of flocculation, or small clumps.

 The results are recorded as reactive (medium

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SEROLOGY APPLICATION

clumps), or nonreactive (no clumps or slight  Antigen is dispensed from a small plastic
roughness). dispensing bottle with a calibrated 20-gauge
needle.
 Tests must be performed at room
temperature within the range of 23o-C to
29oC (73F to 85F), because results may
be affected by temperature changes.

 All sera with reactive or weakly reactive

results must be tested using the
quantitative slide test, in which twofold
dilutions of serum ranging from 1:2 to
1:32 are initially used. Sera yielding
positive results at the 1:32 dilution are
tittered further.

 The VDRL test and some of the newer

ELISA tests are the only ones routinely
used for the testing of spinal fluid.

 For VDRL spinal fluid testing, the

antigen volume used is less than the
serum test and is at a different
concentration. In addition, different
slides are used (Boerner agglutination
slides).

 The test is read microscopically, as in

the VDRL serum test. If a test is
reactive, twofold dilutions are made and
retested following the same protocol.

 A positive VDRL test on spinal fluid is

diagnostic of neurosyphilis, because
false positives are extremely rare.

 The RPR is a modified VDRL test RAPID PLASMA REAGIN (RPR) TEST
involving macroscopic agglutination.

 The cardiolipin-containing antigen

suspension is bound to charcoal
particles, which make the test easier to
read.

 The antigen is similar to the VDRL

antigen with the addition of
ethylenediaminetetraacetic (EDTA),
thimerosal, and choline chloride, which
stabilize the antigen and inactivate
complement so that serum does not
have to be heat- inactivated before use.

 Patient serum (approximately 0.5mL) is

placed in an 18 mm circle on a plastic-
coated disposable card using a capillary
tube or Dispenstir device.

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 One free-falling drop is placed onto each test
area, and the card is mechanically rotated
under humid conditions.
 Cards are read under a high-intensity light
source, and if flocculation is evident, the test is
positive.
 All reactive tests should be confirmed by
retesting using doubling dilutions in a
quantitative procedure.
 The RPR test appears to be more sensitive
than the VDRL in primary syphilis.
STANDARD TREPONEMAL
SEROLOGICAL TESTS
 Treponemal tests detect antibody directed
against the T. pallidum organism or against
specific treponemal antigens.
 The two main types of treponemal tests
include the fluorescent treponemal antibody
absorbed (FTA-ABS) test and agglutination
tests.
 Typically, these tests are more difficult to
perform and are more time-consuming than
nontreponemal tests, so they have been
traditionally used for confirmation rather than
screening.
FLUORESCENT TREPONEMAL
ANTIBODY ABSORPTION TEST
 One of the most used confirmatory tests is the
FTA-ABS test, an indirect fluorescent
antibody test.
 A dilution of heat inactivated patient serum is
incubated with a sorbent consisting of an
extract of nonpathogenic treponemes (Reiter
strain). This removes cross-reactivity with
treponemes other than T. pallidum.
 Slides used for this test have the Nichols
strain
of T. pallidum fixed them.
 They are kept frozen until use and then are
equilibrated at room temperature for 30
minutes.
 Diluted patient samples and controls are
measured and applied to individual wells on
the test slide.
SEROLOGICAL
APPLICATION
 Slides are then incubated in a covered moist
chamber at 37OC for 30 minutes. They are
rinsed with deionized water and placed in a
coplin jar with phosphate-buffered saline for 5
minutes.
 After a second rinsing, the slides are air-dried,
and antibody conjugate is added to each well.
Slides are reincubated as before, and a similar
washing procedure is followed.
 Mounting medium is applied, and coverslips
are placed on the slides. They are examined
under a fluorescence microscope as soon as
possible.
 If specific patient antibody is present, it will
bind to the T. pallidum antigens. The antibody
conjugate will, in turn, only bind where patient
immunoglobulin is present and bound to the
spirochetes.
 When slides are read under a fluorescence
microscope, the intensity of the green color is
reported on a scale of 0 to 4+.
 No fluorescence indicates a negative test, and
a result of 2+ or above is considered
reactive.
 A result of 1+ means that the specimen was
minimally reactive, and the test must be
repeated with a second specimen drawn in 1
to 2 weeks.
 The FTA-ABS is highly sensitive and specific,
but it is time-consuming to perform. This test is
usually positive before reagin tests, although
20 percent of primary syphilis cases are
nonreactive.
 In secondary and latent syphilis, tests are
usually 100 percent reactive. Once a patient is
reactive, that individual remains so for life.
 Although, there are fewer false positive
compared to reagin tests, reactivity is seen
with other treponemal diseases, notably yaws
and pinta.
AGGLUTINATION TESTS
 Several varieties of passive hemagglutination
tests have been used to detect specific
treponemal antibody.
 Particle agglutination tests such as the serodia
TP-PA test have largely replaced
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microhemagglutination tests.

