[CC1] Laboratory Compilation

Transcriber/s: Fatima Jen Sangil

3rd Year - Section 5 – 1st Semester

PRELIM LAB CONVERSIONS AND INTERCONVERSIONS

1. LABORATORY MATHEMATICS Units Conversion Formula

Conc. &
SIGNIFICANT FIGURES C1 V1 = C2 V2
Volume
Normality &
→ Rules for Recording Significant Figures: N= M×V
Molarity
1. All non-zero digits are significant.
Molarity & % w/v × 10
2. All zeros between non-zero digits are significant. M=
%(w/v) mw
3. Zeros to the right of a decimal point and a non-
zero digit are significant. mg/dL & mg/dL × 10
mEq/L =
mEq/L ew
4. Zeros to the left of a non-zero digit and to the right
of a decimal point are not significant if the value mg/dL & mg/dL × 10
mmol/L =
of the number is less than one. mmol/L mw
mmol/L &
mEq/L = mmol/L × V
mEq/L
UNITS OF MEASUREMENT

Base Quantity SI Unit

ACIDS AND BASES
Mass Kilogram
Electrical Current Ampere Parameter Formula
Time Second
Amount of hydrogen released in solution
Temperature Kelvin anti-log of pH
[H+]
Amount of Substance Mole [𝐇 + ] = 𝟏𝟎−𝐩𝐇
Luminous Intensity Candela
Length Meter Amount of hydroxyl released in solution
[OH-] anti-log of pOH
[𝐎𝐇 − ] = 𝟏𝟎−𝐩𝐎𝐇
WAYS OF EXPRESSING SOLUTION pH − 𝐥𝐨𝐠[𝐇 + ]
CONCENTRATION
pOH − 𝐥𝐨𝐠[𝐎𝐇 − ]
Expression Formula
solute Note: pH + pOH = 14
% sol’n w/w, w/v, v/v × 100 (H)(OH) = 1 × 10−14
solution
Molarity g of solute
mol/L
(M) MW of solute × L of sol′n
STRONG AND WEAK ACIDS AND BASES
Normality g of solute
g/L
(N) EW of solute × L of sol′n Parameter Formula
mol of solute
Molality mol/kg Strong Acid [𝐻 + ] = 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑡ℎ𝑎𝑡 𝑠𝑡𝑟𝑜𝑛𝑔 𝑎𝑐𝑖𝑑
kg of solvent

Common Reagents Weak Acid [𝐻 + ] = √𝐾𝑎𝐶𝑎

Chemical MW SG %ASSAY
CH3COOH 60 1.06 99% Strong Base [𝑂𝐻 − ] = 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑡ℎ𝑎𝑡 𝑠𝑡𝑟𝑜𝑛𝑔 𝑏𝑎𝑠𝑒
HNO3 63 1.42 70%
HCl 36 1.19 37% Weak Base [𝑂𝐻 − ] = √𝐾𝑏𝐶𝑏
H2SO4 98 1.84 96%
H2PO4 98 1.69 85%

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 1
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

BUFFER SOLUTIONS Examples of Transfer Pipette:

→ OSTWALD FOLIN
→ Henderson Hasselbalch Equation:
• Bulb like enlargement at the tip
[𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒] • Used to transfer or aspirate VISCOUS fluid/liquid
𝑝𝐻 = 𝑝𝐾𝑎 + log • Drainage Characteristic: Blow out
[𝑤𝑒𝑎𝑘 𝑎𝑐𝑖𝑑]
• Calibration: both TC and TD

[𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑎𝑐𝑖𝑑] → VOLUMETRIC PIPETTE

𝑝𝑂𝐻 = 𝑝𝐾𝑏 + log • Bulb like enlargement at the middle
[𝑤𝑒𝑎𝑘 𝑏𝑎𝑠𝑒]
• AQUEOUS solution
• Drainage Characteristic: Self-draining
• Calibration: To Deliver (TD)
2. PIPETTES

