Clin Chem

Clinical Chemistry Laboratory Exam Reviewer
Dom Tancio, 2013 Page 1

SERUM AMYLASE DETERMINATION
Enzyme in a fixed amount of serum acts on a fixed amount of starch
under fixed conditions.
Amount of starch destroyed is measured by adding IODINE and
comparing amount of BLUE COLOR produced with the blue color of
an iodine treated central starch solution
REAGENTS:
o 0.85% NaCl
o Phosphate buffer pH 7, 0.02 Molar
o Stock Iodine solution (0.1N)
o Working iodine Solution
o 0.1% Starch Solution
PROCEDURE:
1. Prepare 1:10 dilution of SERUM or PLASMA (0.2 mL + 1.8 mL
0.85% NaCl)
2. Set up two tubes: TEST and CONTROL (both with 0.5mL buffer +
0.4 mL 0.1% starch solution)
3. Warm both tubes for 5 mins in 37C water bath
4. Add 0.1 mL serum dilution to TEST tube, return to bath for 15 mins
5. After 15 mins remove from water bath add 8.6 mL of ice cold
distilled water
6. Add 0.4 mL of 1/100 iodine solution mixing well by inversion
7. To the CONTROL add 0.1 mL serum dilution and mix
8. Read optical densities on SPECTROPHOTOMETER at 660-800 mu
or in a PHOTOELECTRIC COLORIMETER using a RED FILTER
setting zero pointin each case.
* READINGS MUST BE MADE within 5 mins AFTER
ADDITION OF IODINE WITHOUT DELAY!
CALCULATION:

* 1 unit amylase = amt enzyme needed to HYDROLYZE 10 mg
starch in 30 mins.
NORMAL VALUES:
60 160 Somogyi units/dL
111 296 U/L
ALKALINE PHOSPHATASE
METHOD: Liquicolor, Colorimetric Test
*optimized std method accdg to GCCA
REACTIONS:
p-Nitrophenylphosphate + H2O Phosphate + p-nitrophenol

REAGENT COMPOSITION
o R1: BUFFER (diethanolamine buffer, MgCl)
o R2: SUBSTRATE SOLN (p-Nitrophenylphosphate)
REAGENT PREPARATION AND STABILITY
o Procedure 1
- Ready to use
- stable till expiry date if stored at 2-8C
o Procedure 2
- 2mL of R2 into R1 mix
- stable for 4 weeks at 2-8C or 5 days at 15-25C
SPECIMEN: Serum/Heparinized Plasma
o Avoid Hemolysis
o LOSS OF ACTIVITY
- 7 days at 4C 0%
- 20-25C 10%
ASSAY
o WAVELENGTH: Hg 405nm or 400-420nm
o OPTICAL PATH: 1 cm
o TEMPERATURE: 25, 30, 37C
o Read against air (increasing absorbance)
o Warm reagent and cuvette till desired temperature
o CONSTANT temperature for the whole test duration

AP


PROCEDURE:
Mix and read absorbance after 1 min, start stop watch
Read absorbance again after 1.2.3 minutes
CALCULATION:
Calculate the mean of the readings
FORMULA:
o Using method 1: A X 3433
o Using method 2: A X 2757
CONVERSION FACTOR
o 1 U/l = 16.67 X 10
-9
kat/L = 16.67 X 10
-3

o 1 kat/l = 60U/L
LINEARITY:
if absorbance exceeds 0.250.. Dilute 0.1mL sample with 0.5 ml
saline and repeat assay using solution. Multiply result by 6
REFERENCE VALUES:
Temperature 25C U/L 30C U/L 37C U/L
Women 40-190 49-232 64-306
Men 50-190 61-232 60-306
Up to 15 Up to 400 Up to 488 Up to 544
Up to 17 Up to 300 Up to 366 Up to 483
QUALITY CONTROL
any control with glucose values, HUMATROL (animal based)
SERODOS (human based)
NOTES
o Buffer and Substrate Solution: w/ Sodium Azide DO NOT
SWALLOW and AVOID CONTACT (skin and mucous membrane)
o P-Nitrophenol is POISONOUS if inhaled, swallowed or absorbed in
skin. If comes into contact wash with water, consult doctor.

