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MUST TO KNOW CC RODRIGUEZ

Terms in this set (439)

Practicability Method is easily repeated

Reliability Maintain accuracy and precision

Intralab/Interlab QC Daily monitoring of accuracy and precision

Proficiency testing (Reference lab) Long-term accuracy

Interlab/External QC
Difference of >2: not in agreement w/ other lab

QC materials Available for a min. of 1 yr

Preferred (Human: biohazard)

Bovine control materials
Not for immunochem, dye-binding and bilirubin

Improper product manufacturing Unpurified analyte

Matrix effect
Altered protein

Precision study First step in method evaluation

Nonlab. personnel 29% of errors (lab results)

SD Dispersion of values from the mean

Index of precision
CV
Relative magnitude of variability (%)

SD2
Variance
Measure of variability

Inferential statistics Compare means or SD of 2 groups of data

 T-test Means of 2 groups of data

F-test SD of 2 groups of data

V-mask
Cumulative Sum Graph (CUSUM)
Earliest indication of systematic errors (trend)

Youden/Twin Plot Compare results obtained from diff. lab

Shewhart Levey-Jennings Chart Graphic representation of the acceptable limits of variation

Gradual loss of reliability

Trend
Cause: Deterioration of reagents (Systematic error)

Values: one side or either side of the mean Cause: Improper calibration
Shift
(Systematic error)

Values: far from the main set of values

Outliers Highly deviating values
Random or systematic errors

Kurtosis Degree of flatness or sharpness

Precision Random error

Accuracy Systematic error

Causes:
-Mislabeling
-Pipetting error
Random error (Imprecision; Indeterminate) -Improper mixing of sample and reagents
-Voltage/Temperature fluctuation
-Dirty optics
Parameters: SD and CV

Causes:
-Improper calibration
-Deterioration of reagents
-Contaminated solution
Systematic error (Inaccuracy/Determinate)
-Sample instability/unstable reagent blanks
-Diminishing lamp power
-Incorrect sample and reagent volume
Parameter: Mean
 Multirule Shewhart procedure Control rules + Control chart

Test method Westgard: at least 40 samples

Reference method Westgard: preferably 100 samples

Analytical Run Control and patient specimens assayed, evaluated, and report together

Physiologic Limit Referred to as absurd value

POCT Performed by nonlab personnel

Tripod:
Quality Assurance Program development
Assessment and monitoring Quality improvement

Quality Patient Care Test request forms, clear instruction for patient prep., specimen handling...

Reference Range/ Interval Range/ Reference At least 120 individuals should be tested in each age and sex category
Values

Distance bet 2 successive peaks (nm)

Wavelength Lower frequency = Longer wavelength (Ex. Red) Higher frequency = Shorter
wavelength (Ex. Violet)

Spectrophotometric meas. Meas. light intensity in a narrower wavelength

Meas. light intensity w/o consideration of wavelength Multiple wavelength

Photometric measurement
(uses filter only)

Light Amplification by Stimulated Emission of Radiation Light source for

LASER
spectrophotometry

Visible region Tungsten light bulb Mercury arc

Deuterium lamp
Mercury arc
UV
Xenon lamp
Hydrogen lamp

Merst glower
IR
Globar (Silicone carbide)
:
 Wavelength outside the band
Stray light
Most common cause of loss of linearity

Most commonly used monochromator

Diffraction gratings
Cutting grooves

Prisms Rotatable

Nickel sulfate Prevents stray light

Cutoff filter Anti-stray light

Bandpass ½ peak transmittance

Alumina silica glass cuvet Most commonly used cuvet

Quartz/plastic cuvet UV

Borosilicate glass cuvet Strong bases

Photodetector Converts transmitted light into photoelectric energy

Simplest detector
Barrier layer cell/ photocell/ photovoltaic cell No external voltage
For filter photometers

Contains anode and cathode

Phototube
Req external voltage

Most common type

Photomultiplier tube Most sensitive
UV and visible region

Galvanometer/Ammeter Meter or read-out device

A = abc (a = absorptivity; b = length of light (1cm); c = concentration)

Absorbance
A = 2 - log%T
:
 Splits monochromatic light into two components:
One beam sample
Double beam spectro.
One beam reference soln or blank (corrects for variation in light source
intensity)

Double-beam in space 2 photodetectors (sample beam and reference beam)

1 photodetector
Double-beam in time
Monochromatic light sample cuvet and reference cuvet

Dydimium filter 600 nm

Holmium oxide filter 360 nm

Reagent blank Color of reagents

Sample blank Optical interference (Hgb)

Meas. light emitted by a single atom burned in a flame Principle: Excitation

FEP Lt. source and cuvette: Flame
For excited ions (Na+, K+)

Cesium and Lithium Internal standards (FEP) Correct variations in flame

Preferred internal std

Lithium
Potent antidepressant

Meas. light absorbed by atoms dissociated by heat Principle: Dissociation

(unionized, unexcited, ground state) Lt. source: Hollow-cathode lamp
AAS
For unexcited trace metals (Ca++ and Mg++)
More sensitive than FEP

Atomizer (nebulizer) Convert ions to atoms

Chopper Modulate the light source

Complex with phosphate

Lanthanum/Strontium chloride
Avoid calcium interference

Unknown sample is made to react with a known solution in the presence of

Volumetric (Titrimetric)
an indicator
:
 Light blocked
Turbidimetry Meas. abundant large particles (Proteins)
Depend on specimen concentration and particle size

Meas. amt of Ag-Ab complexes Scattered light

Nephelometry
Depends on wavelength and particle size

Electrophoresis Migration of charged particles in an electric field

Iontophoresis Migration of small charged ions

Zone electrophoresis Migration of charged macromolecules

Movement of buffer ions and solvent relative to the fixed support Ex: gamma
Endosmosis
globulins