AS TO CALIBRATION / DESIGN

TO CONTAIN (TC)
→ Holds a particular volume
→ Do not dispense the exact volume
→ Specifications:
→ Require rinsing technique • How much liquid will be transferred if the liquid
is drawn up to the calibration line on the neck
• Temperature at which the calibration was made
TO DELIVER (TD) • Whether it is a TD or TC pipette
→ Delivers the exact amount of volume it holds to a
container
MEASURING / GRADUATED PIPETTE
→ Has graduation lines along the stem
AS TO DRAINAGE CHARACTERISTIC
→ Measure and transfer several fractional volume
BLOW-OUT
→ Specifications:
→ With etched ring/double ring (frosted band)
• Maximum volume of liquid that can be
transferred
→ Exact volume is obtained when the last drop is
• Size of the divisions on the pipette
blown out
• Temperature at which calibration were made
• Is the pipette TD or TC
SELF-DRAINING
→ Has NO etched ring/double ring/frosted band or 2 Examples of Measuring/Graduated Pipette:
thin rings
→ SEROLOGIC PIPETTES
• Calibrated up to the tip
→ Liquid is allowed to drain by gravity
• Drainage Characteristic: Blow out
• Calibration: TD
AS TO TYPE
→ MOHR PIPETTES
• Calibrated between two mark
TRANSFER PIPETTE
• No graduation marks up to the tip
→ Designed to dispense one volume without further • Drainage Characteristic: Self-draining
subdivisions • Calibration: TD

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 2
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

MICROPIPETTE → TC pipettes are calibrated using mercury.

Examples of Micropipette: → Gravimetric and Spectrometric methods

• Performed to ensure the accuracy and precision
• WBC pipette of pipettes and how to calibrate them
• RBC pipette
• Sahli Helige pipette → Acid dichromate
• Thoma pipette • Commonly and best to be used in cleaning glass
• Lang Levy pipette – TD/TC pipette.
• Kirk and Overflow pipette

3. SPECTROPHOTOMETER
AUTOMATIC PIPETTES
→ Terms used to describe EMR
→ Mechanism of aspirating and dispensing of fluid is • Amplitude
inherited to the pipet • Energy
• Wavelength – distance between two peaks (nm)
According to MECHANISM, classification includes:
o 400-700 nm – visible spectrum
→ AIR DISPLACEMENT PIPETTE o <400 nm – UV
• Piston for suction creation for drawing sample o >700 nm – IR

→ POSITIVE DISPLACEMENT PIPETTE • Frequency – number of vibrations of wave

• No requirement of different tip for each use motion per second

→ DISPENSER / DILUTOR PIPETTE ANALYTICAL METHODS

• Obtaining liquid from a common reservoir and
dispense it repeatedly → Colorimetry
• Photometry
Automatic Pipette Vol. range & Immersion depth o the measurement of light intensity without
Volume Range Immersion Depth (mm) consideration of wavelength
0.1 – 1 uL 1 – 2 mm • Spectrophotometry
> 1 – 100 uL 2 – 3 mm o the measurement of light intensity in a
>100 – 1,000 uL 2 – 4 mm narrower wavelength
> 1,000 uL 3 – 6 mm
→ Volumetric
→ Turbidimetry
PASTEUR PIPETTES → Nephelometry
→ Electroanalytic methods
→ Transfer of uncalibrated volumes up to 2.5 mL can
be accomplished using glass “transfer” or “Pasteur” • Electrophoresis
pipettes • Potentiometry
• Amperometry
→ These may be sterilized before use.
→ Chromatography – thin layer chromatography, liquid
→ Roughly calibrated volumes of 1 and 2mL can be chromatography, liquid gas chromatography, etc.
transferred with the one-piece plastic transfer • Planar
pipettes which may be purchased as sterile or non- • Column
sterile units.
→ Luminescence
• Fluorometry
NOTES TO REMEMBER • Chemiluminescence