ACID PHOSPATASE
METHOD: Colororimetric Humazym Test
Orthophosphoric Monoester Phosphohydrolase (Acidic Optimum)
PRINCIPLE:
o Increaase of absorbance at 405 nm is PROPORTIONAL to
TOTAL ACID PHOSPATE ACTIVITY
o Prostatic acid phosphatase can be BLOCKED BY TARTRATE
and can be determined indirectly (non-prostatic Phosphatase) by
the activity difference.
REACTIONS:

1 Napthyl phosphate + H2O Phosphate + 1-naphthol
1 Naphthol + FRTR Azo dye
CONTENTS: BUF (Citrate), SUB, STAB
REAGENT PREPARATION:
1. Reagent RA
2. Dissolve one bottle (SUB) with 2 mL of BUF
3. Mark label with RA and date of preparation
STABILITY:
o Stable up to expiry date @ 2-8C
o Reconstitution:
- 5 days @ 2-8C
- 24 hours @ 15-25C (protected from light)
SPECIMEN: Serum (NO PLASMA)
o Avoid Hemolysis
o AVOID Bilirubin > 3mg/100mL
ASSAY
o WAVELENGTH: Hg 405nm or 400-420nm
Ac. P


PROCEDURE:
Mix and read absorbance (A1) after 5 mins, start stop watch
Read absorbance (A2) again after 3 minutes at 30C or 5 minutes
at 25C
CALCULATION:
A2-A1 = A
FORMULA:
o @25C: A X 149
o @30-37C: A X 248
CONVERSION FACTOR
o 1 U/l = 16.67 X 10
-9
kat/L = 16.67 X 10
-3
kat/L = 60U/L
REFERENCE VALUES:
o Men (U/L) 5.0
o Women (U/L) 4.2

SERUM GLUTAMIC PYRUVIC TRANSAMINASE (SGPT)
DETERMINATIONS
METHOD: Kinetic Method for determination of ALAT activity accdg
to Expert panel of IFCC
REACTION PRINCIPLE:

2-oxoglutarate + L-alanine L glutamate + pyruvate

Pyruvate + NADH + H L-lactate + NAD
CONTENTS:
o R1 (enzyme) TRIS buffer, L alanine, LDH, NADH
o R2 (starting) 2 oxoglutarate
REAGENT PREPARATION and STABILITY:
o Reagents ARE READY TO USE
o They are STABLE up to expiry date if stored PROTECTED FROM
LIGHT at 2-8C
o AVOID CONTAMINATION
REAGENT PROCEDURE:
2mL from R2 into R1 mix thoroughly
STABLE for 4 weeks at 2-8C and 5 days at 15-25C
SPECIMEN: Serum, Heparinized/EDTA PLASMA
o Avoid Hemolysis
o Loss of Activity
- 3 days @ 4C: 10%
- @ 20-25C: 17%
ASSAY
o WAVELENGTH: Hg 365nm, 340nm or Hg334nm
PROCEDURE:
Mix and read absorbance (A1) after 1 min, start stop watch
Read absorbance again after 1, 2, 3 minutes at 30C or 5 minutes
at 25C
SEMI MICRO METHOD for macro MULTIPLY VOLUMES
BY 2
CALCULATION:
o For absorbance changes per mins within 0.06-0.08 (hg365) or 0.12-
0.16 (hg334nm, 340nm) only measurements FROM THE FIRST 2
minutes are USED for calculation
Hg 334 nm 340nm Hg 365nm
U/L (25 and 30C) A/min X 971 952 1765
U/L (37C) = A/min X 1780 1745 3235
o CONVERSION FACTOR
- 1 U/l = 16.67 X 10
-9
kat/L = 16.67 X 10
-3