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SEROLOGY APPLICATION

 Particle agglutination tests use colored
gelatin particles coated with treponemal
antigens and are more sensitive in detecting
primary syphilis.
 In the Serodia TP-PA test, patient serum or
plasma is diluted in microtiter plates and
incubated with either T.pallidum – sensitized
gel particles or unsensitized gel particles as a
control.
 Patients would be screened by the EIA test
followed by an RPR or equivalent test.
 If the initial EIA was negative, no further
testing would be done unless early syphilis is
suspected (i.e., prior to seroconversion).
 If the EIA was positive and the subsequent
RPR was positive, then it would be considered
positive for syphilis.

 Presence of T. pallidum antibodies is indicated

by agglutination of the sensitized gel particles,
which form a lattice-like structure that spreads
to produce a smooth mat that covers the well’s
surface.

 In wells containing a sample that is negative

for the antibody, the gel particles settle to the
well’s bottom and form a compact button.

ENZYME IMMUNOASSAY

 Several enzyme immunoassay (EIA) tests

have been developed for serodiagnosis of
syphilis.

 One of these, ReaginII, uses a cardiolipin

antigen similar to that used in the VDRL test.
This method can easily screen large number of
samples, and it is reported to have a sensitivity
of 93 percent in primary syphilis. However, it
also has a slightly higher rate of false positives
than RPR tests.

 Other EIA tests have been developed to

capture a specific class of antibody, either IgM
or IgG.

 Microtiter wells are coated with antibody to IgM

or IgG and are reacted with patient serum.
Treponemal antigens that are labeled with an
enzyme are then added (Fig. 21–3).

 These have a fairly high sensitivity in primary

syphilis, but the sensitivity decreases as the
disease progresses. Specificity is similar to
other treponemal tests. WESTERN BLOT

 EIA tests are especially useful in diagnosing  Immunoblotting, or Western blotting,