Cellulose acetate Molecular size

Agarose gel Electrical charge

Polyacrylamide gel Charge and molecular size 20 fractions (ex. isoenzymes)

Directly proportional to net charge

Electrophoretic mobility Inversely proportional to molecular size & viscosity of the supporting
medium

Isoelectric focusing Molecules migrate through a pH gradient pH = pI

For isoenzymes: same size, different charge

Densitometry Scan & quantitate electrophoretic pattern

Capillary electrophoresis Electro-osmotic flow

Southern blot DNA

Northern blot RNA

Western blot Proteins

Separation by specific differences in physical-chemical characteristics of the

Chromatography
different constituents
:
 Paper chromatography Fractionation of sugar and amino acid Sorbent: Whatman paper

TLC Screening: Drugs

Relative distance of migration from the point of application

Retention factor (Rf) value Rf = Distance leading edge of component moves divided by Total distance
solvent front moves

Separation of steroids, barbiturates, blood, alcohol, and lipids Volatile

compounds
Gas chromatography
Specimens are vaporized
Mobile phase: Inert gases

Gas Solid chromatography Differences in absorption at the solid phase surfaces

Differences in solute partitioning between the gaseous mobile phase and the
Gas Liquid chromatography
liquid stationary phase

Mass Spectrometry Fragmentation and ionization

GC-MS Gold standard for drug testing

MS/MS Detect 20 inborn errors of metabolism from a single blood spot

Most widely used liquid chromatography

HPLC
Fractionation of drugs, hormones, lipids, carbohydrates and proteins

Gel filtration
Hydrophilic gel
Separation of enzymes, antibodies and proteins Ex: Dextran and agarose

Gel permeation
Hydrophobic gel
Separation of triglyceride and fatty acid Ex: Sephadex

Ion exchange Separation depends on the sign and ionic charge density
chromatography

Based on relative solubility in an organic solvent (nonpolar) and an aqueous

Partition chromatography
solvent (polar)

Affinity chromatography For lipoproteins, CHO and glycated hemoglobins

Based on differences between the adsorption and desorption of solutes at

Adsorption chromatography
the surfaces of a solid particle
:
 Det. amt. of lt. emitted by a molecule after excitation by electromagnetic
radiation
Lt. sources: Mercury arc and Xenon lamp (UV) Lt. detector: Photomultiplier
tubes
Fluorometry/Molecular Luminescence Spectro.
2 monochromators:
Primary filter - selects wavelength absorbed by the solution to be measured
Secondary filter - prevents incident light from striking the photodetector
Sensitivity: 1000x than spectro

Major disadvantage of fluorometry

Quenching
pH and temperature changes, chemical contaminants, UVL changes

Borosilicate glasswares For heating and sterilization Ex: Pyrex and Kimax

Boron-free/Soft glasswares High resistance to alkali

Special alumina-silicate glass Strengthened chemically than thermally

Corex (Corning)
6x stronger than borosilicate

For high thermal, drastic heat and shock

Vycor (Corning)
Can be heated to 900OC

Soda-lime glass + Calcium, Silicon, Sodium oxides

Flint glass Easy to melt
For making disposable glasswares

TD: To deliver Exact amount

TC: To contain Does not disperse the exact volume

Blowout w/ etched rings on top of pipet

Self-draining w/ o etched rings Drain by gravity

Volumetric: for non-viscous fluid; self-draining

Ostwald folin: for viscous fluid; w/ etched ring
Transfer pipet
Pasteur: w/o consideration of a specific volume
Automatic macro-/micropipets

Serological: w/ graduations to the tip (blowout) Mohr: w/o graduations to the

tip (self-draining) Bacteriologic
Graduated or measuring pipet
Ball, Kolmer and Kahn
Micropipettes: <1 mL
:
 TC pipets:
Micropipettes Sahli-Hellige pipet Lang-Levy pipet RBC and WBC pipets
Kirk and Overflow pipets

Air displacement pipet Piston: suction Disposable tip

Positive displacement pipet Piston to barrel (like a hypodermic syringe)

Dispenser/Dilutor pipet Liquid: common reservoir dispense repeatedly

Distilled H2O Calibrating medium for TD pipettes

Mercury Calibrating medium for TC pipettes

Acid dichromate Cleaning solution for glasswares

(H2SO4 + K2Cr2O4)

Common reaction vessel

Air bubbles: separates and cleans Glass coil: mix

Continuous flow analyzer

Examples: "STS"
Simultaneous Multiple Analyzer (SMA) Technicon Autoanalyzer II
SMAC

Acceleration and deceleration of the rotor Advantage: Batch analysis

Centrifugal analyzer Examples: "RICC" Cobas-Bio (Roche) IL Monarch

CentrifiChem
RotoChem

Most popular
Req. vol: 2-6 µL
Discrete Analyzer Uses positive-displacement pipets
Run multiple-tests-one-sample-at-a-time
Random access capability (STAT)

4 or 5 layers:
-Spreading layer
-Scavenger layer - Ascorbate Oxidase
Thin-Film Analyzers (Dry slide technology) -Reagent layer
-Indicator layer
-Support layer
Colored reaction Reflectance spectrophotometry

Transport of quantity of analyte or rgt from one specimen rxn into another,
Carry over and
contaminating a subsequent one
:
 Batch testing All samples loaded at the same time Single test is conducted on each sample

One specimen
Parallel testing
More than one test is analyzed

Any sample Any test

Random access testing Any sequence
STAT

Sequential testing Multiple tests analyzed one after another on a given specimen

Open reagent system System other than manufacturer's reagents can be utilized for measurement

