Review in Clinical Chemistry

 Lipid Profile (Cholesterol, Triglyceride, HDL,

LDL, VLDL)
PRINCIPLES OF MEASUREMENT
Fluids Typically Used for Clinical Chemistry Tests
Photometry – relies on measurement of light by a  Blood (Whole Blood in HBA1c, serum or plasma)
photodetector
 Urine
 CSF
Turbidimetry and Nephelometry – based on formation
 Amniotic Fluid
of insoluble particles that interfere with the passage of
 Saliva
lght through the solution
 Synovial Fluid
Example: HDL measurement – we make insoluble
solution first. More turbid=high concentration  Pleural Fluid
 Pericardial Fluid
Fluorescence – certain kinds of chemical structures are  Peritoneal Fluid
able to absorb light of one wavelength and emit light of
another wavelength ANALYTES

Potentiometry – based on measurement of an electrical GLUCOSE

potential between two electrodes. One of the electrodes
is affected by contact with ions in solution.
Example: electrolytes using Ion Selective Electrode
End-Point Reactions – suitable for chemical reactions
that are completed in a relatively short time and are
“stoichiometric”, meaning that they produce one product
of molecule of complex for each molecule of analyte.
Example: Glucose, Uric Acid, Cholesterol
 The standard clinical specimen in venous
plasma glucose
 Fasting glucose in Whole Blood is 15% lower
than in serum or plasma
 A major source of energy for many tissues –
regulated by hormones, such as insulin, cortisol
and glucagon
Why measured? – to test for diabetes; in critical care
setting to test metabolic state

Rate Reactions (Kinetic) – if the analyte is an enzyme,
a molecule which can catalyze the conversion of
unlimited numbers of reagent molecules to product, the
amount of product at the endpoint would not reflect the
amount of enzyme
ROUTINE LABORATORY TESTING
Panel of Tests
- When an individual test alone is not sufficient to
assess a medical condition, a combination of
several tests may be used
- The pattern of results from the combination of
tests may provide better insight into the status of
the patient than any single test result.
Example: FBS and OGTT (patient drinks 75g glucose
load for 5 – 10 minutes) in diagnosing DM
Increased – Diabetes, Cushing’s disease, stress
Decreased – insulin excess, starvation, adrenal
insufficiency
Reference Value: FPG 70-99 mg/dL
100-125 (impaired plasma glucose)
>126 (diabetes mellitus)
Conversion Factor: 0.0555 (mg/dL to mmol/L)
HBA1c GLYCATED HEMOGLOBIN
 Largest subfraction of normal hemoglobin A in
both diabetic and non-diabetic individuals
 Hb molecule with a glucose molecule covalently
bound
Why measured? – a reliable method in the monitoring of
long-term glucose control. Gives a good estimate of
glucose control over a 3-month period

