CLINICAL CHEMISTRY I

LECTURE

King Henry Doesn't Usually Drink Chocolate Milk

FUNDAMENTAL CONCEPTS IN ANALYTICAL
PROCEDURES

UNITS OF MEASURE
● Quantitative lab result consists of 2 components
100 mg/d
○ 100 = related to actual test value
○ mg/d = label identifying the units

S.I. UNITS
● Système International d'Unités CHEMICAL REAGENT
○ Adopted internationally in 1960 ➔ Analytic chemicals exist in varying grades of
○ Preferred in scientific literature and purity
clinical laboratory ◆ Analytic Reagent (AR)
○ Only system employed in many ● Very high purity
countries ● Recommended for qualitative &
○ Devised to provide the global scientific quantitative analysis or most
comm. analytical lab procedure
◆ Ultrapure
● Spectrograde, nanograde, or
HPLC grade
7 BASE LIMITS ● Used for gas chromatography,
HPLC, fluorometry, AAS,
➢ Length - meter (m) immunoassays, molecular
➢ Time - second (s) diagnosis
➢ Amount of substance - mole (mole) ◆ Chemically Pure
➢ Electric current - ampere (A) ● Not recommended for clinical
➢ Temperature - Kelvin (K) laboratories
➢ Luminous intensity - candela (cd) ● Limits of impurities not specified
➢ Mass - kilogram (kg) ◆ United States Pharmacopeia (USP)
National Formulary (NF)
● Not necessarily of sufficient
purity to use as analytic
reagents
REPORTING OF LAB RESULTS ● Used to manufacture drugs
● Not injurious to health
➔ Expressed in terms of substance concentration ◆ Technical or commercial grade
(e.g. moles) for mass of a substance (e.g. ● For industrial use
mg/dL, g/dL, g/L, mEq/L, and IU) rather than in ● Most of sufficient purity to use
SI units. as analytic
➔ Analytes be reported using moles of solute per
volume of solution (substance concentration)
➔ Reference volume (Liter)
CLINICAL CHEMISTRY I
LECTURE

REAGENT WATER REFERENCE MATERIALS

➔ CLSI: water is classified into 1 of 6 categories ● PRIMARY STANDARD
◆ Clinical Laboratory Reagent Water ○ Highly purified chemical that can be
(CLRW) measured directly to produce a
● Pure enough for most routine substance of exact known concentration
testing and purity
● Replaces previously designated ● SECONDARY STANDARD
type I and type II water ○ Substance of lower purity with
◆ Special Reagent Water (SRW) concentration determined by
● For applications that require comparison with primary standard
water of different purity than
CLRW (e.g. DNA/RNA analysis, LABORATORY SUPPLIES
trace metals) ● THERMOMETERS
● Must meet specifications of ➔ Liquid-in-glass
assays ◆ Use a colored liquid (red or
◆ Instrument Feed Water other)
● Used in automated analyzers for ◆ Measure temperature between
rinsing, dilutions, water baths 20°C and 400°C
● Specification set by ◆ Reveal continuous line of liquid,
manufacturer free from separation or bubbles
◆ Water applied by method manufacturer ➔ Electronic thermometer or thermistor
● Water provided by manufacturer probe
for use in particular test system ◆ Accurate, fast-reading
◆ Autoclave and Wash Water ◆ Routinely incorporated in many
● Feed water for autoclaves & devices
dishwashers
● Replacement for previously GLASSWARE
designated type III water ● Kimax/Pyrex (borosilicate)
◆ Commercially bottled purified water ● Corex (aluminosilicate)
● Must meet specifications for ● High silica
intended use & be packaged to ● Vycor (Corning: acid and alkali resistant; can
protect from degradation & withstand very high temperature
contamination (900°C-1200°C)
● Low actinic (amber colored)
➔ TYPE I ● Or flint (soda lime) glass - used for disposable
◆ For procedures that require material
maximum/highest water purity ❖ Consist of high thermal borosilicate or
◆ Used in clinical chemistry (highest aluminosilicate glass
purity)
➔ TYPE II PLASTICWARE
◆ For most laboratory determinations in ● beginning to replace glassware
chemistry, hematology, microbiology, ○ Unique high resistance to corrosion and
immunology, and other clinical breakage
laboratory areas ○ Varying flexibility
➔ TYPE III ○ Relatively inexpensive
◆ For qualitative measurements and most ○ Disposable after each use
procedures in urinalysis, parasitology Major types of resins used in CC laboratory:
and histology, washing glassware ○ Polystyrene
◆ Autoclave wash water and glassware ○ Polyethylene
washing but not for analysis or reagent ○ Polypropylene
preparation ○ Tygon
○ Teflon
CLINICAL CHEMISTRY I
LECTURE