Closed reagent system The operator can only use the manufacturer's reagents

Increased: GU2FT C2L3A5P2

GH
Urea
Urinary protein (Proteinuria) Fatty acid
Testosterone
CPK (muscle) Creatinine (muscle) Lactate
Exercise LH
LD (muscle) ACP
Aldolase (muscle) AST
ALT
Ammonia Pyruvate Prolactin

Decreased: Glucose

Fist clenching Increased: "LPP" Lactate Potassium

Fist clenching
Phosphate

8-16 hours: Glucose Lipids Lipoproteins Increased:

Fasting Bilirubin (48 hours)
Triglyceride (72 hours)

Glucose Cholesterol Triglyceride

Basal state collection
Electrolytes

Increased: "GLUC2H" Glucose

Lipids
Diet
Urea (High protein diet) Caffeine: increases glucose Catecholamines
5-HIAA (From Serotonin)

Turbidity/Lactescence Triglyceride >400mg/dL

:
 Icterisia Bilirubin: 25.2 mg/dL

Interfere with: "TACGu" Total Protein

Icteric samples Albumin Cholesterol
Glucose

Preferred position
Upright/supine (lying) position Patient should be seated/supine at least 20 mins before blood collection to
prevent hemodilution or hemoconcentration

Vasoconstriction Reduced plasma volume Increased: "ECA"

Supine TO Sitting/Standing Enzymes Calcium
Albumin

Hemoconcentration

Increased: "P(u)BLIC" Proteins

Sitting Supine
BUN
Lipids
Iron Calcium

Hemodilution Decreased: "TLC" Triglycerides Lipoproteins

Standing Supine
Cholesterol

Prolonged standing Increased: K+ (muscles)

Prolonged bedrest Decreased: Albumin (Fluid retention)

Tourniquet Recommended: 1 minute application

Hemoconcentration Anaerobiosis Increased: "C2LEA2K" Calcium

Prolonged tourniquet app. Cholesterol Lactate Enzymes Ammonia Albumin
K+

Increased: "TUNG2C3" Triglycerides

Urea
Tobacco smoking (Nicotine) Nonesterified fatty acid Glucose
GH
Catecholamines Cortisol Cholesterol

Increased: "THUG" Triglycerides

Alcohol ingestion Hypoglycemia (chronic alcoholism) Uric acid/Urates
GGT

Ammonia Increases by 100-200µg/L/cigar

:
 Increased: "LAGIC" Lactate
Stress (anxiety) Albumin Glucose Insulin
Cholesterol

Medications affecting plasma volume can affect protein, BUN, iron, calcium
Drugs Hepatotoxic drugs: increased liver function enzymes
Diuretics: decreased sodium and potassium

"CA3PI2TG"
Cortisol
ACTH ACP
Diurnal variation
Aldosterone Prolactin Iron
Insulin
Thyroxine GH

Sleeping patients Must be awakened before blood collection

Unconscious patients Ask nurse or relative Identification bracelet

Venipuncture Median Cubital (1st) Cephalic (2nd) Basilic (3rd)

Velcro or Seraket type

Tourniquet
3-4 inches above the site Not exceed 1 minute

Bevel up
Needle 15-30O angle
Length: 1 or 1.5 inch (Butterfly needle: ½ to ¾ inch)

Cotton site
After blood collection Apply pressure for 3-5 minutesCotton site
Apply pressure for 3-5 minutes

BP cuff as tourniquet Inflate to 60 mmHg

Benzalkonium chloride (Zephiran) Disinfectant for ethanol testing Dilution - 1:750

Discontinue IV for 2 minutes Collect sample below the IV site Initial sample
IV line on both arms
(5mL) discard

Increased:
Glucose (10% contam. w/ 5% dextrose increased bld glucose by 500 mg/dL)
IV fluid contamination Chloride
Potassium Sodium Decreased: Urea
Creatinine
:
 Renin blood level Collected after a 3-day diet, from a peripheral vein

Early morning blood collection

Basal state collection
12 hours after the last ingestion of food

Lancet 1.75mm: preferred length to avoid penetrating the bone

Incision (Skin puncture) <2.0mm (infants and children) 2-3mm (adults)

1.5-2.4mm Distance from the skin surface to bone or cartilage (middle finger)

Earlobe: Preferred site

Arterialized capillary blood
Lateral plantar heel surface: most commonly used site

Minute metal filling which may be inserted into the capillary tube before
Flea collecting blood to help mix the specimen while the blood is entering the
tube

Indwelling umbilical artery Best site for blood gas analysis (newborns)

1000-3000 RCF for 10 mins Centrifugation requirement

Increased:
"KLA6MP ITC2"
-K+
-LDH (150x)
-ACP
-ALP

-Aldolase
Hemolysis
-ALT
-AST
-Albumin
-Mg2+
-Phosphorus
-Iron
-Total protein
Affects bilirubin levels Inhibits lipase

Required for: "ABCGLRP2" Ammonia

Blood gases Catecholamines Gastrin
Refrigeration/Chilling (Low temp) Lactic acid Renin PTH
Pyruvate Decreased: LD 4 and 5 Increased:
ALP
:
 Bilirubin
Photosensitive analytes Beta-carotene Folate Porphyrins
Vitamins A and B6

Insoluble salt
Oxalate
1-2 mg/mL blood

Non-ionized form
Citrate
3.2-3.8 g/dL (1:9 ratio)

Chelation
EDTA 1-2 mg/mL blood Versene: disodium salt
Sequestrene: Dipotassium salt

Weakly dissociated calcium component 2 mg/mL blood: anti-glycolytic

Fluoride
10 mg/mL blood: anticoagulant

A.k.a. Mucoitin polysulfuric acid Universal anticoagulant Antithrombin

Heparin
0.2 mg/mL blood

For glucose, BUN, ionized calcium, electrolyte studies (K+: best) and
Lithium heparin
creatinine

