INTRODUCTION TO CLINICAL CHEMISTRY

CHEMISTRY
• Clinical comes from the Greek word ‘kline,’ meaning ‘bed’
• Chemistry: science that deals with the elements, their
compounds and the chemical structure and interaction of
matter

CLINICAL CHEMISTRY
• Area generally concerned with the quantitative and
qualitative analysis of bodily fluids for diagnostic and
therapeutic purposes
• Originated in the 19th century with the use of simple
chemical reaction tests for various components of blood
and urine
o Other techniques have been applied as science
and technology have advanced including the use
and measurement of enzyme activities,
spectrophotometry, electrophoresis, and
immunoassays
• There are now many blood tests and clinical urine tests with
extensive diagnostic capabilities
• International Associated of Clinical Biochemists was
formed in 1952
o Changed their name to the International
Federation of Clinical Chemistry (IFCC) the
following year

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 INTRODUCTION TO CLINICAL CHEMISTRY
CLINICAL CHEMISTRY and automation, and the difficulty of
• Quantitative science concerned with the measurement of interpretation of results
amounts of biologically important substances or analytes in • Regulated under guidelines that
cover quality standards for
body fluids
proficiency testing, patient test
• Links knowledge of general chemistry, organic chemistry, management, QC and QA, and
and biochemistry with an understanding of human personnel qualifications
physiology
• Produces objective evidence from which medical decisions OSHA
may be made • Occupational Safety and Health Act
• Business that operates under the regulations and practices • Provides guidelines for safe operation of testing processes
that guide commerce • Include guidelines for operating safety equipment and
identifying handling and storing chemical hazards
COMMON ANALYTES
Ion, salts, Sodium, potassium, calcium, chloride, PROFICIENCY TESTING
minerals carbon dioxide, lead, iron
• Method of monitoring accurate outcomes
• Metabolites like glucose, cholesterol
• Test samples from external sources are analyzed and
and uric acid
• Therapeutic drugs like vancomycin, results compared to those reference laboratories and
Small organic theophylline, and digoxin scored for accuracy
molecules • Toxicology involving alcohol,
salicylate, and acetaminophen
• Drugs of abuse like cocaine and
barbiturates
• Transport proteins like albumin,
transferrin, and haptoglobin
• Enzymes like amylase and
Large creatinine kinase
molecules • Specific proteins like
immunoglobulins, CRP, and
complement
• Diabetic markers like HBA1C

REGULATORY GUIDELINES
CLIA 1988
• Clinical Laboratory Improvements of 1988
• Quality standards for all clinical laboratories to ensure
accuracy, reliability, and timeliness of patient test results
regardless of where the test was performed
• Defines clinical laboratories broadly

Simple laboratory examinations and

Waived tests procedures that are cleared by the US
FDA for home use
• Moderately and highly complex tests
Non waived
defined by the requirements for
tests
operator skill, reagent preparation

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 LABORATORY MATHEMATICS
MEASUREMENTS Units that do not take support of other physical
• Determination of the dimensions, capacity, quantity, or extent of Basic units quantities for its measurement; consists of 7
something base units
Quantities derived from the 7 base units, like
• Most common measurements in chemical laboratories include mass, Derived units
meter per second
volume, length, time, temperature, pressure and concentration Widely used and have become acceptable for
the use with SI basic or SI-derived units
Non-SI units
UNITS OF MEASUREMENT including hour, minute, day, gram, liter, and
Measurement system used in many plane angles expressed as degree
English System countries using pounds, yards, miles,
pint, etc SOLUTION
Derived from the Greek word, metron • Homogenous mixture of two or more substances with each
Metric System meaning measuring, used in scientific
substance retaining its own chemical identity
work, and includes gram, meter, and liter

PHYSICAL
METRIC SYSTEM ENGLISH SYSTEM Component of a solution that is present in the
QTY. Solvent
greatest amount
Length Meter Inch, foot, mile Component of a solution that is present in a lesser
Mass Gram Ounce, pound, ton Solute
amount relative to that of the solvent
Volume Liter Pint, quart, gallon
Power Watt Horsepower
MEASUREMENT OF SOLUTIONS
Torque Newton-meter Pound-foot
Temperature Celsius Fahrenheit • Most tests in the clinical laboratory involve measurement of solutes
or analytes in solutions
INTERNATIONAL SYSTEM OF UNITS o Solutions most often being blood, serum, urine, spinal
• Le Systeme International D’Unites / SI units fluid, and other body fluids
• Modern form of the metric system
• World’s most widely used system of measurement !"#$% '( %')*+,
or
3'"$#)2+4
Molarity (M) -./ 0 1')*$, '( %')*+2'3 1#),35,
o Used in both everyday commerce and science 6,2!7+ 23 !"#$%
Moles (mol) -./
!"#$% '( %')*+,
Normality (N) or (𝑚𝑜𝑙𝑎𝑟𝑖𝑡𝑦)(𝑣𝑎𝑙𝑒𝑛𝑐𝑒)
8/ 0 1')*$, 23 )2+,"%
Volume
𝑉1𝐶1 = 𝑉2𝐶2
concentration

PERCENT SOLUTIONS
• Determined in the same number regardless of whether
weight/weight, volume/volume, or weight/volume units
• Implies parts per 100 units represented by ‘%’
• Independent of molecular weight of a substance

• Number of grams of solute per 100 grams of

W/W or solution
mass/mass $#%% '( %')*+, 23 !"#$%
$#%% '( %')*+2'3 23 !"#$%
× 100
• Amount of solute per 100 mL of solution
V/V • Used when both solute and solvent are liquid
$9 '( %')*+,
$9 '( %')*+2'3
× 100
• Number of grams of solute per 100 mL of
solution
W/V • Most common type of solution
!"#$% '( %')*+,
× 100
$9 '( %')*+,

W/W EXAMPLES
1. Consider a 3 g sugar dissolved to make 10 g of sugar solution. What
is the w/w % concentration of the solution?

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 :!
× 100 = 𝟑𝟎% 1. A sample contains 18 g of NaCl. How many moles of NaCl are in the
;< !
sample?
; $')
2. To make up 200 g of a 5% aqueous solution of HCl using 12 M HCl, 𝑚𝑜𝑙𝑒𝑠 = 18 𝑔 × = 𝟎. 𝟑𝟎𝟕 𝒎𝒐𝒍
=B.= !
how much HCl is needed?
=! 0 (= !)(><< !) 2. A staff wants to prepare a solution containing 3 moles of NaCl. How
;<< !
= ><< !
→𝑥= ;<<
= 𝟏𝟎 𝒈
many mg of NaCl does she need?
; $') (0)(; $'))
3. To prepare 9% aqueous NaCl solution using 5 g of NaCl, how many 3 𝑚𝑜𝑙𝑒𝑠 = (𝑥) \=B.= !] → 3 𝑚𝑜𝑙𝑒𝑠 = =B.= !
grams of solvent is needed? What is the total weight of the solution? (0)(; $'))
=
(: $'))(=B.= !)
A! =! ; $') ; $')
= 𝒙 = 𝟏𝟕𝟓. 𝟓 𝒈 → 𝟏𝟕𝟓, 𝟓𝟎𝟎 𝒎𝒈
;<< ! 0
55.56 𝑔 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 → 55.56 = 5 + 𝑥 → 𝑥 = 55.56 − 5
𝒙 = 𝟓𝟎. 𝟓𝟔 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 3. How many moles are in 17.7 g of Na2CO3?
; $')
𝑚𝑜𝑙𝑒𝑠 = 17.7 𝑔 × ;<E !
= 𝟎. 𝟏𝟕 𝒎𝒐𝒍
V/V EXAMPLES
4. Ms. Cookie used 30 mL of pure ethyl alcohol to make 0.3 L of ethyl
MOLARITY
alcohol solution. What is the final v/v concentration of the solution?
:< $9 • Routinely expressed as mol/L, mmol/L, or ‘M’
× 100 = 𝟏𝟎%
:<< $9 • Expressed by dividing the mass of solute in grams by its gram
molecular weight / GMW
5. To make up 100 mL of a 2% v/v concentrated HCl solution, how much 3'.'( $'),% '( %')*+, $#%% '( %')*+, 23 !"#$%
or (%')*+, -./)(1'). '( %')*+2'3 23 9)
HCl is needed? 1').'( %')*+2'3 23 9

> $9 0 (> $9)(;<< $9)

= →𝑥= = 𝟐 𝒎𝑳
;<< $9 ;<< $9 =< $9 % W/V grams of (% '( %')*+2'3 G,%2",G)(+'+#) 1').G,%2",G)
solute ;<<
6. How many mL of distilled water is needed to prepare 50% v/v % V/V mL of (% %')*+2'3 G,%2",G)(+'+#) 1').G,%2",G)
isopropyl solution using 10 mL of isopropyl alcohol? What is the total solute ;<<

volume of the resulting solution? % W/W grams (% %')*+2'3 G,%2",G)(!"#$% '( +'+#) %')*+2'3)
=< $9 ;< $9 (;<< $9)(;< $9) of solute ;<<
= →𝑥= = 𝟐𝟎 𝒎𝑳 1')*$, '( %')*+,
;<< $9 0 =< $9 Ratio 1')*$, '( %')1,3+
1')*$, '( %')*+,
W/V EXAMPLES Dilution 1')*$, '( %')*+2'3
7. Ms. Cookie weighed 18 g of NaCl to make up 2000 mL of NaCl
solution. What is the final w/v concentration of the solution? 1. What is the molarity of a solution containing 20 g of HCl and 1 L of
;B ! water?
× 100 = 𝟎. 𝟎𝟎𝟗 𝒐𝒓 𝟎. 𝟗% 𝑵𝒂𝑪𝒍
><<< $9 ; $') ;
𝑀 = 20 𝑔 × × = 𝟎. 𝟓𝟓 𝒎𝒐𝒍/𝑳
:E.= ! ;9
8. How many grams of NaCl is needed to prepare 2.5 L of 25% w/v
NaCl solution? 2. How many grams of NaOH are required to prepare 1.5 L of a 0.5 M
>= ! 0 (>= !)(>=<< $9)
= →𝑥= = 𝟔𝟐𝟓 𝒈𝒓𝒂𝒎𝒔 solution?
;<< $9 >=<< $9 ;<<
<.= $') ; $') ; <.= $') (0)(; $'))
= (𝑥) \ ]× → =
9 H< ! ;.= 9 9 E< !/9
9. How many mL of 70% w/v glucose solution can be made using 5 g (0)(; $')) (E< !/9)(<.= $'))
=
of glucose dissolved in enough water? ; $') ; $')/9

C< ! =! (;<< $9)(= !) 𝒙 = 𝟑𝟎 𝒈

= →𝑥= = 𝟕. 𝟏𝟒 𝒎𝑳
;<< $9 0 C< !

3. The NSS label reads 5 M containing 0.90 M of NaCl. Determine the

MOLECULAR WEIGHT volume in mL of the solution and the amount of solute used.
• Add the atomic weights of the elements 0.9 𝑚𝑜𝑙 = 𝑥 \
; $')
]→
(0)(; $'))
=
(<.A $'))(=B=.!)
6,2!7+ 23 !"#$% =B.= ! ; $') ; $')
𝑚𝑜𝑙𝑒𝑠 = ./ 𝒙 = 𝟓𝟐. 𝟔𝟓 𝒈

Carbon 12 = $') ; $') ; = $') =>.E= $')/9

= 52.65 𝑔 × × → =
Hydrogen 1 9 =B.= ! 0 9 (=B.=)(0)
Oxygen 16 (𝑥)(58.5)(5 𝑚𝑜𝑙) = 52.65 𝑚𝑜𝑙/𝐿
Nitrogen 14 (0)(>A>.= $'))
= >A>.= $')
=>.E= $')/9
Sodium 23 >A>.= $')

Sulfur 32 1000
𝑥 = 0.18 𝐿 × = 𝟏𝟖𝟎 𝒎𝑳
Chloride 35.5 1𝐿
Calcium 40

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4. A solution of NaOH is contained within a Class A 1 L volumetric flask DILUTION
filled to the calibration mark. The content label reads 24 g of NaOH. • Expression of relative concentration
Determine the molarity. • Simple dilution: volume of sample divided by total volume of
; $') ;
𝑀 = 24 𝑔 × × = 𝟎. 𝟔 𝒎𝒐𝒍/𝑳 solution
H< ! ;9
• Process in which more solvent is added to a solution in order to lower
5. How many grams are needed to make 1 L of a 2 M solution of HCl? its concentration
> $') ; $') ; > $') (0)(; $')) • Representation of the ratio of concentrated or stock material to the
= (𝑥) \:E.= !] × → =
9 ;.= 9 9 :E.= !/9 total final volume of a solution, consisting of the volume or weight of
(0)(; $')) (:E.= !/9)(> $'))
= the concentrate plus the volume of the diluents
; $') ; $')/9
o Ratio: amount of something relative to another, expressed
𝒙 = 𝟕𝟑 𝒈
as part per part or part per whole
o The concentration units remain the same.
MOLALITY
• Molar, normal, percentage solutions: amount of solute contained in
• Amount of solute per 1 kg of the solvent
each volume of solution is equal to the product of volume multiplied
• Expressed in terms of mol/kg or ‘m’
3'.'( $'),% '( %')*+, $#%% '( %')*+, 23 !"#$% by the concentration
or (%')*+, -./)(1'). '( %')*+2'3 23 J!) o When the solution is diluted, the volume is increased and
1').'( %')*+2'3 23 J!
its concentration is decreased, but the total amount of
1. Calculate the molality of the solution prepared from 15.6 g of NaCl in solute remained unchanged.
1 kg of water.
; $') ;
𝑚 = 15.6 𝑔 × =B.= ! × ; J! = 𝟎. 𝟐𝟕 𝒎𝒐𝒍/𝒌𝒈 DILUTION OF STOCK SOLUTIONS
(𝐶; )(𝑉; ) = (𝐶> )(𝑉> )
• C1 = concentration of the stock solution
NORMALITY
• V1 = volume of the stock solution
• The number of equivalent weights per liter or eq/L, or in milli-
• C2 = concentration of the diluted solution
equivalents per mL
• V2 = volume of the diluted solution
o Equivalent weight = GMW divided by valence
• Often used in acid-base calculations
!"#$% '( %')*+, 1. How much 95% alcohol is needed to prepare 3 L of 50% alcohol?
(8/)(9 '( %')*+2'3) C1 = 95%, V1 = x, C2 = 50%, V2 = 3 L
(0.95)(𝑥) = (0.5)(3)
1. What is the normality of a 500 mL solution that contains 7 g of 0.95𝑥 = 1.5
H2SO4? 𝒙 = 𝟏. 𝟓𝟖 𝑳
C!
𝑁 = (HA)(<.= 9) = 𝟎. 𝟐𝟗
CONVERSION FACTORS
Glucose 0.0555
2. What is the normality of a 0.5 M solution of H2SO4? mg/dL to mmol/L
BUN 0.357
𝑁 = (0.5 𝑀)(2) = 𝟏 BUN to Urea 2.14
Urea to BUN 0.467
3. Compute for the normality of 3 M of NaCl. Creatinine mg/dL to µmol/L 88.4
𝑁 = (3)(1) = 𝟑 Uric acid 0.0595
Cholesterol 0.026
mg/dL to mmol/L
4. Compute for the molarity of 6 N HCl. HDL 0.025
𝑀= =𝟔
E Triglycerides 0.0113
; Sodium
Potassium 1
mEq/L to mmol/L
EQUIVALENT WEIGHT Chloride
./
𝐸𝑊 = 1#),35, Magnesium 0.5
Calcium 0.25
• Valence: number of replaceable H or OH atoms mg/dL to mmol/L
Phosphorus 0.323
o NaCl: GMW = 58 g, valence 1 Total protein
§ 58 g per EW Albumin g/dL to g/L 10 (0.01)
o HCl: GMW = 36 g, valence 1 Globulin
§ 36 per EW Bicarbonate mEq/L to mmol/L 1
o H2SO4: GMW = 98 g, valence 2 Ammonia µg/dL to µmol/L 0.587
§ 49 per EW Iron mg/dL to µmol/L 0.179
Bilirubin mg/dL to µmol/L 17.1
Thyroxine µg/dL to nmol/L 12.9

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 LABORATORY SUPPLIES AND EQUIPMENT
GLASSWARES LOW ACTINIC GLASS
HIGH THERMAL RESISTANT GLASS

• Reduces light transmission and has high thermal resistance

• Has materials that usually impart a red color to the glass that reduce
• Borosilicate glass with low alkali content the amount of light passing through the substance inside the
o Type of glass with silica and boron trioxide as the main glassware
glass-forming constituents • Provides protection to reagents highly sensitive to light ranging from
o Known for having very low coefficients of thermal 3000-5000 Angstrom, including bilirubin and Vitamin A
expansion making them more resistant to thermal shock
than any other common glass STANDARD FLINT GLASS / SODA-LIME GLASS
• Resistant to hear, corrosion, and thermal shock
o Corrosion: gradual destruction of materials by chemical
and/or electrochemical reaction with their environment
• Used when heating or sterilization is needed
• Beakers, flasks, pipettes
o Pyrex, Thymax: cannot withstand high pH content; can be
subjected to scratching and may cloud with strong alkali • Used for the manufacture of weighing bottles as it develops less
o Vycor: higher resistant to hear, heat shock resistance up static surface changes
to 900°C • Composed of a mixture of the oxides of silicon, calcium, and sodium
o National Institute of Standards and Technology / NIST:
class A supplies, or those that meet the standards for SPECIAL GLASSES
laboratory use Colored / opal
Used in light fitters, lamp bulbs and lighting lenses
glasses
• Have thin metallic oxide permanently fine-
HIGH SILICA GLASS
bonded to the surface of the glass
Coated glasses
• Have electronic applications as heat shield
to protect against infrared light
• Mostly soda-lime, lead, and borosilicate of
high optical purity
Optical glasses
• Used in making prisms, lenses, and optical
mirrors
• Have high thermal resistance, chemical
stability, and corrosion resistance like
Glass ceramics
• Made by removing all elements from borosilicate glass borosilicate glasses
/ Pyroceram
• Has good optical qualities, temperature capabilities, and is radiation • Useful in hot plates, table tops, and heat
resistant exchanges
• Used for high precision analytical work and for optical reflectors and Radiation- • Made of soda-lime and lead
absorbing • Useful in preventing transmission of huge
mirrors glasses energy radiation like gamma rays and X rays
• Not used for the type of glassware generally used in the laboratory
PLASTIC WARES
HIGH ALKALI-RESISTANT GLASS • Beginning to replace glassware in the laboratory setting
• Partially used for strong alkaline solutions • Has unique high resistance to corrosion and breakage, as well as its
• Often referred to as ‘soft glass’ varying flexibility
o Thermal resistance is much less than of borosilicate • Relatively inexpensive, allowing for most items to be completely
glasses disposable after each use
• Used primarily whenever digestion with strong alkali is made