congenital syphilis in infants, as they look for is a method that, in many systems, is a
presence of IgM, which cannot cross the confirmatory test (e.g., HIV testing).
placenta.  Likewise, in Lyme disease testing, it is
the second test in the CDC-
 Under this scheme, the testing order is recommended two-tier testing scheme.
reversed from the traditional nontreponemal However, the Lyme disease
screening followed by confirmatory treponemal immunoblot
testing strategy.
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IMMUNOLOGY &
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is very complex (Fig. 21–7) nitrocellulose paper.
 These strips are reacted with patient serum
and developed with an antihuman globulin
(either anti-IgG or anti-IgM) to which an
enzyme label is attached.
 The further incubation with the enzyme’s
substrate allows for visualization of any
antibody that has bound to a particular
antigen. The reactivity is then scored and
interpreted.
 Ten proteins are used in the CDC-
recommended interpretation of this test.
 They are designated by their molecular
weights: 18, 23, 28, 30, 39, 41, 45, 58, 66, and
93 kDa.
 For a result to be considered positive for
presence of specific IgM antibody, two of the
following bands must be present: 23(OspC),
39, and 41 (flagellin) kDa.
 An IgG immunoblot is considered positive if any
5 of the 10 bands listed above are positive.
 As previously stated, the currently accepted
testing protocol calls for a two-step approach
with the use of EIA or IFA as initial screening
tools. Then, if a test is equivocal or positive, it
should be further tested by Western blot.
 Current CDC recommendations do not advise
testing seropositive/borderline patients for IgM
antibodies if they have had symptoms for more
than 4 weeks for the reasons outlined above.
 Serological evidence of Lyme disease in these
patients is indicated by a positive result in the
IgG immunoblot.
POLYMERASE CHAIN REACTION
 In testing for Lyme disease, the PCR has
found a niche in certain scenarios. While only
a few organisms need to be present for
detection under optimal conditions, the number
of spirochetes in infected tissues and body
fluids is low, making specimen collection,
transport, and preparation of DNA critical to
these clinical specimens and sensitivity an
issue.
 Several probes for target DNA that is present
only in strains of B. burgdorferi have been
made and used in PCR testing.
SEROLOGICAL
APPLICATION
 The procedure involves extracting DNA from
the patient sample followed by amplification
using specific primers, DNA polymerase, and
nucleotides.
 Once a sufficient amount of the unknown DNA
is made, this is combined with a known DNA
probe to see if hybridization takes place.
 The single-stranded Borrelia DNA probe will
bind only to an exact complementary strand,
thus positively identifying the presence of the
organism’s DNA in the patient sample.
 This is much more specific than testing for
antibody, because there is little cross-
reactivity.
 Specificity of recent PCR studies has ranged
from 93 percent to 100. However, sensitivity
remains an issue.
 In a series of studies, the median sensitivity of
PCR on skin biopsies was 69 percent; of blood
components, 14 percent; of CSF, 38 percent;
and of synovial fluid, 78 percent. However, the
range of sensitivities in any one type of
specimen is quite large, suggesting the testing
remains to be standardized.
 Furthermore, it would be hard to clinically
justify a skin biopsy for PCR as a diagnostic
method for an EM rash in most cases.
However, in difficult diagnostic neurological
and arthritic cases, PCR on CSF and synovial
fluid is often employed.
TYPHOID FEVER
 Widaland Sicard developed one of the earliest
diagnostic tests in 1896 for the detection of
antibodies occurring in typhoid fever,
brucellosis, and tularemia.
 Widaltest, a rapid screening test to help
determinethe possibility of typhoid fever.
 The antigens used in this procedure include
Salmonella O (somatic) and H (flagellar)
antigens.
 A significant finding is a fourfold increase in
antibody titer over time when paired dilutions
of serum samples are tested with any of these
antigens.
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STREPTOCOCCAL INFECTION
 Streptococci are gram-positive spherical,
ovoid, or lancet-shaped organisms that are
catalase negative and are often seen in pairs
or chains.
 They are divided into groups or serotypes on
the basis of certain cell wall components.
 The outermost cell wall component contains
two major proteins known as M and T, and
these determine the serogroup or serotype
(Fig 19-2).
 The serotype is based on minor variations in
the proteins that can be identified
serologically.
 Interior to the protein layer is the group-
specific carbohydrate that divides streptococci
into 20 defined groups, designated A through
H and K through V. These are known as the
Lancefield groups.
 Some strains possess a hyaluronic acid
capsule outside the cell wall that contributes to
the bacterium’s antiphagocytic properties.
GROUP A STREPTOCOCCI
 Streptococcus pyogenes, which belongs to
Lancefield group A, is one of the most
common and ubiquitous pathogenic bacteria
and causes a variety of infections.
SEROLOGICAL
APPLICATION
 The M protein is the major virulence factor of
the group.
 There is a net-negative charge at the amino-
terminal end that helps to inhibit phagocytosis.
 In addition, presence of the M protein limits
deposition of C3 on the bacterial surface,
thereby diminishing complement activation.
 Immunity to group A streptococci appears to
be associated with antibodies to the M protein.
 There are more than 100 serotypes of this
protein, and immunity is serotype specific.
Therefore, infections with one strain will not
provide protection against another strain.
 Additional virulence factors include
exoantigens or exotoxins, proteins excreted
by the bacterial cells as they metabolize during
the course of streptococcal infections.
 Pyrogenic exotoxins A, B, and C are
responsible for the rash seen in scarlet fever
and also appear to contribute to pathogenicity.
 Antibodies are produced to the following
exoantigens: enzymes streptolysin O,
deoxyribonuclease B (DNase B),
hyaluronidase, nicotinamide adenine
dinucleotidase(NADase), and streptokinase.
 Culture and rapid screening methods are
routinely used for diagnostic testing early in
the infection. However, diagnosis of the
sequelae such as glomerulonephritis and
acute rheumatic fever is best achieved by
detection of antibodies.
DETECTION OF GROUP A STREPTOCOCCAL
ANTIGENS
 As an alternative to culture, commercial kits
have been developed to detect group A
streptococcal antigen using throat swabs.
 Antigen is extracted by either enzymatic or
chemical means, and the process takes
anywhere from 2 to 30 minutes, depending on
the particular technique.
 Either enzyme immunoassay (EIA) or latex
agglutination is then used to identify the
antigen.
 Many of these tests require no more than 2 to
5 minutes of hands-on time, providing a
distinct advantage over culture.
 Recent developments using enzyme
immunoassays have improved the test’s
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SEROLOGY APPLICATION

sensitivity, but cultures should be performed

 The onset of clinical symptoms of rheumatic
when rapid test results are negative.
fever or glomerulonephritis typically coincides
with the peak of antibody response, so a
 Molecular methods, including hybridization of serum specimen tested at the time should
specific rRNA sequences and real-time PCR demonstrate an elevation of the concentration
(polymerase chain reaction), have also been of these antibodies.
developed as a means to rapidly detect group
A streptococcal infections.
 If acute and convalescent phase sera are
tested in parallel, a fourfold rise in titer (a two-
 Serotyping and molecular techniques can be tube difference in the doubling serial dilutions)
used to identify a particular strain of group A is considered significant.
streptococcus associated with an epidemic.
 The use of at least two tests for antibodies to
 Serotyping involves identification of M-protein
different exotoxins is recommended, because
antigens by precipitation with type-specific
production of detectable ASO does not occur
antisera.
in all patients.
 However, serotyping has limitations, including
 The most commonly used tests are those for
limited availability of typing sera, new M types
ASO and anti-DNase B.
that do not react with the antisera, and
difficulty in interpreting the results.
ANTISTREPTOLYSIN O (ASO) TESTING
DETECTION OF STREPTOCOCCAL ANTIBODIES
antibodies.
 Culture or rapid screening methods are
extremely useful for diagnosis of acute
pharyngitis. However, serological diagnosis
must be used to identify rheumatic fever and
glomerulonephritis, because the organism is
unlikely to be present in the pharynx or on the
skin at the time symptoms appear.

 Group A streptococci elaborate more than 20

exotoxins, and it is the antibody response to
one or more of these that is used as
documentation of nonsuppurative disease.
Some of the exotoxin products include
streptolysins O and S; deoxyribonucleases A,
B, C, and D; streptokinase; NADase;
hyaluronidase; diphosphopyridine
nucleotidase; and pyrogenic exotoxins (see
Fig. 19–2).

 The most diagnostically important antibodies

are the following: anti-streptolysin O (ASO),
anti-DNase B, anti-NADase, and anti-
hyaluronidase.

 Assays for detection of these antibodies can

be performed individually or through use of the
Streptozymekit which detects antibodies to all
these products.

 During group A streptococcal infections, other

antibodies are made to cellular antigens, such
as the group A carbohydrate and the M
protein.

 Serological evidence of disease is based on

an elevated or rising titer of streptococcal

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 ASO tests detect antibodies to the

streptolysin O enzyme produced by
group A streptococcus, which is able to
lyse red blood cells.

 Presence of antibodies to streptolysin O

indicates recent streptococcal infection in
patients suspected of having acute
rheumatic fever or poststreptococcal
glomerulonephritis following a throat
infection.

 The classic hemolytic method for

determining the ASO titer was the first
test developed to measure streptococcal
antibodies. This test was based on the
ability of antibodies in the patient’s serum
to neutralize the hemolytic activity of
streptolysin O.

 The traditional ASO titer involves dilution

of patient serum, to which a measured
amount of streptolysin O reagent is
added.

 These are allowed to combine during an

incubation period after which reagent red
blood cells are added as an indicator. If
enough antibody is present, the
streptolysin O is neutralized and no
hemolysis occurs.

 The titer is reported as the reciprocal of

the highest dilution demonstrating no
hemolysis.

 ASO testing is currently performed by

nephelometric methods in the routine
clinical laboratory.

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 Nephelometry has the advantage of being an
automated procedure that provides rapid,
quantitative measurement of ASO titers.
 The antigen used in this technique is purified
recombinant streptolysin.
 ASO titers typically increase within 1 to 2
weeks after infection and peak between 3 to 6
weeks following the initial symptoms (e.g.,
sore throat). However, an antibody response
occurs in only about 85 percent of acute
rheumatic fever patients within this period.
 Additionally, ASO titers usually do not increase
in individuals with skin infections.
Anti-DNase B Testing
 Testing for the presence of anti-DNase B is
clinically useful in patients suspected of having
glomerulonephritis preceded by streptococcal
skin infections, as ASO antibodies often are
not stimulated by this type of disease.
 In addition, antibodies to DNase B may be
detected in patients with acute rheumatic fever
who have a negative ASO test result.
 DNase B is mainly produced by group A
streptococci, so testing for anti-DNase B is
highly specific for group A streptococcal
sequelae.
 Macrotiter, microtiter, EIA, and nephelometric
methods have been developed for anti-DNase
testing.
 The classic test for the measurement of anti-
DNase B activity is based on a neutralization
methodology. If anti-DNase B antibodies are
present, they will neutralize reagent DNase B,
preventing it from depolymerizing DNA.
 Presence of DNase is measured by its effect
on a DNAmethyl-green conjugate. This
complex is green in its intact form, but when
hydrolyzed by DNase, the methyl green is
reduced and becomes colorless.
 An overnight incubation at 37°C is required in
some testing methodologies to permit
antibodies to inactivate the enzyme.
 Tubes are graded for color, with a 4+
indicating that the intensity of color is
unchanged, and a 0 indicating a total loss of
color. The result is reported as the reciprocal
of the highest dilution
SEROLOGICAL
APPLICATION
demonstrating a color intensity of between 2+
and 4+.
 Nephelometry provides an automated means
of testing that can be used for rapid
quantitation of anti-DNase B.
 In this method, immune complexes formed
between antibodies in patient serum and
DNase B reagent generate an increase in light
scatter. Results are extrapolated from values
from the classic method and are reported in
international units per mL.
STREPTOZYME TESTING
 The Streptozymetest is a slide agglutination
screening test for the detection of antibodies to
several streptococcal antigens.
 Sheep red blood cells are coated with
streptolysin, streptokinase, hyaluronidase,
DNase, and NADaseso that antibodies to any
of the streptococcal antigens can be detected.
Reagent red blood cells are mixed with a
1:100 dilution of patient serum.
 Hemagglutination represents a positive test,
indicating that antibodies to one or more of
these antigens are present.
 The test is rapid and simple to perform, but it
appears to be less reproducible than other
antibody tests.
 In addition, more false positives and false
negatives have been reported for this test than
for the ASO and anti-DNase B assays.
 However, single-titer determinations are not as
significant as several titrations performed at
weekly or biweekly intervals following the
onset of symptoms.
 The Streptozyme test is an excellent screening
tool, but it should be used in conjunction with
the ASO or DNase when sequelae of group A
streptococcal infection are suspected.
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HELICOBACTER PYLORI
 Helicobacter pylori is a gram-negative,
microaerophilic spiral bacterium is the major
cause of both gastric and duodenal ulcers.
 If untreated, H. pylori infection will last for the
patient’s life and may lead to gastric
carcinoma.
 A variety of techniques have been developed
to diagnose H. pylori infection due to the
prevalence of the organism and the
significance of the infection.
 The genomic sequences of two strains of the
organism have been determined using
molecular techniques. There is a large amount
of genetic heterogeneity due to the frequent
exchange of genetic material between strains.
 One of the proteins of H. pylori, CagA, is highly
immunogenic and is one of the virulence
factors of the bacterium.
 A second virulence factor is vacuolating
cytotoxin, or VacA. The VacAgene codes for a
toxin precursor.
ANTIGEN-DETECTION PROCEDURE
 Since H. pylori is found in the stomach, some
of the methods to determine if the organism is
present require an endoscopy. This includes
culturing for H. pylori, histologically examining
gastric biopsy tissue, and performing a urease
biopsy test.
 The most specific test to detect H. pylori
infection is culture, but the sensitivity is usually
lower than other methods, since the organism
is not evenly distributed throughout the tissue.
 H. pylori produces a large amount of urease,
so a biopsy urease test may be done to detect
the presence of bacteria.
 The organism is present if there is a color
change due to the increase of pH from the
breakdown of urea to ammonia and
bicarbonate.
 The biopsy urease test is easy to use, and
results can be detected within 2 hours in some
test procedures, making it ideal for rapid
diagnosis of H. pylori infections.
 Procedures for detecting H. pylori that do not
require the use of endoscopy include urea
breath testing, enzyme immunoassays for
bacterial antigens in the feces, molecular tests
for H. pylori DNA, and antibody tests.
SEROLOGICAL
APPLICATION
 In the urea breath test, the patient ingests urea
labeled with radioactive carbon (14C) or a
nonradioactive 13C.
 Urea is metabolized to ammonia and
bicarbonate. The bicarbonate is excreted in
the breath, and the labeled carbon dioxide is
measured by detection of radioactivity for
(14C) or mass spectrometry analysis for 13C.
 This breath technique has excellent sensitivity
and specificity, and it is helpful in determining
if the bacteria have been eradicated due to
antimicrobial therapy; however, it involves the
use of radioactivity.
 Analysis of stool samples before and after
antimicrobial therapy for H. pylori antigens is
mainly done to determine if the bacteria have
been eliminated.
 The test currently uses an enzyme-labeled
monoclonal antibody.
 The sensitivity and specificity of this
monoclonal antibody test are 84 to 95 percent
and 97 percent, respectively, as compared to
the urea breath test.
 Noninvasive molecular tests have also been
developed to detect H. pylori DNA. However,
PCR-based methods, which detect the
presence of the organism in fecal samples,
cannot distinguish between living and dead H.
pylori.
 Real-time PCR using TaqMan technology has
been developed to determine the patient’s
bacterial load and has shown good correlation
with the urea breath test.
 Molecular methods are quick and precise and
can determine antibiotic resistance as well.
DETECTION OF HELICOBACTER PYLORI
ANTIBODIES
 Serological testing is a primary screening
method of determining infection with H. pylori.
 Infections from this organism result in antibody
production of IgG, IgA, and IgM.
 The presence of antibody in the blood of an
untreated patient indicates an active infection,
since the bacterium does not spontaneously
clear. Antibody levels in untreated individuals
remain elevated for years.
 In treated individuals, the antibody
concentrations decrease after about 6 months
to about 50 percent of the level the patient had
during the active infection. This means that if
an antibody test is used to detect eradication
of H. pylori, the pretreatment sample should
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be