- 1 kat/l = 60U/L
LINEARITY:
o if absorbance exceeds 0.160 (Hg334nm/340nm) or 0.080 (Hg365nm)
or activity is higher than 150 U/L (25-30C) or 280 U/L (37C)
Dilute 0.1mL sample with 0.9 ml saline and repeat assay using
solution. Multiply result by 10
GPT
LDH


o sera with high activities = LOW ABSORBANCE due to
consumption of NADH RE RUN ON DILUTION
REFERENCE VALUES:
Men Up to 22 U/L Up to 30 U/L Up to 42 U/L
Women Up to 17 U/L Up to 23 U/L Up to 32 U/L
QUALITY CONTROL
any control with GPT values, HUMATROL (animal based)
NOTES
o R1 and Substrate R2: w/ Sodium Azide DO NOT SWALLOW and
AVOID CONTACT (skin and mucous memb)

SERUM GLUTAMIC OXALOACETIC TRANSAMINASE
(SGOT) DETERMINATIONS
METHOD: Kinetic Method for determination of ASAT activity accdg
to Expert panel of IFCC
REACTION PRINCIPLE:

2-oxoglutarate + L-asparatate L glutamate + oxaloacetate

Oxaloacetate + NADH + H L-malate + NAD
CONTENTS:
o R1 (enzyme) TRIS buffer, L aspartate, LDH, MDH,NADH
o R2 (starting) 2 oxoglutarate
o They are STABLE up to expiry date if stored PROTECTED FROM
LIGHT at 2-8C

REAGENT PROCEDURE:
2mL from R2 into R1 mix thoroughly
STABLE for 4 weeks at 2-8C and 5 days at 15-25C
SPECIMEN: Serum, Heparinized/EDTA PLASMA
o Avoid Hemolysis
o Loss of Activity
- 3 days @ 4C: 8%
- @ 20-25C: 10%
ASSAY
o WAVELENGTH: Hg 365nm, 340nm or Hg334nm
PROCEDURE:
Mix and read absorbance (A1) after 1 min, start stop watch
Read absorbance again after 1, 2, 3 minutes at 30C or 5 minutes
at 25C
SEMI MICRO METHOD for macro MULTIPLY
VOLUMES BY 2
CALCULATION:
o For absorbance changes per mins within 0.06-0.08 (hg365) or 0.12-
0.16 (hg334nm, 340nm) only measurements FROM THE FIRST 2
minutes are USED for calculation
Hg 334 nm 340nm Hg 365nm
U/L (25 and 30C) A/min X 971 952 1765
U/L (37C) = A/min X 1780 1745 3235
o CONVERSION FACTOR
- 1 U/l = 16.67 X 10
-9
kat/L = 16.67 X 10
-3

- 1 kat/l = 60U/L

GDT
LDH


LINEARITY:
o if absorbance exceeds 0.160 (Hg334nm/340nm) or 0.080 (Hg365nm)
or activity is higher than 150 U/L (25-30C) or 280 U/L (37C)
Dilute 0.1mL sample with 0.9 ml saline and repeat assay using
solution. Multiply result by 10
o sera with high activities = LOW ABSORBANCE due to
consumption of NADH RE RUN ON DILUTION
REFERENCE VALUES:
Men Up to 18 U/L Up to 25 U/L Up to 37 U/L
Women Up to 15 U/L Up to 21 U/L Up to 31 U/L
QUALITY CONTROL
any control with GOT values, HUMATROL (animal based)
NOTES
o R1 and Substrate R2: w/ Sodium Azide DO NOT SWALLOW and
AVOID CONTACT (skin and mucous membrane)