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 POLYOLEFINS • Tubes are at a fixed angle when rotating
• Unique group of resins with relatively inert properties Fixed-angle / • Capable of higher speeds of 7000 rpm,
• Unaffected by acids, alkalis, salt solutions, and aqueous solutions angle-head with much less heat build-up
• Can be autoclaved • Produces slanted sediments
• Used to separate layers of different
specific gravities
• More vulnerable to attack by oxidizing • Usually refrigerated to counter heat
agents Ultracentrifuge produced due to friction
Polypropylene
• Can withstand higher temperatures • Spins at 100,000 rpm
• Used to make car interior parts • Gold standard for lipoprotein separation
Used primarily to fabricate bottles, beakers, jars, analysis
jugs, funnels, pipette jars, pipette baskets, tanks, Spins between 200-2000 rpm for body fluid cell
burette covers, check valves, disconnect valves, Cytocentrifuge
Polyethylene count/concentration
twistcock connectors, needle valves, hollow
stoppers, dropping pipettes, hydrometer jars,
TYPES OF PIPETTES ACCORDING TO CALIBRATION
stirring rods, tubing, and reagent dispensers
• Holds a particular volume but does not
dispense the exact volume
POLYCARBONATE RESINS
• Calibrated by introducing the exact weight of
• Twice as strong as polypropylene mercury required to give the desired volume
• Maybe used at temperatures ranging from 100-160oC at a specific temperature
• Unsuitable for use with bases like amines, ammonia, alkaline, and o Mercury does not wet glass and
oxidizing agents pipettes calibrated this way; will
contain but not deliver the stated
• Dissolved by chlorinated aliphatic and aromatic hydrocarbons
To contain / TC volume
• Insoluble in aliphatic hydrocarbons, some alcohols, and dilute
aqueous solutions and salts
• Used extensively in centrifuge tubes and graduated cylinders

TYGON

• Dispenses the exact volume

• Calibrated by weighing the volume of water
that will flow from them by gravity
• Rate of delivery must never be hastened by
blowing
• Non toxic, clear, plastic or modified plasticized polyvinyl chloride
• Can be used to handle most chemicals but should not be subjected
to prolonged immersion in aliphatic or aromatic hydrocarbons, To deliver / TD
ketones, and esters
• Flexible at 30oC, brittle at 45oC, resists dry heat to 95oC
• Can be steamed, autoclaved, or chemically sterilized
• Used for the manufacture of tubing like those in auto-analyzers

TEFLON-FLUOROCARBON RESIN
• Same as TD pipettes but drops remaining at
• Pure translucent white and inert to corrosive reagents, boiling aqua the tip after delivery is blown out to receiving
regia, nitric and sulfuric acids, boiling hydrocarbons, ketones, esters, vessel
and alcohols • An etched ring is seen near the mouthpiece
• Can resist extreme temperatures ranging from -270oC to 255oC used
in cryogenic experiments or work at temperatures over extended To blow out
periods
• Used for self-lubricating stopcocks, stirring bars, bottle cap liners and
tubing because of its anti-adhesive properties

TYPES OF CENTRIFUGES
• Tubes attain a horizontal position during ‘Between two Calibration is affected by weighing the water
Horizontal / spinning, and vertical position when at rest marks’ delivered between the two calibration marks
swinging bucket • Produces flat and highly packed
sediments at 3000 rpm

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 TYPES OF PIPETTES ACCORDING TO USE • No calibration
• Has the greatest degree of accuracy and • For biologic fluids with specific volumes
precision needed
• Designed to dispense one volume c/o further
subdivisions
• Calibrated to deliver a fixed volume of liquid Pasteur pipette
• Has a bulb between the mouthpiece and the
tip that decreases the surface area / unit
Volumetric / volume and diminished error from water film
transfer • Self-draining, TD pipette

• Macro: >1 mL, micro: <1 mL

• By far the most routinely used pipette
• Time saving, safer, more stable, provides
Automatic ease of use with increased precision and
pipette lacks required cleaning
• Tips used are often disposable
• TD and to blow out pipette
• Air displacement, positive displacement,
• Used in measuring viscous fluids like whole
dispenser / dilutor
blood
• Measures small volumes of 2.0 mm or less
• Has a bulb near the tip and an etched mark SEMI-AUTOMATIC MICROPIPETTORS
ring near the mouthpiece Uses suction to draw samples into a disposable
Ostwald-Folin Air
• Used with biologic fluids having a viscosity polypropylene tip, where the piston does not come
displacement
greater than that of water in contact with the liquid
Positive Operates like a hypodermic syringe, used for
displacement sampling in most discrete automated systems

BEAKERS
• Used to deliver an amount of liquid between
two calibration marks
• Serologic pipette: blow-out pipette
o The rate of fall of liquid is much
faster
o Has an etched band on the
suction piece
o Has calibration marks to the tip

• Made of glass that is resistant to many chemicals and are resistant

to heat
• Used for general mixing and reagent preparation
• Wide, straight-sided, cylindrical vessels available in many sizes and
Graduated / several forms
measuring
GRADUATED MEASURING CYLINDERS

• Mohr pipette: TD and blow-out pipette

o Calibration lies between two
marks on the stem
o No graduations to the tip
o Self-draining pipette

• Used to measure volumes of liquid when high degree of accuracy is

• TC pipette calibrated with mercury not essential
• Entire content of the pipette must be emptied
Micropipettes
• Used when small amounts of blood or
specimen is needed (<1 mL)
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 VOLUMETRIC FLASKS specifically analyzed for the desired
procedure
• Can be used when higher purity
biochemicals are not available
• USP: United States Pharmacopeia
• NF: National Formulary
• Generally less pure than CP grade as the
USP & NF grade tolerance is specified
• Used to manufacture drugs
• Not injurious to health, but purity standards
may not meet assay requirements
• Pear-shaped flasks Purified / Can be used as starting materials for synthesis of
• Have one calibration mark on the narrow part of the neck practical / pure other chemical of greater purity, but generally
• Used to contain a specific amount or volume of liquid grade should not be used in the clinical laboratory
Technical / • Only for industrial purposes
EQUIPMENT USED IN MEASURING MASS / SOLIDS commercial • Not used in the preparation of reagents for
Analytical • Precision is up to 1/1000 of a gram grade the laboratory
balance • Can be manual/mechanical or electronic • National Bureau of Standards, College of
• Precision is up to 0.1 gram American Pathologists & National
Rough / platform • Torsion balance: for weighing chemicals NCCLS Committee for Clinical Laboratory Standards
balance • Triple beam balance: with 3 beams • The highest grade; purest chemicals
present available

TYPES OF REAGENT WATER

• Substance that occurs naturally or is obtained
through a chemical process • Purest type; recommended for standard
Chemical preparations
• Used to produce a chemical effect or reaction Type I
• Clinical laboratory reagent water
• Produced in various purities or grades
• Colony count: <10 CFU/ml
• Defined as any substance employed to
produce a chemical reaction • Acceptable for most laboratory procedures
Reagent • Substance / compound added to a system to Type II including reagent preparation
cause a chemical reaction, or added to a test if • Colony count: <1000 CFU/ml
a reaction occurs • Can be used for some qualitative tests but not for
routine analysis and reagent preparation
Type III
STANDARDS OF PURITY • Water source for preparing types I and II water
• Wash and autoclave water
• Purity: freedom from adulteration or contamination; absence of
impurity or contaminants in a substance
STANDARDS
• There are many grades of chemicals available and it is essential to An idea or thing used as a measure, norm, or model in a comparative
understand which grade or type should be used for which reagent. evaluation
• When quantitative determinations are to be performed and accurate Highly purified chemicals which may be weighed out
standard solutions are prepared, it is necessary to use pure directly in the preparation of solutions of selected
Primary
chemicals. known concentrations
standard
o The label on the bottle and the supplier catalogue may • ACS primary standard: purity tolerance value of
100 ± 0.02%
give important information like maximum limits of
Prepared solution whose concentration is determined
impurities or an actual analysis of the chemical. Secondary by an analysis of an aliquot amount of the solution
• Very few compounds are available to the clinical laboratory, and are standard using primary standard & acceptable reference
known standards or the clinical type. methods
• Chemicals used in chemistry analysis, available
GRADES OF CHEMICALS in reliable preparations
• Of high degree of purity, used often in the • Reference sample or control material
Reagent grade / o Samples with chemical composition
preparation of reagents
analytic grade and physical characteristic that
• Designed by the American Chemical Society
For specific procedures like chromatography, simulate the specimen being analyzed
Ultrapure grade AAS, immunoassays, molecular diagnostics, and o Either serum or reference pool
Reference
standardization techniques reconstituted or freeze-dried with a
standard
definite volume of diluent before use
• Those sufficiently pure to be used in many
o May be in the form of solution ready
analyses in the clinical laboratory
for use
Chemically • Designation does not reveal limits of
• Many reference samples with normal and
pure grade impurities that are tolerated
abnormal levels are now available commercially.
• May not be acceptable for research and o Can be assayed or unassayed in
various clinical techniques unless
which values are identified
shinggibanggi | RMT March 2024
 • Useful in proficiency testing, inter-laboratory
surveys, and in the calibration of reference
materials like commercial kits and reagent sets

CLASSES OF REFERENCE STANDARDS

Calibration Confidence of the assigned value such that the
Reference overall uncertainty interval does not exceed 8%
Material of the 95% normal range of the constituent
Control Materials
Used as controls as the confidence limit does
with Assigned
not exceed 20% of the normal range
Values
• Similar to the unknown and should be
included in every set of determination
Control Materials
without Assigned • Done to eliminate pre-calibration which
may result from buying new reagents,
Values
deterioration of old reagents, use of
incorrect filters

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 ANALYTICAL METHODS AND INSTRUMENTATION
BASIC DISCIPLINES OF ANALYTICAL CHEMISTRY PARTS OF A SPECTROPHOTOMETER
Spectrophotometry, atomic absorption,
Spectrophotometry
mass spectrometry
Luminescence Fluorescence, chemiluminescence
Electroanalytical Electrophoresis, potentiometry,
methods amperometry
Chromatography Gas, liquid, thin layer

THEORY OF LIGHT WAVES

• Wavelength: distance between 2 successive waves expressed in
nanometers • Light source: provided polychromatic light that the sample modifies
o 1 nm = 10-9 m = 1 mu or attenuates by absorption
o Distance between peak and trough o Visible light: falls between violet at 400 nm, and red at
• The higher the amplitude, the more intense the light; and the more 700 nm as approximate limits of the visible spectrum
light energy produced at that wavelength. o Incandescent tungsten or tungsten-iodide lamp: most
common light source for work in the visible and near-
SPECTROMETRY infrared regions
• Used for measuring wavelengths of light spectra o Deuterium discharge lamp, mercury arc lamp: most
o Light energy, wavelength, and radiant energy spectrum commonly used for UV work
• Energy: transmitted via electromagnetic waves characterized by • Entrance slit: minimizes unwanted stray light; prevents entrance of
their intensity and frequency scattered light into the monochromator system
o Visible spectrum: 400-700 nm • Monochromator: isolation of specific/individual wavelengths
o UV region: <400 nm • Least expensive, passes a relatively wide
o Infrared region: >700 nm Colored glass band of radiant energy, with low
filter transmittance of selected wavelength
• Not precise but is simple and useful
• Produce monochromatic light based on
the principle of constructive interference
Interference waves
filters • Two pieces of glass mirroring on one side,
separated by a transparent spacer that is
precisely ½ of the desired wavelength
• Continuous, non-linear spectrum for better
separation of high frequency light
• Relationship between the energy and wavelength is described by • Wedge-shaped piece of glass, quartz or
Planck’s formula: 𝐸 = ℎ𝑣 NaCl
Prism
• Narrow beam of light is refracted as it
• Frequency: number of vibrations of wave motion per second
enters the denser glass
• Can be rotated, allowing only the desired
SPECTROPHOTOMETRY wavelength to pass through the exit slit
• Most commonly used technique • Most commonly used, with parallel
• Measures solute concentration by measuring light intensity in a grooves etched onto a polished surface
narrower wavelength Diffraction o 15,000-30,000 per inch
• Measurement of the light transmitted by a solution to determine gratings • Based on the principle that wavelengths
bend as they pass a sharp corner
concentration of light absorbing substances in the solution
• Better resolution than prism
o Amount of light absorbed µ analyte concentration • Exit slit: controls the width or bandpass of light beam
• Absorbance: amount of light absorbed o Allows only a narrow fraction of the spectrum to reach the
o 𝐴 = (𝑎)(𝑏)(𝑐) cuvette
o 𝐴 = 2 − 𝑙𝑜𝑔%𝑇 • Sample cell: absorption cell, analytical cell, cuvette
o 𝐴 = 𝑙𝑜𝑔 13𝑇 = −𝑙𝑜𝑔𝑇 o Holds the solution whose concentration is to be measured
• Transmittance: ratio of the radiant energy transmitted ÷ radiant o Path length is 1 cm
energy incident on the sample § To increase sensitivity, some cuvettes are
o Uses blanking technique using blank and sample blank designed to have a path length of 10 cm

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 increasing the absorbance for a given solution • Instrument that splits the monochromatic light
by a factor of 10 into two components: one passing through the
o Borosilicate glass, soft glass sample, and one passing through the reference
Alumina silica Most commonly used, approximately at 350- solution or reagent blank
glass 2000 nm • The additional beam corrects for variation in
Used for the measurement of solutions requiring light source intensity.
Quartz / plastic • Designed to compensate for intensity variations
visible and UV spectra
of the light source by splitting the light beam and
• Photodetector: converts transmitted radiant energy into an
directing one portion to a reference cuvette and
equivalent amount of electrical energy the other to the sample cuvette
• Least expensive, durable, composed of a
film of light sensitive material on a plate of
iron Double beam
Barrier layer • Require no external voltage source, relies
cells, photocell, on internal electron transfer
photovoltaic cell • Used for filter photometers with a wide
bandpass
• Temperature sensitive, nonlinear at very
• Double beam in space: uses 2
low and very high levels of illumination
photodetectors, one for the sample and one for
• Has a negatively charged cathode and a the reference beam
positively charged anode enclosed in a • Double beam in time: uses 1 photodetector
glass case that alternately passes the monochromatic light
Phototube
• Has a photosensitive material giving off through the cuvette and then the reference
electrons when light energy strikes cuvette using a chopper or rotating sector mirror
• Requires external voltage
• Most commonly used detector, measuring BEER’S LAW
visible and UV regions
• Concentration of a substance is directly proportional to the amount
• 200 times more sensitive than phototube
Photomultiplier of light absorbed, or inversely proportional to the logarithm of
• With excellent sensitivity, rapid response
tube transmitted light
detecting very low levels of light
• Should never be used at room light as it • Absorbance / optical density: amount of light absorbed
will burn out o Cannot be measured directly by a spectrophotometer, but
• Not as sensitive as PMT but has excellent is mathematically derived from %T
linearity and speed, and is small in size • Involves measuring absorbance and relating that to the solution’s
• Measures light at a multitude of
Photodiode concentration
wavelengths with less amount of light
• Most useful as simultaneous multi-channel • Expresses relationship between concentration and absorbance
detector o When measuring absorbance and calculating the
• Meter / read-out device: displays output of the detection system concentration from that data, the equation is rearranged to
!
o Galvanometer, ammeter, LED give 𝐶 = "#
§ Since a and b are constants, there is a linear
SPECTROPHOTOMETRY QUALITY ASSURANCE relationship between the absorbance of a
Wavelength set is the actual one selected by the material and the concentration of that material
Wavelength
monochromator, checked using didymium glass or
accuracy in a solution.
holmium oxide
Absorbance Done using glass filters and solutions with known § As the concentration increases, the absorbance
check absorbance values also increases.
Changes in concentration results in straight-line
Linearity calibration curve, determined using neutral density ATOMIC ABSORPTION SPECTROPHOTOMETRY
filters and dichromate solution • Measures concentration by detecting the absorption of
Any wavelength outside the band of interest that electromagnetic radiation by atoms rather than molecules
Stray light causes absorbance errors and loss of linearity;
detected using sharp cutoff filters • Measures light absorbed by atoms dissociated by heat
• Element is not excited by merely dissociated from its chemical bond
TWO TYPES OF SPECTROPHOTOMETERS and place in an unionized, excited, grounded state
• Simplest type of spectrophotometer • Light sources include the following:
• Makes one measurement at a time at one o Hallow cathode tube: separate lamp is required for each
specified wavelength metal like when a copper hollow cathode lamp is used to
Single beam measure Cu
o Electrodeless discharge lamps: relatively new light
source for atomic absorption spectrophotometers
• Interferences: chemical, matrix, ionization
shinggibanggi | RMT March 2024
• Disadvantages: PHOTOMETRY
o Inability of the flame to dissociate samples into free atoms
§ Phosphate may interfere with calcium analysis
by forming calcium phosphate
§ Solved by adding lanthanum or strontium to the
samples to form stable complexes with
phosphate
o Ionization of atoms following dissociation by the flame
§ Solved by reducing flame temperature
o Matrix interference due to enhancement of light absorption
• Measures light intensity without consideration of the wavelength
by atoms in organic solvents or formation of solid droplets
as the solvent evaporates in the flame
FLAME EMISSION PHOTOMETRY
§ Solved with pre-treatment of the sample by
• Measures light emitted by excited atoms
extraction
• Follows the principle of electron excitation from lower to higher state
• Routinely used to measure concentration of trace metals that are not
energy
easily extracted like calcium and magnesium
• Light source: flame
• More sensitive than flame emission photometry; more accurate,
• Method: indirect internal standard method
precise, and very specific
o Internal standard: lithium or cesium; corrects variations
• Monochromator: used to isolate desired emission line from other
in flame and atomizer characteristics
lamp emission lines
o Serves to protect the photodetector from excessive light
CHEMILUMINESCENCE
emanating from flame emissions
• Atomizer: nebulizer or graphite furnace; used to convert ions to
atoms
• Chopper: used to modulate the light source