→ Class A pipettes are available in the laboratory COLORIMETRY

• do not require calibration
→ Spectrophotometry
→ In calibrating pipettes, pipettes that are TD are → Emission Flame Photometry
calibrated with distilled water → Atomic Absorption Spectrophotometry

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 3
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

Wavelengths of Visible Light LIGHT SOURCE

Violet 420 nm (400 in Bishop)
Blue 470 nm → Excitation source or Excitation Lamp
Green 530 nm → This component provides EMR
Yellow 580 nm → It can be
Orange 620 nm • CONTINUUM – emits radiation that changes in
Red 700 nm intensity
• LINE – emits limited radiation and wavelength
→ Notes to Remember:
• The wavelength is inversely related to ➢ Tungsten Iodide lamp,
frequency and energy, Visible
➢ Halogen lamp

• the shorter the wavelength, the higher the UV ➢ Hydrogen lamp/ Deuterium lamp,
frequency and energy and vice versa. (vapour ➢ Mercury lamp,
lamps) ➢ Xenon
• Planck’s formula: E = hv
o E = energy Infrared ➢ Silicon carbide rod heated to 1200ºC
o h = constant
o v = frequency
ENTRANCE SLIT
o The higher the energy, the higher the
frequency, the shorter wavelength → Minimizes unwanted or stray light
• STRAY LIGHT – wavelengths outside the band
o The lower the energy, the lower the transmitted by the monochromator
frequency, the longer wavelength
→ Prevents the entrance of scattered light to the
o Energy and frequency has direct proportional monochromator
relationship

SPECTROPHOTOMETRY MONOCHROMATOR
→ Most commonly used method for measurement of → Spectral isolation / Isolates specific or individual
analytes in the laboratory wavelength of light that is needed for measurement

→ measurement of the light transmitted by a solution → Also have the capacity to isolate the stray light
to determine the concentration of the light-
absorbing substance in the solution. → Kinds of monochromator:

→ Spectrophotometer ➢ wedge shaped pieces of glass,

Prisms
• measures the amount of photons/intensity of light quartz or sodium chloride
absorbed after it passes through sample solution
➢ most commonly used;
Diffraction
➢ made up of grooves etched on a
• This consists of 2 devices: gratings
polished surface
o SPECTROMETER – device that produce,
typically dispense and measure the light ➢ simple, least expensive, not
precise but useful
o PHOTOMETER – indicates the photoelectric ➢ Produces monochromatic light
dictator that measures the light Filters
based on the principle of
constructive interference of
Basic Parts of Spectrophotometer waves
(1) Light Source
(2) Entrance Slit (collimator)
(3) Monochromator
(4) Exit Slit (wavelength selector) EXIT SLIT
(5) Cuvet / Cuvette (sample holder)
(6) Photodetector → Controls the width of light beam (bandpass)
(7) Meter or Read out device → Allows only a narrow fraction of the spectrum to reach
(8) Recorders (sometimes included) the sample cuvette
→ Prevents stray light from hitting the cuvette

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 4
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

CUVETTE DOUBLE BEAM

→ Also called as absorption cell / analytical cell / sample DOUBLE BEAM IN SPACE
cell
→ It holds the solution to be analyzed

Kinds of Cuvettes
(1) Alumina/Silica glass UV region, ≥220 nm
Visible region, strong
(2) Borosilicate glass
alkaline solution
(3) Quartz/plastic UV region
(4) Soft glass strong acidic solution
(5) Polycarbonate plastic UV and visible region
➢ One light source
➢ Two entrance slits
PHOTODETECTOR ➢ Two monochromators
➢ Two exit slits
→ Detects and converts transmitted light into
➢ Two photodetectors
photoelectric energy
→ Detects the amount of light that passes through the DOUBLE BEAM IN TIME
sample in the cuvette

