MLS109 Basic Principles of Clinical Chemistry CLINICAL CHEMISTRY

WILLIAM CHRISTOPHER C. SALAZAR, RMT | AUGUST 26, 2020 LE 1 TRANS 01

OUTLINE
1. Units of Measure III. REAGENTS
2. Reagents  Most instrument manufacturers make the reagents in a ready-to-use
3. Clinical Laboratory Supplies form or “kit” where all necessary reagents and respective storage
4. Centrifugation containers are prepackaged as a unit requiring only the addition of
5. Laboratory Mathematics and Calculations water or buffer to the prepackaged components for reconstitution.
6. Specimen Considerations
A. Chemicals
LEGEND 1. Analytical Reagents
Remember Lecturer Book Previous Presentation  Suitable for use in most analytical laboratory procedures
Trans  Impurities must be stated
2. Ultrapure Reagents
 Chemicals that have been put through additional purification steps
OBJECTIVES:  Used in procedures such as chromatography, atomic absorption,
At the end of the lecture, the student should be able to: immunoassays, molecular diagnostics and other techniques that
 Convert results from one unit format to another using the SI and may require extremely pure chemicals
traditional systems. 3. Chemically Pure Reagents
 Describe the classifications used for reagent grade water.  Indicates that impurity limitations are not stated
 Identify the varying chemical grades used in reagent preparation and  Preparations of these chemicals is not uniform
indicate their correct use.  Not recommended for clinical laboratories unless further
 Define primary standard and standard reference materials. purification or reagent blank is included
 Describe the following terms that are associated with solutions and, 4. USP and NF grade
when appropriate, provide the respective units: percent, molarity,  Used to manufacture drugs
normality, molality, saturation, colligative properties, Redox potential,
 Based on the criterion of not being injurious to individuals
and conductivity.
 Chemicals are pure enough for use in most chemical procedures
 Describe two ways to calibrate a pipetting device.
USP – United States Pharmacopeia, NF – National Formulary
 Identify the preanalytic variables that can adversely affect laboratory
results as presented in this chapter.
5. Technical/Commercial Grade
 Used for manufacturing
I. CLINICAL CHEMISTRY  Should never be used in the clinical laboratory
 Clinical – comes from the greek word kline, meaning “bed”
 Chemistry – is a basic science that utilizes the specialty of IV. REFERENCE MATERIALS
chemistry to study human beings in various stages of health and STANDARD
disease  A substance or solution in which the concentration is determined
 It is an applied science when analysis are performed on body  It is used in the calibration of an instrument of method
fluids or tissue specimens to provide information for the diagnosis
or treatment of disease.
A. Types of Standard – Atomic Weight Standard
II. UNITS OF MEASUREMENT  Pure to the last molecule
Système International d'Unités (SI)  Grade A (IUPAC)
 It consists of seven independent base units, each unit is represented
by a symbol. B. Ultimate Standard
 Adopted internationally in 1960  Grade B (IUPAC)
 It is devised to provide the global scientific community a uniform
method of describing physical quantities. C. Primary Standard
 Highly purified, concentration is exactly known
A. UNITS  At least 99.98% purity
Length meter (m)  Used in clinical laboratories
Mass Kilogram (kg)  Grade C (IUPAC)
Time Second (s)
Quantity of substance Mole (mol) D. Working Standard
Electric current Ampere (A)  <0.05% particulates, 99.95% purity
Temperature Kelvin (K)  Grade D (IUPAC)
Luminous Intensity Candela (cd)
Table 1.01 (Units of Measurement) E. Secondary Standard
 Substance of lower purity
NOTES TO REMEMBER  Grade C (IUPAC)
 It has been recommended that analytes be reported using moles of
solute/volume of solution (mmol/L) V. WATER SPECIFICATIONS
 Enzyme units are given as the international Unit per liter (U/L), WATER
although katal unit has been adopted previously as SI unit, its use is  Considered as the universal solvent
limited  It is the most frequently used reagent in the laboratory
 pH scale is retained for measurement of hydrogen ion concentrations
 Liter is used as the reference volume METHODS OF FILTRATION – A. FILTRATION
 SI unit is used because compounds react on a molar basis, and  Passage through a filter or other material that prevents passage of
expressions of amounts of substances in such terms allows for a certain molecules, particles or substances
better understanding of the relative proportion of compounds.  Can remove particulate matter from municipal water supplies before
any additional treatments