FLUOROMETRY

• Emission of light is created from a chemical or electrochemical

reaction, not from electrochemical energy absorption
• No excitation radiation is required, no monochromators needed
• More sensitive than fluorescence
• The chemical reaction yields an electronically excited compound that
emits light as it returns to its ground state, or that transfers its energy
to another compound, which then produces emission
• Involves oxidation of organic compounds like dioexetane, luminol or
• Molecular luminescence spectrophotometry acridinium ester, by an oxidant like H2O2, hypochlorite or oxygen
• Measures concentrations of solutions containing fluorescing o Reactions may occur in the presence of a catalyst like
molecules enzymes, metal ions or hemin
• Measures light intensity present over a zero background • Used in ELISA and EIA
• Directly determines amount of light emitted by a molecule after • Photodetector: PMT laminator
excitation by electromagnetic radiation • Advantages include the following:
• Light source: gas discharge lamps like mercury arc or xenon lamp o Sub-picomolar detection limits
at 365-366 nm o Has flash type reactions; measures for only 10 seconds
• Light detectors: PMT or phototube o Ease of use, most are one-step procedures
• Uses 2 monochromators: filters, prisms, or gratings o Simple instrumentation
o Primary filter: select the wavelength that is best absorbed • Disadvantages include impurities causing background signals that
by the solution to be measured can degrade sensitivity and specificity
o Secondary filter: prevents incident light from striking the
photodetector TURBIDIMETRY
• About 1000 times more sensitive than spectrophotometry • Determines concentration of solute by measuring amount of light
• Affected by quenching: pH and temperature changes, chemical blocked by the analytes
contaminants, UV light changes o Light blocked depends on the concentration and solute
• Used in measurement of porphyrins, magnesium, calcium, size.
catecholamines
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• Measures abundant large particles like protein in CSF, detection of ELECTROCHEMISTRY
bacterial growth in broth cultures, measuring antibiotic sensitivities,
and detecting clot formation

• For the measurement of blood gas, blood pH, electrolytes, glucose,

urea, ionized calcium, lead, and chloride
• Involves the measurement of current or voltage generated by the
NEPHELOMETRY
activity of specific ions
• Methods include potentiometry using ISE, coulometry, amperometry
used for polarography, and voltammetry

POTENTIOMETRY

• Measures amount of light scattered by small particles

o Scattering depends on the wavelength and particle size
• Detectors placed at various forward angles, as well as at 90o to the
incident light
• For macromolecules with a size close to or larger than the • Measures electrical potential due to free ion activity
wavelength of the incident light • Measures differences in voltage/potential at a constant current
o Sensitivity is increased by measuring the forward light • Follows the Nernst equation
scatter • Reference electrodes: saturated calomel and silver-silver chloride
• More sensitive for protein measurement • Used for pH and pCO2 measurements in blood gases
• Used for measuring the amount of antigen-antibody complexes
• Uses mmol/L
OSMOMETRY • Very sensitive and selective
o Measures the activity of one ion
much more than the other ions
Ion selective present in the sample
electrode • Interference: excess protein
• Direct ISE: without sample dilution, indirect
ISE: with sample dilution
• Ion selectivity depends on the membrane or
barrier composition used
• Glass aluminum silicate or glass ion
exchange membrane
o Reference method for sodium
• Valinomycin gel: potassium
ISE membrane
• Ion exchange membrane: chloride
• Measurement of the osmolality of an aqueous solution like serum, • Organic liquid membrane ion exchanger:
plasma, or urine calcium, lithium
• Based on measuring changes in colligative properties of solutions • Gas and enzyme electrode
occurring owed to particle concentration variation
o ­osmolality, boiling point, osmotic pressure COULOMETRY
o ¯ freezing point, vapor pressure • Measures amount of electricity at a fixed potential
• Freezing point depression osmometry: most commonly used • Electrochemical titration in which the titrant is electrochemically
method in measuring changes in colligative properties generated, and endpoint is detected by amperometry
o used in measuring glucose, urea, nitrogen, sodium • Follows Faraday’s law
• Used for chloride tests for CSF, serum, and sweat
• Interference: bromide, cyanide, cysteine
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 AMPEROMETRY Fat red 7B
• Measures current flow produced by oxidation reaction Glycoproteins Periodic acid Schiff / PAS
• Used in measuring pO2, glucose, chloride, and peroxidase Coomasie blue for CSF proteins to confirm
Others
multiple sclerosis
determination
• Polarography: measures difference in current at a constant voltage
DENSITOMETER
o Follows the Ilkovic equation

VOLTAMMETRY
• Measures current after which a potential is applied to an
electrochemical cell
• Allows samples to be pre-concentrated, utilizing minimal analytes
• Anodic stripping voltammetry: for lead and iron testing

ELECTROPHORESIS

• Detector of an electrophoretic machine

• Measures stain absorption
• Scans and quantifies electrophoretic patterns
• Reads gel and cellulose acetate membranes

THINGS TO CONSIDER DURING ELECTROPHORESIS

• Electrophoretic mobility is directly proportional to net charge, and
inversely proportional to molecular size and viscosity.
• The ionic strength of the buffer determines the amount of current and
the movement of proteins for a fixed voltage.
• Endosmosis: at pH 8.6, the gamma globulins move toward the
• Migration of charged solutes/particles in an electric field
cathode even though they are negatively charged
• Separates proteins based on electric charge densities
• If the electrodes are not properly aligned, the current may be denser
o The acidic and basic amino acids determine the net
on one side of the gel
charge on a protein
• If electrophoresis proceeds too long, the proteins my migrate off the
o Proteins are negatively charged and move towards the
gel into the buffer.
anode
• If there is a break in the electric circuit and no current passes, the
• Iontophoresis: migration of small ions
proteins will not move.
• Zone electrophoresis: migration of charged macromolecules
• Smile artifacts: samples at the center of the gel migrate further than
• Factors affecting rate of migration
those at the edges
o Net electrical charge
o Size and shape of the molecule
MODIFICATIONS OF ELECTROPHORESIS
o Electric field strength
• Separating molecules migrate though pH
o Nature of the supporting medium gradient or constant gradient
o Temperature of the temperature • Ideal for separating proteins of identical
• Supporting media sizes but different net charges
o Paper electrophoresis • Proteins move in the electric field until they
o Starch gel to separate surface charge and molecular size reach a pH equal to their isoelectric point.
o Cellulose acetate to separate by molecular size only Isoelectric • Supporting media: agarose gel,
focusing polyacrylamide gel, cellulose acetate
o Agarose gel separates by electrical charge but does not
• Advantages include:
bind protein; is neutral
o Ability to resolve protein
o Polyacrylamide gel is neutral and separates based on mixtures
charge and molecular size o Detects isoenzymes
§ Can separate into 20 fractions; can study o Genetic variant identification
isoenzymes o Detect CSF oligoclonal bands
• Sample molecules are separated by
STAINS FOR FRACTION VISUALIZATION electro-osmotic flow
Coomasie brilliant blue Capillary o Positively charged ions move
Serum electrophoresis faster, negative ions move
Ponceau S
electrophoresis slower
Amido black
Oil Red O • Uses nanoliter quantities of specimen
Lipoproteins
Sudan black
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 • Used in separating, quantitating and • Substance is first separated by gas
determination of MW or proteins, PCR chromatography
analysis, and drug analysis • Can detect structural information and MV
determination
CHROMATOGRAPHY • Gold standard for drug testing
Gas
• Uses an electron beam to split drug
chromatography
with MS • Used for xenobiotics, anabolic steroids,
and pesticides
• MS/MS
Tandem mass
spectroscopy • Can detect 20 inborn errors of metabolism
from a single blood spot
• Based on the distribution of solutes
between a liquid mobile phase and a
• Techniques used to separate complex mixtures based on different stationary phase
• High performance liquid
physical interactions between the individual compounds and the
chromatography: most commonly used
stationary phase of the system method
o Uses pressure for fast
MODES OF SEPARATION separations, controlled
• Liquid-solid chromatography temperature, in-line detectors,
• Based on the competition between the and gradient elution technique
Adsorption
sample and the mobile phase for o Used in fractioning drugs,
chromatography hormones, lipid, carbohydrates,
adsorptive sites on the solid stationary
Liquid and proteins, separation and
phase
chromatography quantitation of hemoglobin
• Liquid-liquid chromatography
Partition • Based on relative solubility in an organic or associated with specific
chromatography nonpolar solvent and an aqueous or polar diseases like rapid HbA1C
solvent
• A variation of liquid-solid chromatography
Steric exclusion
chromatography • Separates solute molecules based on size
and shape
Ion exchange Solute mixtures are separated by virtue of the
chromatography magnitude and charge of ionic species
• Gel filtration, size exclusion, molecular
sieve chromatography
• Separates molecules based on differences
in size and shape
• As solutes travel through the gel, large
Gel permeation molecules remain in the mobile phase and
are eluted rapidly in the column
• Hydrophilic gel: gel filtration separating
enzymes, antibodies, and proteins
• Hydrophobic gel: gel permeation to
separate triglycerides and fatty acids
• Uses immobilized biochemical ligands as
Affinity stationary phase to separate solutes from
chromatography other unretained solutes
• Uses lock and key binding

• Separates steroids, barbiturates, blood,

alcohol, and lipids
• Useful for naturally volatile compounds, or
for easily convertible to its volatile form
• Specimens are vaporized and swept into
Gas
columns
chromatography
• Detector: flame ionization
• Elution order of volatile is based on their
boiling point
• Mobile phase: nitrogen, helium,
hydrogen, and argon
Mass
• Based on fragmentation and ionization of
spectrometry /
molecules using a suitable energy source
MS
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 LABORATORY AUTOMATION
AUTOMATION Minimum time required to obtain the result after
Dwell time
• Multi-disciplinary strategy to research, develop, optimize, and the initial sampling of the specimen
capitalize on technology to enable new and improved processes Maximum number of samples or tests that can
Throughput be processed in an hour; measures speed of an
• The modern clinical laboratory uses high degree of automation.
analytical system
o Permits operator to focus on manual processes and Labor maintenance, reagents, calibration,
increasing both efficiency and capacity Cost
quality control, consumables, and capital
• Development of new technologies to increase productivity, elevate List of analytes that a laboratory would be able
Test menu
experimental data quality, reduce lab process cycle times, or enable to provide for patient testing
experimentation Number of results a laboratory can generate
Workload
during a given time period
Walk-away Operator’s ability to program the instrument to
• Rapid results capability perform other tasks while processing other tests
• Increase number of tests performed Barcode Providing positive sample identification
• Saves time and effort Range over which patient results can be
• Eliminate need for staff increase Linearity
Advantages reported without sample manipulation
• Economical Values of the highest and lowest calibrations
• Reduced errors in calculation and Linear range
available for a particular instrument
transcription Lowest value that can be reliably detected by a
• Better precision and accuracy Sensitivity
method without providing false positive results
• Limitations in methodology Specificity Ability to measure only the analyte requested
• Personnel is often discouraged from making
observations and using own judgment MAIN TYPES OF AUTOMATIC ANALYZERS
Disadvantages
• Impractical system for a small number of CONTINUOUS FLOW SYSTEM
samples
• Expensive to purchase and maintain

AUTOMATION DESIGNS
Sequential analyzer Performs only one test at a time
Performs only one kind of test, but in multiple
Batch analyzer
specimens
Performs numerous tests but only for a single • All samples are carried through the same analysis pathway
Parallel analyzer
specimen • All samples automatically pass from one step to another without
Random access waiting to bring the samples to the same stage of competition
Performs tests in any order
analyzer • Reactions are not necessarily carried to equilibrium since samples
Each specimen is subjected to a single
Single-channel and standards are treated alike
process so only results for a single analyte are
analyzer • Uses plastic tubes of different diameters and a peristaltic pump for
produced, similar to batch analyzer
Each specimen is subjected to multiple continuous pumping of samples and reagents
Multiple-channel
processes so a set of test results is obtained • Has a maneuver that replaces the pipetting steps in manual
analyzer
for a single specimen procedures
o Introduces air bubbles to separate sample and reagent
All samples are loaded at the same time, and a streams into segments, separate one sample from
Batch testing
single test is conducted on each sample another, for continuous scrubbing of tubing, and prevents
More than one test is analyzed concurrently on
Parallel testing cross contamination or carry-overs
a given specimen
Random access Any test can be performed on any sample in any • Sampler: holds the cup containing the standard and specimen for
testing sequence analysis in a pre-set sequence and pre-selected rate
Sequential Multiple tests analyzed one after another on a • Pumps and manifolds: analogous to pipetting in manual
testing given specimen techniques; used for continuous and proportional delivery of
Open reagent A system other than the manufacturer’s reagent samples, reagents, or gases
system can be used
• Dialyzer: employs dialysis through a semi-permeable membrane to
Closed reagent
Operator can only use manufacturer’s reagent separate proteins from analytes, eliminating the need for manual
system
Number of tests that can be performed on an deproteinization
Test repertoire
instrument • Heating bath: heating and incubation of reaction mixture at fixed
Selective Only performs requested test temperatures

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 Technicon • Results are displayed on a screen and by printer
Can run 3 different tests at 60-80 samples per hr
Autoanalyzer II • Multichannel analyzer is in effect
SMA* 6/60 Runs 6 tests at 60 samples per hr
SMA 12/60 Capable of running 12 tests at 60 samples per hr
AMERICAN MONITOR KDA
SMAC Capable of running 40 tests at 120 samples per hr
*Simultaneous Multiple Analyzer • Computer-controlled, single channel analyzer
• Results are stored with subsequent print-out of collated patient
DISCRETE SAMPLING ANALYZER results
• Each sample reaction is handled in a separate compartment; does
not encounter another sample CENTRIFUGAL FAST ANALYZERS
• Samples and standards are handled on a batch basis and must be
brought before proceeding to the next procedure
• All reactions must be carried out until equilibrium is reached
• Vitros, Dimension Dade, Hitachi, Bayer Advia, Roche Cobas
Integra, Analytics P Module

DUPONT AUTOMATIC CLINICAL ANALYZER

• Centrifugal force moves the reagent and sample to a mixing
chamber, through a small channel into the cuvette, rotating past a
fixed light beam
• Absorbance of the reaction is measured spectrophotometrically
• CentrifiChem, RotoChem, COBAS-BIO, IL-MONARCH

THIN FILM ANALYZERS

• Reagents per test are packaged in a special plastic pack with a rigid
header serving as the reaction chamber and test cuvette for
photometric analysis
o Chromatographic column: removes interfering
substances
o Gel filtration matrix: slows down small molecules
o Protein precipitant column • 16-mm square chip containing several very thin layers, accepting a
metered drop of serum spreading is evenly into a reagent layer
ABBOTT ABA-100 BIOCHROMATIC ANALYZER • Confines the colored product to a fixed are for reflectance
• ABA-200 / VP analyzer spectrophotometry
• Has a single, disposable, plastic, 32-compartment molding so • Kodak ‘EktaChem’
transfer of final solutions is avoided
• With complete immersion of the reaction vessel in a water bath to GENERAL FUNCTIONS OF AUTOMATED ANALYZERS
achieve rapid rise to stable environment Collection and Use of barcoded labels on samples to allow
preparation of electronic identification of the sample and the
• Uses ultramicro samples of about 5 uL the sample tests requested
• Interference: serum and reagent color • Automated measurements measure,
o Solved by taking absorbance readings at 2 wavelengths, Sample and aspirate and introduce samples into the
or in a biochromatic system reagent analyzer reagents.
measurement • Reagent and sample are combined, either by
BECKMAN ASTRA 8 & ASTTRA 4 mixing stirring or agitation, to yield a specific final
concentration
Waiting period of a test mixture is allowed to react;
Incubation done at a specified, constant temperature
controlled by the analyzer
Monitoring or Through optical, thermal, or electrical means in a
sensing the vessel, cell, or cuvette in a process called situ-
reaction result monitoring
• Digital computation: restricted to certain
functions like addition or subtraction
Quantitating the o Needs an analog-to-digital
reaction result converter to process signals
• With microprocessor-controlled instruments, processing ultramicro received from sensing or
samples monitoring devices
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 o Converter changes the voltage
into a digital form which can be
processed by the computer
• Analog computation: uses electrical
signals from the sensor, and compares it
with a reference signal for the blank solution
o Compares the signals and takes
the logarithm of the result as the
final result for the unknown
sample
• Uses a television monitor or cathode-ray
tube or LEDs
• Visualized read-out can be converted to a
hard copy by means of a paper or tape print
out
Visualizing the
result • Data printout is transferred to lab result slips
or other permanent records
o If results are interfaced with a lab
computer, the transcription
process is done quickly and
without errors.