→ Kinds of Detector:
1.) Barrier layer cell / Photocell / Photovoltaic cell
o simplest detector
o least expensive
o temperature sensitive – why it’s not specific

2.) Phototube / Photoemissive tube

3.) Photomultiplier (PM) tube ➢ One light source, entrance slit, and exit slit
o most common type ➢ One monochromator but needs a chopper
o excellent sensitivity ➢ One photodetector
o rapid response with the transmitted light ➢ Similar to single beam but beam is split by chopper
4.) Photodiode
o excellent linearity BEER-LAMBERT’S LAW

→ Law of Colorimetry
METER OR READ OUT DEVICE
→ This states that the concentration of a substance is
→ known as Galvanometer/Ammeter • directly proportional to its absorbance and
→ Displays output of the detection system • inversely proportional to the logarithm of
transmitted light

SINGLE BEAM → Relationship between concentration and absorbance

APPLICATION OF BEER- LAMBERT’S LAW

𝐴𝑐 𝐴𝑢
=
𝐶𝑐 𝐶𝑢
→ Where,
• A = absorbance
➢ The usual spectrophotometer • C = concentration
• c = calibrator or s = standard
• u = unknown

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 5
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

ABSORBANCE
𝟏𝟎𝟎
𝑨 = 𝒂𝒃𝒄 = 𝟐 − 𝐥𝐨𝐠(%𝑻) 𝒐𝒓 𝐥𝐨𝐠 ( )
%𝑻
→ Where,
• A = absorbance
• a = molar absorptivity
o absorptivity of the compound under standard
conditions

• b = length of light through the solution

o light path

• c = conc. of absorbing molecules/solution

SPECTRO IN LAB

➢ C40 IMPLEN Nanophotometer

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 6
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

MIDTERM LAB
Sample 10 uL serum
1. GLUCOSE DETERMINATION
CF 0.357

Trinder Glucose Oxidase CU mg/dL

Method
Enzymatic Method

Principle Glucose Oxidase SI mmol/L

End Color
Red-violet (quinoneimine) • End product of protein catabolism
(red-violet sa demo vid pero violet sabi ni maam) • 5:1 BUN working solution

Wavelength 500 nm • Kinetic

• Reading – decreasing
Std Conc. 100 mg/dL • First reading – 30 seconds
• Second reading – 60 seconds
Sample 0.01 mL serum
→ REFERENCE INTERVAL:
CF 0.0555 • 8 – 23 mg/dL
• 2.9 – 8.2 mmol/L
CU mg/dL

SI mmol/L

→ REFERENCE INTERVAL
• FBS: 70 – 100 mg/dL
• RBS: <200 mg/dL

CREATININE

Fabinay and Eringhausen

Method
Kinetic Jaffe

Principle Jaffe Reaction

Red-orange tautomer
2. NPN DETERMINATION End Color
(creatinine picrate)

BLOOD UREA NITROGEN (BUN) Wavelength 510 nm

Std Conc. 5 mg/dL

Method Kinetic Enzymatic UV Method

Sample 50 uL serum
Principle GLDH Coupled Enzymatic
CF 88.4
Wavelength 340 nm
CU mg/dL
Std Conc. 30 mg/dL
SI umol/L

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 7
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

• End product of muscle metabolism • End product of purine metabolism

• 1:1 working solution • Endpoint

• Kinetic → REFERENCE INTERVAL:

• Reading – increasing • Female: 2.6 – 6.0 mg/dL
• First reading – 20 seconds o 0.16 – 0.36 mmol/L
• Second reading – 60 seconds
• Male: 3.5 – 7.2 mg/dL
→ B:C Ratio – BUN/Creatinine o 0.21 – 0.43 mmol/L
• Normal: 10-20:1

→ REFERENCE INTERVAL:
• Female: 44 – 88 umol/L
• Male: 53 – 106 umol/L

3. LIPID PROFILE DETERMINATION

TOTAL CHOLESTEROL

BLOOD URIC ACID (BUA)

Quantitative Enzymatic
Method
Colorimetric Method
Uricase Quantitative Enzymatic
Method
Colorimetric Method
Principle
Uricase catalyzes the conversion of uric acid
into allantoin and H2O2.

The H2O2 yielded is measured by its reaction End Color Red-violet (quinoneimine)
Principle with 3,5 Dicholoro-2-hydroxybenzane sulfonic
acid (DCHB) where it will be acted upon by
your peroxidase together with Wavelength 500 nm
aminophenazone to form the characteristic
red violet quinoneimine complex Std Conc. 200 mg/dL

End Color Red-violet (quinoneimine) Sample 10 uL serum

Wavelength 520 nm CF 0.026

Std Conc. 8 mg/dL CU mg/dL

Sample 20 uL serum SI mmol/L

CF 0.0595
→ REFERENCE INTERVAL:
• NV: 150 – 250 mg/dL
CU mg/dL
• 3.88 – 6.47 mmol/L
SI mmol/L

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 8
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

HIGH DENSITY LIPOPROTEIN

Polyanion Precipitation Method

Method with Enzymatic
Cholesterol Method

Polyanion Precipitation
Principle (Phosphotungstic acid
and Magnesium ions)

Wavelength 500 nm
TRIGLYCERIDES (TAG)
Std Conc. 50 mg/dL

Quantitative Enzymatic Sample 25 uL supernatant

Method
Colorimetric Method
CF NONE
Lipoprotein Lipase Hydrolysis
CU mg/dL
▪ Hydrolysis by lipase
Principle ▪ Phosphorylation by glycerokinase
▪ Reduction by glycerol-3-phosphate SI NONE
oxidase
▪ Oxidation by peroxidase
→ HDL Separation
• 0.05 mL HDL precipitating reagent to 0.5 mL
End Color Red-violet (quinoneimine) serum
• Resulting clear supernatant = sample
Wavelength 500 nm
→ REFERENCE INTERVAL:
Std Conc. 200 mg/dL • Female: 30 – 85 mg/dL
• Male: 30 – 70 mg/dL
Sample 10 uL serum

CF 0.0113

CU mg/dL

SI mmol/L

→ REFERENCE INTERVAL:
• NV: 10 – 190 mg/dL
• 0.11 – 2.15 mmol/L
• Is the result increase? Decrease? Normal?
LOW DENSITY LIPOPROTEIN

→ LDL = Total Cholesterol – (HDL + VLDL)

→ VLDL?
• Friedewald Method – more commonly used
o TAG / 2.175 (mmol/L) – for SI unit
o Or TAG / 5 (mg/dL) – for conventional unit

• De Long Method
o TAG / 2.825 (mmol/L) – for SI unit
o Or TAG / 6.5 (mg/dL) – for conventional unit

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 9
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

FINAL LAB • Reverse Method

• Kinetic
1. AST DETERMINATION • Reading – increasing
• First reading – 60 seconds
• Second reading – 60 seconds
Method Modified Karmen Method • Third reading – 60 seconds

→ REFERENCE INTERVAL:
Principle • 0 – 24 U/L

Wavelength 340 nm

Sample 50 uL serum

CF 3376 → U/L

• 5:1 working reagent

• Kinetic
• Reading – decreasing
• First reading – 60 seconds 3. LDH DETERMINATION
• Second reading – 60 seconds
• Third reading – 60 seconds
Buhl and Jackson Modification
Method
of Wacker Method
→ REFERENCE INTERVAL:
• 8 – 33 U/L
Principle

Wavelength 340 nm

Sample 50 uL serum

CF 3376 → U/L

• Forward Method
• Kinetic
2. CK-MB DETERMINATION
• Reading – increasing
• First reading – 60 seconds
Quantitative Creatine Kinase MB • Second reading – 60 seconds
Method
Method with Immunoinhibition • Third reading – 60 seconds

→ REFERENCE INTERVAL:
• Male: 80 – 285 U/L
Principle • Female: 103 – 227 U/L

Wavelength 340 nm

Sample 50 uL serum

CF 6752 → U/L

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 10
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

 NADH to NAD+ = loss of hydrogen ions ALBUMIN

• Decreasing absorbance readings
Method Brom Cresol Green Method
 NADP to NADPH = gain of hydrogen ions
• Increasing absorbance readings
Principle Dye Binding
 Involves Peroxidase = 500 nm
Green
 Involves NADH = 340 nm End Color
(albumin + BCG)

 Biuret – peptide bonds Wavelength 550 nm

 Kjeldahl – N content
Std conc. 6 g/dL

4. TPAG DETERMINATION Sample 10 uL serum

TOTAL PROTEIN CF 10

CU g/dL
Biuret Quantitative Colorimetric
Method
Method SI g/L

Principle Biuret Reaction

• Other dyes:
o Brom Cresol Green = most common
Rgt Color Blue (Biuret reagent) o Brom Cresol Purple = most specific
o HABA
Purple / Violet o Methyl orange
End Color
(cuprous protein complex)
→ REFERENCE INTERVAL:
Wavelength 550 nm
• NV: 3.5 – 5 g/dL
Std conc. 10 g/dL

Sample 10 uL serum

CF 10

CU g/dL

SI g/L

→ REFERENCE INTERVAL:
• NV: 6.5 – 8.3 g/dL GLOBULIN

Globulin = Total Protein – Albumin

→ Reference Value:
• 2.3 – 3.5 g/dL

A:G Ratio = Albumin / Globulin

→ Normal Value:
• 1.3 – 3 : 1

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 11
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

5. BILIRUBIN DETERMINATION 6. ALT DETERMINATION

Method Modified Malloy-Evelyn Method Modified IFCC and Bergmeyer

Method
Method
Principle Diazotization Reaction
Principle
Pink-purple
End Color
(azobilirubin)
Wavelength 340 nm
Wavelength 540 nm
Sample 50 uL serum
Std conc. 10 mg/dL
CF 3376 → U/L
Total Bilirubin – 50 uL serum
Sample
Direct Bilirubin – 10 uL serum • 5:1 working reagent
• Kinetic
CF 17.1 • Reading – decreasing
• First reading – 60 seconds
CU mg/dL • Second reading – 60 seconds
• Third reading – 60 seconds
SI umol/L
→ REFERENCE INTERVAL:
• 3 – 35 U/L
• A drop of anti-oxidant
• RT, protected from direct light
o TB – 5 mins.
 Reminder for MSPE
o DB – 3 mins. ▪ “What is the substrate of SGPT?”
o Alanine and keto acids or keto-glutarate
→ INDIRECT BILIRUBIN
• IB = TB – DB ▪ “What is the substrate of AST?”
o Aspartate and keto acids or keto-glutarate
→ REFERENCE INTERVAL:
• DB: 0.05 mg/dL (0 to 88.5 umol/L) ▪ “What is the product of ALT?”
• TB: 0 – 1.20 mg/dL (0 to 20.52 umol/L) o Pyruvate and glutamate
- Why it’s called SGPT
 Reminder for MSPE ▪ “What is the product of AST?”
▪ “What is the end color produced?” o Oxaloacetate and glutamate
o pink to purple - Why it’s called SGOT

▪ “What is the end product produced?”

o azobilirubin

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 12
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

7. ALP DETERMINATION 𝐀𝐮 × 𝐂𝐜 (𝟏𝟎𝟎 𝐦𝐠/𝐝𝐋) × 𝐮𝐫𝐢𝐧𝐞 𝐯𝐨𝐥. (𝐝𝐋)

𝐂𝐮 =
𝐀𝐜
Method Bower’s and McComb Method
→ REFERENCE INTERVAL:
Principle • NV:
o Random urine: 1 – 14 mg/dL
End Color
Yellow o 24-hr urine: 22 – 120 mg/24 hrs
(p-nitrophenol)

Wavelength 405 nm  Reminder for MSPE

▪ “What is the dye used for the measurement of
Sample 20 uL serum your urine protein?”
o Pyrogallol red
CF 2764 → U/L
▪ “What is the end-color produced?”
o Blue purple or blue violet
• 5:1 working reagent
• Kinetic
• First reading – 60 seconds
• Second reading – 60 seconds
• Third reading – 60 seconds

→ REFERENCE INTERVAL:
• 34 – 114 U/L

9. URINE CREA DETERMINATION

Fabinay and Eringhausen

Method
Kinetic Jaffe Method

Principle Dye-binding principle

8. URINE PROTEIN DETERMINATION Red-orange tautomer

End Color
(creatinine picrate)
Watanabe (Dye-Binding
Method Wavelength 510 nm
Colorimetric Method
Std conc. 5 mg/dL
Principle Dye-binding principle
Sample 50 uL urine
Blue-purple
End Color
(pyrogallol red-molybdate complex)
• Urine sample = 1:20 dilution
Wavelength 600 nm

Std conc. 100 mg/dL 𝐀𝐮 × 𝐂𝐜 (𝟓 𝐦𝐠/𝐝𝐋) × 𝟐𝟎 (𝐝𝐢𝐥𝐮𝐭𝐢𝐨𝐧 𝐟𝐚𝐜𝐭𝐨𝐫)

𝐂𝐮 =
𝐀𝐜
Sample 50 uL urine

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 13
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

➢ Two ways to report the result: mg/L or mg/L/24hrs

• Urine Crea (mg/L)
o = Urine Crea (mg/dL) x 10
o Applicable for random urine
o Multiply by 10

• Urine Creatinine (mg/L/24hrs)

o = Urine Crea in mg/L x 24hrs (mL)/100
o Applicable for 24-hour urine sample in mL
- Total urine volume for urine protein is dL

o Multiply by total volume of urine divided by

1000

 DO NOT CONVERT INTO SI UNIT

10. CREATININE CLEARANCE

→ Where:
• U = concentration of analyte in urine
• P/S = concentration of analyte in plasma/serum
• V = volume of urine in mL for 24 hours
• Minutes = time required to collect urine (1440
minutes = 24 hours)
• 1.73 = constant value, average body surface of
an adult individual
• A = body surface of the patient (BSA)
o Obtain height and weight of patient

→ REFERENCE VALUES:
• Male: 85 – 125 mL/min
• Female: 75 – 112 mL/min

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 14
 [CC1] Laboratory Compilation
Transcriber/s: Fatima Jen Sangil
3rd Year - Section 5 – 1st Semester

11. AMYLASE DETERMINATION

Method Modified Wallenfels Method

Principle Amyloclastin Reaction

Yellow
End Color
(p-nitrophenol + glucose)

Wavelength 405 nm

Sample 25 uL serum

CF 4824 → U/L

• Substrate: p-nitrophenyl-D-maltoheptoside
• First reading – immediately after mixing
• Second reading – 60 seconds
• Third reading – 60 seconds

→ REFERENCE INTERVAL:
• Serum: 25 – 125 U/L
• Urine: 1 – 17 U/hr

12. LIPASE DETERMINATION

Tietz-Fiereck Turbidimetric
Method
Method

Principle

Wavelength 550 nm

Std conc. 60 U/L

Sample 50 uL serum

• Substrate: olive oil (500 uL)

→ REFERENCE INTERVAL:
• NV: 89 – 134 IU/L

“So, don’t give up, don’t say when, and just get back on the road again.” – Hairspray 15