Orange top tube Additive: Thrombin

Additives:
None;
Royal blue top tube
Na2EDTA
Sodium heparin

Brown top tube Lead testing

Tan top tube Lead testing

Additive: Buffered sodium citrate

Black top tube
For ESR

Respinning gel tubes Increases potassium

Gel separator (SG: 1.04) Serum: (SG: 1.03)

Thixotropic gel
RBC: (SG: 1.05)

0.179 Conversion factor for iron (mg/dL µmol/L)

0.01 Conversion factor for phospholipid (g/dL to g/L)

:
 2.27 Conversion factor for folate

For qualitative and quantitative analyses For accuracy

Analytical reagent (AR) grade Established by American Chemical Society (ACS)
Uses: Trace metal analysis and preparation of standard solutions

Additional purification steps

Ultrapure reagents Ex: Spectrograde, nanograde, HPLC grade
Uses: Chromatography, atomic absorption, immunoassays

Chemically Pure (CP) or Indicates that the impurity limitations are not stated

Pure Grade Purity is delivered by meas. of melting point or boiling point

In manufacturing
Technical/Commercial grade
Never used in clin. lab. testing

United States Pharmacopoeia (USP) and For human consumption

National Formulery (NF) Not applicable for lab. analysis Purpose: For drug manufacturing

Preparation of reagent grade water Filtration (1st) Distillation, Ion exchange, Reverse Osmosis

Min. interference Max. water purity Used immediately

For ultramicrochemical analyses, measurements of nanogram or
subnanogram concentrations, tissue or cell methods (microscopy) and
Type I Rgt Water preparation of standard solutions
Uses: FEP, AAS, blood gases and pH, enzyme studies, electrolyte testing,
HPLC,
trace metal and iron studies

For clinical laboratory use (hematology, microbiology, immunology,

Type II Rgt Water
chemistry) For prep. of rgts and QC materials

For washing glasswares

Type III
For urinalysis, parasitology and histology

Distilled water Purified to remove almost all organic materials

Free from mineral salts; removed by ion exchange processes Organic

Deionized water
material may still be present

Req. manuf. to indicate lot no., physical or biological health hazard of the
Occupational Safety and Health Act (OSHA)
chem.. rgts, and precautions for safe use and storage
:
 Recommends that a lab. document culture growth, pH and specific water
College of American Pathologists (CAP)
resistance on reagent grade water

Microbiological content pH
Resistivity
Tests for water purity Chemical oxygen demand Ammonia
Ions
Metals

Detergent-contaminated water Alkaline pH

Hard water Contains calcium, iron and other dissolved elements

NCCLS Now: Clinical and Laboratory Standards Institute (CLSI)

Saturated solution Excess of undissolved solute particles

Greater concentration of undissolved solute particles than does a saturated

Super saturated solution
solution of the same substance

Highly purified
Primary standard (IUPAC)
Measured directly to produce a substance of exact known concentration

Low purity
Secondary standard
Concentration is determined by comparison w/ a primary standard

Ordinary combustibles: paper, cloth, rubbish, plastics, wood Extinguisher:

Class A fire
Water (A), Dry chemical (ABC), loaded steam

Flammable liquids: grease, gasoline, paints, oil

Class B fire
Extinguisher: Dry chemical (ABC), carbon dioxide (BC), halon foam (BC)

Electrical equipment and motor switches

Class C fire
Extinguisher: Dry chemical (ABC), Carbon dioxide (BC), halon (BC)

Flammable metals: mercury, magnesium, sodium, lithium Extinguisher: Metal X

Class D fire
Fought be fire fighters only

Detonation (Arsenal fire)

Class E fire
Allowed to burn out and nearby materials protected

Standard Hazards Identification System (Diamond-shaped color coded symbol)

Blue quadrant Health hazard

:
 Red quadrant Flammable hazard

Yellow quadrant Reactivity/Stability hazard

White quadrant Other special information

Chemical spills 1st step: assist/evacuate personnel

1:10 dilution of chlorine bleach (10%) To disinfect and clean bench tops
1:10 dilution of chlorine bleach (10%) In contact with the area for at least 20 minutes HBV: 10 minutes
HIV: 2 minutes

Chloroform Methanol
Carbon tetrachloride Bromide
Poisonous vapors
Ammonia Formaldehyde
Mercury

Acetone Ethanol Toluene Methanol Xylene Benzene Isopropanol

Flammable and combustible solvents
Heptane

Flammable liquids Flash point below 37.8OC

Combustible liquids Flash point at or above 37.8OC

Neutralized before disposal

Strong acids or bases
Water should NEVER be added to concentrated acid

Ether Deteriorate over time hazardous Forms explosive peroxides

Benzidine Known carcinogen

Fumehoods Ventilation: velocity of 100-120 ft/min

Safety showers Deliver 30-50 gal/min of H2O at 20-50 psi

Glycol aldehyde The simplest carbohydrate

Sucrose Most common nonreducing sugar

Exocrine: Enzymes (AMS, LPS)

Pancreas
Endocrine: Hormones (Insulin, glucagon, somatostatin)
:
 "GAG CHET"
Glucagon
ACTH GH
Hyperglycemic Hormones
Cortisol
Human Placental Lactogen Epinephrine
Thyroxine

Hyperglycemia (≥126 mg/dL) Electrolyte Imbalance: Decreased: Sodium, Bicarbonate Increased: Potassium

50-55 mg/dL = Symptoms

Hypoglycemia
≤50 mg/dL = Diagnostic

Low blood glucose concentration Typical symptoms

Whipple's triad (Hypoglycemia)
Symptoms alleviated by glucose administration

6:1 Ratio of BHA to AA in severe DM (Normal = 1:1)