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 QUALITY MANAGEMENT AND QUALITY CONTROL
QUALITY MANAGEMENT QUALITY CONTROL
• Management philosophy and approach focusing on processes and • Ensures accuracy and precision in the laboratory including quality
their improvement as means to satisfy customer needs and control reagents in every series of measurement
improvement • Techniques ensuring with a specified degree of confidence that the
• CLSI and ISO: coordinates activities to direct and control an results obtained from each series is true and correct
organization with regards to quality • Tools used to assess quality of analytical data
• Ensures that an organization, product, or service is consistent • Process ensuring analytical results are correct by testing known
• Quality planning, quality assurance, quality control, quality samples that resemble the patient sample
improvement • Objectives include: checking machine stability, checking reagent
• Focuses on product and service quality, and the means to achieve it quality, checking technical/operator errors
• Uses quality assurance and control of processes, and products to
achieve more consistent quality KINDS OF QUALITY CONTROL
• Internal quality control
ERRORS IN THE TESTING PROCESS • Day to day performance of the laboratory
• Incorrect patient identification • Focused on precision
• Mislabeled specimens • Analysis of at least 2 levels of control every 24
• Incorrect order of draw Intralab QC hours
• Incorrect anticoagulant to blood ratio o Initial control limits are established by
analyzing control materials for at least
• Improper mixing of blood & anticoagulant
20 consecutive days or runs
• Improper patient preparation
Pre-analytical • Controls random and systematic errors
• Incorrect specimen collection
errors • External quality control
• Incorrect use of tubes for blood collection
• Accuracy of tests
• Incorrect specimen preservation
Interlab QC • Used in performance comparison
• Mishandled specimen in transport/storage
• Used to establish comparability and maintain
• Incorrectly interpreted / ordered test
long term accuracy
• Incomplete centrifugation
• Incorrect data log in
• Incorrect sample and reagent volume QUALITY CONTROL MATERIALS
• Incorrect solution incubation • Used for accuracy
• Equipment or instrument malfunction • Usually colorless
Analytical errors • Solution of known characteristics and of known
• Improper equipment calibration;
calibration error value or whose concentration is accurately
Standard known
• Poorly written procedures
solution • Composed of one known constituent only and
• Interfering substances not recognized
used as a basis of reference for the calculation
• Unavailable or delayed results
of the value of unknown
Post-analytical • Wrong transcription of data and results • 100% pure
errors • Long turnaround time • Serves as the reference for the unknown
• Incomplete/missing laboratory results
• Used for precision
• Results submitted to the wrong physician
• Solution that is either commercially or non-
commercially prepared, composed of several
QUALITY ASSURANCE known constituents which can be run
• Overall management plan to guarantee data integrity Control simultaneously with the test
• Talks about the system solution o Assayed: values provided by the
• Six sigma (6 s): reduction of defects to near zero manufacturer
o Unassayed: values determined by
o Define, measure, analyze, improve, control the laboratory
• Lean: reduction of non-valued activities or wastes • Derived from human blood; pooled serum
o Improvement of turn-around time • Solution without the specimen, but with a reagent
o Lessens defects, overproduction, waiting, non-utilized • Reagent blank: corrects for absorbance caused
talent, transport, inventory, motion, and excess processing by reagent color, zeroing the instrument before
• Delta check: algorithm in which a current laboratory result is Blank measuring test samples
compared with results obtained on a previous specimen from the • Sample blank: subtracts intrinsic absorbance
caused by hemolysis, icterus, turbidity, or drug
same patient
interference during analysis
o Detects change in result trends

shinggibanggi | RMT March 2024

 KINDS OF QUALITY CONTROL REAGENTS Distribution of data points around the
• Manufactured by different companies which mean
Standard deviation
may come as lyophilized, pulverized or non- .(/-/̅ )!
Commercially pulverized, or non-lyophilized 𝑆𝐷 = ) 0-2
prepared • Can be assayed or unassayed Best indicator of precision
o Assayed: known and given values Coefficient of variation 34
𝐶𝑉 = /̅ × 100
o Unassayed: values are known only
Variance 𝑉 = 𝑆𝐷5
PARAMETERS / IMPLICATIONS OF QUALITY CONTROL
REFERENCE INTERVAL STUDIES
Ability to measure the smallest concentration of the
Sensitivity • Done to confirm the validity of an existing
analyte of interest; used in screening tests
reference interval for an analyte
Ability to measure only the analyte of interest; used Verifying a
Specificity • Requires at least 20 study individuals, and
in confirmatory tests reference interval
adopted is <10% of the subjects fall
Ability of a method to detect the proportion of
individuals with the disease, generating more true- outside the range
Diagnostic • Done when there is no existing RI for an
sensitivity positive results and few false negative results
!"#$ (') analyte, or when transference studies fail
× 100 Establishing a
!"#$ (')')*+,$ (-) • Requires at least 120 study individuals, set
reference interval
Detects the proportion of individuals without the based on 95% confidence interval of mean
Diagnostic disease, reflecting detection of true negative with ±2SD
specificity few false positive results
!"#$ (-)
(-)')*+,$
× 100 VARIATIONS
!"#$ (')
Nearness / closeness of the assayed value to the • Errors encountered in the collection, preparation, and measurement
Accuracy true value; determines the exact value of the of samples including transcription and release of results
substance of interest o Interpretation of results is either in or out control
Precision / Ability to give repeated results on the same sample
reproducibility that agrees with one another
TYPES OF ERROR
Practicality Degree by which a method is easily repeated
• Due to chance or an unpredictable cause
Ability to maintain accuracy and precision over an
affecting precision; does not recur
Reliability extended period during which equipment, reagents,
o Mislabeling
and personnel may change
o Improper pipetting
Random error o Improper mixing of sample and
BENEFITS OF A GOOD QUALITY CONTROL PROGRAM reagent
• Provision of a continuous record of reliability of results o Voltage or temperature
• Permits valid judgments on the accuracy of results by monitoring fluctuations
precision and permitting comparisons on assay values on known o Dirty optics
control sera with stated values • Recurring error/s in a test procedure
influencing observations in one direction,
• Gives early warning of trends and shifts in control results so remedial
affecting accuracy
actions may be taken before serious loss of precision o Calibration problems
• Monitors the performance and stability of equipment used on the o Reagent / control material
assay deterioration
o Improperly made standard
Systemic error
solutions
STATISTICAL TERMINILOGIES
o Contaminated solutions
Inferential Compares means or standard deviations of two
o Unstable and inadequate reagent
statistics groups of data
blanks
Determines whether there is a statistically significant
o Leaky ion selective electrode
F-test difference between the standard deviations of two
o Failing instrumentation
groups of data
o Poorly written procedures
Determines if there is a statistically significant
T-test • Problems with handwritten labels and
difference between the means of two groups of data
request forms
Linear Compares two methods using the best fit line through Clerical error
• Online computer input: most error-free
regression the data points
means of requesting tests
Value that divides the observations into two groups;
Median
midpoint of the distribution
Mode Most frequent observation
Range Difference between the highest and lowest data score

STATISTICAL TOOLS OF QA AND QC

Arithmetic value / mean ./
𝑥̅ = 0
/ average

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 QUALITY CONTROL CHARTS / HISTOGRAMS • Occurs when data set can be accurately described by the SD and
SHEWHART-LEVEY JENNINGS CHART mean
• Total area under the curve is between 1-100%
o 1 SD: 68%
o 2 SD: 95%
o 3 SD: 99%

• Dot chart; most commonly used QC chart

• Graphic representation of the acceptable limits of variation in
analytical method reports

• Formed by control values that either increase or

decrease for six consecutive days CUMULATIVE SUM GRAPH
• Gradual change in distribution
• Major cause: reagent deterioration

Trend

• CUSUM: calculates difference between QC results and target means

• Plots algebraic sum of the difference of each QC result and the mean
on the y-axis, and the run number on the x-axis

• Formed by control values that distribute on one YOUDEN PLOT

or either side of the mean for six consecutive
days
• Abrupt change in distribution
• Major cause: calibration error or error in
standard preparation

Shift

• Tonks-Youden / Twin plot: displays results of analysis by plotting the

mean values for one specimen on X and Y axis
• For interlaboratory comparison of monthly means
Values that are far from the main set of values • Effective method of comparing within-laboratory and between-
laboratory variability

WESTGARD CONTROL CHART

Outliers • Uses ‘control rule’ to indicate if the analytical process is out of control

Detects imprecision and systematic bias

13S
GAUSSIAN CURVE
• Normal distribution curve: group any series of measurement in the
same sample in a cluster around the mean in a bell-shaped curve

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 • Warning rule: initiates testing of other rules
• Detects imprecision, but run is accepted

12S

22S

R4S

41S

10X

shinggibanggi | RMT March 2024

 INTRODUCTION TO CARBOHYDRATES
CARBOHYDRATES o Location of the CO function group
• Major food source and body’s energy supply o Number of sugar units
• Stored primarily as liver and muscle glycogen o Stereochemistry of the compound
• Compounds of tremendous biological importance
o Provide energy through oxidation SPECIMEN COLLECTION AND HANDLING
o Supply carbon for the synthesis of cell components • Glucose is measured from whole blood, plasma, serum, CSF, pleural
o Serve as form of stored chemical energy fluid, and urine of different types of diagnostic treatment
o Form part of the structures of some cells & tissues o Proper specimen collection & handling: first thing that
• Known as biomolecules as they are closely associated with living needs attention and consideration for accurate and
organisms precise clinical analysis
• Major constituents of physiological systems • Venous plasma glucose: standard clinical specimen
• Disease states are divided into hypoglycemia and hyperglycemia • 0.4 mmol/L/hr: glucose metabolism rate at room temperature
o Early detection of DM is the aim of the American Diabetes o 2 mg/dL/hr: approximate loss at 4OC
Association / ADA guidelines in 1997 o Bacterial contamination and leukocytosis increase rate of
§ Acute and chronic complications may be metabolism
avoided with proper diagnosis, monitoring, and • Serum can be utilized is separated from the cells withing 30 minutes
treatment or a preservative must be used
§ Periodic measurements of glycosylated o Sodium fluoride: utilized as a preservative using 2
hemoglobin like HbA1C and microalbumins mg/mL of blood, preventing glycolysis up to 48 hours in
• Compounds containing carbon, hydrogen, and oxygen the refrigerator
o General formula: CX(H2O)y • -20OC: long term specimen storage
o Glycogen: stored form
o Glucose: principal sugar circulating in the bloodstream GLUCOSE MEASUREMENT
• Can demonstrate several groups of disorders specific in diagnosing
certain conditions like hypo/hyperglycemia
• Most commonly measured from serum or plasma
• Fasting blood glucose: obtained from a person that has not eaten
for 8-10 hours
• In quantitating carbohydrates in body fluids, the ability of glucose as
a reducing agent is useful.
o Can convert cupric ion in an alkaline solution to cuprous
ion, losing its blue color, giving a red precipitate of cuprous
oxide form

• All contain C=O (carboxyl) and =OH (hydroxyl) groups

• Dietary CHO: consist mostly of starch initially hydrolyzed in the
mouth due to amylase secretion, converting starch into maltose and
dextrin
o Further digestion happens in the stomach in the process • Benedict’s and Fehling’s reagents: contains alkaline solution of
of chemical digestion by pepsin cupric ions that are stabilized by citrate and tartrate respectively,
o Most chemical digestion happens in the small intestine used to detect reducing agents in urine and other body fluids
where simple sugars and proteins are absorbed into the o O-toluidine: can also be utilized in quantitating
inner lining; fatty acids and glycerol go to the lymphatic carbohydrates like galactose
system • Glucose oxidase: most specific enzymatic reaction method
o The first section of the SI combines liver and gallbladder o Hexokinase method: more accurate than glucose
bile & stomach acid with pancreatic juice to digest and oxidase as the coupling reaction using G6PD is highly
metabolize carbohydrates into glucose specific
• Classified based on four different properties:
o Size of the base carbon chain
shinggibanggi | RMT March 2024
 METHODS OF DETERMINATION • Maltose, lactose, sucrose
• Glucose oxidase: polarographic / colorimetric Oligosaccharides Chaining of 2-10 sugar units
Enzymatic • Hexokinase with G6PD Linkage of many monosaccharide units yielding
methods • Glucose dehydrogenase: spectrophotometric / Polysaccharides >10 monosaccharides during hydrolysis
electrochemistry • Starch, glycogen
• Copper reduction: Folin-Wu, Nelson Somogyi,
Non- neocuprine CHEMICAL PROPERTIES OF CARBOHYDRATES
enzymatic • Hagedorn Jensen: ferricyanide / ferric • Reducing substances: must contain a ketone or an aldehyde group
methods reduction method o Property used in many methods in the determination of
• Dubowski: condensation / O-toluidine method
carbohydrates
For other • Fructose: Seliwanoff’s / Selivanoff’s test
CHOs • All monosaccharides and disaccharides are reducing agents.
• Pentose: Bial’s test
o A disaccharide remains a reducing agent when the
CLASSIFICATION OF CARBOHYDRATES hemiacetal or ketal hydroxyl group is not linked to another
• Grouped into generic classifications based on the number of carbon molecule
atoms o Maltose and lactose are reducing agents, sucrose is not
o Trioses: 3 atoms
o Tetroses: 4 atoms GLUCOSE METABOLISM
o Pentoses: 5 atoms • Glucose: primary source of energy in humans
o Hexoses: 6 atoms o The nervous system totally depends on glucose from the
• Glyceraldehyde: 3-carbon compound considered the smallest surrounding ECF for energy
carbohydrate in practice o Nervous tissues cannot concentrate or store CHO as it is
critical to maintain a steady supply of glucose in tissues
• Hydrates of aldehyde or ketone derivates based on the location of
the CO functional group • Glucose concentration in the ECF must be maintained in a narrow
o Aldose: CHO with a terminal carbonyl group [O=CH] range
called aldehyde group o When it falls below a certain level, the nervous tissue loses
o Ketose: CHO with a carbonyl group in the middle, [O=C] a primary energy source and is incapable of maintaining
linked to 2 other carbons called ketone group normal function

STEREOISOMERS
• Central atoms of CHO that are asymmetrical / chiral
• Stereogenic centers: four different groups are attached to the
carbon atoms, allowing for various spatial arrangements around each
asymmetric carbon, forming molecules called stereoisomers

• Glucose: only CHO to be directly used for energy, or stored as

glycogen
• Galactose and fructose: converted to glucose before use
• Have the same order and type of bonds but have different spatial o As glucose enters the cell, it is quickly shunted into 1 of 3
arrangements and different properties possible metabolic pathways, depending on the
o For each asymmetric carbon, there are 2n possible availability of substrates, or the cell’s nutritional status
isomers: 2 forms of glyceraldehydes, 16 or 24 possible • The goal of the cell is to convert glucose to CO2 and H2O
forms for aldohexoses • Embden-Meyerhoff pathway: glycolytic pattern for glycogen
breakdown
Simple sugars that cannot be hydrolyzed to • Hexose monophosphate pathway: considered an essential
Monosaccharides simple forms, but can contain 3 or more C atoms pathway; alternative pathway to glycolysis
• Glucose, fructose, galactose • The conversion to glycogen pathway is important for glucose storage
• Formed when 2 monosaccharide units are
joined by a glycosidic linkage
Disaccharides • Will be split into 2 monosaccharides by GLUCOSE METABOLISM PATHWAYS
enzymes in the microvilli of the intestine Metabolism of glucose to pyruvate or lactate for
Glycolysis
during hydrolysis energy production

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 Formation of glucose-6-phosphate from non-
Gluconeogenesis
CHO sources
Glycogenolysis Glycogen breakdown to glucose for energy use
Glycogenesis Glucose conversion to glycogen for storage
Lipogenesis CHO conversion to fatty acids
Lipolysis Fat decomposition

CARBOHYDRATE METABOLISM REGULATION

• Blood glucose control is under 2 major hormones: insulin and
glucagon
o Both are produced by the pancreas, but actions oppose
each other
• Primary hormone responsible for glucose entry into
the cell
• Synthesized by the cells of islets of Langerhans in
the pancreas, releasing insulin once glucose
increase is detected
• Release causes increased movement of glucose
into cells and increased glucose metabolism
Insulin • Normally released when glucose levels are high
• Decreases plasma glucose levels by increasing
receptors
• Regulates glucose by increasing glycogenesis,
lipogenesis, and glycolysis, and inhibiting
glycogenolysis
• Only hormone decreasing glucose levels
• Referred as a hypoglycemic agent
• Primary hormone for increasing glucose levels
o ­ glucose: cortisol, catecholamines,
GH, thyroid hormones, ACTH
• Synthesized by the cells of the islets of
Glucagon Langerhans, released during stress and fasting
states
• Increases plasma glucose by glycogenolysis in the
liver; also increases in gluconeogenesis
• Referred as a hyperglycemic agent
• Other hormones / neuroendocrine substances also exert some
control over blood glucose concentrations
o Permits the body to respond to increased demands for
glucose, or to survive prolonged fasts
o Conserves energy as lipids when excess substrates are
ingested
• Somatostatin: pancreatic cell that inhibits increase and decrease of
glucose levels
o Hormone preventing glucose overproduction

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 CLINICAL CONDITIONS OF CARBOHYDRATE METABOLISM
HYPOGLYCEMIA Occurring after 10 hours without food,
• Decreased glucose levels from the imbalance of glucose utilization Post-absorptive / secondary to hyperinsulinism, hormonal
and production fasting deficiencies, genetic disorders, autoimmunity,
or is drug-induced
o Some can be transient and relatively insignificant; others
Post-prandial /
can be life threatening alimentary / Usually occurs within 4 hours after meal
• 65-70 mg/dL or 3.6-3.9 mmol/L: glucagon and other glycemic reactive
hormones are released into the circulation
• <60 mg/dL: strongly suggests hypoglycemia HYPERGLYCEMIA
o 50-55 mg/dL or 2.8-3.0 mmol/L: appearance of • Increase in blood glucose concentration
observable hypoglycemia symptoms • Toxic to b cell function, impairs insulin secretion
§ Warning signs are either neurogenic or • 260 mg/dL or more: value of fasting blood glucose
neuroglycopenic; all relating to the CNS o >200 mg/dL or 11.1 mmol/L with symptoms of diabetes
Catecholamine mediated tremors, palpitations for random blood glucose
Neurogenic and anxiety, with acetylcholine released • In healthy patients with hyperglycemia episodes, insulin is secreted
symptoms diaphoresis, hunger, and paresthesias
by the cells of the pancreas, enhancing membrane permeability to
• Triggered by the autonomic nervous system
Hunger, sweating, nausea and vomiting, cells in the liver, muscles, and adipose tissue
dizziness, tingling, difficulty concentration, • Caused by hormonal imbalance
Neuroglycopenic nervousness and shaking, blurring of speech and
symptoms sight, mental confusion, behavioral changes, LABORATORY FINDINGS
seizures, comatose • Increased glucose in plasma and urine
• Due to diminished glucose supply to CNS
• Increased urine specific gravity
• Epinephrine is released to the systemic circulation, norepinephrine
• + ketones in serum and urine in Type 1 DM
release at the nerve endings of specific neurons acts with glucagon
o Ketone test: recommended when plasma glucose
to increase plasma glucose
reached 300 mg/dL
o Glucagon: released from the pancreas, inhibits insulin
o DKA/diabetic ketoacidosis: >1:1 ratio of b-
o Epinephrine: from the adrenal glands increasing glucose
hydroxybutyrate to acetoacetic acid
metabolism while inhibiting insulin
§ Gerhardt’s ferric chloride: reacts only with
o Cortisol and GH: increase glucose metabolism
acetoacetate
• Whipple’s triad: important tool in assessing patients with
§ Nitroprusside test: 10x more sensitive to
hypoglycemic episodes
acetoacetate than acetone
o Low blood glucose concentration
§ Acetest tablets: detects acetoacetate and
o Typical symptoms
acetone in lesser degrees
o Symptoms alleviated by glucose administration
§ Ketostix: detects acetoacetate better than
• 5-hour glucose tolerance test: diagnostic test
acetone
o Hypoglycemic dip: often not seen until after 3 hours
• Acidosis: decreased blood and urine pH
o 50 mg/dL or less, or <2.8 mmol/L in infants: considered
• Electrolyte imbalance: ¯sodium, ¯bicarbonate, ­ potassium
abnormal, requires diagnostic assessment
o 180 mg/dL: glucose will be excreted in urine
o 300-500 mg/dL: period of plateau
CAUSES AND CLASSIFICATION OF HYPOGLYCEMIA
• Drug administration: insulin, alcohol, salicylates, sulfonamides,
pentamidine
• Critical illnesses: hepatic failure, sepsis, renal failure, cardiac
failure, malnutrition
• Hormonal deficiency: epinephrine, glucagon, cortisol, GH
• Endogenous hyperinsulinism: pancreatic b cell disorders
• Autoimmune hypoglycemia: insulin antibodies
• Non-b cell tumors: leukemia, hepatoma, pheochromocytoma,
lymphoma
• Hypoglycemia of infancy and childhood: galactosemia, GSD,
Reye’s syndrome