BILIRUBIN IN SERUM
REACTION PRINCIPLE:
Sulphanilic acid + NaNO3 DSA
Water Sol. Bilirubin Gluchorinades + DSA DIRECT Azobilirubin
Albumin Conj. Indirect Bilirubin + DSA + Accelerator
INDIRECT Azobilirubin
o RED AZO DYE absorbance at 456 nm
CONTENTS:
o TBR (white cap) : Sulfanilic Acid, HCl, Caffeine (accelerator),
Sodium Benzoate
o TNR (white cap): Sodium Nitrite
o DBR (red cap) sulfanilic acid, HCl
o DNR (red cap) NaNO3
o They are STABLE up to expiry date if UNOPENED and STORED
at 15-18C
ASSAY
o WAVELENGTH: 546 nm
o TEMPERATURE: 20-25C
o Read against BLANK
PROCEDURE:
o For Total
Mix and incubate for 5 mins
Mix and Incubate at room temp for 10-30 mins
Measure Absorbance against blank (1 drop = 40L)
o For Direct
Mix and incubate for 2 mins
Mix and Incubate at EXACTLY 5 mins
Measure Absorbance against blank (1 drop = 40L)
CALCULATION:
o Bilirubin conc (mg/dL) = A X 13.0
o [mg/dL] 17.1mol/L
REFERENCE VALUES:
Total Bilirubin [mg/dL] [mol/L]
At Birth up to 5 85.5
5 days up to 12 205.0
1 month up to 1.5 25.6
Adults up to 1.1 18.8
Direct Bilirubin
Adults Up To 0.25 4.3

QUALITY CONTROL
any control with Bilirubin values, HUMATROL (animal based)


NOTES
o Bilirubin reagent and nitrite reagent are THOROUGHLY MIXED
o Levels REDUCED IF EXPOSED TO SUNLIGHT
o Hemolysis LOWER VALUE due to INHIBITORY EFFECT of
haemoglobin on diazo reaction

PREGNANCY TEST
(One Step Pregnancy Test Strip)
INTRODUCTION
o Human Chorionic Gonadotropin is a glycoprotein hormone secreted
by DEVELOPING PLACENTA after fertilization
o Normal Pregnancy detected in SERUM
-as early as 7 DAYS
-doubles every 1.3 to 2 days
-reading 100 mlU/mL at FIRST MISSED MENSTRUATION
o Appearance right after conception and rise in early gestation makes
it an EXCELLENT MARKER for pregnancy detection
o OSPTS QUALITATIVE, SANDWICH DYE CONJUGATE
IMMUNOASSAY for detection of HCG
o Employs a unique combination of monoclonal and polyclonal
antibodies to identify hCG with HIGH SENSITIVITY
o <5 mins, high hCG as low as 25 mlU/ml is detected.
PRINCIPLE
o Sample diffuses through the test strip, the labelled antibody-dye
conjugate binds to hCG in the specimen forming an ANTIGEN-
ANTIBODY COMPLEX.
o (+) complex binds to the anti HCG antibody in the positive zone
producing a PINK ROSE COLOR BAND if concentration is
>25mlU/mL
o (-) no line on the positive zone. Mixture continues flowing past the
positive reaction and negative zone. Unbound conjugate binds to
negative zone producing pink rose band.
REAGENTS
o OSPTS
o Strip with POLYCLONAL ANTIBODY COATED MEMBRANE
o Pad with MOUSE MONOCLONAL IgG dye conjugate in protein
matrix with 0.1% sodium azide
STORAGE AND STABILITY
o Stored at room temperature below 25C and stable until expiration
date
o Do not store at hot temperature for a long period of time
PRECAUTIONS:
o IN VITRO USE ONLY
o Contains SODIUM AZIDE which reacts with LEAD or COPPER
PLUMBING (explosive azides). FLUSH LARGE AMT OF
WATER.
SPECIMEN COLLECTION:
o CLEAN DRY CONTAINER (plastic/glass) w/o preservative
o Collected ANYTIME but first morning urine contains HIGHEST
CONCENTRATION
o Maybe refrigerated and stored up to 72 hrs but must be at room
temp before testing
o Precipitates should be filtered
ASSAY PROCEDURE
o Remove test strip from wrapper
o Fill test tube/cup with sample and aspirate 0.5mL urine
o Place sample into hole at tip of test strip
o Read after 5 minutes
PERFORMANCE CHARACTERISTICS
o SENSITIVITY OSPTS detects hCG >25 mIU/ml
HEALTHY MEN/NON PREGANANT WOMEN,
UNDETECTABLE LEVELS OF hCG
(+) on FIRST DAY OF MISSED MENSTRUATION PERIOD
o SPECIFICITY
Cross reaction studies with (ALL NEGATIVE)
TSH (500mlU/mL)
FSH (1000 mlU/ml)
TSH (1000 mlU/mL)


urease
STANDARDIZATION
o Standardized by WHO first international reference preparation
INTERFERENCE TESTING:
o All were negative (Acetamenophen, Acetlysalicylic Acid, Ascorbic
Acid, Atropine, Caffeine, Gentesic Acid, Glucose, Hemoglobin)
BLOOD UREA NITROGEN
METHOD: Enzymatic Colorimetric Test
PRINCIPLE and REACTIONS:

urea + H2O NH4 + CO2
o Berthelot reaction
urea + hypochlorite +salicylate green dye
o Increase in absorbance at 578 nm is PROPORTIONAL to the UREA
CONCENTRATION
REAGENT COMPOSITION, PREPARATION, STABILITY
o R1:Phosphate, Na salicylate, Na nitroprusside, EDTA, urease
- 6 weeks at 2-8C
- 10 days at 15-25C
o R2:Phosphate, NaOH, NaHClO (irritates eyes and skin)
- 6 weeks at 2-25C
o R3: Urea standard
o ALL ARE READY TO USE
o IF UNOPENED LASTS TILL EXPIRY DATE
SPECIMEN:
o Serum/Plasma (dont use AMMONIUM HEPARINATE as
Anti-coagulant)
- NO LIPEMIC SERUM
- stored for 3 days at 4C
o Urine: dilute 1:100(water)

ASSAY
o WAVELENGTH: Hg 578nm
o TEMPERATURE: 20-25, 37C
o Read against BLANK
PROCEDURE:
Mix and incubate for 5 minutes @ 20-25C or 3 mins @ 37C
Add reagent 2
Mix and incubate for 10 minutes @ 20-25C or 5 mins @ 37C
Measure absorbance of sample and standard versus blank
CALCULATION:
o Serum/Plasma
C= 80

mg/dL OR 13.3

mmol/L
o Urine
C= 80.8

mg/dL OR 1340

mmol/L
LINEARITY:
o Serum/Plasma up to 400 mg/dL or 66.6 mmol/L
o Urine up to 400 g/L or 6660 mmol/L
o If higher dilute 1:1 with water, REPET ASSAY multiply to 2
REFERENCE VALUES:
o Serum: 10-50mg/dl or 1.7-8.3 mmol/L
o Urine: 20-35 g/24hr or 333-583 mmol/24hr
QUALITY CONTROL
any control with urea values, HUMATROL (animal based)
NOTES
o All Anticoagulants except Ammonium Heparinate can be used
o Not influenced by haemoglobin up to 200mg/dl and bilirubin up
to 10mg/dL
o Standard has Sodium Azide AVOID CONTACT
o Reagent 2 has NaOH and NaHClO which are IRRITANTS flush
with water


CREATININE DETERMINATION
(Jaffe Reaction)
METHOD: Photometric Colorimetric Test for kinetic Measurements
Method without Deproteinization
PRINCIPLE:
Creatinine + Picric Acid Creatinine-picrate complex
(orange colored compound)
o Done in ALKALINE SOLUTION
o Absorbance is proportional with concentration
REAGENT COMPOSITION: Picric Acid, NaOH STD
REAGENT PREPARATION:
Dilute NaOH with H2O in 1:4
Mix PIC and diluted NaOH 1:1
STD- ready to use
REAGENT STABILITY:
o NaOH Stable till expiry date at 15-25C
o Working Reagent: PROTECTED FROM LIGHT, 4 weeks @15-
25C
ASSAY
o WAVELENGTH: Hg 492nm (490-510)
o TEMPERATURE: 25C
o Read against AIR
PROCEDURE:
Mix and start stop watch after 30 seconds read A1
Read A2 after 2 minutes
CALCULATION:
o A2-A1=A
o Serum/Plasma
C= 2.0

mg/dL OR 176.8

mmol/L
o Urine
C= 80.8

mg/dL
o Creatinine Concentration in 24 hours urine
C= mg/dL X mL urine/24 hrs X 0.01 [mg/24h]
C= mg/24h X 0.00884 [mmol/24h]
o Creatinine Clearance:

[ml/min]
LINEARITY:
o Serum/Plasma up to 13 mg/dL or 1,150 mmol/L
o Urine up to 500 mg/L or 44,200 mmol/L
o If higher dilute 1:5 physiological saline. REPEAT ASSAY, X 6
REFERENCE VALUES:
Serum (mg/dL) (mol/L)
Men 0.6-1.1 53-97
Women 0.5-0.9 44-80
Urine 1000- 1500 mg/24 hrs
Creatinine Clearance
Men 98-156 ml/min
Women 95-160 ml/min

QUALITY CONTROL
any control with creatinine values, HUMATROL (animal based)
AUTOMATION: available on request
NOTES
o Highly Sensitive to TEMPERATURE, must be CONSTANT
@ 25C
o PIC is harmful if inhaled, swallowed or in contact
o Assay affected by REDUCING COMPOUNDS, eliminated by
BOILING URINE shortly
o Slight Precipitate in NaOH is insignificant



ICTERUS IN BLOOD
(Meulengracht 1932)
PRINCIPLE/INTRODUCTION:
o Measures GOLDEN YELLOW COLOR imparted to SERUM by
BILIRUBIN
o Comparing serum color with color of standard solution by
DICHROMATE
o I unit icterus index = 1:10000 soln K2Cr2O7
o Proteins in blood are precipitated with ACETONE and YELLOW
COLOR IMPARTED to the acetone by bilirubin is measured
PHOTOMETRICALLY with K2Cr2O7 as std.
o With this technique HEMOLYSIS and LIPEMIA do not interfere
REAGENTS/ MATERIALS
o SPECIMEN: Unhemolyzed serum (1 mL)
o REAGENTS:
- 5% Sodium Citrate Soln
- Standard (0.01%) KCr2O7
Dissolve 0.1 g KCr2O7 in water + 2 drops conc H2SO4
dilute to 1 L. Eq. Index of 10
PROCEDURES:
1. Test tube with 1 mL unhemolyzed serum + 9mL 5% Na Citrate
2. In another tube + 3 mL 5% Na Citrate (label as B)
3. Wavelength pf 420-460 nm set Optical Density of tube Blank
4. Replace blank tube with sample and read OD scale
5. Prepare another blank + 3 mL water to a new test tube
6. Repeat 3 for this new blank
7. Replace blank in #6 with tube in #2 read OD
CALCULATION:
Icterus Index =

NOTE:
o Limit of accuracy is an ID =50.
o If Higher, greater dilution os sample (#1) is needed
o If this happens substitute 20 to 10 in the formula
SUGAR IN URINE QUANTITATIVE
PRINCIPLE:
Urinary sugar + alkaline NaCO3 + heat Yellow to dark amber
complex
o Measured in SPECTROPHOTOMETER
o Proportional to amount of sugar in urine
REAGENTS/MATERIALS:
o 10% NaCO3
o Standard: 10% glucose solution
o Specimen: 24 hr urine specimen
PROCEDURE:
1. Filter 10-15 mL of well mixed 24 hr urine specimen
2. Into 2 FOLIN WU SUGAR TUBES place the ff:
U B
5mL 10% Na2CO3
1mL filtered urine
5mL distilled water
1mL filtered urine
3. Mix both by inversion
4. Place in boiling water bath for 7 mins
5. Dilute each to 25mL mark with distilled water. Mix by inversion
6. Read %T of U against B at wavelength of 500 mu
STANDARDIZATION
1. Prepare 10% glucose solution
2. Into 6 test tubes, place the following
Tube
glucose
soln
Distilled
Water
Conc of std
in %
B 0 10
1 1 9 1
2 2 8 2
3 3 7 3
4 4 6 4
5 5 5 5
3. Considering B as blank and tubes 1-5 as unknown treat these stds
as urine specimen and carry out steps 2-6 under procedure
4. Plot %T of O.D. against concentration


CALCULATION
o The amount of sugar (g%) in urine is obtained directly from the
calibration curve.
PROTEINS IN URINE QUALITATIVE
METHOD: Sulfosalicylic Acid Method
REAGENTS/MATERIALS:
o 3% Sulfosalicylic Acid
o 10% Acetic Acid
PROCEDURE:
1. Filter specimen
2. If urine is alkaline or neutral, add 10% acetic acid drop by drop until
it is ACID
3. Place 2mL clear urine in a tube
4. Add 2mL sulfosalicylic acid and mix. FORMATION OF WHITE
PRECIPITATE is POSITIVE
5. Report as:
(-) urine remains CLEAR
(+) faintly VISIBLE TURBID
When observed against dark background (<10mg/100mL)
Value Turbidity Amount (mg/100mL)
1+ Definite turbidity 10-50
2+ Heavy turbidity but NO flocculates 50-200
3+ Heavy cloud W/FLOCCULATION 200-300
4+ Heavy cloud W/HEAVY FLOCCULATION Above 500
6. IF specimen not cleared b y centrifugation, DILUTE 2mL urine with
2 mL water and compare with sulfosalicylic acid-urine mixture. (+)
SHOWS A DISTINCT DIFFERENCE.
UROBILINOGEN IN URINE
PRINCIPLE:
Urobilinogen + Ehrlich reagent RED COLOR
(p dimethyl amino benzaldehyde in HCl)
* BaCl2 removes BILE PIGMENTS

MATERIALS:
o Fresh urine
o Filter Paper
o REAGENTS
- 100mL 10% BaCl2
- Ehrlichs Reagent (p dimethylamnobenzaldehyde, HCl, H2O)
PROCEDURE
1. Fresh urine + 2mL 10% BaCl2 in test tube, mix and filter
2. 5mL filtrate + 1 mL Ehrlichs reagent mix
3. Stand 3-5 mins
4. Look solution from top to bottom against white background
5. RED COLOR (+) for UROBILINOGEN
BILE PIGMENTS IN URINE
METHOD: Fouchets Method
PRINCIPLE:
BaCl2 + SO4 BaSO4 precipitate
o BaSO4 absorbs Bile Pigments
o Wash precipitates
BaSO4 precipitate + FeCL3 in TCA soln (+) GREEN- BLUE
DERIVATIVE
REAGENTS:
o 10% BaCl2
o Fouchets Reagent (10% FeCl2, TCA, DW grad)
o Filter paper
PROCEDURE
1. 10mL urine + 5mL 10% BaCl2, Mix and Centrifuge
2. Decant supernate + 5 mL D/W Mix and Filter
3. Place filter paper on dry paper
4. + 2 drops Fouchets reagent to precipitate
(+) GREEN-BLUE color



CHOLINESTERASE IN BLOOD
(Determination according to ELLMAN)
EQUIPMENT:
o Laboratory Centrifuge
o Photometer
BLOOD SAMPLES
o Cleanse ear/finger pad with ETHANOL
o Collect blood in 10L disposable micropipettes
o Wipe both ends of pipette and place in a reaction vessel containing
1.0 mL of 0.9% NaCl solution
o Close vessel immediately and shake blood out of pipette
o Centrifuge vessel
o Siphon off the clear supernatant and store in a cool place (Serum-
CHE)
o Wash blood cake 2x with NaCl, centrifuge it and separate it from
supernatant (Ery-CHE)
o Both samples can be refrigerated and stored for several days
PREPARATION OF SOLUTION
o Use DOUBLE DISTILLED WATER which has been BOILED for 5
mins and COOLED
1. 0.1M Phosphate Buffer, pH 7.5
- Dissolve 13.6g KH2PO4 in 900 mL water and adjust solution
to pH 7.5 with KOH.
2. NaCl solution
- Dissolve 9g NaCl in 1000 mL water
3. Saponin Solution
-Dissolve 10 mg saponin in 100 mL water. Prepare fresh daily
4. S- acetylthiocholine iodide (Athi)
- Dissolve 225 mg S-acethylthiocholine iodide in 5mL water
(good for a week)
5. 5,5- Dithiobisnitrobenzoic acid (DTNB)
- Dissolve 20 mg DTNB in 100 mL Phosphate buffer. Prepare
fresh daily