○ Polycarbonate ■ Hollow cathode

○ Polyvinyl chloride ○ UV Region
■ Mercury/sodium vapor
❖ LAB VESSELS ■ Xenon lamp
➢ Flasks, beakers, graduated cylinders ■ Deuterium lamp
are used to hold solution ■ Hydrogen lamp
■ Hollow cathode
➔ VOLUMETRIC FLASK ○ Infrared Region
◆ Hold one exact volume of liquid ■ Nernst glower
(TC) ■ Globar (silicon carbide)
◆ Round lower portion with flat ENTRANCE SLIT
bottom and long, thin neck ● Minimize unwanted or straylight and prevents
◆ Bring a given reagent to its final scattered light into the monochromator system
volume with prescribed diluent
➔ ERLENMEYER FLASK AND GRIFFIN STRAYLIGHT
BEAKERS ● Any wavelength outside the band transmitted by
◆ hold different volumes rather the monochromator and does not originate from
than one exact amount the polychromatic light source.
◆ ERLENMEYER FLASK
● Wide bottom evolves MONOCHROMATOR
into smaller, short neck ● Disperses the light into isolated wavelengths
◆ GRIFFIN BEAKER 1. Glass filters and interfere filters (used in
● Flat bottom, straight photometers)
sides opening as wide ● Simple, least expensive, not
as the flat base, with precise
small spout in lip ● Produces monochromatic light
2. Diffraction gratings and prisms (used in
spectrophotometers)
GRADUATED CYLINDER ● Diffraction grating: most
● Long, cylindrical tubes, held upright by commonly used
octagonal or
● Has calibration marks along its length used to EXIT SLIT
measure of liquids ● Selects bandpass of selected wavelength to
● Do not have accuracy of volumetric glassware pass through cuvette on detector
➔ Bandpass/Spectral bandwidth
SPECTROPHOTOMETER ◆ Narrower the bandpass, the
● Measure light transmitted by solution to greater the resolution and
determine concentration of light-absorbing spectral purity of instrument
substance in solution
PHOTODETECTORS
❖ BEER’S LAW ● Converts transmitted light onto photoelectric
● Concentration of substance is directly energy
proportional to the amount of light ➢ Barrier layer cell/ photovoltaic cell/
absorbed or inversely proportional to the photocell
logarithm of the transmitted light ○ Simplest, least expensive,
temperature sensitive
LIGHT SOURCE/LAMP ○ Used in filter photometers with
● Produces an intense, constant beam of wide bandpass
polychromatic light ➢ Phototube
○ Visible Region ○ Contains cathode and anode
■ Tungsten light bulb enclosed in glass case
■ Mercury/sodium vapor ○ Requires external voltage
CLINICAL CHEMISTRY I
LECTURE

➢ Photomultiplier tube ❖ MOLARITY (M)

○ Most commonly used ➢ # of moles per 1 liter of solution
○ UV visible region (moles/liter)
○ Excellent sensitivity ➢ 1 mol of substance is equal to gmw of
○ Detects very low levels of light, substance
should never be exposed to ➢ Formula:
from light 𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
M=
➢ Photodiode 𝑀𝑊 𝑥 𝐿
○ Excellent linearity
○ Most useful as simultaneous ➢ Calculate molarity of solution that has
multichannel detector volume of 375 mL and contains 42.5 g
of NaCl
METER/READOUT DEVICE g of solute: 42.5 g
● Displays output of detection system MW: 22.99 + 35.44
● Ex. galvanometer, ammeter, light-emitting diode Volume (L): 375mL = 0.375 L
(LED) display
42.5 𝑔
LABORATORY MATHEMATICS M=
58.44 𝑥 0.375 𝐿
➢ ANALYTE
○ Biologic solute or constituent (e.g.
calcium, glucose, sodium) M = 1. 94 𝑀
➢ ANALYTE CONCENTRATION in solution
○ Can be expressed in many ways
➢ PERCENT SOLUTION ➢ # of moles per 1L of solution (moles/L)
○ Amount of solute per 100 total units of ■ Remember: 1 mol of substance
solution is equal to gmw of that
○ Percent implies “parts per 100” - substance
represented as percent (%)
➔ 3 ways:
◆ Weight per weight (% W/W)
◆ Volume per volume (% V/V)
◆ Weight per volume (% W/V)

❖ Weight per weight (% W/W) 𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

➢ # of g of solute per 100 moles =
𝑀𝑊
g of solution
❖ Volume per volume (% V/V)
➢ Milliliters of solute in ❖ MOLALITY (m)
100 mL of solution ➢ Amount of solute per 1 kg of solvent
❖ Weight per volume (% W/V)
➢ # of g of solute in 100 𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
mL of solution M=
𝑀𝑊 𝑥 𝑘𝑔
➔ Percent Solution
◆ Weight per volume (% W/V)
● Most frequently used ❖ NORMALITY (N)
term for % solution ➢ # of g equivalent weight per 1L of
solution
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
➢ Normality = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 (𝐿)
CLINICAL CHEMISTRY I
LECTURE