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 • Glucosuria: occurs after the renal tubular transported system for
glucose becomes saturated
o Glucose concentration exceeds 180 mg/dL in an individual
with normal renal function and urine output
• Plateau period: concentration of 300-500 mg/dL, or 17-28 mmol/L
during hepatic glucose overproduction
o Provided that renal output is maintained, glucose
excretion will match overproduction, causing the plateau
• Ketosis: develops from excessive synthesis of acetyl-CoA in an
attempt to obtain required energy from stored fat, in the absence of
adequate supply of CHO metabolites
o (+) ketones: frequent finding in severe, uncontrolled DM
o 6:1 ratio of b-hydroxybutyrate to acetone
DIABETES MELLITUS
o Can be reversed by insulin administration
• Group of metabolic diseases characterized by hyperglycemia
resulting from defects in insulin secretion, insulin action, or both
RISKS AND PRE-DISPOSING FACTORS
• National Diabetes Data Group, 1979: development of the
• Age
classification and diagnosis scheme for DM
• Being overweight; habitual physical inactivity
• ADA / WHO guidelines recommended the following categories of DM
• Family history; history of impaired glucose tolerance
• b cell destruction
• High risk population
• Absolute insulin deficiency
• (+) autoantibodies • History of GDM
o Islet cell autoantibodies • Hypertension: usually a BP of >140/90 mmHg
Type 1 o Insulin autoantibodies • History of cardiovascular disease
o Glutamic acid decarboxylase • Low HDL concentration: <35 mg/dL or 0.90 mmol/L
autoantibodies
• Elevated triglycerides: >250 mg/dL or 2.82 mmol/L
o Tyrosine phosphatase IA-2 and IA-2B
autoantibodies • Women with PCOS
• Insulin resistance with an insulin secretory defect • Other clinical conditions associated with insulin resistance like
Type 2
• Relative insulin deficiency acanthosis nigricans
• Associated with any secondary condition
o Genetic defects of b cell function TYPE 1 DIABETES MELLITUS
o Pancreatic diseases
Others o Endocrine diseases
o Drug or chemical-induced
o Insulin receptor abnormalities
o Other genetic syndromes
• Glucose intolerance during pregnancy
GDM
• Metabolic or hormonal changes
• FBS concentration of >126 mg/dL on more than one testing: • IDDM, juvenile diabetes, brittle diabetes, ketosis-prone diabetes
diagnostic of DM • Inappropriate hyperglycemia resulting from pancreatic islet cell
destruction and a tendency to ketoacidosis
• Cellular-mediated autoimmune destruction of pancreatic cells
causing absolute deficiency in insulin secretion
• 110 mg/dL: FBS upper limit
• Constitutes only 10-20% of all cases; commonly during childhood
and adolescence
• Usually initiated by environmental factors or infection, usually viral,
in individuals with genetic predisposition causing immune destruction
of pancreatic cells leading to decreased insulin production
• Abrupt onset and insulin dependence
• Signs and symptoms include the following
o Polydipsia / excessive thirst
o Polyphagia / excessive food intake
PATHOPHYSIOLOGY o Polyuria / excessive urine production
• DM patients will be hyperglycemic in both types, which can be severe o Rapid weight loss
o Hyperventilation

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 o Mental confusion; possible loss of consciousness due to GESTATIONAL DIABETES MELLITUS
increased glucose in the brain
• Complications include microvascular problems like nephropathy,
neuropathy, and retinopathy

IDIOPATHIC TYPE 1 DIABETES

• Form of type 1 DM with no known etiology
• Strongly inherited, with no cell autoimmunity
o Not associated with autoantibodies, but insulin treatment
is required for survival
• Have episodic requirements for insulin replacement
• Any degree of glucose intolerance with onset or first recognition
TYPE 2 DIABETES MELLITUS
during pregnancy
• Include metabolic and hormonal changes; with patients frequently
returning to normal post-partum
• Increased perinatal complications, increased risk for diabetes
development in later years
• Infants born to mothers with diabetes are at increased risk for
respiratory distress syndrome, hypocalcemia, and hyperbilirubinemia
o Fetal insulin secretion is stimulated in the neonate of a
• NIDDM, adult type diabetes, maturity onset diabetes, stable mother with diabetes
diabetes, ketosis-resistant diabetes, receptor-resistant diabetes o When the infant is born and the umbilical cord is severed,
• Hyperglycemia resulting from an individual’s insulin resistance with the infant’s oversupply of glucose is abruptly terminated
an insulin secretory defect, resulting in a relative deficiency causing severe hypoglycemia
• Constitutes majority of all cases • Diagnostic criteria include:
o Most patients are obese, or have an increased percentage o FBS of >92 mg/dL
of body fat distribution in the abdominal region o 1-hr GCT: >180 mg/dL
• Often goes undiagnosed for many years o 2-hr OGTT: >153 mg/dL
• Associated with a strong genetic predisposition, with patients at
increased risk with increasing age, obesity, and lack of exercise OTHER SPECIFIC TYPES OF DIABETES
• Adult onset and has milder symptoms, with ketoacidosis seldom • Associated with certain or secondary conditions including:
occurring o Genetic defects of cell function or insulin action
o Patients more likely go into hyperosmolar comatose, and o Pancreatic diseases
has an increased risk in developing macro and o Diseases of endocrine origin like Cushing’s syndrome,
microvascular complications acromegaly, and hyperthyroidism
• Geneticist’s nightmare: presented as a triad of atherosclerosis, o Drug or chemical-induced insulin receptor abnormalities
diabetes, and hypertension o Certain genetic syndromes: Down syndrome,
• Not related to any autoimmune disease Klinefelter’s syndrome, Rabson-Mendel, Huntington’s and
• Risk factors: obesity, family history, advanced age, hypertension, Turner’s syndrome
lack of exercise, GDM, and impaired glucose metabolism
• It is recommended that >45 years old adults be screened every 3 GLUCOSE METHODOLOGIES
years, but should be performed earlier and more frequent if the • Venous plasma glucose: standard specimen
individual is high risk. • FBS level in whole blood is 15% lower than in serum or plasma
o Venous blood glucose is 7 mg/dL lower than capillary
Type 1 DM Type 2 DM blood
Pathogenesis b cell destruction Insulin resistance • CSF glucose: 60% of plasma concentration
Incidence rate 5-10% 90-95% • Peritoneal fluid glucose is same with plasma glucose
• Used to monitor individual responses to • Lower plasma glucose levels are seen more often in infants than in
pancreatic surgery adults; concentration increasing with age
• Evaluates hypoglycemia and continuous
C-peptide assessment of b cell function SPECIMEN STORAGE AND HANDLING
• Detectable in increased levels during
ingestion of hypoglycemic drugs • Room temperature, 20-25OC: glycolysis decreases glucose by 7
• Uses fasting serum mg/dL/hr in normal uncentrifuged coagulated blood
Medication Insulin (absolute) Oral agents • Refrigerated temperature, 4OC: glucose is metabolized at the rate
of 2 mg/dL/hr
shinggibanggi | RMT March 2024
 SAMPLES FOR GLUCOSE MEASUREMENT
Random blood Requested during insulin shock and
sugar / RBS hyperglycemic ketonic comatose
Fasting blood
Measures overall glucose hemostasis
sugar / FBS
Measures how well the body metabolizes glucose
2 hour PPBS
over a period of time
Multiple blood sugar test measuring how well the
body metabolized glucose
GTT • Oral GTT: Janney-Isaacson / single dose, or
Exton Rose / divided oral dose
• Intravenous GTT
INBORN ERRORS OF CARBOHYDRATE METABOLISM
Reliable method in monitoring long-term glucose
control, performed 3-6 months • Cause of failure to thrive syndrome in
infants
• Specimen: EDTA whole blood
Glycosylated • Congenital deficiency of 1 of 3 enzymes
• Methods: electrophoresis, immunoassays,
hemoglobin / involved in galactose metabolism resulting
HPLC, affinity chromatography Galactosemia
HbA1C in increased levels of plasma galactose
• 5.7-6.4%: indicates increased risk for DM
o Galactose-1-phosphaye
For every 1% change in value, 35 mg/dL is added
uridyl transferase: most
to plasma glucose
common deficiency
• Any change in blood sugar is reflected in the
Essential • Fructokinase deficiency
CSF approximately 1 hour later due to lag in
fructosuria • Presence of fructose in urine
equilibrium time
• Normal values: • Defect of fructose-1,6-biphosphate
CSF glucose Hereditary aldolase B activity in the liver, kidney, and
o Adults: 40-70 mg/dL
o Child: 60-80 mg/dL fructose intestine
intolerance • Irritability, lethargy, seizures, and
• Bacterial meningitis: markedly decreased
hepatomegaly
CSF glucose of <40 mg/dL, with increased
WBC / neutrophil count • Failure of hepatic glucose generation by
Glycosylated albumin or plasma protein Fructose-1,6- gluconeogenic precursors like lactate and
ketoamine used for short-term glucose control of biphosphate glycerol
Fructosamine 3-6 weeks, useful for monitoring individuals with deficiency • Hypoglycemia, lactic acidosis,
chronic hemolytic anemia and hemoglobin convulsions, comatose
variants like HbS and HbC
GLYCOGEN STORAGE DISEASES
OXIDATION REDUCTION / CHEMICAL METHODS • Inherited deficiencies of enzymes that control glycogen synthesis
Reduction of cupric ions forming cuprous o Deficiency of a specific enzyme that causes an alternation
oxide in hot alkaline solution by glucose of glycogen metabolism
• Folin-Wu: blue end product • Von Gierke’s disease: most common congenital form of GSD,
Alkaline Copper • Nelson Somogyi: blue end product o Glucose-6-phosphatase deficiency; autosomal recessive
Reduction Method • Neocuprine method: yellow-orange
in nature
end product
• Benedict’s method: modification of o Several hypoglycemia that coincides with metabolic
Folin-Wu acidosis, ketonemia, and elevated lactate and alanine
Inverse colorimetry: reduction of a yellow • Other enzyme defects or deficiencies that cause hypoglycemia
ferricyanide to a colorless ferrocyanide by include the following:
glucose o Glycogen synthase
Alkaline Ferric
• Hagedorn-Jensen: colorless end o Fructose-1,6-biphosphatase
Reduction Method
product
o Phosphoenolpyruvate carboxykinase
• Dubowski condensation: green end
product o Pyruvate carboxylase
• Glycogen debrancher enzyme deficiency does not cause
hypoglycemia, but can cause hepatomegaly

PRINCIPAL
GLYCOGEN GLYCOGEN
TYPE DISEASE NAME DEFECTIVE ENZYME TISSUE RELATED CONDITIONS
LEVELS STRUCTURE
AFFECTED
Hepatomegaly, retarded
I Von Gierke’s disease Glucose-6-phosphatase High Normal Liver, kidney
growth, seizures
Cardiomegaly, infantile
II Pompe’s disease a-1,4 glucosidase Very high Normal All organs
death
Short outer Liver, heart, Hepatomegaly,
III Cori Forbes’ disease Debranching enzyme High
branches muscle cardiomyopathy, muscle
shinggibanggi | RMT March 2024
 weakness, retarded
growth
Long outer Liver, muscle, Cirrhosis, esophageal
IV Andersen’s disease Branching enzyme Normal
branches spleen varices, ascites
Myoglobinuria, muscle
V McArdle’s disease Muscle phosphorylase High Normal Muscles
cramps
Hepatomegaly,
VI Hers’ disease Liver phosphorylase High Normal Liver
hypoglycemia
Pain and stiffness on
VII Tarui’s disease Phosphofructokinase High Normal Muscle
exertion
Hepatomegaly,
Hepatic phosphorylase
VIII Phosphorylase kinase High Normal Liver hypoglycemia, delayed
kinase deficiency
motor development

shinggibanggi | RMT March 2024

 LIPIDS AND LIPOPROTEINS
LIPIDS Lp(a) Lipoprotein (a); sinking pre-b lipoprotein

ABNORMAL LIPOPROTEINS
Found in obstructive jaundice and LCAT
Lipoprotein X
deficiency; used as a cholestasis indicator
Floating and abnormally migrating b lipoprotein,
considered lighter than Lp(a) usually found in type
b VLDL
3 hyperproteinemia and dysbetalipoproteinemia
• VLDL rich in cholesterol

• Commonly referred to as fats; composed of mostly C-H bonds

o Rich source of energy, efficient way of storing excess
calories
• Ubiquitous constituents of all living cells
• Integral part of cell membranes, playing an important structural role
in cells
• Transported by lipoproteins like TAG, phospholipids, cholesterol, and
cholesteryl esters

LIPOPROTEINS

• Biochemical assembly with the main function of transporting

hydrophobic lipid molecules in water, like in blood plasma or other
extracellular fluids
• Large molecular complexes of lipids with specialized proteins called
apolipoproteins
• Typically spherical in shape, ranges from 10 nm to >1 um in size
• Transports TAG and cholesterol to energy sites for storage and
utilization
o Travel in plasma not as free-floating molecules, but as part
of water-soluble complexes FATTY ACIDS
• Building blocks of fat in the body and the food we eat
MAJOR LIPOPROTEINS • Linear chains of C-H bonds grouped based on:
Chylomicrons Largest, least dense, non-atherogenic o Number of carbon atoms
Very Low-Density Lipoprotein o Presence of double bonds
VLDL
Pre-b lipoprotein, atherogenic • During digestion, the body breaks down fat into fatty acids which can
High Density Lipoprotein be absorbed into the blood.
HDL
Good and reverse cholesterol, cardioprotective
o Fatty acid molecules are usually joined together in groups
Low Density Lipoprotein
LDL of 3, forming a molecule called triglyceride
Bad cholesterol, most atherogenic

MAJOR LIPOPROTEINS TRIGLYCERIDES

Considered VLDL remnants due to VLDL catabolism, • 3 fatty acid molecules attached to one molecule of glycerol by ester
IDL
which can be converted to LDL bonds

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 • Has a single C-H side chain tail similar to fatty acids in physical
properties

• Corn, sunflower seeds, safflower seeds: rich in polyunsaturated

fatty acids, considered oils
o Those from animal sources contain mostly saturated fatty • Found on the surface of lipid layers along with phospholipids
acids, and are usually solid at room temperature • Do not serve as fuel source as they are not readily catabolized by
• Good source of energy compared to sugar glycolysis most cells
• Transport and excretion are promoted by estrogen
PHOSPHOLIPIDS • Can be converted in the liver to primary bile acids like cholic acid and
chenodeoxycholic acid to promote fat absorption in the intestine by
acting as detergents
• Precursor of the major classes of steroids: progestin, glucocorticoid,
mineralocorticoid, androgen, estrogen
• Important constituent in the assembly of cell membranes and bile
acids
o 7-dehydrocholesterol: Vitamin D3 for sunlight protection
• Evaluates risk for atherosclerosis, myocardial infarction, coronary
• Structurally similar to TAG, but only has 2 esterified fatty acids arterial inclusions
• Contain both hydrophobic fatty acid C-H chains, and a hydrophilic • Used for thyroid, liver, renal function tests, for diabetic studies,
head group diagnosis and management of lipoprotein disorders, and in
o Polar hydrophilic head: faces outward toward the monitoring effectiveness of lifestyle changes and stress
aqueous environment management
o Fatty acid chains: face inward away from the water,
perpendicular in orientation with respect to the lipid FORMS OF CHOLESTEROL
surface Cholesterol • 70% of total cholesterol
• Considered amphipathic lipid molecules, found in the surface of lipid ester • Bound to fatty acids, found in plasma or serum
layers • 30% of total cholesterol
Free
• Synthesized in cytosolic compartments of all body organs cholesterol • Bound to surfaces of lipoproteins found in
o Phosphatidylcholine, phosphatidylethanolamine: plasma, serum, and RBCs
most abundant phospholipids in the body
LIPID & LIPOPROTEIN TRANSPORT AND METABOLISM
• Liver and intestine: source of the most abundant lipids
• Absorption of TAG and cholesterol through the
o Produced in the lungs by type 2 pneumocytes in the form
intestine with formation and release of
of Lamellar bodies chylomicrons into the lymph and into the blood
§ Allows effective gas exchange; prevents through the thoracic duct
Dietary /
alveolar collapse during expiration • Chylomicrons release TAG to adipose tissues
exogenous
• Participates in cellular metabolism and blood coagulation pathway • Lipoprotein lipase liberates fatty acids from TAG,
• Important substrate for a number of lipoprotein-metabolizing reducing chylomicron size to become remnants
taken up by the liver
enzymes
• Free fatty acids liberated by TAG are taken up
• Has 3 forms, which deficiencies can lead to neonatal respiratory by muscles and adipose tissues
distress syndrome: • TAG production from fatty acids by the liver take
o Lecithin: 70%, lung surfactant place with the synthesis of VLDL particles
o Sphingomyelin: 20%, lung surfactant containing apo-B100 and apo E
§ L:S ratio should be 2-2.5:1 for a mature fetal • VLDL are then converted by lipoprotein lipase to
Endogenous
lung IDL that can be removed by the liver through apo
pathway
o Cephalin: 10% E or converted to LDL
• The cholesterol-rich properties can be taken up
by the liver, or into other tissues for steroid
CHOLESTEROL synthesis or part of cell membranes
• 3-hydroxy-5,6-cholestene
• Unsaturated steroid alcohol containing 4 rings, A, B, C, D ENZYMES FOR TRANSPORT AND METABOLISM
o A-ring hydroxyl group: only hydrophilic part • Lipoprotein lipase / LPL
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• Hepatic lipase
• Lecithin cholesterol acyl transferase / LCAT
• Endothelial lipase
• ATP-binding cassette protein A1 / ABCA1