IDDM
Juvenile Onset Brittle
Ketosis-prone
Type 1 DM 80-90% reduction of beta-cells Symptomatic Type 1 DM HLA-DR3 and DR4
(+) Glutamic acid decarboxylase (GAD65) (+) Insulin autoantibodies
(+) Microalbuminuria: 50-200 mg/24 hours = Diabetic nephropathy
(-) C-peptide

Microvascular disorders: Nephropathy Neuropathy

Complications of Type I DM
Retinopathy

NIDDM
Adult type/Maturity Onset Stable
Ketosis-resistant Receptor-deficient
Type 2
Insulin resistance: relative insulin deficiency Strong genetic predisposition
Geneticist's nightmare
If untreated glucose: >500 mg/dL nonketotic hyperosmolar coma

Screening: 1hr GCT (50g) - bet. 24 and 28 weeks of gestation Confirmatory:

3-hr GTT (100g)
Infants: at risk for respiratory distress syndrome, hypocalcemia,
Gestational DM
hyperbilirubinemia
After giving birth, evaluate 6-12 weeks postpartum
Converts to DM w/in 10 years in 30-40% of cases

MUST TO KNOW CC RODRIGUEZ Study

:
 FBS = ≥95 mg/dL
1-Hr = ≥ 180 mg/dL
OGTT (GDM) 2-Hr = ≥ 155 mg/dL
3-Hr = ≥ 140 mg/dL
GDM = 2 plasma values of the above glucose levels are exceeded

Impaired fasting glucose (Pre-diabetes) FBS = 100-125 mg/dL

FBS = <126 mg/dL

Impaired glucose tolerance
2-Hr OGTT = 140-199 mg/dL

WB = 15% lower than in serum or plasma

FBS
VB = 7 mg/dL lower than capillary and arterial blood

CSF glucose 60-70% of the plasma glucose

Peritoneal fluid glucose Same with plasma glucose

Fasting: 2 mg/dL/decade
Plasma glucose increases w/ age Postprandial: 4 mg/dL/decade
Glucose challenge: 8-13 mg/dL/decade

w/in 1 hour Separate serum/plasma from the cells

(Preferably w/in 30 mins)

5-7%/hr Glycolysis at room temperature

1-2 mg%/hr Glycolysis at refrigerated temperature

Copper reduction methods Cupric Cuprous Cuprous oxide

Folin Wu Cuprous ions + phosphomolybdate phosphomolybdenum blue

Nelson-Somogyi Cuprous ions + arsenomolybdate arsenomolybdenum blue

Neocuproine method Cuprous ions + neocuproine Cuprous-neocuproine complex (yellow)

Benedict's method Reducing substances in blood and urine

(Hagedorn-Jensen) Ferricyanide ---(Glucose)--> Ferrocyanide (Yellow)

Alkaline Ferric Reduction method
(Colorless)

Ortho-toluidine (Dubowski method) Schiff's base

:
 Glucose oxidase Measures beta-D-glucose (65%)

Mutarotase Converts alpha-D-glucose (35%) to beta-D-glucose (65%)

NADH/NADPH Absorbance at 340nm

Consumption of oxygen on an oxygen-sensing electrode

Polarographic glucose oxidase
O2 consumption α glucose concentration

Most specific method Reference method

Hexokinase method
Uses G-6-PD

G-6-PD Most specific enzyme rgt for glucose testing

False-decreased Bilirubin
Interfering substances (Glucose oxidase) Uric acid
Ascorbate

Hemolysis (>0.5 g/dL Hgb) Major interfering substance in hexokinase method (false-decreased)

Cellular strip
Dextrostics
Strip w/ glucose oxidase, peroxidase and chromogen

Janney-Isaacson method (Single dose) = most common Exton Rose (Double

OGTT dose)
Drink the glucose load within 5 mins

For patients with gastrointestinal disorders (malabsorption) Glucose: 0.5 g/kg

body weight
IVGTT
Given w/in 3 mins
1st blood collection: after 5 mins of IV glucose

Ambulatory Fasting: 8-14 hours

Requirements for OGTT Unrestricted diet of 150g CHO/day for 3 days
Do not smoke or drink alcohol

75 g = adult (WHO std) 100 g = pregnant

Glucose load
1.75 g glucose/kg BW = children
:
 2-3 months
Glucose = beta-chain of HbA1

1% increase in HbA1c = 35 mg/dL increase in plasma glucose 18-20% =

HbA1c
prolonged hyperglycemia
7% = cutoff
Specimen: EDTA whole blood
Test: Affinity chromatography (preferred)

IDA and older RBCs High HbA1c

RBC lifespan disorders Low HbA1c

2-3 weeks
Fructosamine (Glycosylated albumin/ plasma Useful for patients w/ hemolytic anemias and Hgb variants Not used in cases
protein ketoamine) of low albumin
Specimen: Serum

Congenital deficiency of 1 of 3 enzymes in galactose metabolism Galactose-

Galactosemia 1-phosphate uridyl transferase (most common) Galactokinase
Uridine diphosphate galactose-4-epimerase

Essential fructosuria Autosomal recessive Fructokinase deficiency

Hereditary fructose Defective fructose-1,6-biphosphate aldolase B activity

intolerance

Failure of hepatic glucose generation by gluconeogenic precursors such as

Fructose-1,6-biphosphate deficiency
lactate and glycerol

Autosomal recessive
Glycogen Storage Disease
Defective glycogen metabolism Test: IVGTT (Type I GSD)

Ia = Von Gierke Glucose-6-Phosphatase deficiency (most common worldwide)

II = Pompe Alpha-1,4-glucosidase deficiency (most common in the Philippines)