𝑔𝑚𝑤
❖ Equivalent weight =
𝑣𝑎𝑙𝑒𝑛𝑐𝑒
❖ Valence = # of H+ or OH- ions DILUTION
➔ Ratio of concentrated or stock material to total
➢ # of g equivalent weight per 1L of final volume of a solution
solution Dilution = C1V1 = C2V2
𝑠𝑜𝑙𝑢𝑡𝑒
➢ Other formula: Dilution =
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 (solute to solvent)
N=
𝑒𝑞. 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑣𝑜𝑙. 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 (𝐿)
Dilution factor = total volume of solution / sample
aliquot volume
➢ Calculate normality of a solution that
contains 50 g of H2SO4 dissolved in 15 𝑝𝑒𝑟𝑐𝑒𝑛𝑡 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
L. Solute =
100
g = 50 g
Vol. of solution = 15 L
Eq. weight = gmw/valence = 98/2

H2SO4:
H=1X2 =2
S = 32
O = 16 X 4 = 64
Total = 98

𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
N=
𝑒𝑞. 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑣𝑜𝑙. 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

50 𝑔
N=
49 𝑥 15

N = 0.068 N

❖ NORMALITY
➢ # of g equivalent weight per 1L of
solution

N = Molarity x Valence

𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 50
M=
𝑀𝑊 𝑥 𝐿
= 98 𝑥 15
= 0.034 M

N = Molarity x Valence
= 0.034 x 2
= 0.068

** valency = 2 (2H ions)

CLINICAL CHEMISTRY I
LABORATORY

EVACUATED TUBES MICROCOLLECTION TUBES

● Single-use only, contains vacuum (facilitate ● AKA Microtainer Tubes

exact blood draw volume) has expiration date ○ For collection of capillary blood
● Various sizes and volumes with color-coded ○ Similar additives with evacuated tubes
stoppers (caps/tops/closures) denote the special ○ Plasma and hematology tubes: invert
property of the tube and the type of additive in 8-10x
the tube (anticoagulants, clot activator, gel, other ○ Serum tubes: allow to clot for at least
additives) 30 mins.
● Require gentle tube inversion to achieve proper ○ May also be centrifuged for
mixing of additive and blood, otherwise, delayed serum/plasma separation
clotting (for non-anticoagulated tubes) or platelet
clumping, clotting or incorrect test results may
occur.
CAPILLARY BLOOD (ORDER OF DRAW)
Silica particles - clot activator red top
25°C = temperature for tubes ● Capillary blood gas
Low temperature = high vacuum ● Slides (for blood smear)
Thrombin = enhances clotting ● EDTA Tube
● Other additive tube
● Non-additive tube
ORDER OF DRAW
● Multiple blood tests are usually ordered
simultaneously for a patient, hence multiple
tubes with different additives may be needed.
PIPETTING
● When drawing blood, reflux (backflow of blood
from tube to needle) can occur. This may lead to
contamination or additive carry-over to the next ➔ Another type of volumetric glassware used to
tube. measure fluids
● Hence, in order to prevent this, proper order of ➔ Filled by using mechanical suction on an
draw is important. aspiration bulb
➔ Mouth suction is never used
➔ Blood culture (SPS) ➔ Each manual pipette has at least one calibration
➔ Citrate tube (light blue) or graduation mark on it
➔ Serum separator tubes (gold/tiger) ➔ Opening (orifice) at delivery tip of pipette is of
➔ Serum tube (red) certain size to allow a specific time to drainage
➔ Rapid serum tube (orange) when pipette is held vertically.
➔ Plasma serum tube (orange)
➔ Plasma separator tube CLASSIFICATION
➔ Heparin tube (green) I. DESIGN
➔ EDTA tube (lavender) A. To Contain (TC)
➔ PPT separator tube (pearl) B. To Deliver (TD)
➔ Fluoride tube (gray) II. DRAINAGE CHARACTERISTICS
◆ Antiglycolytic - sodium fluoride A. Blowout
◆ Anticoagulate - potassium oxalate B. Self-draining
(affects rbc morphology) III. TYPE
A. Measuring
Calcium - important for clotting B. Transferring
CLINICAL CHEMISTRY I
LABORATORY

TO CONTAIN VS TO DELIVER
MOHR To deliver (TD) Calibrated
(Smaller) between 2
TO CONTAIN TO DELIVER marks on stem
(1-10)
Not necessarily calibrated Calibrated to deliver the
to deliver exact amount amount of fluid Serologic To contain (TC) Graduation
designated on pipette (Larger) marks up to the
(drains faster) tip (10-0)
Volume will flow out of the
pipette by gravity
READING MENISCUS
● Must be made with eye at the level of meniscus
● When clear solutions are used, the bottom of
meniscus is read
BLOWOUT VS SELF-DRAINING
AUTOMATED PIPETTED
● Depress the thumb knob to the first
● Immerse tip in 3mm or halfway into the sample
BLOWOUT SELF-DRAINING

Has continuous etched Has no continuous

rings/ 2 small, close, etched rings/ 2 small,
continuous rings close, continuous rings

MEASURING VS TRANSFERRING

MEASURING TRANSFERRING

● Serologic ● Calibrated
● Mohr ● Consist of
● Used in cylindrical bulb
measuring
reagents
● Straight piece of
glass
● Not calibrated
 COLOR ADDITIVE MODE OF ACTION LABORATORY USES

Yellow Tube Anticoagulant: Binds calcium Microbiology testing - for

Sodium Polyanethol Sulfonate cultivation of microorganisms
(SPS) Inhibits complement, present in blood
Inversion: 8-10x phagocytosis, and
Growth media for activity of certain
microorganisms antibiotics

Blood culture bottles come in diff. colors

BaCT/ Alert bottles: Yellow for pediatric; green for adult (aerobic); and orange for adult
(anaerobic)

Light Blue Tube 3.2% or 3.8% Chelates calcium Hematology specifically for
coagulation testing
Anticoagulant: Minimizes platelet
Inversion: 3-4x Buffered sodium citrate activation (sensitive light) PT - evaluates extrinsic system of
coagulation
Others:
CTAD (citric acid, theophylline, Activated Partial Thromboplastin
adenosine, dipyridamole) Time (APTT/PTT)

FDP (Fibrinogen Degradation

Products)

TT (Thrombin Time) and Factor

assays

Gold/ Red/ Black No anticoagulant Clot activator promotes Require serum such as chemistry
(Tiger) blood clotting with glass and serology tests
Serum separator Thixothropic gel as Serum
tube separator Specific gravity of gel: Not recommended for
1.03-1.09 g/cm3 Therapeutic Drug Monitoring
Inversion: 5x Glass as clot activator (TDM) due to slow absorption of
Clotting time: 30 the drug
mins

Red Top No anticoagulant

Inversion: 5x (for Plastic tube: silica particls as

plastic) clot activator
None
(glass) Glass tube

Clotting time: 30-60

mins

Orange No anticoagulant Clot activator promotes STAT (emergency)

blood clotting
Inversion: 5-6x Thrombin (clot activator)

Clotting time: 5
mins

Light Green Top Anticoaglant: Inhibits thrombin Chemistry testing (plasma

Heparin (lithium) determinations)
Inversion: 8-10x Formation to prevent
Has gel as plasma separator clotting
Green Top Anticoagulant: Inhibits thrombin Chemistry testing
Heparin (sodium, lithium, Ammonia, Carboxyhemoglobiin
Inversion: 8-10x ammonium) Formation to prevent
clotting

Lavender Top Ethylenediaminetetraacetic Chelates calcium Hematology testing (CBC), ESR

acid) EDTA
Clin. Chem.: HBA1C
Inversion: 8-10x Disodium EDTA
• Versene Blood films

Tripotassium EDTA Blood bank (routine testing and

• Sequestrene donor screening)

Gray Top Anticoagulant: Anticoagulant chelates Glucose test - not going to be

Potassium oxalate calcium tested immediately
Inversion: 8-10x
Sodium fluoride (anti-glycolytic Inhibits enolase, a late
additive) enzyme in the glycolytic
pathway
Sodium iodoacetate
Inhibits glyceraldehyde-
3-phosphate
dehydrogenase

Black Top 3.2% sodium citrate Chelates calcium Hematology , erythrocyte

sedimentation rates (ESR)
For whole blood
collection

Pink Top Spray-dried K2 EDTA Blood banking, molecular

diagnosis

White/Pearl K2EDTA Gel Molecular diagnosis (PCR,

bDNA)

Royal Blue Top May not contain anticoagulant Chemistry, toxicology or Trace
(free from trace (for serum testing) Element
element (sodium heparin or K2EDTA)
contamination)

Inversion: 8-10x

Tan Top (glass) Sodium Heparin: Tube contains less than 0.01
Inhibits thrombin microgram/mL, thus it is used for
Inversion: 8-10x lead testing
(plastic) K2EDTA:
Chelates calcium

Yellow Top Acid citrate dextrose (ACD) Blood bank studies, HLA
phenotyping, DNA and paternity
Inversion: 8-10x testing