Introduction to Clinical Chemistry

A.Y. 2014-2015
MT-IA
1A-MT
Prepared by Group 3
De Guzman, Elisha Jhoanna
De Lara, Harissa Katrina R.
Deiparine, Jesmae Anril N.
Diaz, Jorina P.
Espina, Doxa Charisse L.
Fajardo, Ma. Pamela Rica M.

Introduction to CLINICAL CHEMISTRY

HISTORY:
> 19th Century Application of chemistry to medicine was more used on
understanding the diseases rather than its cure/relief.
>During the French Revolution Era, Fourcroy proposed that chemical laboratory
to be located near the wards.
>Justus Liebig, wrote the book of Clinical Chemistry, which was important in the
development of the clinical chemistry since it introduced quantitative method of
observation into physiological
chemistry.
- The use of the Duboscq
colorimeter for color comparison in
the analysis for creatinine in urine
pushed the modern
era of clinical chemistry.
WHAT IS CLINICAL
CHEMISTRY?
> Also known as clinical pathology,
clinical biochemistry, or
medical biochemistry.
> Roles in the Laboratory: The area
of clinical pathology concerned
with the analysis of bodily
fluids. It uses chemical processes to
measure levels of chemical
components in bodily fluids.
ROUTINE TESTS IN CLINICAL CHEMISTRY
Glucose
BUN [Blood Urea Nitrogen]
Creatinine
Lipids Profile Tests
Cardiac Markers
Liver Function Tests
1.) GLUCOSE
> Definition: A simple sugar (monosaccharide) by product of carbohydrates.
> Insulin
-Insulins role in ones body is to regulate the glucose present in one the
blood, by storing these glucose into muscles
or fats (glycogen) for use in obtaining energy (anabolic) .
- Glucagon (produced by the alpha cells) opposes the insulin by breaking
down glucose and releasing it to the
bloodstream (catabolic)
Metabolic Disorders caused by abnormally high or low blood glucose
*Hypoglycemia abnormally low blood glucose than the normal glucose level.
*Hyperglycemia abnormally high blood glucose than the normal glucose level.
27

* Diabetes chronic metabolic disease caused by perennially high blood glucose.

Types of Diabetes:
1. Type 1 Beta Cells (producers of insulin) are destroyed by the immune
system.
-Insulin Dependent, can be genetically acquired.
2. Type 2 The pancreas can still produce insulin, but it may not be
enough or the immune system blocks it.
-Non-Insulin Dependent"
3. Gestational Diabetes During pregnancy, the placenta secretes a
hormone that blocks insulin, if the pancreas
cant match the amount of
glucose, then it will cause Gestational Diabetes.
> Tests for Blood Glucose:
*All tests performed are usually done with venipuncture, with
blood as the specimen.
TEST
USE
SPECIMEN &
COLLECTION
- measures amount of
- Patient must not eat for
sugar in blood.
8-10 hrs before the test.
Fast Blood Sugar
Test
Random Blood
Sugar Test

Oral glucose
tolerance test

- It is used to measure the

bodys ability to use sugar
after drinking a standard
amount of glucose.
- It is used to check if a
pregnant woman acquired
Gestational Diabetes.

- blood testing done

anytime of the day.
- Steps:
1. Get the FBS as
baseline
2. Drink 75-100 g of
glucose
3. Blood samples will be
obtained again

VA

- Normal Value
<100mg/dL g
- hyperglycemia
100mg/dL- 1
- diabetes
126mg/dL =
- Normal value
<140mg/dL
Amount of
glucose intake
50g (1hr)
75g (2hrs)

100g (3hrs)

2-Hour PostPrandial Glucose

Blood Sugar Test
(2Hr PPBS)
A1cTest for HbA1c
- Glycosylated
Hemoglobin test

- It is used to check how

the body responds with
sugar and starch after
eating.

- Steps:
1. Get the FBS for
baseline
2. Get another sample
after 2 hrs after eating
- It is to measure average
- Test is usually done
level of blood sugar
with venipuncture with
(glucose) over 3 months to blood as the specimen or
monitor control with
using finger stick for

Age
Newborn-50yrs
50-60yrs
60yrs and
above
- Normal (no dia
<5.7%
- Pre-diabetes (h
5.7%-6.4%
28

diabetes.
tests at home.
- For this test we can also
use electrophoresis,
enzymatic assay (Diazyme
A1c), ion-exchange
chromatography or highperformance
chromatography.

