Manual On Standard Operation Procedures, Sample Collection and Reference Ranges For Clinical Chemistry

MANUAL ON
STANDARD OPERATION PROCEDURES, SAMPLE COLLECTION AND

REFERENCE RANGES FOR
CLINICAL CHEMISTRY
WORLD HEALTH ORGANISATION,

MINISTRY OF HEALTH
AND
THE DEPARTMENT OF BIOCHEMISTRY, MEDICAL RESEARCH INSTITUTE
SRI LANKA
MANUAL ON
STANDARD OPERATION PROCEDURES, SAMPLE COLLECTION
AND REFERENCE RANGES FOR
CLINICAL CHEMISTRY
Dr. Meliyanthi M. Gunatillaka

Consultant Chemical Pathologist and Head, Department of Biochemistry
Medical Research Institute, Colombo
Ms. D. K. Daya Silva

Superintendent Grade Medical Laboratory Technologist
Mr. M. Muhammed Hunais

Medical Laboratory Technologist
ISBN 978-955-1647-00-1
This document is NOT for sale.
The document may, however, be freely reviewed, abstracted, reproduced or translated, in
part or in whole for non commercial purposes.
i
CONTENTS
Contents ............................................................................................................................................i
Acknowledgements.........................................................................................................................ii
Preface.............................................................................................................................................iii
General introduction .....................................................................................................................iv
1. Format of a technical procedure manual ............................................................................1
2. Albumin ..................................................................................................................................3
3. Amylase ...................................................................................................................................7
4. Alkaline phosphatase.......................................................................................................... 10
5. Aspartate amino transferase .............................................................................................. 13
6. Alanine amino transferase.................................................................................................. 18
7. Bilirubin................................................................................................................................ 21
8. Calcium ................................................................................................................................ 27
Calcium in urine .................................................................................................................. 31
9. Creatinine............................................................................................................................. 32
10. Urine Creatinine ............................................................................................................. 35
Creatinine clearance........................................................................................................ 36
11. Cholesterol ...................................................................................................................... 37
12. Glucose............................................................................................................................ 40
13. Inorganic phosphate ...................................................................................................... 46
14. Inorganic phosphate in urine ........................................................................................ 49
15. Total protein ................................................................................................................... 50
16. Urea.................................................................................................................................. 54
17. Uric acid .......................................................................................................................... 58
18. Urine uric acid................................................................................................................. 61
19. Electrolytes...................................................................................................................... 62
20. Urine sodium and potassium ........................................................................................ 67
21. Appendix 1 - Sample Collection and Transportation ............................................. 68
22. Appendix 2 - Diabetes mellitus ................................................................................. 71
23 Appendix 3 - Reference ranges.................................................................................. 73
References: .................................................................................................................................... 94
Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

ii
ACKNOWLEDGEMENTS
We would like to acknowledge the WHO representative to Sri Lanka, Dr. Kan
Tun, for identifying the need for quality assurance in local laboratories and offering
us the opportunity to publish this handbook.
We thank the Director General of Health Services Dr. Athula Kahandaliyanage,

the Deputy Director General (Planning) Dr. T. S. B. Tennekoon and the Deputy
Director General (Education, Training and Research) Dr. Stanley De Silva, Deputy
Director General (Laboratory Services) Dr. Ajith Mendis and Director Laboratory
Services Dr. Jayasundara Bandara, for approval and facilitation of this project.
We are grateful to the Director of the MRI Dr. G. S. S. K. Colombage and the
Deputy Director of MRI Dr. Lulu Raschid, for all their support and
encouragement in bringing this project to fruition.
We appreciate the assistance of administrative staff of World Health Organisation

and colleagues, resource persons, administrative staff of the Medical Research
Institute and staff of the Department of Biochemistry.

iii
PREFACE
Clinical laboratory services have become an important component of modern

medicine. Clinical laboratories play a major role in the diagnosis, treatment,
prognosis and monitoring of diseases. Quality assurance in laboratory services,
aimed at improving reliability, efficiency and facilitating inter – laboratory
comparability in testing is the backbone of quality health care delivery. The use of
standard operating procedures in laboratory testing is one of the major factors in
achieving quality. This manual provides guidelines on standard operating
procedures, sample collection and reference ranges for routine biochemical
methods. The publication aims at popularising the use of biochemical methods
recommended by the world health organization, which may be adapted to local
needs, based on the experience of the editors. It is hoped that this publication will
be useful in achieving its objective of improving the quality of laboratory results
using the available resources.
.

iv
GENERAL INTRODUCTION
Standard Operating Procedure (SOP) manuals are required in the laboratory and
are a key element in internal Quality Control within the laboratory. Hence, assuring
quality health care, method manuals should include properly authenticated
methods, or methods recommended by relevant professional organisations. A
methods manual containing all methods and procedures needs to be authorised for
use in the laboratory. The manuals shall be available in the appropriate work areas
as bench copies and it is recommended that a master copy be maintained by the
head of the unit. These manuals should be review at least annually, by the
competent member of the technical staff and the head of the unit.
The first consideration is the selection of the test methods best suited to full fill the
function of the laboratory. Each test performed in the laboratory (for e.g.
screening, routine clinical testing, reference laboratory service) must be evaluated
and appropriate methods should be selected depending on the local needs and
resources. Each method should be evaluated in terms of sensitivity, specificity,
accuracy, simplicity, speed, reliability and economy.
The methods that are included in this manual are based on world health
organization recommended biochemical tests, adapted and evaluated by the editors
taking into consideration the clinical and technical experience, quality control
measures and the available resources. This standard operating procedure manual
includes the routine biochemical tests based on manual spectrophotometric
methods. The clinical significance of each analyte has been described in detail for
the benefit of the users. The precautions to be followed are included for each
analyte to maximize the performance. This is based largely on the experience of the
editors.
The manual contains the collection procedures of blood and urine samples. It
includes the method of collection, selection of containers and preservatives,
storage, transport and stability. There must be vigorous control of the procedures
involved in the collection and identification of specimens to ensure the quality of
the specimen to be examined in the laboratory. Copies of collection procedures
should be available at all collection areas. The collection manual should be updated
regularly as for laboratory procedure manual by the competent member of the
technical staff.
Specimen which do not conform to the requirements or are of inadequate quality

for the test involved may be rejected with the consultation of the head of the unit
and the sender informed. Appropriate techniques shall be used for the correct
identification of specimens and release of results. Specimens shall be retained by
the laboratory for a time appropriate to the nature and origin of the specimen.
The manual includes a section for reference ranges. The reference ranges are given
for many biochemical analytes considering the age and the sex. Standard adult and
paediatric text books along with the information available on the internet were
taken into consideration in accepting the relevant reference ranges.

1
1. FORMAT OF A TECHNICAL PROCEDURE MANUAL
The following format for technical procedures should be adhered to as closely as possible
and double spaced typing is recommended.
TITLE
Name of test or procedure.
INTRODUCTION
A brief, concise introduction of the analyte is recommended.
(In this manual a detailed description is included for the benefit of the user to prepare their
own local S.O.P)
CLINICAL SIGNIFICANCE
Give reasons as to why the test is usually requested and indicates the significance of low or
high results using the reference
PRINCIPLE
A short but informative statement which describe the basis of the method.
REFERENCE(S)
A list of primary reference(s) as well as modifications.
SPECIMEN
List the types of specimens which are appropriate and indicate stability limits if known
GLASSWARE AND EQUIPMENT

A detailed description of required glassware and equipment should be provided.
REAGENTS
State concentration or use descriptive name where appropriate (e.g. Biuret reagent) and
give detailed stepwise instructions for preparing each reagent. Specify chemical brand and
grade where it is known to be critical. Unusual reagents/components should have source
or company stated. Expiry of reagents should be specified.
PROCEDURE
Present a concise stepwise description of the method.
CALCULATIONS
Explain the use of formulae and indicate, briefly, the derivation of factors.
UNITS
Units used and abbreviations.
REFERENCE RANGE
Adult reference ranges for males and females and paediatric ranges where applicable.
QUALITY CONTROL
Quality control procedures, including documentation and methods of statistical analysis
NOTES
Elaborate on any particular points which may require further explanation

2
APPROVAL
Each procedure to be dated and signed at the bottom of the final page by the appropriate
senior authorised qualified staff. Any amendments should be authorised with date and
signature.
METHOD HISTORY
This should be in a separate sheet and include date notations of method changes and
review
REPORTING
Format and procedure of reporting, including procedures for urgent and especially
clinically significant results, must be described.
COPIES
Any copies of method for bench use are to be made in full from the master copy and
should include method history and approval sections
SUPPLEMENTARY INFORMATION
Certain information to be used at the bench to implement procedures may be extracted
from the procedure manual. This information may include flow diagrams, index cards, and
manufacturer product literature. Such supplementary information must be current and be
referenced to the procedure manual by date, procedure and reviewer’s initials.

3
2. ALBUMIN
2.1 INTRODUCTION
Albumin is the most abundant protein in human plasma from 20 weeks of gestation,
representing 40-60% of the total protein. It is synthesized exclusively in the liver. The rate
of synthesis is depended on protein intake and subject to feed back regulation by the
plasma albumin level. The half life of albumin is estimated at 15-19 days. Traces of
albumin can be found in almost all extra vascular body fluids. The loss of albumin via the
glomerular filtrate is very small as almost all the albumin is reabsorbed by the proximal
tubular cells. Albumin is catabolized in various tissues where it is taken up by cells by
pinocytosis. Its constituent amino acids are released by intracellular proteolysis and
returned to the body pool. Albumin has a molecular weight of approximately 66,000.
Albumin is an anion at pH 7.4 with more than 200 negative charges per molecule. The
chief biological functions of albumin are to transport and store a wide variety of ligands, to
maintain the plasma osmotic pressure and to serve as a source of endogenous amino acids.
The capacity of albumin to act as a binding protein is due to the large numbers of charges
of each molecule as well as very large no of molecules available. Albumin binds nonpolar
compounds such as bilirubin & long chain fatty acids. Albumin binds hormones such as
thyroxine, triiodothyronin, cortisol and aldosterone, thus act as a reservoir in which these
compounds are stored in inactive form but from which they are readily mobilised. Some
40% of serum calcium is bound to albumin. Many drugs such as phenylbutazone, warfarin
and salicylates are also strongly bounded to albumin. Albumin concentration is the major
determinant of plasma oncotic pressure, one of the factors that regulate partition of water
between intra and extra vascular compartments.
2.2 CLINICAL SIGNIFICANCE
 Hypoalbuminaemia is very common and may result due to the following factors
o Impaired synthesis: Diminished protein intake or liver disease
o Increased catabolism: Due to tissue damage and inflammation
o Reduced absorption of amino acids: Malabsorption syndromes or malnutrition.
o Protein loss in urine: Due to nephrotic syndrome,chronic glomerular nephritis,
diabetes,or systemic lupus erythromatosis
o Protein loss in faeces: Due to protein losing enteropathy
o Protein loss through the skin: Burns
o Altered distribution: Ascites(high pressure in the portal circulation drives albumin
into the peritoneal fluid)
Most severe hypoalbuminaemia is caused by protein loss by way of urine or faeces. When
plasma albumin levels are less than 2.0 g/l oedema is usually present.
 Hyperalbuminemia: is of little diagnostic significance except in dehydration.
Albumin has more than 20 genetic variants, which are not associated with disease but
which cause two bands or a single band in the albumin region on electrophoresis. The
condition is called bisalbuminaemia. A transient form is sometimes caused by the intake of
drugs. Congenital absence of albumin or analbuminaemia is asymptomatic except for
occasional slight oedema.
2.3 PRINCIPLE OF THE METHOD
The requirements of a dye binding method for albumin include specific binding of the dye
to albumin in the presence of other plasma or serum protein, high binding affinity between
dye and albumin so that small changes in ionic strength, pH or the presence of competing

4
ligands do not break the dye-protein complex; a substantial shift in the absorption
wavelength of the dye in the bound form so that it remains spectrally distinct from the free
form present in excess, and absorption maximum for the bound form at a wavelength
distinct from those at which Bilirubin and haemoglobin can interfere. Serum albumin and
buffered BCG (Bromocresol green) are allowed to bind at pH 4.2, and absorption of the
BCG/Albumin complex is determined spectrometrically at 632 nm (filter No 607)
Albumin act as a cation to bind the anionic dye. Absorption reading taken within 30
seconds of mixing the serum and BCG avoids the problem of non specific reaction of
BCG with globulin. The manual method described here for albumin by BCG is adaptable
to automated analysis.
2.4 SPECIMEN TYPE, COLLECTION AND STORAGE
Serum is preferred. Fasting specimen is not an absolute requirement but it may be desirable
because marked lipaemia interferes in the assay. Avoid, applying a tourniquet in specimen
collection because haemoconcentration due to venous stasis increases the apparent
concentration of albumin and other plasma proteins. Storage: Separate the serum within 2
hours of collection; separated serum in a tightly stoppered container is stable for 24 hours
at room temperature 25-30 0 C, for 1 week at 2-8 0C, for 3 months at -20 0C.
APPARATUS AND CHEMICALS

APPARATUS:
Visible spectrophotometer, wavelength 632 nm or Colorimeter, orange filter, Ilford 607
(600nm)
pH meter
GLASSWARE:
Volumetric flasks (1 litre and 100 ml volumes)
Micro pipette (20 µl)
Graduated pipettes (10 ml in 0.1 ml)
Beakers (5ml, 50ml, 500 ml and 1 litre)
Amber colour reagent bottles (1 litre)
Test tubes (125 mm x 16 mm)
Measuring cylinders (1 litre)
CHEMICALS:
(All chemicals must be analytical grade)
Bromocresol green sodium salt, also called BCG, water soluble
Sodium azide caution: handle with care
Sodium chloride
Succinic acid
Sodium hydroxide pellets
Brij-35 (polyoxy7ethylene (23) lauryl ether) solid or solution 30% w/v
Standard buffers for pH meter
Bovine albumin or other available calibrator (fraction v powder)
REAGENTS
1. Succinic acid solution 50 g/l: weigh out 1.0 g of Succinic acid, dissolve and make up to
about 20 ml with distilled water. Prepare as required, discard after use.
2. Sodium hydroxide solution 10 g/l: Weigh out 1.0 g of sodium hydroxide in a glass beaker,
dissolve and make up to 100 ml with distilled water. This solution is stable for several
months at 20-25 C. Store in polypropylene bottle.

5
3. Brij-35 solution 250 g/l: Warm 25 g solid Brij-35 in a small volume of distilled water to
dissolve and make up to 100 ml with distilled water or use the 30 % w/v Brij-35
solution.
4. Working dye solution: Dissolve 5.6 g of Succinic acid, 58 mg of Bromocresol green
(Sodium salt) and 100 mg of sodium azide in about 900 ml of distilled water in a clean
beaker. Add 1.0 g of sodium hydroxide and dissolve, and then add 2.5 ml of Brij-35
250 g/l solution. (If 30% Brij solution is used add 2.1 ml)Adjust to pH 4.2 using small
volumes of sodium hydroxide solution or Succinic acid solution if necessary. Transfer
slowly (avoid frothing) to 1 litre volumetric flask and make up to volume with distilled
water, mix gently and transfer into a clean brown bottle. This solution is stable for
several months at 2-8 0C.
NOTE: The BCG working dye solution requires careful preparation. Some laboratories
may find it economical to purchase this solution. There are several different BCG reagents
available. Make sure you select a BCG reagent in Succinate buffer at pH 4.2.
In 10 mm light path cuvette (cell) the working dye solution should have the following
absorbance readings (zero the instrument with distilled water).
Spectrometer Colorimeter
Wavelength Absorbance Filter Absorbance
430 nm About 1.4 601 About 1.1
615 nm About 0.25 607 About 0.2
5. Succinate buffer solution: Prepare in exactly the same way as the working dye solution but
do not add any Bromocresol green. This solution is stable for several months at 2-8 0C
6. Albumin standard 40 g/l: Using a 5ml volumetric pipette dilute 5.0 ml of the bovine
albumin standard (80 g/l) with 5.0 ml of sodium chloride/sodium azide solution to
prepare an albumin standard containing 40 g/l. This standard is stable for 6 months at
2-8 0C. (Bovine albumin standard 80 g/l provide by the Department of Biochemistry,
MRI)
7. Sodium chloride /Sodium azide solution: Weigh out 9.0 g of sodium chloride and 1.0 g of
sodium azide, dissolve and make up to 1 litre with distilled water. This solution is
stable indefinitely at 20-25 0C (room temperature)
2.5 PROCEDURE
1. Pipette 4.0 ml of working dye solution into test tubes.
2. Add 20 µl of standard or control or test sample, mix and measure the absorbance
immediately (within 30 seconds).
3. Read the absorbance at 632 nm or filter No 607 after setting the instrument to zero
absorbance with the working dye solution.
PREPARATION OF CALIBRATION GRAPH

Preparation of working albumin standard solutions: They are prepared by dilution of the
albumin standard (40 g/l) in sodium chloride/sodium azide solution as follows:
Working standard No (01) (02) (03) (04)

Albumin standard 40 g/l (ml) 0.5 )1.0 1.5 2.0
Sodium chloride/Sodium azide solution (ml) 1.5 1.0 0.5 0.0
Concentration of working standards (g/l) 10 20 30 40

6
Working dye solution (ml) 4.0 4.0 4.0 4.0
Working standard No (20 µl) (01) (02) (03) (04)
Mix each tube thoroughly )
Read the absorbance of each tube immediately at 632 nm (or filter No 607) after setting
the instrument to zero with working dye solution.
Plot the absorbance of each tube against the concentrations of working standards solution
on the graph. The calibration graph should be linear up to 40 g/l. If the graph is linear then
a single standard (40g/l) may be used for routine analysis but linearity should be confirmed
for each new batch of working dye solution and at least once a month.
The recommended method proposes the use of a bovine albumin solution as a calibrator.
Alternative commercial bovine albumin solutions are available.
2.6 CALCULATION
Albumin concentration = T/S x 40 g/l
Where T=the absorbance of the test

S=the absorbance of the standard
NOTE: Always include a standard (40 g/l) in each and every batch of samples.
QUALITY CONTROL
OPTIMAL CONDITIONS VARIANCE : A coefficient of variation of around 3%
should be attainable.
ROUTINE CONDITIONS VARIANCE : The value obtained for the RCV should not
exceed 6%
REFERENCE VALUES
New born : 25-50 g/l
1 Year : 35-50 g/l
2-3 Year : 36-50 g/l
4th Year and after: 37-50 g/l
Adult : 30-45 g/l
2.7 LIMITATIONS
If a serum sample is extremely lipaemic a serum blank should be used. (Moderate lipaemia
does not affect the results) The blank is prepared by adding 20 µl of sample to 4.0 ml of
Succinate buffer solution. The absorbance of this blank, with distilled water as reference is
subtracted from the test. Grossly haemolysed specimens are unsuitable for albumin
determination.
REFERENCES
Ann.clin.Biochem.14 (1977)105-115
Tietz text book of clinical chemistry

7
3. AMYLASE
3.1 INTRODUCTION
Amylases are a group of hydrolases that split complex carbohydrates constituted of α-D-
glucose units linked through carbon atoms 1 and 4 located on adjacent glucose residues.
Both straight-chain (linear) polyglucans, such as amylose, and branched polyglucans, such
as amylopectin and glycogen, are hydrolyzed, but at different rates. In the case of amylose,
the enzyme splits the chains at alternate α-1, 4-hemiacetal (-C-O-C-) links, forming maltose
and some residual glucose; maltose, glucose, and a residue of limit dextrins are formed if
branched –chain polyglucans are used as substrate. The α-1, 6- linkages at the branch
points are not attacked by the enzyme. Two types of amylases are recognized. Beta-amylase
(e.g. plant and bacterial exoamylase) acts only at the terminal-reducing end of polyglucan
chain; it splits off two glucose units (maltose) at a time. Animal amylases, including those
present in human tissues, are α-amylases. They are also called endoamylases because they
attack α-1, 4-linkages in a random manner anywhere along the polyglucan chain. Linear
starch chains in helical form react with molecular iodine to form the well-known deep blue
starch-iodine complex. The enzyme present in normal serum and urine is predominantly of
pancreatic (p-type) and salivary gland (S-type) origin. These isoenzymes are products of
two closely linked loci on chromosome 1. Each gene is allelic; thus there are 12 distinct
phenotypes for the salivary isoenzyme and 6 for the pancreatic isoenzyme.

Assays of amylase activity in serum and urine are largely of use in the diagnosis of diseases
of the pancreas. In acute pancreatitis a transient rise in serum amylase activity occurs in 2-
12 hours of the onset; levels return to normal by the third or the fourth day. A four to six
fold elevation in amylase activity above the reference limit is usual, with maximal levels
attained in 12 to 72 hours. The magnitude of the elevation of serum enzyme activity is not
related to the severity of pancreas involvement. However the greater the rise, greater the
probability of acute pancreatitis. A significant amount of serum amylase is excreted in urine
and therefore elevation of serum activity is reflected in the rise of urine amylase activity.
Urine amylase as compared with serum amylase appears to be more frequently elevated,
reaches higher levels and persist for longer periods. In quiescent chronic pancreatitis both
serum and urine activity are usually subnormal. Acute pancreatitis is sometimes difficult to
diagnose because it must be differentiated from other acute intra abdominal disorders and
because an increase serum amylase activity may not necessary due to acute pancreatitis.

In solution iodine reacts with starch to give an intense blue – violet complex. Amylase
hydrolyses starch, forming maltose and other fragments which do not react with iodine.
After incubation of serum with buffered starch solution, the amount of starch remaining is
assayed by measuring the absorbance at 660 nm after the addition of iodine

3-5 ml of clotted blood in a clean dry bottle; avoid haemolysis; Separate serum as early as
possible. Enzyme activity loss is negligible in sterile serum stored at 2-8 0C for a week (free
of bacterial contamination).
3.5 APPARATUS AND CHEMICALS

APPARATUS:
Hot plate or Bunsen burner
Water bath at 37 0C, pH meter
Visible Spectrometer wavelength at 660nm or Colorimeter with red filter Ilford No: 608
(680 nm)

8
GLASSWARE: CHEMICALS:
Volumetric flasks (1litre volume) Soluble starch pharmaceutical grade
Automatic micro pipette 20 µl Potassium iodide AR
Graduated pipettes (1ml, 5ml, 10 ml Potassium iodate AR
in 0.1 ml) Disodium hydrogen ortho-
Beakers (50ml, 1 litre) phosphate (anhydrous) AR
Graduated cylinders (100 ml and 1 Sodium chloride AR
litre) Benzoic acid AR
Amber colour reagents bottles (100 Hydrochloric acid (concentrated (37
ml and 1 litre) % w/v) caution: highly corrosive)
Buffers for pH meter
REAGENTS
1. Buffered starch substrate: Dissolve 26.6 g of anhydrous disodium hydrogen phosphate,
1.75 g of sodium chloride and 8.6 g of benzoic acid in about 500 ml of distilled water
in a large beaker. Heat to boil. In a 50 ml beaker, mix separately 0.4 g of soluble starch
in 10 ml of cold distilled water to form a paste. Add the paste with stirring to the
boiling mixture, rinsing the beaker with distilled water. Continue to boil for one
minute. Cool to room temperature, and transfer to volumetric flask and dilute to 1 litre
with distilled water. This solution is stable for at least one year at 20- 25 0C and should
have a pH of 6.9-7.1; the stability is monitored by noting the absorbance of the reagent
blank with each set of tests. We recommend that the solution is stored at 4-8 0C in the
refrigerator. Aliquot the required amount for daily use. If 1 litre substrate is excess
then prepare 500 ml for use.
2. Stock iodine solution 50 mmol/l: Dissolve 3.57 g of potassium iodate and 45 g of
potassium iodide in about 800 ml of water in a volumetric flask. Slowly and with
mixing add 9.0 ml of concentrated hydrochloric acid. Dilute to 1 litre with distilled
water. This solution should be stored in a dark bottle and is stable for a year at 4 -8 0C.
3. Working iodine solution: Dilute 10 ml of stock iodine solution with 90 ml distilled water
in a graduated 100 ml volumetric flask. This solution should be stored in a dark bottle
and is stable for 2 months at 2-8 0C.
3.6 PROCEDURE
1. Pipette 1.0 ml of buffered starch substrate into 150 x 16 mm test tubes. You will need
1 tube for each patient and control sample and 1 tube for a reagent blank.
2. Place all of the tubes in a water bath at 37 0C for 5 minutes to warm the contents.
3. Pipette 20 µl of patient’s or control serum into the bottom of the test tubes, mix and
incubate at 37 0C for exactly 7 minutes and 30 seconds. (No serum is added to the
reagent blank).
4. After 7 minutes and 30 seconds remove the test tubes from the water bath
immediately add 1.0 ml of working iodine solution to each tube (samples and reagent
blank) then add 8 ml of distilled water.
5. Mix the contents of each tube well then measure the absorbance without delay at 660
nm (red filter Ilford No. 608) setting the spectrometer to zero with distilled water.
NOTE: Avoid contamination of the pipette with saliva
3.7 CALCULATION
Amylase activity U/L = B-T x 1470
B
B = absorbance of reagent blank
T = absorbance of test
1470 = factor to express values in U/L

9
If the result is greater than 735 U/L (i.e. there is no blue colour in tube T) then the
sample must be diluted with saline (20 µl serum + 100 µl saline) and the analysis repeated
using 20 µl of the diluted sample. The measured value must be multiplied by 6 to calculate
the amylase activity of the sample to take into account the dilution factor.
QUALITY CONTROL
A quality control sample with a value in the range 200 – 700 U/L should be analysed with
each batch of specimens. If single specimens are analysed a control specimen should
always be included.
OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 6% should be

attainable.
ROUTINE CONDITIONS VARIANCE: This value should not exceed 12 %
REFERENCE VALUES: Approximate reference values: 70 – 340 U/L
REFERENCES
WHO Manual LAB/86.3

10
4. ALKALINE PHOSPHATASE
4.1 INTRODUCTION
The alkaline phosphatases (ALP) are a group of glycoprotein enzymes that act as
phosphotransferases by hydrolysing various types of monophosphate bonds at alkaline
pH. ALP activity is found in virtually all tissues, particularly bone, liver, kidney, intestine,
adrenal and placenta. The protein moieties comprise about 510 amino acid residues, to
which is attached various amounts of carbohydrate and sialic acid. Tissue-specific
posttranslational modifications occur to the carbohydrate content, leading to the formation
of isoforms, e.g. bone, liver and kidney ALP, each of which contains the same tissue non-
specific protein. ALP is found attached to the outer lipid bilayer of cell membranes by a
glycosyl-phosphatidylinositol group, in the case of liver ALP possibly as a tetramer of
identical subunits; if released from cell membranes, ALP is dimeric. Liver ALP is located in
the cell membranes of the hepatcoyte, and particularly in the outer layer of the cells
adjacent to the bile canaliculi and also in the cells lining the sinusoids. Adult intestinal ALP
lacks sialic acid and is found in the epithelial cells of the intestinal brush border. The
placental enzyme is formed by the syncitiotrophoblast cells lining the microvilli that
interface the placental and fetal blood circulations but the placental ALP does not cross
into the fetal circulation.

Serum ALP measurements are of particular interest in the investigation of two groups of
conditions: hepatobiliary and bone disease associated with increased osteoblastic activity.
The response of the liver to any form of biliary tree obstruction is to synthesize more ALP.
The main site of new enzyme synthesis is the hepatocytes adjacent to the biliary canaliculi.
Some of the newly formed enzyme enters the circulation to raise the enzyme level in
serum. The elevation tends to be more marked (more than three-fold) in extrahepatic
obstruction (e.g. by stone or by cancer of the head of the pancreas) than in intrahepatic
obstruction and is greater the more complete the obstruction. Serum enzyme activities may
reach 10 to 12 times the upper limit of normal, returning to normal on surgical removal of
the obstruction. Inappropriate ALP synthesis is observed in some types of tumours. In
children the ALP activity is due to the physiological response of osteoblasts during growth.
ALP activity is pathologically high in children with rickets.

4 - Nitophenylphosphate is hydrolysed by alkaline phosphatase at pH 10.3 at 37 0 C and 4 -
Nitrophenol is liberated. Alkali is added to stop the enzyme activity at the end of the timed
incubation period and the increase in absorbance due to the 4-Nitrophenol released is
measured at 410 nm.

Collect about 2-3 ml blood, separate serum as soon as possible, avoid haemolysis. Freshly
collected serum sample should be kept at room temperature and assayed as soon as
possible. If there is a delay in the assay, store the serum at -20 C. Sample should be
completely thawed before the assay. Refrigeration of the serum at 40 C will increase the
ALP activity.
APPARATUS:
Water bath at 37 0C
pH meter
Spectrometer wavelength at 410 nm or Colorimeter with violet filter, Ilford 600 (410 nm)

11
GLASSWARE: Automatic pipette (50µl and 100µl)

Volumetric flasks (100 ml and 1
litre volumes) CHEMICALS:
Beakers (1 litre) All chemicals must be analar grade
Measuring cylinders (100 ml and 1 Standard buffer solutions for pH
litre) meter
Test tubes (125 mm x 16 mm or Hydrochloric acid (Concentrated
150 mm x 16 mm) (37% w/v); caution: highly
Volumetric pipettes (5 ml in 0.1 corrosive)
ml) 2-Amino-2-methyl-1-propanol
Polyethylene reagent bottles (1 Magnesium chloride hexahydrate-
litre) Disodium 4 – Nitrophenyl
Amber colour bottles phosphate hexahydrate
Graduated pipettes (1ml, 5ml, 10ml) Sodium hydroxide pellets
4 - Nitrophenol
REAGENTS
1. AMP Buffer pH 10.3: Dissolve 78.5 g of 2-amino-2-methyl-1-propanol (CH3)2 C (NH2)
CH2 OH in about 900 ml of distilled water. Adjust to pH 10.3 with concentrated
hydrochloric acid (about 18 ml) and make up to 1 litre with distilled water. Store in an
Amber colour reagent bottle. This solution is stable for 1 month at 20-25 0C.
Method done at MRI: Use the chemical with the specifications of 95 % (CH3)2C
(NH2)CH2OH with MW(89.14) and specific gravity of 0.93 g/ml (BDH). Add 84.4 ml
of the above liquid to 800 ml of distilled water.
2. Magnesium chloride solution 1.5mmol/l: Dissolve 300 mg of magnesium chloride
hexahydrate in water and make up to 1 litre. This solution is stable indefinitely at 20 -
250C. MgCl2.6H2O chemical and the solution (1.5mmol/l) are best stored in
refrigerator.
3. Substrate solution 225 mmol/l in the magnesium chloride solution: Dissolve 83.5 mg of
disodium 4 -Nitrophenyl phosphate hexahydrate (store the chemical in freezing
compartment) in 1.0 ml of magnesium chloride solution as required. This solution is
stable for one working day. It’s best to keep the solution in refrigerator since at room
temperature colour development may occur.
4. Sodium hydroxide solution 250 mmol/l: Dissolve 10 g of sodium hydroxide in distilled
water and make up to 1 litre. Store in a tightly Stopperd polyethylene bottle. This
solution is stable indefinitely at 20-25 0C. We have observed that this solution is stable
even at room temperature at 20-30 0C.
5. 4 – Nitrophenol stock solution 10.8 mmol/l: Weigh out 150 mg of 4 – Nitrophenol
accurately in a beaker & transfer the chemical from beaker to a 100 ml volumetric flask
using a funnel and wash any chemical remaining in the container into the volumetric
flask with distilled water. Make up to the mark with distilled water. This solution is
stable for about 6 months in an amber colour bottle at 4 0C.
6. 4 – Nitrophenol working solution 54 µmol/l: Pipette 0.5 ml of the 4 – Nitrophenol stock
solution into a 100 ml volumetric flask, make up to 100 ml with sodium hydroxide
solution (250mmol/l) Prepare this solution freshly before use.
NOTE: The quality of the disodium 4 – Nitrophenyl phosphate should be checked to