TYPICAL PANELS OF TEST Reference Value: <5.7%

 Electrolyte Panel (Na, K, Cl, PCO2, HCO3,
anion gap)
 Hepatic Panel (Liver Profile) – Bilirubin test
 Comprehensive Metabolic Profile
 Basic Metabolic Panel (Na, K, Cl, CO2, Glucose,
Creatinine, BUN)
5.7-6.4% (prediabetic)
> 6.5% (DM)
TOTAL PROTEIN (serum/plasma)
 Measures the amount of proteins, primarily
albumin and globulins, found in serum or plasma
 Maintains circulatory system oncotic pressure
Why measured? – it is a screening test to see if protein
levels are at expected value
Increased – dehydration, infections, some cancers like
myelomas and lymphomas
Decreased – protein loss (from kidney or GI tract), liver
disease, malnutrition
Reference Value: 6.4-5.3 g/dL
Conversion factor: 10 (g/dL to g/L)
ALBUMIN
 Protein present in highest concentration in
plasma (1/2 the plasma protein mass)
 Major protein in blood made in the liver, it binds
and transports many substances
 Sensitive and highly prognostic marker in cases
of cystic fibrosis
Why measured? – albumin level is a general indicator of
health and nutritional status
Increased – dehydration, infection, malignancy
Decreased – starvation, burns, kidney disease, liver
disease
Reference Value: 3.5-5.0 g/dL
Conversion factor: 10 (g/dL to g/L)
GLOBULINS
 Group of proteins consists of a1, a2, beta, and
gamma fractions
Why measured? – calculated as total protein minus
albumin, globulins are the other major protein fractions
Increased – infections, multiple myeloma
Decreased – leukemias, immunosuppression
Reference Value: 2.3-3.5 g/dL
Conversion Factor: 10 (g/dL to g/L)
UREA NITROGEN (BUN)
 Waste product from protein breakdown formed
by the liver and excreted by the kidney
 It is the major end product of protein and amino
acid catabolism – 45% of the total NPN
 The concentration of urea is expressed only the
the nitrogen content of urea
 To obtain the concentration of urea from BUN:
2.14 (f) x BUN = urea (mg%)
Why measured? – often ordered with creatinine to
evaluate kidney function; also used to monitor dialysis
patients
Increased – kidney dysfunction, stress, high protein diets
Decreased – low protein diets, liver disease
Reference Value: 8-23 mg/dL
Conversion factor: 0.357
Urea to BUN: Urea/2.14
CREATININE
 A waste product from the muscle breakdown of
a compound called creatine, which is excreted
into urine by kidneys
 Produced by three amino acids such as
METHIONINE, ARGININE, LYSINE
 A measure of completeness of 24-hour urine
collection
 Used to evaluate the fetal kidney maturity
Why measured? – to evaluate kidney function and
monitor therapy for kidney disease; creatinine may be
measured I blood, urine or both to evaluate kidney
function
Increased – kidney dysfunction (drug toxicity, poorly
controlled diabetes, inadequate blood flow – shock or
congestive heart failure)
Decreased – decreased muscle mass, advanced and
severe liver disease
Reference Value: Male 0.9-1.3 mg/dL
Female 0.6-1.1 mg/dL
Conversion Factor: 88.4 (mg/dL to mmol/L)
Elevated plasma creatinine concentration is associated
with abnormal renal function, especially as it relates to
glomerular function
CREATININE CLEARANCE
 Provides an estimate of an amount of plasma
that must flowed through the kidney
glomeruli/minute. Major limitation: accurate urine
collection
 Clearance (ml/min) = UV(ml/minute)/P x 1.73/A
Reference Value: Male = 85 – 125 ml/min
Female = 75 – 112 ml/min
Conversion Factor: 0.0167 (mL/min to mL/s)
URIC ACID
 A waste product from breakdown of purines
(DNA components) that is excreted by the
kidneys; too much uric acid can lead to deposits
or urate crystals in joints (gout) or in kidneys
(stones)
 Freely filtered, partially reabsorbed and secreted
in the renal tubules
 About 1 gram of uric acid is excreted normally
Why measured? – to evaluate joint inflammation that
may be due to gout; often ordered to monitor uric acid
production in patients undergoing chemotherapy or
radiation treatments
Increased – gout, kidney disease, leukemia
Decreased – Fanconi’s syndrome, Wilson’s disease,
Hodgkin’s disease
Reference Value: Male 3.5 – 7.2 mg/dL; Female 2.6 –
6/0 mg/dL
Conversion Factor: 0.0595
AMMONIA
 A waste product of amino acid breakdown
converted to urea by the liver; increased
ammonia levels an cause mental and neurologic
changes in the brain
Why measured? – to evaluate disorientation, confusion
and coma in adults; to evaluate irritability, lethargy and
seizures in newborns
Increased – severe liver disease, cirrhosis, severe
hepatitis, Reye’s syndrome, inherited genetic
deficiencies
Reference Value – 40 – 80 µg/dL
Conversion Factor: 0.587
CHOLESTEROL
 Found on the surface of lipid layers; synthesized
in the liver
 Its transport and excretion is promoted by
estrogen
 Precursors of five major classes of steroids:
progestins, glucocorticoids, mineralocorticoids,
androgens and estrogens
 Cholesterol ester (70%) and Free Cholesterol
(30%)
Why measured? – high cholesterol has been implicated
as a risj factor for coronary artery disease. Evaluates the
risk for atherosclerosis, myocardial and coronary arterial
occlusions
Increased – Hypothyroidism, uncontrolled diabetes,
kidney disease
Decreased – Liver diseases, starvation, anemia
Reference Value: <200 mg/dL (desirable)
200-239 (borderline high)
>240 (high cholesterol)
Conversion Factor: 0.0260
TRIGLYCERIDES (fasting required – 10 - 14hrs)
 Chemical form of fatty acids for transport and
storage in adipose tissue – constitutes 95% of
stored fat and the predominant form of glyceryl
ester found in plasma
 The breakdown of triglycerides are facilitated by
lipoprotein lipase, epinephrine, and cortisol
 People with low calorie intake have relatively low
triglyceride levels
Why measured? – part of the cardiovascular risk profile.
It evaluates suspected atherosclerosis and measured
the body’s ability to metabolize fat
Increased – hypothyroidism, alcoholism, liver disease,
uncontrolled diabetes
Decreased – malabsorption syndrome, hyperthyroidism,
malnutrition, brain infarction
Reference Value: <150 mg/dL (normal)
150-199 (borderline high)
200 – 499 (high triglycerides)
>500 (very high triglycerides)
Conversion Factor: 0.0113
HIGH DENSITY LIPOPROTEIN/ALPHA LPP
 HDL removes excess cholesterol rom tissue for
disposal; elevated HDL has been found to
protect against coronary artery disease
 Transports excess cholesterol from the tissues
and return it to the liver (reverse cholesterol
transport)
Why measure? Part of the cardiovascular risk profile
Increased – estrogen therapy, alcohol consumption
Decreased – smoking
Reference Value: Male >37 mg/dL
Female >40 mg/dL
Conversion Factor: 0.0260
LOW DENSITY LIPOPROTEIN/BETA LPP
 Carries cholesterol from the liver to peripheral
tissue; contributes to formation of plaques that
clog arteries and lead to coronary heart disease
 Synthesized in the liver; major end product from
the catabolism of VLDL
 Most atherogenic LPP; target of therapy
Why measured? – part of the cardiovascular risk profile
Increased – high saturated fat diets, inherited disorders
of cholesterol metabolism
Decreased – high fiber intake, drug treatment
Reference Value: <100 mg/dL
Conversion Factor: 0.0260
VERY LOW DENSITY LIPOPROTEIN/PRE-BETA LPP
 Triglyceride-rich lipoprotein that is secreted by
the liver and is the precursor to LDL
 Transports endogenous triglycerides from the
liver to muscle, fat depots, and peripheral
tissues
 Prolonged consumption of high fat diets –
elevated triglycerides in the VLDL particles
Why measured? – part of the cardiovascular risk profile
Increased – high saturated fat diets, inherited disorders
of cholesterol metabolism
Decreased – high fiber intake, drug treatment
Reference Value:<30 mg/dL(same conversion factor w/
LPPs)
Formula for LDL and VLDL
LDL = Total cholesterol – HDL – VLDL
Friedewald Method
 VLDL (mmol/L) = plasma TAG/2.175
 VLDL (mg/dL) = Plasma TAG/5.0
De Long Method:
 VLDL (mmol/L) = Plasma TAG/2.825
 VLDL (mg/dL) = Plasma TAG/6.5
TOTAL BILIRUBIN
 This breakdown product of hemoglobin is
excreted by the liver into the bile – it circulated in
blood in two forms referred to as conjugated and
unconjugated, reflecting whether the carboxyl
groups are free (unconjugated) or esterified
(conjugated)
Why measured? – to assess liver function
Increased – Hepatitis, cirrhosis, hemolytic diseases and
obstruction of biliary or hepatic ducts
DIRECT BILIRUBIN (CONJUGATED BILIRUBIN)
 Water-soluble, made in the liver and excreted in
the blood, it reacts directly with diazo dye, so it is
called direct reacting
Why measured? – to test the liver’s ability to conjugate
bilirubin and excrete it
Increased – Obstruction of biliary of hepatic ducts, and in
hereditary conditions like Dubin-Johnson syndrome
(bilirubin excretion deficit)
INDIRECT BILIRUBIN (UNCONJUGATED BILIRUBIN)
 Fat-soluble and the product of hemoglobin
breakdown; it reacts with diazo dyes only in the
presence of activators so it is called indirect
reacting
Why measured? – calculated as Total Bilirubin – Direct
Bilirubin; it reflects the difference between the total and
direct forms
Increased – Hereditary conditions like Gilbert’s disease
(bilirubin transport deficit; impaired cellular uptake of
bilirubin; elevated B1) and Crigler-Najjar syndrome (type
1 – deficiency of enzyme glucoroneotransferase, total
absence of B2 production; type 2 – partial deficiency of
the enzyme)
 Van den Berg reaction is diazotization of bilirubin
to produce azobilirubin
Reference Value:
 Total Bilirubin = 0.2 – 1.0 mg/dL
 Conjugated Biliruin = 0.0 – 0.2 mg/dL
 Unconjugated Bilirubin = 0.2 – 0.8 mg/dL
Conversion Factor: 17.1 (mg/dL to µmol/L)
AUTOMATION
 Increase the number of tests to be performed in
a given period
 Minimizes variation of result from one
laboratorian to another
 Eliminated the potential error in manual analyses
such as pipetting, calculation and transcription of
results
o Continuous Flow Analyzer
o Centrifugal Analyzer
o Discrete Analyzer
Definition of terms:
 Batch Testing – all samples are loaded at the
same time, and a single test is conducted on
each sample
 Parallel Testing – more than one test is
analyzed concurrently on a given clinical
specimen
 Random access testing – any test can be
performed on any sample, in any sequence
 Sequential testing – multiple test analyzed one
after another on a given specimen
 Open reagent system – other than
manufacturer’s reagent
 Closed reagent system – only the
manufacturer’s reagent
 Pneumatic tube delivery system – point-to-
point delivery of specimens
CONTINUOUS FLOW ANALYZER
 Liquids are pumped through a system of
continuous tubing
 Sample flow through a common reaction vessel
or pathway
  Disadvantage: all tests are performed in parallel
CENTRIFUGAL FORCE ANALYZER
 It uses force generated by centrifugation to
transfer specimen and reagents
 Liquids are placed in separate cuvettes for
measurement at the perimeter of a spinning
rotor (1000 rom)
 Major advantage: Batch analysis (discrete-batch
type system)
and assay processing, giving you accurate,
efficient result reporting
 Method: Colorimetric/Rate, Potentiometric (direct
ISEs), Immuno-rate, Turbidimetric, Enhanced
Chemiluminiscence
 Sample Types: Serum, Plasma, Whole Blood,
Urine, CSF, Amniotic Fluid
 Sample Volume: 2 – 80 uL (per assay)
 Dead Volume: minimum of 35 uL

DISCRETE ANALYZER
 Most popular and versatile analyzer – measures
only the tests requested on a sample
 Requires 2-6 uL of the sample (minimum
volume)
 Major Advantage: Random access capability –
allows STAT samples to be easily tested

ABBOTT DIAGNOSTICS ARCHITECT c4000

 Demonstrates high-quality testing results and
rapid STAT turnaround time. The ARCHITECT
c4000 enhances laboratory productivity and
provides users high confidence in clinical results.
 Offers a maximum throughput of up to 800 tests
per hour. Featuring a load-up capacity of 100
samples with 35 priority positions, the
ARCHITECT c4000 allows for up to 90
refrigerated reagent positions on board, plus
Integrated Chip Technology (Na+, K+ and Cl-).
 Wet chemistry analyzer
 Methods: Photometric, Potentiometric,
Turbidemetric
 Sample Types: Serum, Plasma, Whole Blood,
Urine, CSF
 Sample Cup: 50 uL (dead volume)
 Sample Volume: 1.5 – 35 u: (average of 6 uL)

VITROS 5600 INTEGRATED SYSTEM (dry chemistry

analyzer)
 VITROS Microslide – design filters out protein
and lipids when appropriate, minimizing sample
interferences
 VITROS Microtip – eliminated carry-over and
contamination risk of water-based systems
 VITROS Microwell – delivers precise results,
with fewer unnecessary dilutions, repeats and
redraws
 VITROS Microsensor – detects endogenous
interferences without the need for extra sample
or reagent – and with no effect on turnaround
time
 VITROS Intellicheck – monitors, verifies, and
documents diagnostic checks throughout sample
 ANALYTE REFERENCE RANGE UNIT CONVERSION FACTOR
70 – 99 (normal)
GLUCOSE 100 – 125 (prediabetic) mg/dL 0.0555 (to mmol/L)
>126 (DM)
<5.7 (normal)
HBA1c 5.7 – 6.4 (prediabetic) % N/A
>6.5 (DM)
TOTAL PROTEIN 6.4 – 8.3 g/dL 10 (to g/L)

ALBUMIN 3.5 – 5.0 g/dL 10 (to g/L)

GLOBULIN 2.3 – 3.5 g/dL 10 (to g/L)

BUN to Urea (mg%) = BUN x 2.14
UREA NITROGEN (BUN) 8 - 23 mg/dL UREA to BUN = Urea/2.14
BUN CU to SI unit = 0.357
Male: 0.9 – 1.3
CREATININE mg/dL 88.4
Female: 0.6 – 1.1
Male: 85 – 125
CREATININE CLEARANCE mL/min 0.0167 (to mL/s)
Female: 75 - 112
Male: 3.5 – 7.2
URIC ACID mg/dL 0.0595
Female: 2.6 – 6.0
AMMONIA 40 – 80 µg/dL 0.587
<200 (desirable)
CHOLESTEROL 200 – 239 (borderline high) mg/dL 0.0260
>240 (high cholesterol)
<150 (normal)
150 – 199 (borderline high)
TRIGLYCERIDES mg/dL 0.0113
200 – 499 (high TG)
>500 (very high TG)
Male: >37
HDL mg/dL 0.0260
Female: >40
LDL <100 mg/dL 0.0260

VLDL <30 mg/dL 0.0260

TOTAL BILIRUBIN 0.2 – 1.0 mg/dL 17.1
CONJUGATED (DIRECT) BILIRUBIN 0.0 – 0.2 mg/dL 17.1
UNCONJUGATED (INDIRECT) BILIRUBIN 0.2 – 0.8 mg/dL 17.1