- Diabetes
>6.5%

2.) BUN [Blood Urea Nitrogen]

> Urea/Urea Nitrogen
- is the chief component of the NPN (non-protein nitrogen) material in the
blood.
- A waste product of protein metabolism
> Liver site of urea formation.
> Normal Value of Urea:
Urea nitrogen, serum
Urea, serum
Urea Nitrogen, urine
Adult (7-18
mg/dL)

2.5 6.4
mmol
urea/L

Adult (5-39
mg/dL)

2.5 6.4
mmol/L

12-20g/24 hrs

0.428
-0.714
mmol
urea/24 hr

> Method:
1. Addition of enzyme, Urease, to the whole blood, serum, or plasma
during incubation
- Urease hydrolyzes urea in the sample and the ammonium ion produced in
the reaction is quantified.
2. Analysis of reaction through Classical and Indirect Method.
> Importance of Urea Tests:
a. helps us see the rough estimate of renal function
b. evaluates renal function
c. assesses hydration status
d. determine nitrogen balance
e. aid in diagnosis of renal disease
f. verify adequacy of dialysis
> Uremia abnormal or high urea nitrogen in the blood due to impaired
kidney function since urea is not removed from the blood and excreted in the
urine.
3.) CREATININE
> Creatinine in the blood results in the spontaneous formation of creatinine
from creatine (is synthesized primarily in the liver
and then transported to other tissue where it serves as high energy source
to drive metabolic reactions) and creatine phosphate.
Its formation and release into the body fluids occur at a constant rate and
have a direct relationship to muscle mass.
29

 Creatinine concentration varies with age and gender and is measured in

the blood and in the timed urine specimen
How is creatinine produced?
- Creatinine is made from creatine, serves as high energy source to drive
metabolic reactions.
- Creatinine Phosphate loses phosphoric acid and creatine loses to water to
form creatinine.
Specimen: Serum or heparinized plasma
> Methods:
1. Jaffe reaction
- The oldest clinical chemistry method in use.
- Creatinine reacts with alkaline picrate to form an orange-red solution
that is measured in spectrophotometer
- To improve the specifity of the reaction and to eliminate the
interference from the many non-creatinine in blood, acidification step is
added.
- Noncreatinine Jaffe-reacting choromogens include proteins,
glucose,ascorbic acid and pyruvate.
> Normal Values:

2.) Serum Creatinine

o blood test that is performed as a part of a physical examination if a
person has blood work done.
o Serum creatinine concentration is relatively constant and is
somewhat higher in males than in females.
Glomerular Filtration Rate
o measures of a kidney function.
o The quantity of glomerular filtrate per unit time in all nephrons of
both kidneys.
o Various substances in plasma and urine can be used to estimate the
GFR.
o Inulin has historically been the gold standard for measuring GFR.
> Clinical Significance:
- Creatinine in the blood results from creatinine originating in the
muscles of the body.
- Creatinine is freely filtered by the glomeruli of the kidney but it is not
reabsorbed under normal circumstances.
- The concentration of creatinine is not affected by dietary intake, degree
dehydration in the body or protein metabolism.
4.) LIPID PROFILE TEST
30

> Lipids - a class of biochemical compounds of fats, oils, hormones, and

certain components of membranes that are grouped
together because they do not interact appreciably with water.
> Lipid Profile Testing - a collection of tests carried out in order to assess
the levels of different types of cholesterols in the
blood stream. Its objective is to assess the risk of coronary heart disease
that these different types of cholesterol pose to
the patient under examination.
Preferred lipid testing specimen: A serum collected in a serum separator
evacuated tube from a patient who has been fasting for 12-15 hours. if analysis
must be delayed, the serum can be refrigerated at 4C for several days. Acute
illness, high fever, starvation or recent surgery lowers the blood cholesterol and
triglyceride levels. Patients should stop taking medications that may affect the
accuracy of the test and avoid alcohol intake for 24 hours.
Parameter

Cholesterol

Triglycerides

Method/Routine
Enzymatic Assay
- Most common method used to determine cholesterol
- The common lipid panel, including measurements of
total, LDL, HDL cholesterol, together with triglycerides
can be completed routinely using chemistry analyzers.
Enzymatic reagents are mild compared with the acid
reagent and better suited for automated chemistry
analyzers.
- The enzyme, cholesterol esterase, hydrolyzes
cholesteryl esters to free cholesterol. The free
cholesterol is reacted by the second enzyme, cholesterol
oxidase producing hydrogen peroxide, which oxidizes
various compounds to form a colored product which is
measured photometrically.
- The intensity of the resulting color, proportional to the
amount of cholesterol, can be measured by a
spectrophotometer.
-Measurement of serum triglycerides in conjunction with
cholesterol is useful in detecting certain genetic and
other types of metabolic disorders, as well as in
characterizing risk of CVDs. The triglyceride value is
also commonly used in the estimation of LDL
cholesterol by the Friedewald equation.
Routine:
-The appearance of plasma or serum can be observed
after a minimum of 12-hour fast.
-Plasma clear triglyceride level < 200 mg/dL
Plasma hazy and turbid triglyceride level >
300 mg/dL
Plasma opaque and milky (lipemic)
31

