Jereza, Reyzeil Rose

Saberon, Daiden P.
SPECHE (Laboratory)
1. Total Cholesterol
a. Cholesterol Oxidase Method
i. Name: Cholesterol Oxidase Method
ii.Reagents/Equipments: 4 aminophenazone Cholesteroesterase, Phenol
Cholesterol oxidase, Peroxidase Sodium azide
iii. Sample: Fasting blood sample either plasma or serum
iv. Principle: Cholesterol esters in serum are hydrolysed by cholesterol esterase.
The Iree cholesterol is then oxidized by cholesterol oxidase to the
corresponding ketone liberating hydrogen peroxide, which is then
converted to water and oxygen by the
enzyme peroxidase. Para aminophenazone (4 aminophenazone) takes up
the oxygen and together with phenol Iorms a
pink coloured quinoneimine dye, which can be measured at 515nm/
yellow green Iilter
v. Chemical Reaction:




vi. InterIering Substances: Serum bilirubin and Ascorbic acid
b. Lipid ProIile
i. Name: Piccolo Lipid Panel Reagent Disc
iii. Sample: Whole blood or serum
iv. Principle: CE hydrolyzes cholesterol esters to Iorm cholesterol and Iatty acids.
The CHDH reaction converts cholesterol to cholest-4-en-3-one. The
NADH is measured bichromatically at 340 nm and 405 nm. NADH


production is directly proportional to the amount oI cholesterol present.
An assay-speciIic blank is also monitored to ensure no extraneous
reactions interIere with the calculations oI CHOL levels.
v. Chemical Reaction:
Cholesterol esters H2O -------- Cholesterol Fatty Acids
Cholesterol NAD -------------Cholest-4-en-3-one NADH H
vi. InterIering Substances:
2. Triglycerides
a. GPO Reagent Set
i. Name: GPO Reagent Set
ii.Reagents/Equipments: ATP, Magnesium Salt, TBHB, GPO, Lipase , GK 1,000
U/L, Peroxidase, BuIIer, SurIactant, Stabilizers, Fillers, with Sodium
Azide
iii. Sample: Serum, EDTA, or heparinized plasma
iv. Principle:Triglycerides in the sample are hydrolyzed by lipase to glycerol and
Iatty acids. The glycerol is then phosphorylated by adenosine-5-
triphosphate (ATP) to glycerol phosphate (G3P) and adenosine-5-
diphosphate in a reaction catalyzed by glycerol kinase (GK). Glycerol-3-
phosphate is then converted to dihydroxyacetone phosphate (DAP) and
hydrogen peroxide by glycerophosphate oxidase (GPO). The hydrogen
peroxide then reacts with 4-aminoantipyrine (4-AAP) and 3-hydroxy-
2,4,6-tribomobenzoic acid (TBHB) in a reaction catalyzed by peroxidase
to yield a red colored quinoneimine dye. The intensity oI the color
produced is directly proportional to the concentration oI Triglycerides in
the sample when measured at 540nm.

v. Chemical Reaction:
Triglycerides -------------------------/ Glycerol Fatty Acids
Glycerol ATP ------------------------/ G3P ADP
G3P O2 -------------------------------/ DAP H2O2
H2O2 TBHB ------------------------/ Quinoneimine Dye 2H2O


vi. InterIering Substances: Serum bilirubin and Ascorbic acid
b. Lipid ProIile
i. Name: Piccolo Lipid Panel Reagent Disc
ii. Reagents/Equipments: 4-Aminoantipyrine, Adenosine 5'-triphosphate, disodium
salt, Ascorbate oxidase , Cholesterol dehydrogenase, Cholesterol esterase
(Genzyme-N), Cholesterol esterase (Genzyme-P), Dextran sulIate,
Diaphorase, N-Ethyl-N-(2-hydroxy-3-sulIopropyl)-3-methylaniline,
sodium salt, dihydrate (TOOS), Glycerol kinase, Glycerol-3-phosphate
dehydrogenase, Iodonitrotetrazolium chloride (INT), Lipase, Magnesium
chloride, hexahydrate, Magnesium sulIate, heptahydrate, Nicotinamide
adenine dinucleotide, monosodium salt (NAD), PEG-cholesterol esterase,
PEG-cholesterol oxidase, Peroxidase
iii. Sample: Whole blood or serum
v. Chemical Reaction:
Triglycerides 3H2 ---------Glycerol 3Fatty Acids
Glycerol AT -------------G-3-P ADP
G-3-P NAD ------------ DAP NADH H
NADH H IN -------- NAD Formazan
G-3-P Glycerol-3-Phosphate
G-3-PDH Glycerol-3-Phosphate Dehydrogenase
DAP Dihydroxyacetone Phosphate
INT p-Iodonitrotetrazolium Violet

vi. InterIering Substances:
3. Bilirubin
a. Jendrassik & GroI method
i. Name: Jendrassik & GroI method
ii.Reagents/Equipments: CaIIeine-Benzoate, Suphanilic acid, Sodium Nitrite,
Diazo Reagent, Alkaline Tartrate, Ascorbic acid
iii.Sample: clear, non-haemolysed samples oI serum
iv.Principle:Conjugated (direct) bilirubin in serum is coupled
with diazotised sulphanilic acid to Iorm a red coloured compound.
Ascorbic acid is used to stop the coupling reaction, and to eliminate


interIerence by haemoglobin. CaIIeine benzoate solution is used to split
the unconjugated bilirubin protein complex releasing the bilirubin so that
it can react with diazotised sulphanilicacid.The tartrate buIIer makes the
mixture alkaline and converts the red acid bilirubin to a
green coloured compound which shows peak absorbance at 607 nm. At
this wavelength the absorbance due to haemoglobin or carotene is
minimal.
v. Chemical Reaction:
vi. InterIering Substances: strong haemolysis oI serum
b. Fouchet`s Test
i. Name: Fouchet`s Test (Harison Spot Test)
ii. Reagents/Equipments: Fouchet's reagent (Dissolve 25g oI trichloroacetic acid in
about 50ml oI distilled water, then add 1g Ierric chloride, mix to dissolve and
then make up to 100ml with distilled water.)

iii. Sample: Urine

iv. Principle: When Ierric chloride in acid solution is added to a precipitate (ReI
: Urobilinogen procedure) oI urine containing bilirubin, a greencolour is
produced as the bilirubin in the urine is oxidized to biliverdin.
v. Chemical Reaction:

vi. InterIering Substances:
4. ALT
a. ALT (SGPT) Reagent (Colorimetric, Endpoint Method)
i. Name: ALT (SGPT) Reagent (Colorimetric, Endpoint Method)
iv. Principle: The method used here is a modiIication oI the classical Reitman
Frankel colorimetric endpoint reaction. In this procedure ALT (SGPT)
catalyzes L-alanine and u-ketoglutarate to Iorm pyruvate and glutamate.
The pyruvate is then reacted with 2, 4-dinitrophemyl-hydrazine (2,4-
DNPH-ine) to Iorm 2,4-DNPH-one. The addition oI sodium hydroxide
dissolves this complex, allows 2, 4-DNPH-one to be measured at 505 nm.
v. Chemical Reaction:
L-Alanine u-ketoglutarat ---------Pyruvate Glutamate
pyruvate 2,4 - DNPH-in ---------Pyruvate 2, 4-DNPH-on
vi. InterIering Substances: Pyridoxal phosphate


b. ALT (GPT) Reagent
i. Name: ALT (GPT) Reagent
ii. Reagents/Equipments: L-alanine, 2-ketoglutarate, NADH, LDH (microbial),
buIIer, surIactant, preservative
iii. Sample: Serum
v. Chemical Reaction:
L-alanine 2-ketoglutarate --------L-glutamate pyruvate
pyruvate NADH ------------------L-lactate NA
vi. InterIering Substance: Triglyceride concentration oI 1000 mg/dL
5. Sodium
a. Sodium- Flame Photometry
i. Name: Sodium- Flame Photometry
ii.Reagents/Equipments: Stock Sodium 1000 mmol/L
iii.Sample: Serum
iv.Principle: When a solution oI an inorganic salt such as sodium chloride is
sprayed into the Ilame, the elements in the compound are partly converted into
the atomic state. Due to the heat energy oI the Ilame a very small proportion oI
these atoms is excited and the electrons move to a higher energy level. The
proportion oI the atoms that are excited depends upon the concentration oI the
particular element and on the temperature oI the Ilame. In the excited state the
electrons are unstable and they rapidly revert back to their Iormer lower energy
level. As they change Irom the excited state or higher energy level back to the
lower energy level, they emit the light in the Iorm oI a Iixed wavelength, to
produce a spectrum. Under careIully controlled conditions the amount oI light
emitted is directly proportional to the number oI atoms that are excited, which
in turn is proportional to the concentration oI the substance in the sample.
v. Chemical Reaction:
vi. InterIering Substances: haemolysed serum
B. ISE Method
i. Name: ISE Method
ii.Reagents/Equipments: Glass Ion-Exchange Membrane


iii.Sample: Lithium Heparin Plasma
iv.Principle: ISE method uses a semipermeable membrane to develop a potential
produced by having a diIIerent ion concentrations on either side oI the
membrane. In this type oI system, two electrodes are used. One electrode has a
constant potential, making it the reIerence electrode. The diIIerence between
the reIerence and measuring electrodes can be used to calculate the
'concentration oI the ion in the solution. However, it is the activity oI the ion,
not the concentration that is being measured.
v. Chemical Reaction:
vi. InterIering Substances: protein buildup on the membrane through continuous
use
5. Calcium
a. Calcium Test
i. Name: Calcium Test
ii.Reagents/Equipments:
iii.Sample: Plasma
iv.Principle: Calcium reacts with red Arsenazo III to produce a calcium-Arsenazo
III complex which has an intense inIrared-absorbing blue color under the
conditions oI the test. The chromophore produced has a molar absorbance which
is twice that produced by traditional Arsenazo III reagents.The absorbance oI
the blue calcium-Arsenazo III complex can be measured at either 600 nm or at
650-660 nm and is proportional to the calcium concentration.
v. Chemical Reaction:
Arsenazo III Ca
2
-~ Arsenazo III - Ca
2
complex
(red) (blue)
5. Potassium
a. Piccolo Electrolyte Reagent Disc
i. Name: Piccolo Electrolyte Reagent Disc
ii.Reagents/Equipments: Adenosine-5`-diphosphate, Amylase, Calcium acetate, 2-
Chloro-4-nitrophenyl alpha-maltotrioside (CNPG3), Citric acid,
trisodium salt, Ethylenediaminetetraacetic acid (EDTA), Ethylene glycol-
bis(-aminoethyl ether)-N,N,N`,N`-tetraacetic acid (EGTA), -
Galactosidase, 4,7,13,16,21,24-Hexaoxa-1,10
diazabicyclo|8.8.8|hexacosane (KryptoIix 222), Lactate dehydrogenase,


Magnesium acetate, Malate dehydrogenase (porcine heart), N-acetyl
cysteine, -Nicotinamide adenine dinucleotide, reduced (NADH), u-
Oxoglutarate, 4,7,13,16,21-Pentaoxa-1,10-diazabicyclo|8.8.5|tricosane
(KryptoIix 221), Phosphoenol pyruvate, Phosphoenol pyruvate
carboxylase, Pyruvate kinase,BuIIers, surIactants, excipients and
preservatives
iii.Sample: Heparinized whole blood, heparinized plasma, or serum
iv.Principle: In the coupled-enzyme reaction, pyruvate kinase (PK)
dephosphorylates phosphoenolpyruvate (PEP) to Iorm pyruvate. Lactate
dehydrogenase (LDH) catalyzes conversion oI pyruvate to lactate.
Concomitantly, NADH is oxidized to NAD

. v. Chemical Reaction:




vi. InterIering Substances: Sodium heparin