SPECIMEN CONSIDERATIONS
• Fasting: requirement when TAG and LDL are being measured
o Usually 10-12 hours
• Collected using red or gold tops
• Diet: LDL and HDL concentrations temporarily decline after eating
• Posture: recommend patients be seated for 5 minutes before
sampling to prevent hemoconcentration

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 MEASUREMENT AND CLINICAL SIGNIFICANCE OF LIPIDS AND LIPOPROTEINS
CHOLESTEROL Hyperlipoproteinemia V Severe burns
Biliary cirrhosis Hyperthyroidism
Nephrotic syndrome Malabsorption syndrome
Poorly controlled DM
Alcoholism
Primary hypothyroidism

TRIGLYCERIDES / TRIACYLGLYCEROL
• Presents as milky serum in specimens
• 10-12 hours fasting requirement
• <200 mg/dL: reference value o Avoidance of postural changes
• 200-239 mg/dL: borderline values o Interferences: high Vit. C levels and bilirubin
• >240 mg/dL: high cholesterol values • Reference levels:
• Patient preparation: should be on usual diet 2 weeks before testing o <150 mg/dL: normal
• Measured using both chemical and enzymatic methods o 150-199 mg/dL: borderline high
o Enzymatic methods: more commonly used, more rapid, o >200 mg/dL: risk for coronary artery disease
uses less amounts of samples, and do not require o 200-499 mg/dL: high TAG
preliminary sample collection o >500 mg/dL: very high TAG
§ Cholesterol oxidase reaction § Seen in acute and recurrent pancreatitis and
§ Vitamin C: interference severe hypertriglyceridemia
• Diagnostic significance: atherosclerosis, CHD
CHEMICAL METHODS
Dehydration and oxidation of cholesterol to form a colored compound CLINICAL METHODS
• Most commonly used Van Handel & Colorimetric method
Liebermann • Color developers: glacial acetic acid, acetic Zilversmith End product: blue-colored compound
Burchardt anhydride, concentrated H2SO4 Hantzsch Fluorometric method
Reaction • End product: green-colored cholestadienyl condensation End product: diacetyl lutidine compound
monosulfonic acid
Abell-Levy ENZYMATIC METHODS
Brodie CDC reference method for cholesterol Glycerol kinase reaction; most commonly used method
Method The disappearance of NADH measured at 340 nm
Salkowski End product: red-colored cholestadienyl disulfonic • TAG ® lipase (enzyme) ® glycerol + FA
reaction acid • Glycerol + ATP ® glycerol kinase ® glycerol
Reaction A
PO4 + ADP
GENERAL METHODS • ADP + phosphoenol pyruvate ® pyruvate
Colorimetry kinase ® ATP + pyrivate
One-step
Pearson, Stern and Mac Gavack • TAG ® lipase ® glycerol + FA
Colorimetry + extraction • Glycerol + ATP ® glycerol kinase ® glycerol
Two-step
Bloors PO4 + ADP
Colorimetry + extraction + saponification • Glycerol + PO4 + NAD ® glycerol PO4
Three-step Reaction B
Abell-Kendall dehydrogenase ® dihydroacetone PO4 +
Colorimtery + extraction + saponification + NADPH
Four-step precipitation • NADPH + tetrazolium dye ® diaphorase ®
Parekh and Jung, Sperry, Schoenheimer formazan + NAD

NCEP GUIDELINES OF ACCEPTABLE MEASUREMENT ERROR MODIFIED VAN HANDEL AND ZILVERSMITH
ANALYTE CV BIAS TOTAL ERROR
• CDC reference method for TAG measurement
Cholesterol <3% <3% <9%
Triglycerides <5% <5% <15% • Time consuming manual method, cannot be automated
LDL <4% <4% <12% • Involves saponification using alcoholic KOH, solvent extraction with
HDL <4% <5% <13% chloroform
o The extract is treated with silisic acid to isolate TAG
Increased cholesterol Decreased cholesterol through chromatography
Hyperlipoproteinemia IIa Severe hepatocellular disease • Color reaction with chromotropic acid will give rise to a pink color
Hyperlipoproteinemia III Malnutrition

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 Increased TAG Decreased TAG
Hyperlipoproteinemia I Malabsorption syndrome
Hyperlipoproteinemia IIb Hyperthyroidism
Hyperlipoproteinemia III Malnutrition
Hyperlipoproteinemia IV Brain infarction
Hyperlipoproteinemia V
Alcoholism
Nephrotic syndrome
Hypothyroidism
Pancreatitis
Diabetes mellitus

APOLIPOPROTEINS
• Help keep lipids in solution during circulation through the blood
• Amphipathic helix: structural motif that binds proteins to lipids
• Maintain structural integrity of the lipoprotein complex
o Constitutes the main function of apolipoproteins

LIPOPROTEINS AND THEIR APOLIPOPROTEINS

Creamy layer in specimens
Chylomicrons • APO B48: unique
• APO A7, APO C, APO E
Present in turbid serum
VLDL
• APO B100, APO C, APO E
Most dense and smallest
• APO A1, APO A2, APO C
HDL
<35 mg/dL: high risk CHD
>60 mg/dL: cardioprotective
Primary CHD marker
• APO B100, APO E
<100 mg/dL: optimum LDL level
LDL o 100-192 mg/dL: above optimum
o 130-159 mg/dL: borderline
o 160-189 mg/dL: high LDL
o >190 mg/dL: very high LDL
IDL APO B100
Lp(a) APO B100, APO(a)
Lp X APO C, albumin METHODOLOGIES
b VLDL APO B100 • Reference method for quantification
• Expressed in Svedberg proteins
• Frozen samples are inappropriate as
Ultracentrifugation TAG-rich lipoproteins do not withstand
freezing
• Reagent: potassium bromide solution
with 1.063 density
• Fastest measurement method
• Pattern: HDL, VLDL, LDL,
chylomicrons
Electrophoresis • Agarose gel: preferred supporting
medium due to speed and sensitivity
• Lipid staining dyes: Oil Red O, Fat
Red 7B, Sudan Black B
• Uses polyanions like heparin sulfate,
dextran sulfate or phosphotungstate,
and divalent cations like Mg, Ca, and
Mn
Chemical • Enzymatic coupled with detergent
precipitation precipitation: most useful lipoprotein
tests
• Presence of APO B-containing
lipoproteins: most consistent
analytical error involved
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 • HDL method: a yellow/orange discoloration of the
o CDC reference 3-step tonsils and pharynx
method: ultracentrifugation, Type 1 chylomicronemia syndrome,
heparin Mn precipitation, resulting in the inability to clear CM
LPL deficiency
Abell-Kendall assay particles, presenting as childhood
o Homogenous assay: most abdominal pain and pancreatitis
popular method for HDL-C LCAT deficiency Fish eye disease
measurement Hexosaminidase A deficiency; and a
• LDL method: Tay-Sach’s disease neurodegenerative disease resulting in
o EDTA plasma: preferred sphingolipid accumulation in the brain
sample for b quantification; CM retention disease related to APO
18 hours centrifugation B48 resulting to hypocholesterolemia,
o VLDL and CM accumulate Anderson’s disease chronic diarrhea, failure to thrive,
at the top layer deficiency of fat-soluble vitamins like
Chromatographic Vit. E and neurologic deficits
Gel or affinity chromatography
methods High LDL-C during childhood
Sitosterolemia
Immunochemical connected to chromosome 2P27
Antibody specific epitopes
methods
Immunoassay / FREDRICKSON CLASSIFICATION
Apolipoprotein assay
immunonephelometry Familial LPL deficiency presented as CM
Type 1
accumulation leading blocking of CM
hyperchylomicronemia
LDL CHOLESTEROL metabolism to its remnants
• Most important value assessing cardiac risk and directing therapy Type 2 Blocking of LDL metabolism and defective
• Can be measured directly; usually directed using the Friedewald hyperlipoproteinemia APO B
Type 3
formula Presence of floating VLDL
dysbetalipoproteinemia
o Calculated values require evaluation of fasting samples Hypertriglyceridemia presented as
o Unsuitable for non-fasting samples that contain CM or Type 4 blocking of conversion of VLDL to IDL and
samples that can contain b-VLDL hyperlipoproteinemia LDL, with ­ TAG and VLDL and normal
• 𝐿𝐷𝐿 𝐶 = 𝑡𝑜𝑡𝑎𝑙 𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 − 𝐻𝐷𝐿 − 𝑉𝐿𝐷𝐿 LDL
• Friedewald method: Type 5 LPL deficiency with the inability to
!"#$%# '() hyperlipoproteinemia breakdown TAG
o 𝑉𝐿𝐷𝐿 (𝑚𝑚𝑜𝑙/𝐿) =
*.,-.
!"#$%# '()
o 𝑉𝐿𝐷𝐿 (𝑚𝑔/𝑑𝐿) =
../
• De Long Method:
!"#$%# '()
o 𝑉𝐿𝐷𝐿 (𝑚𝑚𝑜𝑙/𝐿) =
*.0*.
!"#$%# '()
o 𝑉𝐿𝐷𝐿 (𝑚𝑔/𝑑𝐿) =
1..

DISEASES ASSOCIATED WITH LIPIDS AND LIPOPROTEINS

Familial
Autosomal dominant with deficient /
Hypercholesterolemia
defective LDL receptors
Type 2a
Familial
Accumulation of VLDL-rich cholesterol
Dysbetalipoproteinemia
and chylomicron remnants
Type 3
Bassen-Kornweig Syndrome
Abetalipoproteinemia • Defective APO B synthesis
• No VLDL, LDL, and CM in plasma
Decreased fats and APO B deficiency;
Hypobetalipoproteinemia
¯ LDL-C and total cholesterol
Common autosomal dominant
disorder characterized by HDL levels
Hypoalphalipoproteinemia
of <30 mg/dL for men, and <40 mg/dL
in women
Lipid storage disease connected to
sphingomyelin accumulation that is
Niemann-Pick disease
inherited, and deficiency in the
sphingomyelinase enzyme
Rare, autosomal recessive disease in
Tangier disease chromosome 9, connected with the
complete absence of HDL, resulting in

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 PROTEINS
AMINO ACIDS • 2-3 band forms
• a helix: 3D conformation
o R groups radiate outward
o 3.6 aa per 360° turn
o Forms a right-handed /
counterclockwise helix
Secondary • b sheet: short segments, containing 5-8 residues
structure that fold and H bonded into pleated sheets

• Building blocks of proteins

• Differ from one another by chemical composition of the R group
• Electric charge depends on the pH of the solution and type of R group
• A single amino acid contains at least 1 amino group and 1 carboxyl
functional group
o N-terminal end (-NH2) and C-terminal end (-COOH) are • Electrovalent linkages
bonded to the a-carbon forming an amino acid. • 3D orientation of proteins in space
• Most stable conformation of a protein
AMINO ACIDS REQUIRED IN PROTEIN SYNTHESIS • Responsible for many physical and chemical
properties of proteins
Glycine Valine Isoleucine
Methionine Phenylalanine Glutamine Tertiary
Threonine Lysine Histidine structure
Glutamate Alanine Leucine
Cysteine Tryptophan Asparagine
Serine Tyrosine Arginine
Aspartic acid Proline
Association of 2 or more polypeptide chains to form a
PROTEINS functional protein
• Macromolecules providing 10-20% of total body energy requirement
o First rank of importance
Quaternary
• Can bear (+) and (-) charges due to amphoteric characteristics, or its
structure
basic and acidic amino acid compositions
• Widely essential in a lot of body functions like:
o Catalyzing biochemical reactions as enzymes
o Transporting metals like iron and copper
o Acting as receptors for hormones PROTEIN CLASSES ACCORDING TO FUNCTION
o Providing structure and support to cells • For catalytic activity and function, normally
o Participating in immune response as antibodies intracellular
• Contain nitrogen that sets them apart from pure CHO and lipids • Released into the bloodstream as results of
• Contains about 200-300 amino acids tissue damage
Enzymes o Amylase: earliest pancreatic
• Functions in transport, blood coagulation, and maintenance of
marker
osmotic pressure and blood pH o Lipase: most specific pancreatic
marker
PROTEIN STRUCTURE o Trypsin
• Linear sequence of amino acids Chemical messenger proteins controlling the
• One end has an amine group or N-terminus, while action/s of specific cells or organs that directly
Hormones
the other end has a free carboxyl group affect growth and development, metabolism,
• Size is specified by mass: MW in Daltons = 1 mu sexual function, reproduction, and behavior
Primary Transport Transport substances across biological
structure proteins membranes
Contractile Involved in the contraction and relaxation of
proteins muscles; usually seen as long, fibrous materials
Structural Provide structural support for the body, a tissue
proteins or cell, usually as long fibrous molecules

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 • Collagen of tendons and cartilage • One of the serum glycoproteins that rise in
• Elastin of ligaments response to acute inflammations
• Keratin of hair and nails • Can destroy the alveoli and cause
Antibodies / Mediate humoral response to identify and emphysema
immunoglobulins neutralize foreign antigens • Protease inhibitor
Regulate metabolic processes, hormones, • Deficiency is related to SERPINA1 gene
Regulatory
transcription factors and enhancers, and growth mutation, leading to pulmonary emphysema
proteins
factor proteins and juvenile hepatic cirrhosis
• Reference value: 145-270 mg/dL
CLASSIFICATIONS OF PROTEINS o ­ inflammation, pregnancy,
• Contain peptide chains yielding only amino acids contraceptive use
during hydrolysis o ¯ juvenile hepatic cirrhosis,
• May be fibrous or globular emphysema
Simple
o Fibrous: fibrinogen, troponins, • Largest major non-immunoglobulin protein in
proteins
collagen plasma
o Globular: hemoglobin, plasma • Women have higher levels than men, due to
proteins, enzymes, peptide hormones a-2 estrogen
Can yield amino acids and other molecules on macroglobulin • Concentration rises 10-fold or more when
hydrolysis other lower MW proteins are lost in nephrotic
• Lipoproteins: blood, membrane, and transport syndrome
proteins • Reference value: 150-420 mg/dL
Conjugated • Glycoproteins: antibodies, cell surface proteins • Light chain component of the MHC found on
proteins • Metalloproteins: ferritin, ceruloplasmin, the surface of lymphocytes; used in CD8
hemoglobin, flavoproteins b-2 production
• Mucoproteins or proteoglycans: mucin microgobulin • Reference value: 0.2-2.8 µL/dL
• Nucleoproteins: ribosomes and organelles, • Seen in inflammatory diseases like RA, SLE,
chromatins, nucleic acids multiple myeloma, renal failure, HIV
• a2 glycoprotein considered as acute phase
MAJOR COMPONENTS OF PLASMA PROTEINS reactant
• Fraction migrating in a position faster than • Binds free hemoglobin by its a chain
albumin towards the anode • Prevents hemoglobin loss and its iron
• Has a b pleated configuration constituent into urine
• Half-life: 2 days • Half-life: 4 days
Haptoglobin
• Landmark for CSF specimen confirmation • Normal value: 26-185 mg/dL
• Transport protein for T4 and retinol o ­ stress response, infection, acute
Pre-albumin
• Detects malnutrition and response to dietary inflammation, tissue necrosis,
supplements myoglobinuria
• Normal range: 18-45 mg/dL o ¯ hemoglobinuria, intravenous
o ­ alcoholism, chronic renal failure, hemolysis
steroid treatment • Major b globulin fraction; negative APR
o ¯ poor nutrition • Transports iron to its storage sites
• Most abundant protein in serum, highest • Transports ferric irons from storage of
concentration in plasma intracellular or mucosal ferritin to the bone
• General transport protein or carrier; major marrow
transporter of lipoproteins o Cause of anemia and loss of iron
• Mobile repository of amino acids for Transferrin / carrying capacity of the blood
incorporation into other proteins siderophilin • Prevents iron loss through the kidney
• Maintains osmotic pressure and nutritional • Normal values: 215-365 mg/dL in males, 250-
status 380 mg/dL in females
• Concentration is important in interpreting Ca o ­ hemochromatosis, IDA
Albumin and Mg levels o ¯ liver disease, malnutrition,
• Marker for cystic fibrosis nephrotic syndrome
• Half-life: 17 days o
• Normal range: 3-5 mg/dL; 23-35 g/L • One of the largest proteins in the blood
o ­ dehydration and prolonged • Most abundant of the coagulation factors
application of tourniquet • Forms the fibrin clot when activated by
o ¯ nephrotic syndrome thrombin
Fibrinogen • May serve as marker for long term prognosis
• Dye binding methods: bromcresol green,
hydroxyozobenzene benzoic acid of cardiovascular disease
• Major component of the a1 globulin: 90% • Normal value: 200-400 mg/dL
a-1
antitrypsin • Can combine with trypsin and inactivate it o ­ pregnancy, contraceptive
medications, inflammation