III = Cori Forbes Debrancher enzyme deficiency

IV = Andersen Brancher enzyme deficiency

V = McArdle Muscle phosphorylase deficiency

VI = Hers Liver phosphorylase deficiency

:
 VII = Tarui Phosphofructokinase deficiency

XII = Fanconi-Bickel Glucose transporter 2 deficiency

Collect blood glucose at least 60 mins (to 2 hrs) before the lumbar puncture
CSF glucose
(Because of the lag in CSF glucose equilibrium time)

< 0.5 Normal CSF : serum glucose ratio

C-peptide Formed during conversion of pro-insulin to insulin

5:1 to 15:1 Normal C-peptide : insulin ratio

Differentiate pancreatic insufficiency from malabsorption (low blood or urine

D-xylose absorption test
xylose)

Gerhardt's ferric chloride test Acetoacetate

Nitroprusside test 10x more sensitive to acetoacetate than to acetone

Acetest tablets Acetoacetate and acetone

Ketostix Detects acetoacetate better than acetone

KetoSite assay Detects beta-hydroxybutyrate but not widely used

RBS = <140 mg/dL FBS = 70-100 mg/dL HbA1c = 3-6%

Fructosamine = 205-285 µmol/L 2-Hr PPBS = <140 mg/dL
GTT:
Normal Values (Carbohydrates)
30 mins = 30-60 mg/dL above fasting
1-Hr = 20-50 mg/dL above fasting
2-Hr = 5-15 mg/dL above fasting 3-Hr = fasting level or below

Most abundant lipid

Phospholipids
Amphipathic: polar (hydrophilic head) and nonpolar (hydrophobic side chain)

Reference material during 3rd trimester of pregnancy Concentration is

Sphingomyelin constant as opposed to lecithin
Not derived from glycerol but from sphingosine (amino alcohol)

70% Lecithin/Phosphatidyl choline

Forms of phospholipids 20% Sphingomyelin
10% Cephalin
:
 TLC + Densitometric quantitation Method for L/S ratio

Microviscosity Measured by fluorescence polarization

Not a source of fuel Not affected by fasting

Cholesterol 70% Cholesterol ester (plasma/serum)
30% Free cholesterol (plasma/serum and RBC)

LCAT Esterification of cholesterol

Apo A-1 Activator of LCAT

Cholesterol increases after the age of 50 2 mg/dL/year between 50 and 60 years old

Cholestadienyl Monosulfonic acid

Liebermann Burchardt
Green end color

Salkowski Cholestadienyl Disulfonic acid Red end color

Glacial acetic acid Acetic anhydride

Color developer mixture (Cholesterol)
Conc. H2SO4

One-step method Colorimetry (Pearson, Stern and Mac Gavack)

Two-step method Color. + Extraction (Bloor's)

Three-step method Color. + Extract. + Saponification (Abell-Kendall)

Color. +Extract. + Sapon. + Precipitation (Schaenheimer Sperry, Parekh and

Four-step method
Jung)

Abell, Levy and Brodie mtd (Chemical method) CDC reference method for
cholesterol:
Abell, Levy and Brodie mtd (Chemical method) -Hydrolysis/saponification (Alc. KOH)
-Hexane extraction
-Colorimetry (Liebermann-Burchardt)

Most insoluble lipid

Triglycerides
Main storage lipid in man (adipose tissue) - 95% Fasting: 12 hours

Triglyceride increases after 2 mg/dL/year between 50 and 60 years old

the age of 50
:
 Chromotropic acid
Van Handel & Zilversmith (Colorimetric)
(+) Blue color compound

Diacetyl acetone
Hantzsch Condensation (Fluorometric)
(+) Diacetyl lutidine compound

(Chemical method) CDC reference method for triglycerides:

-Alkaline hydrolysis
-Chloroform extraction extract treated w/ silicic acid
Modified Van Handel and Zilversmith
-Color reaction w/ chromotropic acid - meas. HCHO

(+) Pink colored

Short chain = 4-6 C atoms Medium chain = 8-12 C atoms Long chain = >12 C
atoms Saturated = w/o double bonds Unsaturated = w/ double bonds
Fatty acids
Substrate for gluconeogenesis
Most is bound to albumin

Palmitic acid 16:0

Arachidonic acid 20:4

Stearic acid 18:0

Oleic acid 18:1
Linoleic acid 18:2

Lipoprotein lipase (Lipemia clearing factor) Hydrolyzes TAG in lipoproteins, releasing fatty acid and glycerol

Hydrolyzes TAG and phospholipids from HDL Hydrolyzes lipids on VLDL and
Hepatic lipase
IDL

Endothelial lipase Hydrolyzes phospholipids and TAG in HDL

Protein component of lipoprotein

Apolipoprotein
Amphipathic helix - ability of proteins to bind to lipids

Largest and least dense Produced by the intestine SG: <0.95

Chylomicrons 80-95% TAG (exogenous) Apo B-48 (Major)
EP: Origin

Secreted by the liver SG: 0.95-1.006

VLDL 65% TAG (endogenous) Apo B-100 (Major)
EP: pre-beta
:
 Synthesized by the liver SG: 1.006-1.063
50% CE
LDL Apo B-100 (Major) EP: beta
Cholesterol transport: LiverTissues Target of cholesterol lowering therapy
Better marker for CHD risk

Smallest but dense SG: 1.063-1.21

45-55% protein
26-32% phospholipid Apo A-1 (Major)
HDL
EP: alpha
Produced by the liver and intestine
Reverse cholesterol transport: TissueLiver

Product of VLDL catabolism

IDL Seen in Type 3 hyperlipoproteinemia (Apo E-III def.; beta-VLDL) SG: 1.006-
1.019

Sinking pre-beta lipoprotein SG: 1.045-1.080

Apo B-100
Lp(a)
EP: pre-beta (VLDL) UC: like LDL
Independent risk factor for atherosclerosis