TRANS GROUP #1 Marquez, Datinggaling, Daya EDITORS 1 of 7

MLS109 Basic Principles of Clinical Chemistry LE 1 TRANS 01

 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 the  For urinalysis, parasitology and histology
filtrate.  For washing glasswares not requiring Type I or II reagent grade
water (RGW)
B. Distillation  Silicate content ≤1.0 mg/L
 The process of vaporizing and condensing a liquid to purify or  CO2 free water
separate a volatile substance from a non volatile substrate  Obtained by boiling Type II RGW
 Limitations  Used if CO2, amonia, or O2 may affect analysis
 Carryover of volatile impurities and entrapped water droplets may
contain impurities such as: VI. LABORATORY EQUIPMENT – GLASSWARE
 Volatiles A. Borosilicate
 Sodium, potassium, magnesium, carbonates and sulfates  General purpose, for test tube and disposable glassware
 Most common type encountered in volume measurement
C. Ion Exchange  With high thermal resistance and low alkali content
 A process that removes ions to produce mineral-free water  Should not be heated above strain point because rapid cooling
 Accomplished by passing feed water through columns containing cracks the glass easily when heated again
insoluble resin polymers that exchange H+ and OH- ions for the  Examples:
impurities present in ionized from in the water  Pyrex – strain point 512 °C
 Limitations  kimax
 Only ions are removed, therefore it is neither pure nor sterile
B. Alumina-silicate
D. Reverse Osmosis  6x stronger than borosilicate
 A process in which water is forced through a semipermeable  Strengthened chemically rather than thermally
membrane that acts as a molecular filter
 Resist clouding and scratching better
 Removes 95-99 % of organic compounds, bacteria and other
 Only half of the thermal resistance of borosilicate glass
particulate matter and 90-97% of all ionized and dissolved minerals
 Example : corex
 Limitations
 Used for
 Does not efficiently remove gasses
 Graduated cylinder
 Thermometers
E. Ultraviolet Oxidation
 Test tube for high speed centrifugation
 Uses ultraviolet radiation at the biocidal wavelength of 254 nm
 Eliminates many bacteria and cleaves many ionizing organics that
C. Low Actinic Glassware
are then removed by deionization
 High thermal resistance
 Limitations
 With red color to permit adequate visibility of contents yet give
 Requires further purification methods
maximum protection from light
 Used for photosensitive materials like bilirubin standards
TYPES OF WATER – A. DISTILLED
 Purified to remove almost all organic materials
D. Corning Boron Free Glass
 Distillation does not remove volatile organic impurities
 Highly resistant to alkali
 Water may be distilled more than once to remove impurities
 Soft, poor heat resistance
B. Deionized
VII. LABORATORY EQUIPMENT – PLASTICWARE
 Produced from distilled water using either an anion or a cation
exchange resin followed by replacement of the removed particles
A. Polyethylene
with hydroxyl or hydrogen ions respectively  For inexpensive, disposable tubes
 Generally, it is purified from previously treated water such as pre-  Used for storage of alkaline solutions
filtered or distilled water
B. Polypropylene
C. Reagent Grade  Withstands higher temperature
 Type I  Becomes discolored by solvents
 Used for the procedures that require maximum water purity
 Preparation of standard solutions C. Polycarbonate
 Ultra micro chemical analysis  Stronger, more heat resistant
 Enzyme analysis  Lower chemical resistance
 Immunoassays  For centrifugal tubes and graduated cylinders
 Fluorescence quantitation  Autoclavable
 HPLC
 Measurement of nanogram or subnanogram concentration D. Teflon
 Tissue or cell culture  Almost chemically inert, very heat resistant
 Final rinsing of glassware  For stopcocks, stirring bars, cap liners and tubings
 Microbial content ≤ 10 cfu/mL  Autoclavable
 Silicate ≤ 0.05mg/L
 Water is passed through

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MLS109 Basic Principles of Clinical Chemistry LE 1 TRANS 01

E. Polyolefins I. VOLUMETRIC PIPET

 Strong and resistant to elevated temperature  Used for non-viscous fluids
 Autoclavable  Standard, reagents, dilution liquids
 Plasma, serum, urine
F. Polyvinyl Chloride  Drains by gravity by placing tip vertically against the side
 Soft and flexible of accepting vessel but not touching the liquid
 Used to construct tubings  Distinguished by bulb-like enlargement partway up the
stem
VIII. ANALYTICAL BALANCE  Has the highest degree of accuracy and precision
A. Mechanical Balance  TD Pipette (TD)- To Deliver
 Double pan analytical balance  Drains freely with the pipette touching the inner
 Two pans of equal mass suspended from ends of a beam surface of the receiving vessel
supported at its center by a knife-edge fulcrum  Calibrated for the volume delivered
 Useful for weighing to the nearest 0.1 gram Do not attempt to wash out the film that adhere
 Not to be used for measurement of standards to the inside of the glass surface
 Single pan analytical balance  Calibrated to blow out
 Substitution balance; weights are remove from the side of the Remaining liquid after free delivery must be
beam holding the unknown mass until equilibrium is restored blown out and added to the initial volume
Indicated by opaque ring near mouthpiece of
 Unequal arms; single pan, provides greater precision and
accuracy the pipet.
 TC Pipette (TC)- To Contain
 Digital readout to 0.01 mg
 Calibrated for the total volume of liquid held in the
 Used for measuring primary standards, small amount of
pipette
chemicals and for gravimetric calibration of pipets
 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”

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 Volumetric Pipette

 Have built-in provisions for tarring, so that the weight of the
container can be subtracted II. OSTWALD-FOLIN PIPETTE
 They have a single pan, top loading or analytical, fast and can be
 Similar to volumetric pipette but have their bulb closer to
computer interfaced
the delivery tip
 Operate on the principle of electronic force compensation.
 Used for viscous fluids like whole blood
 With etched rings at the top = blow out, TD

IX. VOLUMETRIC SAMPLING AND DISPENSING

Ostwald-Folin Pipette
PIPETTE
 Used in the transfer of a volume of liquid from one container to
another B. Measuring/Graduated Pipette
 Types of pipettes  Delivers variable volume
 Transfer pipet  Long, cylindrical tubes drawn out a tip and calibrated in uniform
 Measuring/Graduated pipet fractional volume measurement
 Micropipettes  Principally used for measurement of reagents and not considered
sufficiently accurate for measuring samples and calibrators.

A. Transfer Pipette TYPES OF MEASURING/GRADUATED PIPETTE

 Delivers fixed volume (more accurate and precise)
 Serologic Pipette
 They consist of cylindrical bulb joined at both ends to a narrower
 Has graduated marks up to the tip
glass tubing
 Has etched rings
 A calibration mark is etched around the upper section of the tube
 Calibrated to blow out
 The lower delivery tube is drawn out to a gradual taper.
 Has larger orifice than Mohr Pipette

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MLS109 Basic Principles of Clinical Chemistry LE 1 TRANS 01

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 deviation.
Serologic Pipette  Accuracy
 Determined by correcting the mean for temperature (20 –
 Mohr Pipette 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
 Determined by comparison to a reference solution
Measuring/Graduted Pipette
B. Centrifugation
X. MICROPIPETTE  The process using centrifugal force to separate the lighter portions
 Pipettes used for measurements of microliters of volume of a solution, mixture or suspension from heavier portions of a
 Piston operated, positive displacement solution
 Uses disposable tips and detipers  Used to
 Remove cellular elements from biological fluids to provide cell-
 Blue Tip – 100 to 1000 uL or 1mL free specimen for analysis
 Yellow Tip – 2 to 200 uL  To concentrate cellular elements for microscopic examination
 White Tip – 0.5 to 10 uL  RPM
 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
XI. QUALITY CONTROL AND PREVENTIVE MAINTENANCE separate substances in a solution
 Routine  Formula
 RCF = 1.118 x 10-5 x r x (rpm)2
 Rinse immediately after using
Wash with low alkali, non-ion detergent  Where : r = radius in cm from the center of the head to the
bottom of the tube shield or bucket
 Rinse with tap water, then 3-5x with Type I water
 Final rinse should have a pH of 5.5 – 5.7 TYPES OF CETRIFUGES
 For Blood Clots
1. Horizontal Head Centrifuge
 Soak in 1 0% NaOH
 For New Pipettes  Swinging bucket type
 Soak in 5% HCl or 5% nitric acid  The centrifuge tubes are held in a vertical position when not
moving but are horizontal when the centrifuge is fully in motion
 For Grease
 Soak in  Generate low speeds only but can produce a tighter pellet of
 Any organic solvent precipitate or clotted cells at the bottom of the tube
 50% KOH or Contrad 70  Generates a lot of friction
 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