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stored for a year so that parallel testing can be
performed.
 A decrease in antibody concentration of more
than 25 percent must occur for treatment to be
considered successful.
 Most serological tests in clinical use detect H.
pylori–specific antibodies of the IgG class.
Although IgM antibody is produced in H. pylori
infections, testing for its presence lacks clinical
value, since most infections have become
chronic before diagnosis. Thus, IgG is the
primary antibody found.
 IgA testing has a lower sensitivity and
specificity than IgG testing, but it may increase
sensitivity of detection when used in
conjunction with IgG testing.
 Measurement of the antibodies may be done
by several techniques, including enzyme-
linked immunosorbent assays (ELISA),
immunoblot, and rapid tests using latex
agglutination or flow- through
membrane-based enzyme
immunoassay.
 The test methodology of choice for the
detection of H. pylori antibodies is the ELISA
technique, which is reliable and accurate.
 Tests employing antigens from a pooled
extract from multiple and genetically diverse
strains yield the best sensitivity, since H. pylori
is so heterogeneous.
 Very few, if any, patients produce antibodies to
all of the H. pylori antigens; most patients
produce antibodies against the CagAand
VacAproteins. Antibodies to these two proteins
indicate a more severe case of gastritis or
gastric carcinoma
 It is recommended that samples with positive
rapid test results be tested by an ELISA test
for correlation.
 When compared to other techniques for
antibody detection of H. pylori, ELISA tests are
sensitive, specific, and cost-effective for
determining the organism’s presence in
untreated individuals. However, since
antibodies are not rapidly cleared after
treatment, antibody testing is not as well suited
for determining eradication of infection as are
other methods.
 Additionally, individuals who are
immunocompromised (the elderly or
immunosuppressed individuals) may have a
false-negative result with antibody testing.
RICKETTSIAL INFECTIONS
SEROLOGICAL
APPLICATION
 The rickettsiae are short rods, or coccobacilli,
that are obligate, intracellular, gram-negative
bacteria.
 The genus Rickettsia is made up of two
distinct groups: the spotted fever group (SFG)
and the typhus group (TG).
 Rickettsial diseases are associated with
arthropods, as they spend at least part of their
life cycle in an arthropod host before being
transmitted to humans. Arthropod hosts
include ticks, mites, lice, or fleas.
 Humans are accidental hosts for rickettsiae
except for Rickettsia prowazekii, which causes
epidemic typhus.
SEROLOGICAL DIAGNOSIS
 Serodiagnosis is currently the method of
choice for detecting rickettsialinfections.
Methods that may be used are probe-based
immunoassays and agglutination assays.
 For many years, antibodies produced in
patients with rickettsialinfections were detected
by an agglutination test known as the Weil-
Felix test, which was based on cross-reactivity
of the patient’s antibodies with polysaccharide
antigens present on the OX-19 and OX-2
strains of Proteus vulgaris and the OX-K strain
of Proteus mirabilis.
 Current serological assays for
rickettsialantibodies are organism-specific and
include indirect fluorescent assays (IFA),
microimmunofluorescentassays (micro-IF),
immunoperoxidaseassays (IPA), ELISA, and
immunoblot assays (IBA).
 IFA and IPA require the whole bacterium as
the reagent, while ELISA and IBA use
rickettsialantigens adsorbed onto a solid phase
or nitrocellulose membrane.
 The IFA test and the micro-IF are currently
considered the gold standard for detecting
rickettsialantibodies.
 The IPA, which uses a light microscope
instead of a fluorescent microscope to read the
slides, has a sensitivity and specificity similar
to that of IFA.
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 Testing by both of these methods can detect  Testing for cold agglutinins is no longer
significant titers of antibodies in Rocky recommended for the detection of M.
Mountain spotted fever by the second week of
infection.
 However, anti rickettsial treatment needs to be
started by the fifth day of illness in order to be
successful. Thus, serological diagnosis tends
to be more retrospective, after treatment has
begun.

MOLECULAR TECHNIQUES

 Molecular detection of rickettsial infections can

be accomplished by performing a PCR using
primers that target genes of the TG and SFG
groups of organisms.

 To detect both SFG and TG rickettsiae, the

citrate synthase gene and the17 kDa antigen
gene are used, while the 190 kDa antigen
(OmpA) gene and the 120 kDa antigen
(OmpB) gene are used only to detect the SFG
group.

 PCR can then be followed by restriction digest

or nucleotide sequence analysis of the
amplicon, or real-time PCR can be used to
definitively identify the rickettsialspecies.

MYCOPLASMA PNEUMONIAE

 Mycoplasma is a member of a unique group of

organisms that belong to the class Mollicutes.
 These organisms lack cell walls and have a
small genome, sterols in their cell membrane,
and complex growth requirements. This makes
culture of these organisms difficult and time-
consuming.
 The best-known organism of this class is
Mycoplasma pneumoniae, which is a leading
cause of upper respiratory infections
worldwide.

DETECTION OF ANTIBODIES TO MYCOPLASMA

PNEUMONIAE

 For many years, prior to the development of

antigen-specific serological tests, laboratory
diagnosis of Mycoplasma pneumoniae
involved testing for cold agglutinins, because
these are present in about 50 percent of
patients with the infection.

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pneumoniae infections, since some viral
infections and collagen vascular diseases
also cause production of cold agglutinins.
 More recently, M. pneumoniae–specific
serological tests have been developed.
These include enzyme immunoassay,
particle agglutination, indirect
immunofluorescence, and complement
fixation.
 Depending on the technique used, IgM, IgG,
and IgA classes of antibody to M.
pneumoniae may be detected.
 Enzyme immunoassays (EIA) are the most
widely used method of detection, because
they require a small sample volume and can
test large numbers of specimens in an
automated format.
 The EIA procedure may be as sensitive as
PCR if enough time has elapsed for
antibodies to be formed.
 It is better to test for the presence of both the
IgG and IgM classes of antibody for greater
accuracy, but each class of antibody must
be tested for separately.
 The indirect fluorescent assay (IFA) can also
detect IgM or IgG classes of antibody to M.
SEROLOGICAL
APPLICATION
pneumoniae.
 In this test, Mycoplasma antigen is attached to
a glass slide. Patient serum is placed on the
slide and incubated.
 The slide is then washed, and fluorescent-
labeled anti-IgM or anti-IgG is added to the
slide and allowed to incubate. After incubation,
the slide is washed and observed under a
fluorescent microscope.
 If the screening test is positive, then a serial
dilution may be done to determine the
concentration of antibody present.
MOLECULAR TECHNIQUES
 Molecular techniques provide a more rapid
means of detecting Mycoplasma infection than
culture or serological testing.
 PCR testing for Mycoplasma may be
performed using throat swabs,
nasophayngealswabs, sputum, fixed tissue,
and cultures.
 In addition to obtaining rapid results, PCR
tests are also advantageous in that only one
specimen is required, tissues that have
already
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been processed for histology can be used, and
live organisms are not required.
 RNA-based amplification methods are highly
sensitive due to the large number of rRNA
(ribosomal RNA) copies in the M. pneumoniae
cell, which also indicates that the organism is
alive.
 Nucleic acid amplification methods have been
developed that detect several different gene
targets, including the ATPase operon gene,
the PI adhesion gene, the 16S RNA gene, and
the tufgene.
TOXOPLASMOSIS
 Toxoplasmosis results from infection with
Toxoplasma gondii, a ubiquitous protozoan
parasite that infects humans by ingestion of
infective cysts (oocysts).
 It is thought that cysts are transferred by hand-
to-mouth contact from contaminated soil or cat
litter or by ingesting oocysts in raw or partially
cooked pork, mutton, or beef.
 Transmission may also occur through blood
transfusions or organ transplantation.
 Cats are known to be definitive hosts for this
parasite, but other definitive hosts must also
exist.
 This disease is nearly always asymptomatic or
may present with a mild lymphadenopathy.
 Encysted organisms can be found in any
tissue.These cysts may remain viable for
years, which explains why immunosuppressed
patients can exhibit symptoms long after initial
infection.
 The greatest concern, however, is that
Toxoplasma species can cross the placenta. If
the fetus is exposed during the first trimester,
death nearly always occurs as a result of CNS
damage.
 Infection during the second trimester may
result in hydrocephaly, blindness, or other
nervous system damage.
SEROLOGICAL
APPLICATION
 Later infection may result in blindness or mild
CNS defects. However, women who are
exposed to this parasite before pregnancy do
not transmit the infection to the fetus.
 Serological testing plays an important role in
diagnosing toxoplasmosis, because isolation
of the organism requires tissue biopsy.
 Generally, seroconversion to an antibody-
positive state, a fourfold increase in the titer of
IgG, or high levels of IgM and IgG antibodies
are considered diagnostic.
 IgA levels can also be detected in early
infection and can persist for 3 to 9 months.
 Concurrent elevations of IgM and IgA indicate
early infection and are used to screen
pregnant women.
 Enzyme immunoassays (EIA) for IgM, IgG, or
IgA and indirect fluorescent antibody (IFA)
assays for IgG are available and should be
performed when congenital toxoplasmosis is
suspected.
 IFA testing has been widely used, but EIA is
the method of choice, as it is more sensitive,
less difficult to perform, and easier to interpret.
 Elevated titers of IgG, IgM, or IgA antibody
classes suggest that infection has occurred
within the previous 3 to 9 months.
 Elevated IgG titers without IgM antibody
suggest an older infection.
 Paired samples whose collection is separated
by 3 weeks are tested to confirm the presence
of recent infection. In a recent infection, titers
of both of these antibody classes will rise.
 Newborns with congenital toxoplasmosis
usually have detectable IgM antibody.
Presence of elevated IgM titers in the mother’s
serum further supports the diagnosis (Table
20–1).
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the parasite, and procedures to detect

 Specific IgA antibody assays provide even circulating antigen appear to lack sensitivity.
more sensitive methods for early detection and  PCR is, therefore, the method of choice to
confirmation of congenital toxoplasmosis and detect T. gondii DNA in CSF.
should be performed on newborns suspected
of harboring the parasite. [TABLE / FIGURE COMPILATION]
 Early diagnosis and maternofetal treatment
can reduce fetal infection to mild or subclinical.
 Prenatal congenital toxoplasmosis can be
diagnosed by performing polymerase chain
reaction (PCR) technology on amniotic fluid to
detect T. gondii DNA.

 Currently, there are no useful serological

procedures for diagnosing CNS infection in
immunocompromised patients.
 These individuals often do not produce
detectable levels of specific antibody against

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