Found in obstructive jaundice (cholestasis) and LCAT deficiency 90% FC and

LpX PL
Apo C and albumin

Floating beta-lipoprotein SG: <1.006

EP: beta (LDL) UC: like VLDL
Beta-VLDL
Found in type 3 hyperlipoproteinemia (Apo E-III def; IDL)
Rich in cholesterol content than VLDL

Specimen: sample from serum separator tubes (preferred) EDTA plasma:

Lipoprotein methodologies choice for research studies of LPP fractions Fasting state: TAG VLDL
Nonfasting state: TAG CM

Reference method for LPP quantitation Reagent: Potassium bromide (SG:

Ultracentrifugation 1.063) Ultracentrifugation of plasma for 24 hours
Expressed in Svedberg units

Electrophoretic pattern:
(+) HDL VLDL LDL CM (Origin) (-)
Electrophoresis
Agarose gel: sensitive medium
VLDL: migrates w/ alpha2-globulin (pre-beta)

Uses polyanions (heparin and divalent cations) and polyethylene glycol

Chemical precipitation Dextran sulfate-Mg2+
Heparin-Mn2+
:
 CDC Reference method for HDL
3-step procedure: Ultracentrifugation Precipitation
Abell-Kendall assay

Beta quantification + Ultracentrifugation + Method for LDL Sample: EDTA plasma

Chemical precipitation

Immunoturbidimetric assay Measures Lipoprotein (a)

LDL Cholesterol Total Cholesterol - HDL - VLDL

Most commonly used

Friedewald method VLDL = TAG/2.175 (mmol/L) VLDL = TAG/5 (mg/dL)
Not applicable if TAG is >400 mg/dL

De Long method VLDL = TAG/2.825 (mmol/L) VDL = TAG/6.5 (mg/dL)

Apo A-1 Activates LCAT

Apo B-100 LDL LDL receptor

CM (major)
Apo B-48
Not recognized by LDL receptor

Apo C-II Activates LPL

Apo D Activates LCAT

Apo E Apo E-4: associated w/ high LDL, higher risk of CHD and Alzheimer's disease

Lp(a)
Apo(a)
Homologous to plasminogen

Autosomal recessive
Abetalipoproteinemia (Basses-Kornzweig syn.)
Defective apo B synthesis Deficient fat soluble vitamins

Niemann-Pick disease Sphingomyelinase deficiency

Deficiency of HDL (1-2 mg/dL)

Tangier's disease
Defects in the gene for the ABCA1 transporter

TAG = 10,000 mg/dL

LPL deficiency (Chylomicronemia) Do not develop premature coronary disease (CM are not atherogenic)
Abdominal pain and pancreatitis
:
 LCAT deficiency Fish-eye disease Low HDL

Tay-Sachs disease Hexosaminidase A deficiency

Fredrickson Classification Type 1 LPL deficiency (Chylomicronemia) Increased: CM (TAG)

Type 2a Familial hypercholesterolemia Increased: LDL (cholesterol)

Combined hyperlipidemia (most common primary hyperlipidemia) Increased:

Type 2b
LDL (cholesterol), VLDL (TAG)

Dysbetalipoproteinemia Increased: IDL, (+) beta-VLDL (+) Apo E-II

Type 3
(+) Eruptive and palmar xanthomas

Type 4 Hypertriglyceridemia Increased: VLDL (TAG)

Type 5 Increased: VLDL (Endo.TAG), CM (Exo.TAG)

Cholesterol:
Desirable = <200 mg/dL Borderline high = 200-239 mg/dL High = >240 mg/dL
Triglycerides:
Desirable = <150 mg/dL Borderline high = 150-199 mg/dL High = 200-499
Very high = >500 mg/dL HDL:
Normal Values (Lipids) Low = <40 mg/dL (Cutoff) High = >60 mg/dL
LDL:
Optimal = <100 mg/dL
Near/above optimal = 100-129 mg/dL Borderline high = 130-159 mg/dL High =
160-189 mg/dL
Very high = >190 mg/dL

Proteis First rank of importance

Amphoteric: positive and negative charges Effective blood buffers

Synthesized by the liver except immunoglobulins (plasma cells) Provide 12-
Proteins
20% of total daily body energy requirement Composed of 50-70% of the
cell's dry weight

Amino acid sequence

Primary structure Det. the identity of protein, molecular structure, function binding capacity,
recognition ability

Winding of polypeptide chain

Secondary structure
Specific 3-D conformations: alpha-helix, beta-pleated sheath, bend form
:
 Actual 3-D configuration Folding pattern
Tertiary structure
Physical and chemical properties of proteins

Quarternary structure Association of 2 or more polypeptide chains protein

Albumin No quarternary structure

Alanine (pyruvate)
Glucogenic amino acids
Arginine (alpha-ketoglutarate) Aspartate (oxaloacetate)

Degraded to acetyl-CoA Leucine

Ketogenic amino acids
Lysine

Hydrolysis Amino acids

Simple proteins Fibrous: fibrinogen, troponins, collagen
Globular: hemoglobin, plasma proteins, enzymes, peptide hormones

Protein (apoprotein) + nonprotein moiety (prosthetic group) Metalloproteins:

ferritin, ceruloplasmin, hemoglobin, flavoproteins Lipoproteins: VLDL, HDL,
LDL, CM
Conjugated proteins
Glycoproteins: haptoglobin, alpha1-antitrypsin (10-40% CHO) Mucoproteins
or proteoglycans: Mucin (CHO > CHON)
Nucleoproteins: Chromatin (combined w/ nucleic acids)

Nitrogen balance Balance bet. anabolism and catabolism

Negative nitrogen balance Catabolism > anabolism Excessive tissue destruction

Positive nitrogen balance Anabolism > catabolism Growth and repair processes

Transports thyroxine and retinol (Vit. A)