Ad

- Desirab
199mg/dL or

- Borderli
200-239mg

- Higher R
240mg/dL
greater

Adult Values:
- Desirable
149 mg/dL

- Borderline
150-199 mg

- Higher Risk
200-499 mg

MALE:
40-160 mg/

High-Density
Lipoprotein (HDL)
Cholesterol (HDL
parin po ito )

Low-Density
Lipoprotein (LDL)
Cholesterol

triglyceride level
> 600 mg/dL
-Triglyceride methods are usually enzymatic. In most
automated enzymatic methods, hydrolysis of the
triglyceride present in the sample is usually achieved by
lipase. The resulting glycerol is assayed by various
coupled-enzyme methods.
- The measurement of HDL cholesterol has assumed
progressively greater importance in the NCEP treatment
guidelines. In the earliest guidelines, HDL cholesterol
was measured as a risk factor but otherwise was not
considered in treatment decisions. Because the risk
associated with HDL cholesterol is expressed over a
relatively small concentration range, accuracy in the
measurement is especially important.
Routine:
-Chemical Precipitation
-involves a two-step procedure with manual pretreatment. A precipitation reagent added to serum or
plasma aggregated non-HDL lipoproteins, which were
sedimented by centrifugation, at forces of
approximately1500g (gravity) with lengthy
centrifugation times of 10to 30 minutes or higher forces
of 10,000 to 15,000g, decreasing centrifugation times to
3 minutes.
- LDL cholesterol, well validated as a treatable risk
factor for CHD, is the primary basis for treatment
decisions in the NCEP clinical guidelines.
Routine:
- Beta-quantification combines ultracentrifugation and
chemical precipitation. Ultracentrifugation of serum at
the native density of 1.006 g/L is used to float VLDL
and any chylomicrons for separation. The fractions are
recovered by pipetting.

5.) CARDIAC MARKERS

> Cardiac marker tests identify blood chemicals associated with myocardial
infarction (MI), commonly known as a heart
attack.
> Collection of specimen:
*Homocysteine tests require the patient to fast.
32

FEMALE:
35-135 mg/

- Desirable:
40mg/dL or
- Higher Risk:
39 mg/dL o

Adult Values (2

-Desirable if p
has CHD
99 mg/dL or
- Desirable
129mg/dL o
- Borderline
130-159mg/
- Higher Risk
160mg/dL o
greater

*Blood Collection after the onset of Myocardial Infarction:

1.) On admission
3.) 6-8 hours
2.) 2-4 hours
4.) 12 hours
*if earlier specimens are found negative but high MI
suspection collected again after 24 hours
> Current tests used for acute MI and myocardial injury are:
a.) Myoglobin
b.) Troponins
c.) CK isoenzyme (CK-MB)
TESTS

Myoglobin

Creatinine Clearance
USES
- Heme protein found in
striated skeletal and cardiac
muscles.
-released rapidly after
tissue injury and may be
elevated as early as one
hour after myocardial
injury
-Can be measured by:
>Latex agglutination
>Enzyme-linked
immunosorbent
assay (ELISA)

VALUES
NORMAL
CHANGES IN
VALUES
VALUE
- Adult men
-rise in
30-90
concentration:
mg/Dl
2-6 hours period
- Adult women after MI symptoms
< 50 mg/dL -return to normal:
1224 hours.

>Immunonephelometry
>Fluoroimmunoassay
>Spot test using
Troponins

immunochromatography
- gold standard of cardiac
markers
- is a complex of 3 proteins
that form the thin filaments
of muscle fibers and
regulate muscle
contraction:
1. Troponin T (TnT)
2. Troponin I (TnI);
3. Troponin C (TnC)

Troponin T:
< 0.1 ng/mL
- rise in
concentration:

-rise in
concentration:
2-4 hours and
peak at 1024
hours
after MI
- return to normal:
withing 5-14
days.
Troponin I:
- rise:
< 1.5 ng/mL
2-4 hours and
peak at 1024
hours
after MI
- return to normal:
33

within 5-10
days.