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 o ¯ excessive coagulation • ¯ heart disease, type 2 DM, metabolic
syndrome, obesity
MINOR COMPONENTS OF PLASMA PROTEINS • Prostaglandin D synthase
• Copper-binding protein • Accurate marker for CSF leakage
b trace
• Responsible in oxidizing iron from ferrous to • Potential marker in detecting impaired renal
protein
ferric function
• Wilson’s disease: ceruloplasmin deficiency • Diagnosis of perilymphatic fluid fistulas
Ceruloplasmin Low MW protein used as a new marker in the early
due to disordered copper metabolism Cystatin C
• Normal value: 18-45 mg/dL assessment of changes to GFR
o ­ oral contraceptive therapy, • Heme protein found in striated skeletal
pregnancy, cancer Myoglobin muscles and cardiac muscles
• Binds heme released by hemoglobin • Accounts for 2% of total muscle protein
degradation • Protein polysaccharide complex produced and
Hemopexin • Most profoundly decreased in intravascular deposited in tissues during chronic infections,
hemolysis Amyloid malignancies, and rheumatologic disorders
• Normal value: 50-115 mg/dL • Homogenous substance staining readily with
• Orosomucoid: 45% CHO, 11-12% cyalic acid Congo red
• Binds to progesterone; important to transport
a1-acid and metabolism METHODS OF DETERMINATION
glycoprotein • Normal value: 55-140 mg/dL TOTAL PROTEIN DETERMINATION
o ­ pregnancy, bacterial infection in • Reference range: 6.5-8.5 mg/dL
neonates, cancer, pneumonia, RA
• Scavenger molecule appearing in blood of Decreased levels
patients with diverse inflammatory diseases Increased levels
Gastrointestinal cancers
• Gamma migrating globulin Dehydration
Liver disease
• Earliest indicator of inflammation; serves as Severe exercise
Malnutrition
C-reactive cardiac marker Infection
Low thiamine
protein • Used to monitor progression/remission of Cancer
Glomerulonephritis
autoimmune diseases
• Rapid test for presumptive diagnosis of KJELDAHL METHOD
bacterial vs viral infection
• Reference method
• Normal value: <1.0 mg/dL
• Most abundant in children, usually • Acid digestion to release ammonium ions from nitrogen-containing
synthesized by the fetal yolk sac and then by compounds
fetal parenchymal cells of the liver o Ammonium can be quantitated by conversion to ammonia
• Tumor marker for hepatic and gonodal cancer gas and titration as a base by Nesslerization
a1 fetoprotein
• Reference value: 5 ng/dL • Total serum protein is obtained by multiplying the value of total
o ­ amniotic and serum in neural tube protein nitrogen/TPN by 6.25
defects, twin pregnancy
o ¯ Down syndrome
REFRACTIVE INDEX
• Accurate for measuring serum protein concentration for levels >2.5
OTHER PROTEINS OF CLINICAL SIGNIFICANCE
• Complex of 3 proteins that bind to thin g/dL
filaments of cardiac muscles • Cannot be used for urine protein measurements due to excess
• Regulator of actin and myosin amounts of solutes in relation to the protein
Troponin
• Most important marker for cardiac injury
• Gold standard in the diagnosis of myocardial SPECIFIC GRAVITY
infarction • Simple; has been used as a screening test for hemoglobin
• With N-terminal BNP concentration in whole blood
• Marker for congestive heart failure
• Estimated by pipetting drops of serum or blow into graded series of
Brain • Neurohormone affecting body fluid hemostasis
through natriuresis and dieresis, and blood copper sulfate solutions
natriuretic • Protein concentration is estimated from the SG of the copper sulfate
peptide pressure through decreased angiotensin II,
norepinephrine synthesis solution in which the drop remains stationary
o Major components in CHF
pathology TURBIDIMETRIC METHOD
Fetal fibronectin: glycoprotein used to help predict
Fibronectin • Measures protein concentration in CSF or urine
short-term risk of premature delivery
• 247-amino acid fat hormone with an N-terminal • Protein forms precipitate in the addition of trichloroacetic acid,
Adiponectin collagen-like domain and a C-terminal globular sulfosalicylic acid, or other acid reagents
domain produced by adipocytes • Can be measured by optical density

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• Not specific to proteins as other acid insoluble substance like nucleic SPECIFIC PROTEIN QUANTIFICATION
acids can also precipitate ELECTROPHORESIS
• Protein quantification and identification
APPEARANCE PROTEIN CONTENT • Performed when an abnormality in total protein or albumin
Negative Clear sample, no turbidity <20 mg/dL concentration is found
Trace Very faint precipitate 20-200 mg/dL • Separates proteins on the basis of their electric charge and density
+ Small degree of turbidity 100-1000 mg/dL
• When placed on an electric current, proteins will move according to
++ Moderate turbidity 1000-2500 mg/dL
charge determined by the pH of the surrounding buffer
+++ Heavy turbidity 2500-4500 mg/dL
++++ Heavy flocculation / clumping >4500 mg/dL

COLORIMETRIC METHOD
• Highly specific for proteins and peptides
• Depends on the presence of 2 or more
peptide bonds that form a purple complex
with copper salts in alkaline solution
Biuret method
• Interferences: ammonium ion, hemoglobin,
bilirubin
• Reagents: copper sulfate, tartrate salt,
potassium oxide
Involves oxidation of phenolic compounds like MOVING BOUNDARY / FRONTAL ELETROPHORESIS
Folin-Ciocalteu
tyrosine, tryptophan, and histidine to give a deep • Electrophoresis using an aqueous medium
reagent
blue color • pH 7.6: four serum protein fractions (albumin, a, b, g) are identified
Coomasie Sensitive for detecting down to 1 pg of protein;
and quantified optically by change in refractive index at the
brilliant blue free of interferences from a very wide range of
dye substances boundaries
Produces a violet color by reacting with primary o Separation is achieved in a homogenous solution without
Ninhydrin a solid support medium so convective forces prevent
amines
resolution into distinct zones
ALBUMIN DETERMINATION
• Reference range: 3.5-5.0 mg/dL
o ­ dehydration, sunstroke, exercise, multiple sclerosis,
hypothyroidism
o ¯ pregnancy, malnutrition, malabsorption, liver &
kidney diseases, burns
METHOD PRINCIPLE COMMENT
Globulins are precipitated
in high salt concentrations.
Salt
Albumin in the supernatant Labor intensive
precipitation
is quantitated by biuret
reaction
DYE BINDING
Nonspecific for
Methyl orange
albumin
Many
interferences like ZONE ELECTROPHORESIS
HABA*
salicylates and
Albumin binds to dye and bilirubin • Paper: solid medium to permit separation of protein fractions into
causes shift in maximum Most commonly discrete bands or zones
absorption used dye but is • Solid support medium: filter paper, at pH 8.6
Bromcresol
sensitive and can o a fraction further splits into 2 groups of proteins: a1 and
green
overestimate low a2
levels
Bromcresol Specific, • Other support media: cellulose acetate membrane, agarose gel,
purple sensitive, precise starch gel, polyacrylamide gel
Accurate and can o Cellulose acetate & agarose: predominate due to ease
give an overview of use, low cost, commercial availability
Proteins separated based of relative
Electrophoresis
on electric charge changes in At a pH greater Protein is negatively charged, will migrate towards
different protein than their pI the anode, which is the positive terminal
fractions
*HABA: 2,4’-hydroxyazobenzene-benzoic acid

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 The protein is positively charged, will migrate CAPILLARY ELECTROPHORESIS
At a pH less
toward the negatively charged terminal, the • Separation method based on flow through a capillary tube that can
than their pI
cathode be tailored to a resolution of different molecules based on size,
hydrophobicity, or stereospecificity
STANDARD DYES USED IN ELECTROPHORESIS
Coomasie brilliant blue
Serum
Ponceau S
electrophoresis
Amido black
Oil Red O
Lipoproteins
Sudan black
Glycoproteins Periodic acid Schiff / PAS

PRECIPITATION
• Globulins precipitated using sodium sulfate, sodium sulfite,
ammonium sulfate, or methanol leaves albumin in the solution AMINOACIDOPATHIES
• Class of inherited errors of metabolism where there is an enzyme
• Values for albumin and globulin can be derived by measuring total
defect inhibiting the body’s ability to metabolize certain amino
protein in the original serum and protein in either the precipitate or acids
supernatant • Can cause severe medical complications due to toxic amino acid
• Not as accurate as zone electrophoresis; some a globulins may fail accumulation or their by-products in the blood and tissues
to precipitate and can lead to an overestimate of the albumin fraction • Autosomal recessive trait
• Classic form due to absence of
COLUMN SEPARATION: GEL FILTRATION phenylalanine hydroxylase / PAH from PAH
gene mutation; causing urine to have a
• Order of protein elution: by molecular weight or size, from largest to
musty odor
smallest • Can cause mental retardation
• Necessary to apply the sample in a small and uniform volume; all Phenylketonuria
• Guthrie bacterial inhibition assay:
proteins species continuously move through the gel filtration column screening procedure; for B. subtilis spores
all at the same time but with different rates with >4 mg/dL growth
• HPLC: reference method
• Urine testing: FeCl3 reacts with
phenylpyruvic acid producing a green color
• Excretion of tyrosine and tyrosine
catabolites in urine
• Defects in fumarylacetoacetase: ­
Tyrosinemia methionine leading to liver damage and
cirrhosis
ION EXCHANGE CHROMATOGRAPHY • Tested through ion exchange column
• Based on protein charges which bind to the heads of a charge chromatography
support medium • Deficiency in homogentisate oxidase in the
tyrosine catabolic pathway
HYDROPHOBIC CHROMATOGRAPHY • Build-up of homogentisic acid leading to
• Samples are applied at high salt, eluted at low salt tissue pigmentation and ochornosis
o Accumulates in connective
• Support medium interacts with proteins according to hydrophobic tissues causing generalized
nature Alkaptonuria
pigmentation and arthritis-like
• Good complementary technique: follow exchange chromatography in degeneration
which the sample was eluted at high salt • Causes urine to darken upon air exposure,
presenting as brown to black color
AFFINITY CHROMATOGRAPHY • Tested by adding ferric chloride to urine to
produce a blue color
• Based on specific binding between the protein of interest and another
• Deficiency of the branched chain ketoacid
protein that has been covalently linked to the solid support medium decarboxylase: ¯ in a-ketoacid
of a column dehydrogenase
• Build-up of leucine, isoleucine, and valine
Maple acid • Causes mental retardation, convulsions,
acidosis, hypoglycemia, failure to thrive, and
urine disease
death
• Presented as a burnt sugar odor in urine,
breath, and skin
• Modified Guthrie test: screening test
detecting elevated plasma leucine

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 • Deficiency of isovaleryl-CoA dehydrogenase
in leucine degradation pathway
Isovaleric
• Causes sweaty feet urine odor
acidemia
• Tested through chromatography and mass
spectrometry
• Impaired activity of cystathionine b-
synthase
• Results in elevated plasma and urine levels
of homocysteine and methionine
• Causes thrombosis, osteoporosis,
Homocystinuria dislocated lenses, and mental retardation
during late childhood
• Guthrie test: neonatal screening test using
L-methionine sulfoximine
• HPLC: confirmatory testing should have a
value of >2 mg/dL
• Deficiency in arginosuccinic acid lyase and
a decrease in its activity, causing
Arginosuccinic citrullinemia
aciduria, • Symptoms: vomiting, high ammonia levels,
Citrullinemia mental retardation
• Citrulline: detection marker used
• Tested through mass spectrometry
• Increased cysteine excretion due to defects
in renal absorption
Cystinuria • Tested through urine cyanide-nitroprusside
giving a red-purple color when specimen is
positive

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 NON-PROTEIN NITROGEN COMPOUNDS
KIDNEYS o Calculated in terms of mm of blood per unit of time
! #$%&'( -./0
• 2 bean-shaped organs in the posterior wall of the abdomen § 𝑚𝐿/𝑚𝑖𝑛: " × ')*&+(, × 1
• Adult kidney: about 12 cm, or 5 in in length § U: concentration of analyte in urine
• Approximately ¼ of the total blood supply passes through the kidneys § P: concentration of analyte in plasma
from the paired renal arteries § Volume: in mL, 24-hr urine collection
• Each kidney is attached to a ureter, a tube that carries excreted urine § Minutes: time required to collect urine, usually
to the bladder 1440 mins
• 20-35 mg/dL of urine comes from plasma § 1.73: constant value; average body surface of
o Urea: 45% an adult individual
o Amino and uric acids: 20% each § A: body surface of the patient
o Creatinine: 5%
o Creatine: 1-2% • Excellent measurement of clearance; best
o Ammonia: 0-2% alternative method
• Kidney function tests: for measuring glomerular filtration rate / • Those occurring through metabolic
GFR, for measuring tubular functions, and for measuring renal blood production is eliminated from the plasma by
flow glomerular filtration
Creatinine
clearance test • Specimen: 24-hr urine
o Stable and normal excretion due
to muscle mass
• Normal value: 107-139 mL/min
o Males: 85-125 mL/min
o Females: 75-115 mL/min
• Reference method, but not routinely used
• Considered the most accurate GFR measure
• Freely passes the glomeruli, but is neither
secreted nor reabsorbed by the nephric
Inulin clearance
tubules
test
• Usually increased in males
• Specimen: intravenous and timed urine
PARTS OF THE KIDNEY • Normal value: 250 mL/min in males, 110
Cortex Outer layer mL/min in females
Medulla Inner layer • Used in monitoring renal disease
Functional units of kidneys progression and therapy response
• Glomerulus • Freely filtered by the glomeruli, but is
• Proximal and distal convoluted tubules variably reabsorbed in the tubules
Nephrons Urea clearance
• Loop of Henle: ascending part contains 25 test depending upon the transit time of urea
L of diluted urine filtrate
• Collecting tubes o Transit time: rate of urine flow
along the course of nephric
GENERAL FUNCTIONS OF KIDNEYS tubules
• Elimination of metabolic waste products through urine formation
TESTS MEASURING TUBULAR FUNCTIONS
• Regulation of plasma and water volume
• Excretory tests
• Regulation of ionic equilibrium
o Para-amino Hippurate test / PAH or Diodrast test
• Maintenance of acid base balance
o Phenolsulfonphthalein / PSP dye excretion test
• Endocrine function, and release of erythropoietin
NON-PROTEIN NITROGEN COMPOUNDS
TESTS FOR GLOMERULAR FILTRATION RATE UREA / BLOOD UREA NITROGEN
• Best overall indicator kidney function • Concentration in the blood is historically measured as nitrogen
• Clearances: term used for tests measuring GFR remaining from a protein-free filtrate of the blood
o Tests kidney capacity to clear waste products/foreign • Major nitrogen-containing compound in the blood
materials like inulin from the blood to be excreted in the
• Product of protein catabolism
urine during a given period
• Major organic solute in urine
§ Plasma concentration is inversely proportional
with clearance
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• Synthesized in the liver from amino groups and free ammonia, • Direct measurement of urea
generated during protein catabolism • Fearon’s reaction: urea is made to extract
Diacyl with diacetyl monoxime producing a yellow
monoxime diaxine derivative
method • Arsenic thiosemicarbazide is added to
enhance color formation and to exclude protein
interference
Isotope
Considered as gold standard; only used as a
dilution mass
reference method as it is expensive
spectrometry