METHOD:
* both are measured KINETICALLY with aid of a simple
PHOTOMETER or an AUTO ANALYZER
o Serum CHE
- 1500l DTNB buffer into 1 cm cuvette
- 100l DTNB buffer + 100l NaCl supernatant +50l
acetylthiocholine iodide solution.
- Measure for 1 min at 25C
o Ery CHE
- Hemolyze Blood Cake with 1000l saponin
- 1500l DTNB solution + 50l of hemolyzate + 50 l
acetylthiocholine
- Measure for 1 min at 25 C
CALCULATION
o Serum CHE
Dilution factor from 10l blood in 1000l NaCl
LIMIT: 0.90 U/mL
E 124 = U/mL

o Ery CHE
Factor 4 results from additional dilution of human
erythrocytes for determination of autoanalyser
LIMIT: 0.95 U/mL
E 60.2 = U/mL



SALICYLATES
ANALYSIS
Serum + acid soln FeNO3+ MgCl (Trinders) Violet supernatant
o Centrifuge the mixture and measure the absorbance at 540 nm
EQUIPMENT
o Spectrophotometer/photometer with 540 nm filter
o Centrifuge
REAGENTS
o Ferric Nitrate (Trinders reagent)
- Dissolve 40 g HgCl2 and 40 g Fe(NO3)39H2O in 900 mL water
- + 10 mL conc HCl, dilute to 1L with water.
- Filter with whatman #1 before use, Store at room temperature.
o Salicylic Acid, Stock Std, 200 mg/dL
- Dissolve 580 mg sodium salicyclate in water
- dilute to 250 mL + chloroform, refrigerate
- stable for 2 months
o Salicylic Acid, working ref soln
- Dilute 0.5, 1.0, 2.0 and 3.0 stock std to 10.0mL with water
- provides stds of 10,20,40,and 60 mg/dL respectively
- Refrigerate
PROCEDURE:
Tube 1 Tube 2
2 mL reagent
0.2 mL serum
2 mL reagent
0.2 mL water
1. Mix well, FLOCCULENT PRECIPITATE will be formed
2. Centrifuge for 10 minutes and decant supernatant into cuvette.
Salicylates will YIELD A VIOLET COLOR
3. Read Absorbance against blank at 540 nm
4. Plot absorbance of standards against their concentration and
determine concentration based on standard curves.
COMMENTS:
o Use of Plasma
Compounds can react with ferric salts to yield colored products
but they do not interfere when plasma is used
Nonspecific chromogens provide background as high as 4mg/dl.
Levels <4 mg/dl should be reported NEGATIVE
Phenothiazines do not reach high concentration to interfere
High concentrations of ACETOACETATE may produce
slight interference
o Use of Urine
MORE LIKELY TO CONTAIN INTERFERENCE (like
phenothiazines)
Analysis with salicylates is LIMITED
Interferences can be eliminated if if extractiuon procedure is
used PRIOR to reaction with Ferric salt.
Should be used if desired accuracy in range <5mg/dL of plasma
or if analysis of urine is required.
INTERPRETATION
o >30mg/dL EVIDENCE OF TOXICITY (tinnitus)
o Treatment of RHEUMATIC DISEASES <30mg/dL
o Concentration 20-30 mg/dL are REQUIRED
o >100 mg/dL FATAL
o Concentrations achieved after usual single HEADACHE DOSE of
650 mg may NOT BE DETECTABLE or directed towards detecting
low levels resulting from occasional salicylate use.

Clin Chem

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Clinical Chemistry Laboratory Exam Reviewer

Dom Tancio, 2013 Page 1

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