- Creatine kinase is an
enzyme responsible for
transferring a phosphate
group from ATP to creatine.
Creatine Kinase
But it is not cardiacMB
specific.
- but the MB isoenzyme is
cardiac-specific (CK-2);
excellent sensitivity after
MI onset
- an amino acid in the
blood
- Laboratory testing for
plasma homocysteine
levels can improve the
assessment of
cardiovascular disease risk.
Homocysteine - The only cardiac marker
test that requires
FASTING.
- High levels of
homocysteine are the result
of a lack of certain B
vitamins, inheritance, or
dietary excess and have
been implicated in
vascular-wall injury.

1013 units/L

- abnormal/rise:
4-6 hours and
peaks at 1024
hours after MI
- Returns to
normal:
within 72
hours.

*Normal Fasting Level: 515

micromol/L
- Hyperhomocysteinemia
Concentrations:
Moderate
- 16-30
micromol/L
Intermediate - 31-100
micromol/L
Severe
- < 100
micromol/L

6.) LIVER TESTING

Bilirubin
taken from the non-contaminating heme portion of hemoglobin, released
from the breakdown of red blood cells.
Specimen: serum or plasma
must be protected from light
must be use within 2-3 hrs
can be stored in a dark refrigerator for 1 week or in a freezer for 3 months
without significant loss of albumin.
> Methods:
-Diazo Reaction
first described by Ehrlich in 1883 with the use of urine.
used diazotized sulfanillic acid to produce azobilirubin dye,
color: red-purple
34

most commonly used in determining assays of serum

biliburin
modified by van der Bergh and Muller
to be used on serum specimens.
uses accelerators(solubilizer)

> Procedures:
- Malloy Evelyn Procedure
-diazotized sulfanilic acid reaction forms two molecules of
azobiliburin.
developed the first clinically useful methodology for the
quantitation of bilirubin.
uses 50% methanol solution as an accelerator.
-Jendrassik-Grof Method for Total and Conjugated Bilirubin
Determination
- diazo reagent and an accelerator is used to determine the total
bilirubin value. Thus, determining conjugated, unconjugated &
delta bilirubin.
- uses caffeine-benzoate-acetate as an accelerator.
- conjugated bilirubin - polar compound that is found in the
plasma
Population
Type of Biliburin
Reference Range
Adults

Conjugated bilirubin

0.00.2 mg/dL (03 mol/L)

Unconjugated bilirubin

0.20.8 mg/dL (314

mol/L)

Total bilirubin

0.21.0 mg/dL (317

mol/L)
- unconjugated bilirubin nonpolar compound that is found in the
plasma bound to the albumin.
- delta blirubin conjugated bilirubin found in albumin.
(significant in hepatic obstruction)
> Normal Values:
c. Proteins
> a large polypeptide (a chain of amino acids)
> make up antibodies (aka immunoglobulins, w/c are synthesized in
plasma cells) , a major component of our immune system.
>Different Classification of Protein Acc. to Function:
a) Enzymes - catalyzed chemical reactions
b) Hormones chemical messengers that control actions of specific
cells/organs.
35

c) Structural Proteins structure of cells and tissues in muscle,

tendons, and cone matrix
d) Storage proteins energy source
>Plasma Proteins (albumins and globulin) are more frequently analyzed
for protein.
in a blood panel, 4 measurements of determination is given total protein, Albumin, Globulin and Alubimin/Globulin Ratio
> Methods:
Biuret Method
o To determine Total protein in the body.

Hypoproteinemia total protein level less than the reference

interval. Indicative of renal disease and liver damage.
o Cupric ions piled with the groups involved in the peptide bond. In
an alkaline medium, & in presence of two peptide bonds, a violetcolored chelate is formed.
o The Biuret reagent also contains sodium-potassium tartrate which
complexes cupric ions to prevent their
precipitation in the
alkaline solution and potassium iodide which acts as an antioxidant
o measured photometrically, darker the solution, the higher the
concentration of protein
Dye-binding Method
o For determining Albumin (binds various substances in the blood)
- Indicative of Liver damage when Albumin level is low
(Hypoalbuminemia).
o based on the ability of most proteins in serum to bind to dyes such
as bromphenol blue.
o Coomassie brilliant blue 250 binding to protein causing a gift in
the absorbance maximum of the dye from 465-595 nm. the
increase in absorbance at 595 is used to determine protein
concentration.
o used to stain protein bands after electrophoresis *when
abnormality is found in total proteins or albumin;
separates proteins on the basis of their electric charge densities.
>Normal Values:
Serum = 6.5 8.3 g/dL
Albumin = 3.5 5.5 g/dL
d. Enzymes
>Methods:
measurement of catalytic activity
-Detects increase in product concentration, decrease in subtrate
concentration, decrease in coenzyme concentration, or increase in
the concentration of an altered coenzyme.
-Performed in zero-order kinetics

36