CLINICAL SIGNIFICANCE
• Azotemia/azo: nitrogen-containing biochemical abnormality
• By custom, its concentration is expressed by urea’s nitrogen content
referring to an increase in BUN and creatinine levels that is largely
o Urea nitrogen: appropriately used to refer to urea
related to decrease in GFR
determination
• Uremia: increase in urea and creatinine with accompanying signs
• Urea concentration depends not only on the renal function and
and symptoms of renal failure like:
perfusion, but also on the rate of protein catabolism depending
o Metabolic acidosis due to kidney failure in eliminating
largely on protein intake
acidic products of metabolism
• 90% is excreted, 10% remains in the blod
o Hyperkalemia due to failure of potassium excretion
• Decreases in liver damage, but is first to increase in kidney disease
o Generalized edema due to water retention: ­ plasma urea
• Readily removed by dialysis
and electrolyte imbalance
• Normal value: 6-20 mg/dL, or 2.1-7.1 mmol/L
o BUN:creatinine ratio should be 10-20:1 MAIN CATEGORIES OF INCREASED PLASMA UREA
• Can be converted to urea concentration by multiplying it by 2.14 • Hemorrhage or blood loss
o Urea nitrogen concentration expressed in mg/dL can be • Cardiac decompression
converted to mmol/L by multiplying it by 0.357 Pre-renal
• Increased protein catabolism
causes
• Heatstroke or dehydration
TEST METHODOLOGIES • Burns and fluid loss
• Urease hydrolyzes urea which produces Presence of lesions on the parenchyma; tubular
Indirect ammonia and CO2 injuries
method • Ammonia: often used by several methods to • Chronic nephritis
calculate urea concentration • Acute glomerulonephritis: follows an infection
• Urea in the protein-free filtrate is made to react by sudden onset of inflammation and damage of
with urease glycerol extract in the presence of Renal the glomerulus resulting to hematuria and
buffer and heat, forming ammonium carbonate causes proteinuria
which is then reacted with Nessler’s reagent to • Polycystic kidneys
form a yellow dimercuric ammonium iodide • Nephrosclerosis
Enzymatic & o Urease-Nessler method • Tubular necrosis: presents with BUN of >100
Nesslerization § Urea + CO2 + NH3 mg/dL, creatinine of 20 mg/dL, and BUA of 12
§ NH3 + Nessler’s reagent mg/dL
+ NH2HgI3 Post-renal Due to obstruction in the urinary tract due to stones,
o Micro-Kjeldahl method: oldest causes prostatic enlargement, or tumors
method; mixture of sulfuric acid and
phosphoric acid CREATININE
• Urea is hydrolyzed by urease forming
ammonia, which is then titrated with a weak
Enzymatic &
acid
acid titration
o Van Slyke Cullen
o Urograph
• Urea is hydrolyzed to ammonium carbonate by
Urease urease
Berthelot • Ammonia reacts with phenol and sodium
method hypochlorite in an alkaline medium forming a
blue indophenol measured at 630 nm
GLDH The ammonia produced from urea hydrolysis by
coupled urease reacts with 2-oxoglutarate and NADH to
enzymatic produce glutamate and NAD+ by glutamate • Most commonly used NPN for overall renal function
method dehydrogenase, measured at 340 nm • Principal waste product of muscular metabolism derived mainly from
creatine or a-methyl guanidoacetic acid
• Synthesized from 3 amino acids: methionine, arginine, and lysine
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 JAFFE METHOD ENZYMATIC METHOD • Normal values: 3.5-7.2 mg/dL or 0.21-0.43 mmol/L in males, 2.6-6.0
0.9-1/3 mg/dL 0.6-1.1 mg/dL mg/dL or 0.16-0.36 mmol/L in females
Male
80-115 µmol/L 53-97 µmol/L
0.6-1.1 mg/dL 0.5-0.8 mg/dL TEST METHODOLOGIES
Female
53-97 µmol/L 44-71 µmol/L • Chemical method
• Uric acid is oxidized to allantoin and CO2 by
TEST METHODOLOGIES phosphotungstic acid reagent, a protein
• Enzymatic determination precipitant and color reagent in alkaline
• Available on Ektachem analyzer solution
• Creatinine is hydrolyzed to N-methyldantolin o Phosphotungstic acid is reduced to
and ammonia by creatinase Direct redox tungsten blue
Creatinase o Ammonia is then made to react methods • Caraway method: oldest method of BUA
method with a-ketoglutarate and NADH in determination
the presence of glutamate o Measured at 650-700 nm
dehydrogenase forming o Uses sodium cyanide as color
glutamate and NAD stabilizer
o NADH decrease is followed • Henry’s method: Caraway modification which
fluorometrically uses Na2CO3 as color stabilizer
• Enzymatic method • Enzymatic method
• Creatinine is hydrolyzed to creatine by • Routine method, using differential or
Creatinine creatinine aminohydrolase, followed by a absorption spectrophotometry
aminohydrolase series of coupled enzyme reactions in which Blaunch and • Uric acid is destroyed by the action of uricase
method creatine reacts with creatinine kinase, Kock / to form allantoin, hydrogen peroxide, and CO2
pyruvate kinase, and LDH, culminating in the uricase o Allantoin has no absorption at 290-
oxidation of NADH method 293 nm, BUA’s maximum peak,
• Chemical method decrease in absorbance is positively
• Formation of red tautomer of creatinine related to BUA present in the
Direct Jaffe
picrate when creatinine in serum is made to sample
reaction
react with freshly prepared alkaline sodium • Enzymatic method
picrate solution/Jaffe’s reagent • Uricase method followed by the secondary
reaction of peroxidase to catalyze a chemical
CLINICAL SIGNIFICANCE Peroxidase indicator reaction
• Aside from renal diseases, creatinine is also elevated in myopathies coupled o The color produced is proportional
enzyme to the BUA concentration present in
o Muscular dystrophy
method the sample
o Familial periodic paralysis • Used on traditional wet chemistry analyzers,
o Myasthenia gravis and for dry chemistry slide analyzers
o Dermatomyositis • Major interferences: bilirubin, ascorbic acid
• Normal creatinine and high BUN: GI hemorrhage and high protein
diets CLINICAL SIGNIFICANCE
• Increased creatinine and normal BUN: post-renal azotemia, and • Gout: uric acid metabolism causing an excess of the acid and salts
pre-renal azotemia with renal disease and renal failure to accumulate in the blood stream and joints
o Birefringent crystals: gout definitive diagnosis
Low BUN:crea ratio High BUN:crea ratio • Chronic alcoholism: ­ uric acid in blood due to excretion inhibition
Low protein diet Pre-renal azotemia by alcohol
Acute tubular necrosis Dehydration • Leukemia and other malignant conditions: increased
Repeated dialysis Catabolic states
Hepatic disease nucleoprotein turnover
• Elevated in decreased renal functions either due to overproduction
BLOOD URIC ACID or decreased excretion
• Major end product of purine metabolism formed in the liver and o Low levels are found in Fanconi’s syndrome, Wilson’s
intestinal mucosa from xanthine by the action of xanthine oxidase disease, and Hodgkin’s lymphoma
• Weakly acidic, with a pH of 7.4 • Fetal poisoning with chloroform and methanol, excessive exposure
• Relatively insoluble in plasma to X-rays and radioactive radiators
• 89% of filtered BUA is reabsorbed o Due to excessive cell breakdown and nucleic acid
o When accumulated, it may be deposited in the joints or metabolism
tophi, or in the genitourinary tract as uric acid stones • Genetic diseases like Lesch-Nyhan syndrome and Von Gierke’s
§ Tophi: deposit of uric acid crystals and other diseases
substances at surfaces of joints or cartilage,
typically as feature of gout AMMONIA
• Deamination of amino acids during protein metabolism
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• Determination contributes very little or none in renal impairment
• Applied in hepatic failure, Reye’s syndrome, and inherited
deficiencies of urea cycle enzymes
o Reye’s syndrome: rare, but rapidly worsening liver and
brain disease usually seen in children recovering from viral
infections like influenza and chickenpox
• Significant in hepatic comatose and terminal stages of hepatic
cirrhosis
• Known to be neurotoxic
• Normal value: 19-60 µg/dL or 11-35 µmol/L

AMMONIA MEASUREMENT
Ammonia formed reacts with phenol and alkaline
Berthelot’s
hypochlorite using sodium nitroprusside as a
reaction
catalyst to form indophenol blue
Ammonia is formed when it reacts with Nessler’s
reagent in the presence of a colloidal stabilizer,
forming a colloidal suspension of dimercuric
Nesslerization ammonium iodide or NH2Hg2I2
• Yellow: nitrogen is present in low to moderate
concentration
• Orange-brown: present in high concentration
Formation of glutamate and NADP+ from NH4+, 2-
oxoglutarate and NADPH catalyzed by glutamate
dehydrogenase
• Adenosine diphosphate is added to the
GLDH method
reaction mixture, increasing the rate of the
reaction and to stabilize GDLH
• Decrease in absorbance of NADP+ at 340 nm
is proportional to ammonia concentration

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 LIVER FUNCTION
LIVER • Site of storage for fat soluble vitamins
• Very large and complex organ responsible for performing vital tasks • Synthesizes nitric oxide to regulate intrahepatic
impacting all body functions blood flow
o Metabolism of carbohydrates, lipids, proteins, and bilirubin
Stellate / Ito
o Detoxification of harmful substances
cells
o Storage of essential compounds
o Excretion of substances to prevent harm
• Reddish-brown in color, considered as the largest and most versatile
organ in the body
• Has 2 main lobes, weighing from 1.2-1.5 kg in a normal adult Liver stem cells involved in the regeneration of
hepatocytes and bile ducts after liver injury
• Located under the diaphragm in the upper right quadrant of the
abdomen
• Only organ in mammals that can regenerate as much as 80% after
surgical removal of even a large portion Oval cells
• Total loss of the liver usually results in death from hypoglycemia
within 24 hours

LIVER BLOOD SUPPLY

• Receives approximately 15 mL/min from 2 major vessels
o Hepatic artery: branch of the aorta contributing to 20% of LIVER SYSTEMS
the blood supply, providing most of the O2 requirement Biochemical / Responsible for the vast majority of all
hepatocytic metabolic activities
o Portal vein: drains GI tract and transports most recently
Hepatobiliary Concerned with the metabolism of bilirubin
absorbed material from the intestine to the liver
Reticuloendothelial Involved with the immune system

LIVER METABOLIC FUNCTIONS

• Glycogenolysis: converts glycogen to
glucose during times of high energy demand or
lowered plasma glucose concentration
Carbohydrate • Glycogenesis: converts glucose to glycogen
metabolism during excess CHO intake
• Gluconeogenesis: synthesis of glucose from
the metabolism of some amino acids
• Cori cycle: converts lactate back to glucose
LIVER CELLS • Synthesizes almost all plasma proteins, except
• Phagocytic cells derived from monocytes immunoglobulins and adult hemoglobin
• Contains lysosomes with hydrolytic enzymes, o Albumin: one of the most important
immunoglobulin, and complement receptors proteins synthesized by the liver
• Secretes interleukins, TNF, collagenase, and Protein • Synthesis of positive and negative APR and
prostaglandin synthesis coagulation proteins
Kupffer • Stores amino acid pools through protein
cells degradation
• Transamination and deamination of amino
acids
• Active center of lipid metabolism
• Synthesis of endogenous lipids
• Major functioning cells • Catabolizes fatty acids to acetyl CoA and
cholesterol to primary bile acids
• Performs most of the metabolic and synthetic
Lipid o Bile acids and their conjugates act
liver functions
metabolism like detergents and essential
emulsifiers of ingested lipids
Hepatocytes
• Other lipid transformation includes
transamination of ketoacid to amino acid, and
acetyl CoA to ketone bodies

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 OTHER LIVER FUNCTIONS JAUNDICE CLASSIFICATIONS
Storage Storage site for glycogen, Vit. A, D, B12, E, and K, and • Excessive bilirubin production due to excessive
function iron from RE cells RBC production
• Detoxification of blood by excretion • ­ B1, normal or ¯ B2
o Phagocytosis by Kupffer cells • Can be caused by any of the following:
Pre-hepatic
o Metabolism and excretion of steroid o Hemolytic disease of the newborn
Protective hormones, drugs, and foreign o Malaria
functions compounds o Extensive hematoma
o Production of urea, uric acid, and o Hemolytic transfusion reaction
other molecules that are less toxic • Implies damage to hepatocytes where both types
than their parent compound of bilirubin are increased
• Play in immunologic defense through its RE • Can be due to jaundice retention or inability to
system and Kupffer cells conjugate as seen in:
Circulatory • Helps regulate blood volume by serving as blood o Physiologic jaundice of the newborn
functions storage area o Gilbert’s syndrome
• Means of mixing blood from the portal vein with o Crigler-Najjar syndrome types I and II
that of the systemic circulation • May be caused by hepatocyte injury as seen in:
• Processing and excretion of endogenous and o Viral hepatitis
exogenous substances into the bile or urine o Cirrhosis and alcoholic hepatitis
o Bile: complex fluid composed of bile o Toxic liver injury
acids, lecithin, cholesterol, bilirubin, o Parasitism
urobilinogen, and electrolytes Hepatic • May be due to impaired excretion of products
§ Important in lipid digestion, from hepatocytes:
and in formation of micelles o Dubin-Johnson syndrome
o Bilirubin: bile pigment resulting from o Rotor’s syndrome
heme catabolism due to old age or o Viral hepatitis
Excretory trauma o Cirrhosis
functions § Orange-yellow pigment
§ Degrades in the spleen,
bone marrow, and liver
§ Increased at birth, peaks at
the 6th day
§ Overproduction presents
as increase in plasma
levels
§ Total bilirubin: 0.2-1.0 • Failure of bile to flow due to obstruction of the
mg/dL biliary tree
• Normal B1, ­ B2 and ALP
CHARACTERISTIC B1 B2 o ­ ALP: marker for obstructive
jaundice
Slow reacting Prompt
Post • Can be cause by:
Normal value 0.2-0.8 mg/dL 0.0-0.2 mg/dL hepatic o Choledocholithiasis, or stones in the
Water solubility - +
common bile duct
Alcohol solubility + +
o Structures and spasm caused by
Affinity to serum albumin + - bacteria
Lipid membrane o Pancreatic sarcoma
+ -
permeability o Parasitism
Renal excretion - +
Van den Bergh reaction Indirect Direct
INHERITED DISORDERS OF BILIRUBIN METABOLISM
Non-polar Polar
Unconjugated Conjugated • Insertion of two bases into the promoter region
Indirect-reacting Diglucuronide of the UGT1A1 gene resulting in lower
Synonyms transcriptional rates and an overall lower
Hemobilirubin Direct-reacting
Hemolytic bilirubin Water soluble enzymatic activity
Water insoluble Cholestatic • Bilirubin transport deficit characterized by mild
unconjugated hyperbilirubinemia
• ¯ conjugation and uptake: pre-conjugation
JAUNDICE Gilbert’s
• Icterus: yellow discoloration or pigmentation of the skin, sclera, and disease failure, ­ B1
mucus membrane caused by bilirubin deposition
• Signifies hyperbilirubinemia that becomes clinically evident with
serum bilirubin levels exceed 2 mg/dL

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 • Conjugation deficit
• Type 1: autosomal recessive due to UDPGT
absence, with a severe increase of B1 leading
to death
• Type 2: autosomal dominant due to a partial
defect of the conjugating enzyme leading to
increased B1, but can survive to adulthood
Crigler-Najjar
syndrome

• Decreased hepatic excretion of bilirubin

leading to a black-colored liver
• ­ B2, with increased hepatic pigmentation due
to melanin
• Bilirubin excretion deficit
Dubin-
Johnson
syndrome

• ­ B2 and total bilirubin, but cause still remains

Rotor unknown
syndrome • Similar to Dubin-Johnson, but without the
hepatic pigmentation
Lucey- Familial form of unconjugated hyperbilirubinemia
Driscoll caused by a circulating inhibitor of bilirubin
syndrome conjugation

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 LABORATORY DIAGNOSIS OF LIVER DISEASE
CLINICAL SIGNIFICANCE OF LIVER DISORDERS HEPATITIS
JAUNDICE Inflammation of the liver that may have been caused by heavy alcohol
use, toxins, some medications, but is often caused by a virus

• Hyperbilirubinemia: result of RBC destruction, but with normal liver

• Fecal transmission
function Hepatitis A • Prevented through pre- and post-exposure
• Hypercarotenemia: skin discoloration immunization
• Percutaneous / mucosal transmission from blood
CIRRHOSIS and body fluids
Hepatitis B
• Also prevented by pre- and post-exposure
immunization
• Percutaneous / mucosal transmission from blood
Hepatitis C and body fluids
• Prevented by modification of risk behaviors
• Percutaneous / mucosal transmission from blood
and body fluids
Hepatitis D
• Prevented by pre- and post-exposure
immunization
• Clinical condition where scar tissue replaces normal, healthy liver
• Transmitted by the fecal-oral route, primarily
tissue from fecal specimens
Hepatitis E
• Blocks the blood flow through the organ and prevents the liver from • Prevented by ensuring proper food handling
functioning properly techniques
• Signs and symptoms: fatigue, nausea, unintended weight loss,
jaundice, GI tract bleeding, intense itching and swelling in the legs TUMORS
and abdomen Primary liver
Cancer that begins from liver cells
cancer
• Irreversible scarring process starts with a fatty liver, progressing to
Metastatic When tumors from other parts of the body spread
liver fibrosis to cirrhosis cancer to the liver
• May be presented with abnormal nodules Benign Hepatocellular adenoma, and hemangiomas
• Can cause the following disorders: Hepatocellular carcinoma, hepatocarcinoma and
Malignant
o Portal hypertension: blocked passageways hepatoma
o Splenomegaly: spleen enlargement
o Esophageal varices: blood vessel rupturing REYE’S SYNDROME
o Affected synthetic activity
§ Reduced organ productivity
§ Hypoalbuminemia
§ Clotting factor deficiencies
§ Accumulation of ascitic fluid in the abdomen

• Macronodular cirrhosis
According to
• Micronodular cirrhosis
size
• Possible mixed forms • Preceded by a viral syndrome like varicella, gastroenteritis, or upper
• Alcohol abuse respiratory tract infection like influenza
• Hemochromatosis: iron overload • With non-inflammatory encephalopathy and fatty liver degeneration
According to
o Deposition of hemosiderin in the
etiology • Presented with mild hyperbilirubinemia, and 3-fold increase in
tissues leading to dark discoloration
• Biliary cirrhosis ammonia and aminotransferases

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• Can proceed to neurologic abnormalities • Dilution of serum with saline until it visually matches the color of 0.1%
• Liver function is always abnormal, but bilirubin level is not usually potassium dichromate solution
elevated o The number of times the serum must be diluted is the
• Accumulation of ammonia can cause edema and death sample’s icterus index.

DRUG AND ALCOHOL-RELATED DISORDERS

• Immune-mediated injury to hepatocytes
• Adverse immune response directed against the organ itself, resulting
in hepatic and/or cholestatic disease
• Ethanol: very mild, transient, and unnoticed liver injuries
o Can lead to alcoholic cirrhosis with heavier and prolonged
• Has a limited clinical usefulness, and can be affected by carotene,
consumption
xantophyll, and hemoglobin
• Can occasionally cause massive hepatic failure or cirrhosis
o Form of hepatic necrosis leading to comatose and death
QUANTITATION BY DILUTION
• Classes of drugs causing hepatic damage include:
• Direct measurement of absorption by a spectrophotometer
o Barbiturates
• Used in pediatric laboratories to test newborns
o Tricyclic anti-depressants
• Hemolysis: ‘blanked-out’
o Anti-epileptics
o Acetaminophen • Presently reported separately as conjugated and unconjugated
o Chemotherapeutic drugs like vincristine, vinblastine, bilirubin
actinomycin D, and 5-fluorourcail o More commonly reported as conjugated and total bilirubin
o UB = TB - CB
EARLY DEVELOPMENT OF BILIRUBIN ASSAY
• Ehrlich, 1883 Fraction producing a color in the Van den Bergh
Direct bilirubin
method in an aqueous solution
• First described reaction
Fraction producing a color only after alcohol is
• Formation of red to blue pigment in the urine when bilirubin is coupled Indirect bilirubin
added
with diazotized sulfanilic acid solution
Best method for measuring only small
HPLC
Modification of the Ehrlich’s reaction and amounts of conjugated serum bilirubin
Van den Bergh applies it to serum bilirubin, using alcohol as an • Used in determining total bilirubin
accelerator for the coupling reaction • Follows absorbance of bilirubin in serum
• First quantitative technique developed at 455 nm
• Voided precipitation of protein that causes o Absorbance is proportional to
a false increase in bilirubin concentration
Malloy and Direct
• Uses 50% methanol solution as • Corrected value is achieved by
Evelyn spectrophotometric
accelerator method subtracting hemoglobin absorbance at
• Azobilirubin: end product, changing from 575 nm by absorbance at 455 nm
pink to purple • Causes of errors: turbid buffer,
• Reference method developed in 1938 inaccurate volume, presence of
• More sensitive test producing pink to blue lipochromes, insensitivity to hemolysis in
color change in azobilirubin infants
• Uses caffeine-benzoate-acetate as
accelerator
• Sensitive to sample pH changes
Jendrassik and • Sensitive to 50-fold variations in protein
Grof concentrations
• Adequate optical sensitivity for low
bilirubin concentration
• Has minimal turbidity and a relatively
constant serum blank
• Not affected by hemoglobin concentration
up to 750 mg/dL

ICTERUS INDEX
• Developed in 1919, when results are subjective
• Obsolete test
• Involves direct measurement of the sample’s natural color

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