Prealbumin (Transthyretin)
Landmark to confirm that the specimen is really CSF

Albumin Maintains osmotic pressure Negative acute phase reactant

Acute phase reactant

Alpha1-antitrypsin
Major inhibitor of protease activity 90% of alpha1-globulin band

Gestational marker
Tumor marker: hepatic and gonodal cancers
Screening test for fetal conditions (Spx: maternal serum) Amniotic fluid:
Alpha1-fetoprotein
confirmatory test
Increased: Hepatoma, spina bifida, neural tube defects
Decreased: Down Syndrome (Trisomy 21)
:
 Low pI (2.7)
Alpha1-acid glycoprotein/ orosomucoid
Negatively charged even in acid solution

Acute phase reactant Binds and inactivates PSA

Alpha1-antichymotrypsin
Increased: Alzheimer's disease, AMI, infection, malignancy, burns

Acute phase reactant

Haptoglobin (alpha2)
Binds free hemoglobin (alpha chain)

Copper binding (6-8 atoms of copper are attached to it) Has enzymatic
activities
Ceruloplasmin (alpha2)
Decreased: Wilson's disease (copper skin, liver, brain, cornea [Kayser-
Fleisher rings])

Larges major nonimmunoglobulin protein Increased: Nephrotic syndrome

Alpha2-macroglobulin (10x)
Forms a complex w/ PSA

Group-specific component (Gc)-globulin (bet. Affinity w/ vitamin D and actin

alpha1 and alpha2)

Hemopexin (beta) Binds free heme

HLA
Beta2-microglobulin
Filtered by glomeruli but reabsorbed

Negative acute phase reactant

Major component of beta2-globulin fraction Pseudoparaproteinemia in
Transferrin/Siderophilin (beta)
severe IDA
Increased: Hemochromatosis (bronze-skin), IDA

Complement (beta) C3: major

Acute phase reactant

Fibrinogen (bet. beta and gamma)
Between beta and gamma globulins

General scavenger molecule Undetectable in healthy individuals

CRP (gamma)
hsCRP: warning test to persons at risk of CAD

Immunoglobulins (gamma) Synthesized by the plasma cells IgG>IgA>IgM>IgD>IgE

Myoglobin Marker: Ischemic muscle cells, chest pain (angina), AMI

Troponins Most important marker for AMI

:
 Specific for heart muscle
TnT (Tropomyosin-binding subunit)
Det. unstable angina (angina at rest)

Only found in the myocardium Greater cardiac specificity than TnT Highly
specific for AMI
TnI (Inhibitory subunit or Actin-binding unit)
13x more abundant in the myocardium than CK-MB
Very sensitive indicator of even minor amount of cardiac necrosis

TnC Binds calcium ions and regulate muscle contractions

Glomerular proteinuria Most common and serious type Often called albuminuria

Defective reabsorption
Tubular proteinuria
Slightly increased albumin excretion

Hemoglobinuria Myoglobinuria
Overload proteinuria
Bence-Jones proteinuria

Postrenal proteinuria Urinary tract infection, bleeding, malignancy

Type 1 DM
Albumin excretion ≥30 mg/g creatinine (cutoff: DM) but ≤300 mg/g
Microalbuminuria creatinine Microalbuminuria: 2 out of 3 specimens submitted are w/ abnormal
findings
(w/in 6 months)

2 or more IgG bands in the gamma region: Multiple sclerosis

CSF Oligoclonal banding Encephalitis Neurosyphilis
Guillain-Barre syndrome Neoplastic disorders

Leukemia
Serum Oligoclonal banding
Lymphoma Viral infections

Alkaptonuria Ochronosis (tissue pigmentation)

Impaired activity of cystathione beta-synthetase

Homocystinuria Elevated homocysteine and methionine in blood and urine
Screen: Modified Guthrie test (Antagonist: L-methionine sulfoximine)

Markedly reduced or absence of alpha-ketoacid decarboxylase 4 mg/dL of

leucine is indicative of MSUD
MSUD
Screen: Modified Guthrie test (Antagonist: 4-azaleucine)
Diagnostic: Amino acid analysis (HPLC)

PKU Deficiency of tetrahydrobiopterin (BH4) elevated blood phenylalanine

:
 Total protein = 6.5-8.3 g/dL
Normal Values (Proteins)
Albumin = 3.5-5.0 g/dL Globulin = 2.3-3.5 g/dL

-Inulin clearance
-Creatinine clearance
Tests for GFR Clearance:
-Urea clearance Phenolsulfonphthalein dye test
Cystatin C

BUN
Tests for Renal Blood Flow
Creatinine Uric acid

Excretion:
-Para-amino hippurate test (Diodrast test)
Tests Measuring Tubular Function -Phenolsulfonphthalein dye test Concentration:
-Specific gravity
-Osmolality

Decreases by 1.0 mL/min/year after age 20-30 years 150 L of glomerular

GFR
filtrate is produced daily

Inulin clearance Reference method

Best alternative method

Creatinine clearance Measure of the completeness of a 24-hour urine collection Excretion: 1.2-1.5
g creatinine/day

Urea clearance Demonstrate progression of renal disease or response to therapy

Low MW protease inhibitor

Cystatin C FilteredNot secretedCompletely reabsorbed (PCT) Indirect estimates of GFR
Its presence in urine denotes damage to PCT

Synthesized from Ornithine or Kreb's Henseleit cycle First metabolite to

BUN elevate in kidney diseases
Better indicator of nitrogen intake and state of hydration

2.14 BUN Urea (mg/dL)

Fluoride or citrate Inhibit urease

Thiosemicarbazide Ferric ions Enhance color development (BUN mtd)

Diacetyl monoxime method Yellow diazine derivative

:
 Routinely used
Urease method urease: prepared from jack beans
Urea ---(Urease)--> NH4 + Berthelot reagent (Measure ammonia)
: