ENZYMOLOGY DETERMINATION 1. For each sample add 1.

0 mL CK – MB working
reagent into a cuvette / test tube and warm for
UNIT OUTCOMES approximately 5 minutes at 37˚C.
• To achieve this unit a learner must: 2. Add 50µL of sample to its respective tube, mix
• Define concepts and principles of various
well and incubate for 5 minutes at 37˚C
Instruments used in the performance of liver
3. Set the wavelength of the instrument at 340
function test
• Explain the principles of liver function test nm. Zero the instrument with distilled water.
• Demonstrate how to operate the basic instruments 4. After 5 minutes has elapsed, read and record
and equipment’s in the clinical laboratory. the absorbance
• Develop expertise in the different laboratory works 5. Record the increase in absorbance at 60
that deals mainly with clinical preparation including seconds intervals for the next 2 minutes
collection and preparation of aspartate (ΔA/minute). The rate of change should be
aminotransferase specimen. constant

CALCULATION:
CREATINE KINASE – MB DETERMINATION
In this procedure CK activity is measured in the
Pre-Analytical Phase presence of an antibody to CK – M monomer. This
antibody completely inhibits the activity of CK – MM
Materials:
and half of the activity of CK – MB, while not affecting
• Spectrophotometer the B subunit activity of CK – MB and CK – BB. Due to
• Accurate pipetting devices negligible concentrations of CK – BB in the circulation,
• Cuvets the remaining activity, multiplied by a factor of 2,
• Centrifuge represents the activity of the CK – MB isoenzyme.
• Interval Timer
CK-B Activity: Values are derived based on the
• Test Tubes
absorptivity micromolar extinction coefficient of NADP
• Water Bath
at 340 nm (0.00622). A unit per liter (U/L) of CK-B
CK-MB Reagent (powder): activity is that amount of enzyme that oxidizes one
µmol/L of NADP per minute.
 ADP
 G6PDH
 Creatinine phosphate
 NAD
 Hexokinase (yeast)
 D-glucose
 Anti-human CK-M antibody (mouse)

Reagent preparation and stability:

 Reconstitute working reagent with distilled water as

indicated on vial label. Invert gently and swirl to
dissolve completely.

Specimen collection:

 Clear un-hemolyzed serum is the recommended

specimen sample. No special additives or
preservatives are required REFERENCE VALUE:

Analytical Phase:  0 – 24 U/L (37˚C)

 0 – 14 U/L (30˚C)
Procedure:  % CK- MB < 5.5%
 ALKALINE PHOSPHATASE DETERMINATION CALCULATION:

Pre-Analytical Phase Values are derived based on the absorptivity micromolar

extinction coefficient of 4-nitrophenol at 405 nm
Materials:
(0.01845). A unit per liter (U/L) of ALP activity is that
• Spectrophotometer amount of enzyme which produces one mmol/L of 4-
• Accurate pipetting devices nitrophenol per minute.
• Cuvets
• Centrifuge
• Interval Timer
• Test Tubes
• Water Bath

Reagent 1

 Diethanolamine buffer, pH 10.2

 Magnesium Chloride REFERENCE VALUE:
Reagent 2 Temperatur 25˚C (U/L) 30˚C (U/L) 37˚C (U/L)
e
• p-nitrophenylphosphate

Reagent preparation and stability: Children: 110 – 720 145 – 950 180 – 2000

 Dissolve the reagent 2 in the suitable volume of Adults: 60 – 170 80 – 220 100 – 290
reagent 1

Stability:
BY STANBIO (LAB PROCEDURE INSTRACTION)
• 24 hours at 20 - 25˚C
• 7 days at 2 - 8˚C SUMMARY
Specimen collection: • In diagnosis of hepatobiliary disorders and bone
disease associated with osteoblastic activity.
• Serum
• Serum ALP activity can be measured using
• Heparinized plasma
various phosphate esters as substrates.
Analytical Phase: • 4-nitrophenyl phosphate as substrate
• 4-nitrophenol (yellow in color at pH 10.4)
Procedure:
REAGENT:
1. .For each sample add 1.0 mL ALP working
reagent into a cuvette / test tube and warm for Alkaline Phosphate Buffer (R1)
approximately 3 minutes at 37˚C.
• 2-amino-2-methyl-1-propanol, pH 10.4 --- 0.35
2. Add 20µL of sample to its respective tube, mix
mmol/L
well gently.
• Magnesium Chloride --- 2.0 mmol/L
3. Set the wavelength of the instrument at 405
• Zinc Sulfine ---- 1.0 mmol/L
nm. Zero the instrument with distilled water.
• HEDTA ---- 2.0 mmol/L
4. Read and record the absorbance at 1 minute.
Continue incubating at 37 degrees Celsius and Alkaline Phosphate Substrate (R2)
record absorbance again at 2 and 3 minutes.
Rate should be constant. • 4-Nitrophenyl Phosphate ---- 16 mmol/L
5. Determine the average absorption per minute REAGENT PREPARATION:
(ΔA/minute). Multiply by the factor 2764 in U/L.
Prepare working reagent in the ratio of 5 parts buffer to
1 part substrate (25ml buffer to 5 ml substrate)
MANUAL PROCEDURE: Analytical Phase:

1. Prepare Alkaline Phosphate working reagent A. TOTAL ACP:

2. 405 nm spectro, zero with distilled water 1. Label tubes as “control” and “patient”
3. For each sample and control, add 1.0 ml working 2. Pipet 1.0mL of reagent in all tubes
reagent to cuvet and warm to 37C for 3 minutes 3. Zero spectrophotometer with water at 405 nm. Set
4. Add 20 uL (o.020 ml) serum to its respective tube cuvette temperature at 37˚C
and mix 4. Add 100µL (0.10mL) of sample to respective tube
5. Read and record for 1 mins. Continue incubating at and allow incubating at 37˚C for 5 minutes.
37C and record absorbance again at 2 and 3 5. Ater incubation, read and record absorbance every
minutes. Rate should be constant. minute for 5 minutes to determine (ΔA/minute)
6. Determine the average absorbance per minute, 6. Repeat procedure for each sample
multiply by factor 2764 for result in U/L. 7. Values U/L are obtained by multiplying (ΔA/minute)
by the factor
RANGE:

Normal Range (Adult): 34-114 U/L (37C) B. NON-PROSTATIC ACP

1. Add 1.0 mL of reagent to appropriately labelled
tubes.
ACID PHOSPHATASE DETERMINATION 2. Add 10 µl (0.01 mL) of L-Tartrate Reagent and mix.
3. Zero spectrophotometer with water at 405 nm. Set
Pre-Analytical Phase cuvette temperature at 37˚C
Materials: 4. Add 100µL (0.10mL) of sample to respective tube
and allow incubating at 37˚C for 5 minutes.
• Spectrophotometer 5. After incubation, read and record absorbance every
• Accurate pipetting devices minute for 5 minutes to determine (ΔA/minute)
• Cuvets 6. Repeat procedure for each sample
• Centrifuge 7. Values U/L are obtained by multiplying (ΔA/minute)
• Interval Timer by the factor
• Test Tubes
• Water Bath C. PROSTATIC ACP
• The value is obtained by subtracting the result
ACP Reagent
of the non prostatic acid phosphatase assay (B)
• L-tartrate reagent from the total acid phosphatase assay (A)
• Acetate Buffer: 5M, pH 5.0
CALCULATION:
Reagent preparation and stability:

• Reconstitute acid phosphatase reagent with

distilled water stated on the level. Swirl to
dissolve
• Reconstitute L-Tartrate Reagent with 5.0 mL
distilled water. Warm reagent to aid dissolution,
if necessary
• Acetate buffer is ready to use.

Specimen collection:

• Use only clear, unhemolyzed serum

REFERENCE VALUE:
• Serum must be separated from clot within two
hours after collection. • Total Acid Phosphatase: 0-9 U/L
• Don’t use plasma • Prostatic Acid Phosphatase: 0-3 U/L
 • Preservatives (Sodium Azide)
• Distilled Water
PROFAME DIAGNOSTIC REAGENT:
Analytical Phase:
Test and Quality control assurance procedure:
MANUAL PROCEDURE:
1. Place 50 uL of unhemolyzed test sample in the
bottom of separate test tube • Wavelength: 340nm
2. 50 uL od p-Nitrophenol standard in separate • Temperature: 37 degree Celsius
tube
REAGENT PREPARATION AND STABILITY
3. 250 uL od acid phosphate substrate into test
sample ONLY • Working Reagent - Prepare working reagent in
4. Mix and stand for 10 mins at room temperature the ratio of 5 parts Buffer (R1) to 1 part Enzyme
or 5 mins at 37 C (R2) (i.e., 25 mL Buffer and 5 mL Enzyme)
5. Add 1500 uL of acid phosphate diluent to each • Reagent Storage: 2-8°C. Protected from light.
tube • Reagent Stability: Stable until the expiration
6. Mix and read at 450 nm against water blank date.
• Working Reagent Storage and Stability: 4 weeks
CALCULATE:
at 2-8°Celsius. 5 days at room temperature (20-
|of|sample 22°C)
Value of Unknown= x Valueof Standard(15.6UL)
|of|Standard
MANUAL PROCEDURE
NORMAL RANGE:
1. Prepare AST Working Reagent according to
• Male: 4.7 – 13.6 U/L instructions.
• Female: 5.0 – 11.0 U/L 2. For each sample, add 1.0 mL Working Reagent
to test tube and warm to 37°C at 3 mins
3. Add 100 uL serum to its respective tube and mix
ASPARTATE AMINOTRANSFERASE DETERMINATION gently. Incubate for 1 minute at 37°C.
4. Zero spectrophotometer at 340 nm with
Pre-Analytical Phase Distilled water.
Materials: 5. Read and record decrease in absorbance at 1
minute. Continue incubating at37°C and record
• Spectrophotometer absorbance again at 2 and 3 minutes to get the
• Accurate pipetting devices change in absorbance per minute (ΔA/minute).
• Cuvets Rate should be constant.
• Centrifuge
• Interval Timer CALCULATION:
• Test Tubes • Values are derived based on the “Absorptivity
AST Reagent (Buffer) – R1 micromolar extinction coefficient” of NADH at 340
nm (0.00622).
• L-Aspartate • Units per liter (U/L) of AST/GOT activity is that
• MDH amount of enzyme which oxidizes one umol/L NADH
• Tris Buffer, pH 7.5 per minute.
• Stabilizers
• Preservatives (Sodium Azide)

AST Reagent (Enzyme) – R2

• 2-Oxoglutarate
• NADH
• Stabilizers
 1:10 sample to reagent ratio

Same as in the ppt.

1:20 sample to reagent ratio:

U/L = (abs/mns / 0.00622) x (1.05/0.05)

U/L = abs/mns x 3376

REFERENCE VALUE:

Normal Value:
ALANINE AMINOTRANSFERASE DETERMINATION
• 0-40 U/L (37°C)
• 0-28 U/L (30°C) Pre-Analytical Phase

The range should serve only as a guideline. It is Materials:

recommended that each laboratory establish its own
• Spectrophotometer
range of expected values, since differences exist
• Accurate pipetting devices
between instruments, laboratories and local populations
• Cuvets
BY STANBIO (LAB PROCEDURE INSTRACTION) • Centrifuge
• Interval Timer
SUMMARY
• Test Tubes
• One of the several enzyme that catalyze the
ALT Reagent (Buffer) – R1
exchange of amino acid and oxo groups between
alpha-oxo acids.  L-Alanine
• AST catalyzes the transfer of the amino group  LDH
aspartate to 2-oxoglutarate to yield oxalacetate  Tris Buffer, pH 7.5
and glutamate.  Stabilizers
 Preservatives (Sodium Azide)
REAGENT:
ALT Reagent (Enzyme) – R2
AST Buffer (R1)
• Alpha-ketoglutarate
• L-Aspartate – 240 mmol/L
• NADH
• MDH – 600 U/L
• Stabilizers
• LDH – 600 U/L
• Preservatives (Sodium Azide)
• Tris Buffer, pH 7.5 – 80 mmol/L
• Distilled Water
AST Enzyme (R2)
SPECIMEN COLLECTION
• 2-Oxaglutarate – 12 mmol/L
• Serum is the specimen of choice. As concentration
• NADH (Disodium salt) – 0.18 mmol/L
of ALT in red cells is roughly 5 times that of serum,
REAGENT PREPARATION: hemolysis must be avoided.

Prepare working reagent in the ratio of 5 parts buffer to Analytical Phase:

1 part substrate (25ml buffer to 5 ml substrate)
MANUAL PROCEDURE:
MANUAL PROCEDURE
• Wavelength: 340nm
1. Prepare AST Working Reagent according to • Temperature: 37 degree Celsius
instructions.
REAGENT PREPARATION AND STABILITY
2. Determine the average absorbance per minute,
multiply by factor 1768 (1:10) or 3376 (1:20).

CALCULATION:
Working Reagent - Prepare working reagent in the ratio between instruments, laboratories and local
of 5 parts Buffer (R1) to 1 part Enzyme (R2) (i.e., 25 mL populations.
Buffer and 5 mL Enzyme)
BY STANBIO (LAB PROCEDURE INSTRACTION)
Reagent Storage: 2-8°C. Protected from light.
SUMMARY
Reagent Stability: Stable until the expiration date.
• The enzyme alanine aminotransferase is widely
Working Reagent Storage and Stability: 4 weeks at 2- reported in a variety of tissue source.
8°Celsius. 5 days at room temperature (20-22°C).
REAGENT:
MANUAL PROCEDURE:
ALT Reagent (Buffer) – R1
1. Prepare ALT Working Reagent according to
 L-Alanine – 500 mmol/L
instructions.
 LDH – 1200 U/L
2. For each sample, add 1.0 mL Working Reagent to
test tube and warm to 37°C.  Tris Buffer, pH 7.5 – 100 mmol/L
3. Add 100 uL serum to its respective tube and mix ALT Reagent (Enzyme) – R2
gently. Incubate for 1 minute at 37°C
4. Zero spectrophotometer at 340 nm with Distilled  2-oxaglutanate – 15 mmol/L
water.  NADH (discount salt) – 0.18 mmol/L
5. Read and record decrease in absorbance at 1 REAGENT PREPARATION:
minute. Continue incubating at37°C and record
absorbance again at 2 and 3 minutes to get the Prepare working reagent in the ratio of 5 parts buffer to
change in absorbance per minute (ΔA/minute). Rate 1 part substrate (25ml buffer to 5 ml substrate)
should be constant
MANUAL PROCEDURE
CALCULATION:
3. Prepare AST Working Reagent according to
Values are derived based on the “Absorptivity instructions.
micromolar extinction coefficient” of NADH at 340 nm 4. Determine the average absorbance per minute,
(0.00622). multiply by factor 1768 (1:10) or 3376 (1:20).

Units per liter (U/L) of ALT/GPT activity is that amount of CALCULATION:

enzyme which oxidizes one umol/L NADH per minute.
1:10 sample to reagent ratio

Same as in the ppt.

1:20 sample to reagent ratio:

U/L = (abs/mns / 0.00622) x (1.05/0.05)

U/L = abs/mns x 3376

REFERENCE VALUE:

Normal Value: AMYLASE DETERMINATION

• 0-38 U/L (37°C) Pre-Analytical Phase

• 0-26 U/L (30°C)
METHOD: Quantitative
Newborns: 12.0 U/L
PRINCIPLE:
The range should serve only as a guideline. It is
 Amylase hydrolyzes p-Nitrophenyl-D-
recommended that each laboratory establish its own
maltohepatoside(PNPG7) to p-Nitrophenyl-
range of expected values, since differences exist
maltostriose (PNPG3) and maltotetraose.
 Glucoamylase hydrolyzes PNPG3 to p- COMPUTATION:
Nitrophenylglycoside (PNPG1) and glucose.
Values are determined based on the millimolar
 Then is hydrolyzed by glucosidase to glucose and p-
absorptivity of p-Nitrophenol which is 8.5 at 405 nm
Nitrophenol, which produce a yellow color, the rate
under the test conditions described.
of increase in absorbance is measured at 405 nm is
proportional to the amylase activity in the sample.

MATERIALS:

 Spectrophotometer
 Accurate pipetting devices
 Cuvets
 Interval timer
 Test tubes REFERENCE:
REAGENTS:  Serum: 25 – 125 U/L
 Amylase Reagent (powder)  Urine: 1 – 17 U/Hour
 Composition when reconstituted: PROFAME DIAGNOSTIC REAGENT
 p- Nitrophenyl D- Maltohepatoside
 glucosidase(microbial) REAGENT:
 Glucoamylase (Microbial) Reagent Blank Sample/Control
 Sodium chloride Amylase 250 ul 250 ul
 Calcium chloride substrate
 Buffer, pH 6.9 ± 0.1 Test Sera/N-AB 10 ul
Controls
SPECIAL COLLECTION AND PEREPARATION
Incubate test & control only for 5 mins
Unhemolyzed serum is the specimen of choice. Plasma Color developer 250 ul 250 ul
from heparin tubes may be used. Other anticoagulants, De ionized 2000 ul 2000 ul
such as citrate and EDTA, bind calcium, an ion needed water
for amylase activity. Therefore, plasma with any Invert to mix and read % absorbance t 610/640 nm
anticoagulant other than heparin should not be used. against water blank

Analytical Phase:
CALCULATION:
MANUAL PROCEDURE
Factor: 750 u/l
 Wavelength: 405 nm
|Blank|−|Sample|
 Temperature: 37 degrees Celsius amylase activity ( UL )= x 750=UL
|Blank|
1. For each sample add 1.0 ml reconstituted reagent to NORMAL RANGE: 60-180 U/L
cuvette or test tube and pre warm at 37°C for at
least 3 minutes.
2. Zero spectrophotometer with water at 405 nm. LIPASE DETERMINATION
3. Add 0.025 ml (25µL) serum to its respective tube
and read immediately. Pre-Analytical Phase
4. Record increase in absorbance at 30 second METHOD: Quantitative Turbidimetric Determination of
intervals for 2 minutes. Pancreatic Lipase in Human Serum
5. Determine the mean absorbance difference per
minute (ΔAbs/min). PRINCIPLE:
6. Multiply the Δabs/min by 4824 to obtain the result
in U/L
Triglyceride + H2o ---------> mono + di-glycerides + fatty Read the absorbance of the blank and each
acids sample tube against the distilled water.

MATERIALS: COMPUTATION:

• Spectrophotometer
• Accurate pipetting devices
• Cuvets
• Interval timer
• Test tubes

REAGENTS: REFERENCE:
• Substrate: 0.8% (w/v) olive oil in alcohol Adults 10 – 150 U/L (age more than 60 old: 18 – 180
• Buffer: Tris Buffer 69 mM, Sodium deoxycholate U/L)
10mM,
• pH 9.0 TECO DIAGNOSTICS

Reagent Preparation: REAGENT:

1. Add lipase buffer to a 50mL Erlenmeyer flask. Add Substrate: 0.8% (w/v) olive oil in alcohol.
25 mL distilled water and swirl to dissolve Buffer: Tris Buffer 69 Mm, Sodium Deoxycholate 10 mM,
2. Pipette 1mL of well-mixed lipase substrate into pH 9.0 (37C)
buffer solution
REAGENT PREPARATION:
SPECIMEN COLLECTION AND PREPARATION
1. Add lipase buffer to a 50 ml Erlenmeyer flask.
1. Use fresh serum specimen. Add 25 ml distilled water and swirl to dissolve
2. Lipase activity in serum is stable at room 2. Pipette 1 ml of well-mixed lipase substrate into
temperature for 1 week; sera may be stored for 3 buffer solution. Note: the absorbance of the
weeks in the refrigerator (4-8˚C) and for several emulsion prior to use must be greater than 1.0.
months if frozen. due to variation in regional temperature the
ANALYTICAL PHASE absorbance may be less than 1.0. if this occur,
add 0.5-1.0 ml mor subrate until absorbance is
MANUAL PROCEDURE greater than 1.0.
1. Reconstitute lipase reagent according to MANUAL PROCEDURE:
instruction
2. Label test tubes “blank”, “control”, “patient”, etc. 1. Reconstitute lipase reagent according to
3. Pipette 3.0 mL of reagent into appropriate test instructions.
tubes and pre-warm at 37˚C for at least 5 minutes 2. Label test tubes “blank”, “control”, “patient”
4. Zero spectrophotometer with distilled water at 3. Pipette 3.0 ml of reagent into test tube and pre-
400nm (390-420) warm at 37C for 5 mins.
5. Read and record absorbance of blank and place 4. Wavelength range: 390-420 nm
back in heating bath. 5. Using timed intervals, add 0.1 ml (100 ul) of sample
6. Using timed intervals, add 0.1 mL (100µL) of to each tube, mis, and read initial absorbance.
sample of each tube, mix, and read initial Return each tube to heating bath after initial
absorbance. Return each tube to heating bath reading
after initial reading. 6. Exactly 5 minutes after the initial absorbance
7. Exactly 5 minutes after the initial absorbance reading, using the same timed intervals, remove
reading, using the same timed intervals, remove each tube from the heating bath and mix each tube.
each tube from heating bath and mix each tube. Read the absorbance of the blank and each sample
tube against distilled water
PROCEDURE NOTES: REAGENT STORAGE AND STABILITY:

1. If the abs. of the blank is a negative value, • Working Reagent is stable for 2 weeks at 2-8°C
consider it zero. or 1 day at room temperature (15-30°C).
2. Elevate blank rates i.e. (0.005) and above may
SPECIMEN COLLECTION AND PREPARATION:
be caused by olive oil coating on cuvette
surface. Periodically rinse with acetone followed • Clear unhemolyzed serum is the recommended
by water flush. specimen sample. Plasma may be used if
3. Turbid samples should be diluted with distilled collected with EDTA.
water (1:5). Multiply final answer by dilution
factor. SAMPLE STABILITY:
4. Use fresh sera, when possible, for greatest • Serum LDH activity is reportedly stable at 2-8°C
accuracy. for 3 days. Frozen samples show decreased
LACTATE DEHYDROGENASE DETERMINATION activity of isoenzymes LD-4 and LD-5 and,
Pre-Analytical Phase therefore of total LDH values

METHOD: Quantitative Determination of Lactate ANALYTICAL PHASE

dehydrogenase in Human Serum MANUAL PROCEDURE:
PRINCIPLE: • Wavelength: 340nm
LDH specifically catalyzes the oxidation of lactate to • Temperature: 37˚C
pyruvate with the subsequent reduction of NAD to
NADH. The rate at which NADH form is proportional to 1. For each sample add 1.0 mL working reagent into
LDH activity. The method described determines NADH a cuvette/test tube. Warm for 37˚C for 3 minutes.
absorbance increase per minute at 340 nm. 2. Add 0.050 mL (50µL) serum to its respective tube
and mix gently. Incubate for 1 minute at 37˚C.
MATERIALS 3. Read and record absorbance at 1 minute. Continue
incubating at 37 degrees Celsius and record
• Spectrophotometer
absorbance again at 2 and 3 minutes. Rate should
• Centrifuge
be constant.
• Accurate pipetting devices
4. Determine average abs. per min. (∆ A/min),
• Test tubes
multiply by factor 3367 for results in U/L.
• Cuvette
• Interval timer COMPUTATION
• Water bath
Values derived based on the “absorptivity micromolar
REAGENTS extinction coefficient:” of NADH at 340 nm (0.00622). A
unit per liter (U/L) of LDH activity is that amount of
LDH Buffer (R1):
enzyme which produces one umol/L of NADH per
• L-lithium Lactate minute.
• 2-Methyl-2-Amino-1-Propanol; pH 8.8

LDH Enzyme (R2):

• NAD

Reagent Preparation and Stability

• Prepare Working Reagent in the ratio of 5 parts

Buffer (R1) to 1 part
• Enzyme(R2) (i.e., 25 mL Buffer and 5 mL
REFERENCE VALUES
Enzyme).
Males absorbance of the supernate-color reagent
mixture à sodium content of the specimen
• 50 – 166 U/L (30˚C)
• 80 – 285 U/L (37˚C) Materials and Instrument

Females • Spectrophotometer
• Centrifuge
• 60 – 132 U/L (30˚C)
• Pipette
• 103 – 227 U/L (37˚C)
• Cuvettes and test tubes
BY STANBIO (LAB PROCEDURE INSTRACTION) • Timer

REAGENT PREPARATION: Reagents

Prepare working reagent in the ratio of 5 parts buffer to • Sodium color reagent – solution of uranyl
1 part ENZYME (25ml buffer to 5 ml substrate) acetate and zinc
• acetate in aqueous acetic acid-ethanol
ELECTROLYTES DETERMINATION • mixture.
• Precipitating Reagent – aqueous solution of TCA
UNIT OUTCOMES
• Sodium standard – sodium chloride in aqueous
• To achieve this unit a learner must:
TCA
• Define concepts and principles of various
Instruments used in the performance of liver Specimen Collection and Preparation
function test
• Explain the principles of liver function test • Serum: Remove from clot promptly and
• Demonstrate how to operate the basic instruments carefully to prevent hemolysis
and equipment’s in the clinical laboratory. • Plasma: Use lithium heparinate, ammonium
• Develop expertise in the different laboratory works heparinate, or lithium oxalate as anticoagulant.
that deals mainly with clinical preparation including • Sample stability: Sodium levels remain stable for
collection and preparation of aspartate at least days at 15-25 degrees Celsius
aminotransferase specimen. • Interfering substances: Contaminated glassware
is the greatest source of error. All glassware
SODIUM DETERMINATION should be washed with 10-20% nitric acid, rinse
thoroughly with dis tilled water, dried and
Methods of Determination stored in dust-free area.
• Ion-specific Electrodes Preparation of Protein-Free Supernate
• Atomic Absorption Spectrophotometry (AAS)
• Flame Emission Spectrophotometry (FES) / 1. Label test tube as sample and add 0.5 mL serum or
Emission Flame Photometry (FEP) plasma
• Colorimetric Method – Albanese Lein 2. Add 0.5 mL Precipitating Reagent. Mix vigorously
• Combining sodium with zinc uranyl acetate à 3. Allow to stand at room temperature for 5 minutes,
sodium uranyl acetate precipitate à addition of then centrifuge at high speed for 5-10 minutes
water produces yellow solution 4. Collect supernate in separate test tube.

Principle TEST PROCEDURE

• Kolthoff – 1927 à Precipitation of sodium as 1. Pipette into labeled tubes the following volumes
triple salt à Sodium uranyl zinc acetate (mL). Mix each tubes promptly after addition of
• Albanese and Lein à colorimetric measurement color reagent
of the solubilized residue
RB Standard Sample
• Bradbury à Monitoring the color fade of the
Distilled 0.5
yellow supernate after precipitation water
• Stanbio à sodium is precipitated from a protein- Standard 0.5
free supernate as the triple salt à decrease in
 Supernate 0.5 • Potassium Boron Reagent – aqueous solution of
Color 2.5 2.5 2.5 sodium tetraphenylboron acetic acid-ethanol
reagent mixture.
• Sodium Hydroxide Reagent – aqueous sodium
hydroxide
2. Re-mix contents of tubes, then incubate for 10
• TCA Precipitating Reagent – aqueous solution of TCA
minutes at room temperature
• Potassium Standard – solution of potassium chloride
3. Mix the tubes thoroughly and centrifuge at high
in aqueous TCA
speed for 5 minutes
4. Transfer supernate of each tube to appropriate Preparation of working reagent
cuvette
5. With the spectrophotometer set at 420 nm, zero • Mix 1 volume of Potassium Boron Reagent wit 1
the instrument with water. Read and record volume of Sodium Hydroxide. Allow to stand for at least
absorbace of Reagent Blank (RB), Standard (S) and 15-30 minutes before use.
Sample (U) within 30 minutes Specimen Collection and Preparation:
CALCULATION: • Serum: Remove from clot promptly and carefully to
prevent hemolysis
• Plasma: use lithium heparinate or lithium oxalate as
anticoagulant
REFERENCE VALUE:
• Sample stability: sodium levels remain stable for at
Normal Range: Serum/Plasma = 135-155 mmol/L least 14 days at 20-25 degrees Celsius
• Interfering substances: Contaminated glassware is
the greatest source or error. All glassware should be
Potassium Determination washed with 10-20% nitric acid. Hemolysis will
falsely elevate serum potassium levels due to high
ANALYTICAL METHODS
potassium content of erythrocytes
• Ion Selective Electrode (ISE) - method of choice
Preparation of Protein-Free Supernate
• Atomic Absorption. Spectrophotometry (AAS)
• Flame Emission. Spectrophotometry (FES) • Label test tube as sample and add 0.5 mL serum or
plasma
PRINCIPLE
• Add 0.5 mL Precipitating Reagent. Mix vigorously
 Turbidimetric Technique (Hillmann and Beyer) à • Allow to stand at room temperature for 5 minutes,
Potassium ions in a protein-free alkaline medium then centrifuge at high speed for 5-10 minutes
react with sodium tetraphenyl boron à produce • Collect supernate in separate test tube.
finely dispersed turbid suspension of potassium TEST PROCEDURE:
tetraphenylboron
 Turbidity  proportional to potassium 1. Pipette into labeled tubes the following volumes
concentration (mL). Mix each tubes promptly after addition of
color reagent
MATERIALS AND INSTRUMENT
RB Standard Sample
• Spectrophotometer Working 1.0 1.0 1.0
• Centrifuge reagent
• Pipette Standard 0.1
• Cuvettes and test tubes Supernate 0.1
• Timer Distilled 0.1
water
REAGENTS
 2. Incubate all cuvettes at room temperature for 5 • Timer
minutes.
REAGENTS
3. Read S and U vs RB at 580 nm within 60
minutes. • Direct Chloride Color Reagent – Solution of Mercuric
Thiocyanate, Ferric Nitrate, Mercuric Nitrate in
CALCULATION:
methanol and water. Also contains nitric acid and
surfactants
• Direct Chloride Standard (100 meq/L) – Aqueous
solution of Sodium Chloride

REFERENCE VALUE: Specimen Collection and Preparation:

Normal range: • Blood should be drawn with a minimum ofvenous

stasis and the serum or plasmaseparated from
• Serum: 3.6 – 5.5 mmol/L cells as soon as possible toprevent diffusion of
• Plasma 3.5 – 4.8 mmol/L chloride into the red cells

CHLORIDE DETERMINATION PROCEDURE:

Analytical Methods 1. Pipette into labeled tubes or cuvettes the

following volumes (mL). Mix each tubes
• Ion-selective electrode promptly after addition of color reagent
• Mercurimetric Titration (Schales-Schales method)
• Colorimetric method uses mercuric thiocyanate and RB Standard Sample
ferric nitrate to form a reddish-colored complex Color 1.0 1.0 1.0
with a peak at 480 nm. reagent
• Coulometric-Amperometric Titration (Cotlove Chloride 0.01
standard
Chloridometer)
Specimen 0.01
PRINCIPLE

• Classical Method: combination of chloride with 2. Incubate all cuvettes at room temperature (25-
either silver or mercury  undissociated chloride 30 ℃) for 5 minutes
compound. 3. Read S and U vs RB at 500 nm within 30 minutes
• Most Popular Method: Titrimetric (Schales and
CALCULATION:
Schales)  standard solution of mercuric nitrate
and diphenycarbazone as indicator
• Colorimetric Method: (Zall et. al, Skeggs and
Hochtrasser)
• Chloride reacts directly with mercuric thiocyanate to REFERENCE VALUES
release thiocyanate ions. The latter immediately Normal range:
combines with ferric thiocyanate. The absorbance of
this stable colored compound is measured at 500 • Serum/Plasma 98-106 meq/L
nm and compared to that of a chloride standard Hyperchloremia
similarly treated
• It can be seen in the following conditions:
Materials and Instrument • Dehydration
• Spectrophotometer • Renal tubular acidosis
• Centrifuge • Acute renal failure
• Pipette • Metabolic acidosis associated with prolonged
• Cuvettes and test tubes diarrhea
Hypochloremia • Timer

It is seen in: REAGENTS

• Prolonged vomiting Magnesium Reagent

• Profuse sweating
• Tris Buffer 0.2 mol/L
• Increased gastric juice secretion
• Potassium Carbonate 70 mmol/L
• Salt-losing nephritis
• Gedta 40 mmol/L
• Addison’s disease
• Xylidyl Blue 0.1 mmol/L
• Sodim Azide 0.1%
• Activators

Magnesium Standard (2.0 mEq/L)

• Aqueous solution of Magnesium Chloride with

sodium azide as preservative.

MAGNESIUM DETERMINATION
SPECIMEN COLLECTION AND PREPARATION
ANALYTICAL METHODS
• Sample Stability: Serum and plasma are reportedly
• Total Magnesium stable for 7 days at 2-8 ˚C. Treated urine is
 Atomic Absorption Spectrophotometry (AAS) is reportedly stable for 7 days at 2-8˚C.
the reference method but it is not routinely
Interfering Substances:
done in the clinical laboratory.
 Photometric methods on automated analyzers - • EDTA - unsatisfactory anticoagulant for this
These methods employ metallochromic procedure.
indicators or dyes such as calmagite, formazan • Do not use serum that has visible hemolysis à
dye, magon, and titan yellow dye. erythrocytes contain higher levels of magnesium.
• Ionized (Free) Magnesium • The test is not influenced by bilirubin concentrations
• Ion-Selective Electrode for magnesium up to 20 mg/dL, nor by lipemic serum.
* Intracellular Magnesium • Contaminated glassware is the greatest source of
• Fluorescence measurement using furapta error.
(magnesium binder) • It is recommended that disposable tubes and
• Nuclear magnetic resonance spectroscopy pipette tips be used in this procedure
• Ion selective microelectrode
PROCEDURE:
• Electroprobe microanalysis
1. Pipette into labeled tubes or cuvettes the following
PRINCIPLE
volumes (mL). Mix each tubes promptly after
• Magnesium and Xylidyl Blue-1 à combine under addition of color reagent
alkaline conditions  form water soluble red-purple
RB Standard Sample
chelate à 520 nm
Color 1.0 1.0 1.0
• GEDTA (Glycoletherdiamine-N, N, N’.N’–tetraacetic
reagent
acid) – prevent interference from Calcium
Magnesium 0.01
Materials and Instrument standard
Specimen 0.01
• Spectrophotometer
• Centrifuge
• Pipette 2. Incubate all cuvettes at room temperature (25-30℃)
• Cuvettes and test tubes for 10 minutes or at 37℃ for 3 minutes
3. Read S and U vs RB at 520 nm within 3 hours
CALCULATION:

REFERENCE VALUES

Normal range:

• Serum, Plasma: 1.3 – 2.1 mEq/L

• 24-hour Urine: 6.0 – 10.0 mEq

Hypermagnesemia

• It is a condition with high level of serum

magnesium.
• Increased magnesium level in the blood is rare and
usually iatrogenic.
• Elderly and patients with bowel disorder and renal
insufficiency are the most at risk.
• Clinical manifestations include hypotension,
bradycardia, respiratory depression, depressed
mental status and electrocardiographic (ECG)
abnormalities. Hypomagnasemia

• It is a condition with low serum magnesium level

• The most common causes of hypomagnesemia are:
 Loss of magnesium in the GI tract as in chronic
diarrhea and malabsorption steatorrhea
 Diabetes mellitus secondary to glycosuria and
osmotic diuresis
 Alcohol
 Stress
 METHOD

• Quantitative Enzymatic-Colorimetric Determination of

Calcium in Serum or Urine.

PRINCIPLE

 Calcium reacts with cresolphtalein complexone in

8-hydroxyquinoline to form a colored complex
(purple color) that absorbs at 570 nm (550-580).
The intensity of the color is proportional to the
calcium concentration. Color interfiers and a
stabilizer are present to minimize interference by
other metallic ions.

MATERIALS

• Spectrophotometer
• Centrifuge
• Accurate pipetting devices
• Cuvettes and test tubes
• Interval timer
• Graduated cylinder or beaker

REAGENTS

• Calcium Color Reagent (A): O-Cresolphtalein

Complexone 0.14 mM, 8-Hydroxyquinone 13 mM
• Calcium Buffer : Diethylamide 363 mM, Potassium
Cyanide, 2 mM, Nonreactive ingredients and
stabilizers in both reagent A and B.
• Calcium Standard: Calcium Carbonate in dilute
hydrochloric acid. (10mg/dl).

REAGENT PREPARATION

• Combine equal volumes of Calcium Color Reagent

(A) and Calcium Buffer (B), mix and let stand for 10
minutes at room temperature before use.
• Reagents should be combined in clean plastic
vessels. Water and glassware containing calcium will
react with the reagent. All glassware should be
rinsed with diluted hydrochloric acid before use.

SPECIMEN COLLECTION AND PREPARATION

• Fasting nonhemolyzed serum is specimen of choice.

• Anticoagulants other than Heparin should not be
used.
• Remove serum from clot as soon as possible since
CALCIUM DETERMINATION
red blood cells can absorb calcium
• Older serum specimens containing visible BLUE SCREEN ONE-STEP SALIVA ALCOHOL TEST
precipitate should not be used STRIP
• Tubes with cork stoppers should not be used
MATERIALS AND PRINCIPLE OF TEST
• Serum calcium is stable for twenty-four (24) hours at
room temperature, one (1) week refrigerated (2- • Test strips
8˚C) and up to five (5) months frozen, and protected • Collection cups
from evaporation. • Package insert
• Chemical assay based on alcohol sensitive enzymatic
URINE
reaction
• Collect 24 hours urine in a dry clean container • Color change is directly proportional to the
containing 20-30 ml of 6N HCl concentration of alcohol in the saliva specimen
• Alternatively use 1-2 ml of 6N HCl for random
PROCEDURE OF THE TEST
sample
1. Allow the strip and specimen to equilibrate to
MANUAL PROCEDURE
room temperature prior to testing.
1. Prepare working reagent. 2. Do not place anything in the mouth for 15
2. Label tubes Blank, Standard, Controls, Patients, etc. minutes before beginning the test.
3. Transfer 1.0 mL of working reagent into each tube. 3. Bring the pouch to room temperature before
4. Add 0.02 mL (20µl) of sample to respective tubes opening it. Remove the test strip from the
and mix. sealed pouch and use it as soon as possible after
5. Let stand for atleast sixty (60) seconds at room observing the reaction pad on the end of the
temperature. test strip.
6. Zero spectrophotometer with blank at 570 nm. 4. Saturate the reaction pad with saliva from the
(Wavelength ranges:550-580) collection cup. Start the timer immediately after
7. Read and record absorbance of all tubes. Final color saturating the reaction pad with saliva.
is stable for twenty (20) minutes. 5. Read results at 2 minutes by visually comparing
the color of the reaction pad to the
COMPUTATION:
corresponding color blocks printed on the
pouch to determine the relative blood alcohol
concentration.
6. Do not interpret the result after 3 minutes
REFERENCE VALUES:
INTERPRETATION OF THE TEST
Serum

• 8.5-10.5 mg/dl
• Children under 12, usually have high values which
decrease with aging

QUALITY CONTROL

CONTROL TEST - qualitatively verified (5 drops of 80

proof distilled spirits + 30 ml water)

solution: COLOR CHANGE on the reaction pad

Color reaction in saliva: SLOWER and LESS INTENSE ONE-STEP PREGNANCY (hCG) TEST

* DO NOT perform control test with undiluted alcohol. MATERIALS AND PRINCIPLE OF THE TEST

LIMITATIONS: • IMMUNOCHROMATOGRAPHY

1. Failure to wait 15 mins after smoking, or placing

food, drink, or other materials
2. Test strip cannot be used to determine the presence
of alcohol in beverages, in undiluted alcohol, or in
other liquid solutions.
3. Test strip is highly sensitive to the presence of
alcohol. Alcohol vapors in the air are sometimes
detected by the strip.
4. Ingestion or general use of over-thecounter
medications and products containing alcohol can
produce positive result

ASSAY SPECIFICITY:

will react with methyl, ethyl and allyl alcohols produce

positive result

INTERFERING SUBSTANCES:

These don’t normally appear in sufficient quantity in PROCEDURE OF THE TEST:

saliva to interfere.
• Collect the urine specimen in clean disposable
• Peroxidase container without preservatives. Do not reuse
• Mercaptans container treated with pregnant urine.
• Bilirubin • Remove the test device from the foil pouch, and
• Strong oxidizers place it in a flat, dry surface.
• Tosylates • Holding the urine dropper above the test device,
• L-dopa squeeze 3 to 4 drops of urine into the urine well.
• Ascorbic Acid • As the test begins to work, you will see a purple
• Oxalic acid color band across the result window in the center of
• L-methyldopa the test device.
• Tannic acid • Interpret test results at 3 minutes
• Uric acid
• Methampyrone
• Pyrogallol

SPECIMEN CONSIDERATION

• First morning urine sample

• Urine samples containing visible precipitate
• Test will not be performed immediately
• Prolonged storage
QUESTIONS:

• If I see two band, but one band is darker than the

other, am I pregnant?
• Can the result change after standing for a certain
length of time?
• If the test is positive, what do I do? If negative?
• Can I get a false misleading result?