 Bromsulhalein Dye
 used to check random pieces of residual alkaline detergent
 Pink = incomplete removal of detergent

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MLS109 Basic Principles of Clinical Chemistry LE 1 TRANS 01

The major difference between plasma and serum is that

B. Plasma
serum does not contain fibrinogen and some potassium is
released from platelets.  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
Properly balance Cetrifuge. Colored Circles represent  Used in measuring blood gases (partial pressure of oxygen and
counterbalanced positions for sample tubes. carbon dioxide) and pH
 Syringes containing heparin anticoagulant are used instead of
2. Angle Head Centrifuge evacuated tubes because of the pressure in an arterial blood vessel
 Has a fixed 25 to 52 degree angle at which tubes are held during
centrifugation E. Other Body Fluids
 The sediment packs at an angle bit not as tightly as with a  Cerebrospinal fluid
horizontal head centrifuge  Paracentesis fluids
 Since the sediment is formed at an angle and not tightly packed,  Pleural
decantation is not recommended  Pericardial
 Less prone to heat build up due to air friction than horizontal head  Peritoneal
centrifuge heads  Amniotic fluid
 Urine

F. Hemolysis
 Ruptured or destruction of RBC

G. Icterus
 Increased bilirubin pigment

H. Lipemia
 Increased lipids
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 XIII. FACTORS AFFECTING COMMON SERUM CONSTITUENTS
to occur after that time, samples should be appropriately stored. A. Prolonged Torniquet Application
For most, this means refrigeration at 4°C for 8 hours.  Increased:
3. Ultracentrifuge  Cholesterol
 Centrifuge head is held at a fixed angle but generates tight  Albumin
sediments due to high speeds generated  Potassium
 Generates the highest speeds  Ammonia
 Refrigerated to reduce the heat generated by the friction due to  Lactic Acid
high centrifugal speeds B. Posture – Supine to Standing
 Especially used for lipoprotein separation since refrigeration  Increased:
enhances separation of lipoproteins  Albumin
 Cholesterol
C. CENTRIFUGATION – QC and MAINTENANCE  Calcium
 Timer and speed to be checked every 3 months
 Speed is checked using a tachometer C. Hemolysis
 Timer is checked using a stopwatch  Increased:
 Routinely cleaned everyday  Potassium,
 Must be cleaned immediately if there are spills  Lactate Dehydrogenase (LDH)
 Alkaline Phosphatase (ALP)
XII. SPECIMEN CONSIDERATIONS  Acid Phosphatase (ACP)
 The process of specimen collection, handling, and processing  Creatine Kinase (CK)
remains one of the primary areas of pre-analytic error.
 Aldolase
 Phosphate
A. Phlebotomy or Venipuncture
 Magnesium
 23- or 21- gauge needle , routinely used needle
 Ammonia
 IV infusion set or butterfly is used when veins are fragile, small, or
 Iron
hard to reach or find
 Sites adjacent to IV therapy should be avoided; however if both
D. Alcohol
arms are involved in IV therapy and the IV cannot be discontinued
Increased Decreased
for a short time, a site below the IV site should be sought.
Gamma-Glutamyl Transferase
 The initial sample drawn (5mL) should be discarded because it is Glucose
(GGT)
most likely contaminated with IV fluid and only subsequent sample
tubes should be used for analytic purposes Triglycerides
 In addition to venipuncture, blood specimens can be collected using
a skin puncture technique ( hell stick, finger stick) E. Diurnal Variation

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MLS109 Basic Principles of Clinical Chemistry LE 1 TRANS 01

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

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