ensure that it does not contain excessive amounts of free 4 – Nitrophenol. Prepare sodium
hydroxide solution (10mmol/l) by diluting 4.0 ml of sodium hydroxide solution
(250mmol/l) to 100 ml with distilled water. Add 200 µl of substrate solution (reagent 3
above) to 3.8 ml of sodium hydroxide solution (10 mmol/l) Mix and measure the
absorbance at 410 nm after setting the spectrometer to zero with sodium hydroxide
solution (10 mmol/l). The quality of the substrate is satisfactory if its absorbance after
dilution in sodium hydroxide solution is less than 0.25 (10 mm light path cuvette at room

12
temperature and 410 nm) In a colorimetric the absorbance should be less than 0.12
(10mm light path and Ilford filter No 600) This procedure should be carried out when a
new batch of chemical/bottle is introduced to the bench.
4.6 PROCEDURE
1. Pipette 1.4 ml of AMP buffer into sufficient test tubes for patients’ samples, controls
and reagent blank and pre incubate in the water bath at 37 0C for about 5 minutes.
2. To each tube add 50 µl of serum, to the blank; add 50 µl of distilled water .Mix well.
3. To each tube in sequence add 100 µl of substrate solution at timed intervals. Mix well.
4. Incubate for exactly 15 minutes at 37 0C then add 4.0 ml of sodium hydroxide solution
(250mmol/l) to each tube in sequence, maintaining timed intervals. Mix each tube and
allow them to cool to room temperature.
5. Measure the absorbance of each test solution at 410 nm ( violet filter ,Ilford No :600)
setting the spectrometer to zero with the blank
6. If the absorbance is greater than the absorbance of a 400 U/L standard, then repeat
the procedures, but at step 4, incubate for exactly 5 minutes (instead of 15 minutes)
then add 4.0 ml sodium hydroxide solution (250mmol/l) and complete the procedure
described in steps 4 and 5. Multiply the activity by 3 before reporting the result.

Tube No (1) (2) (3) (4) (5) (6)
4 – Nitrophenol solution 54 µmol/l (ml) 1 2 4 6 8 10
Sodium hydroxide 250 mmol/l (ml) 9 8 6 4 2 0
Activity (U/L) 40 80 160 240 320 400
Mix well and measure the absorbance of each tube at 410 nm (violet filter Ilford No 600)
setting the spectrometer to zero with sodium hydroxide solution (250mmol/l) Plot the
absorbance of each tube on the graph. Prepare a new calibration graph every 3 months.
4.7 CALCULATION
Read off the activities of alkaline phosphatase in the unknown and control samples from
the calibration graph. Multiply by 3 if you used 5 minute incubation instead of 15 minutes
incubation.
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 20-350 U/L, one
of which is unknown to the operator should be included with each batch of specimens. If
single specimens are analysed a control specimen should always be included.

attainable
ROUTINE CONDITIONS VARIANCE: The value should be less than 20%
APPROXIMATE REFERENCE VALUES

Males (age 20 -60 years) : 20- 90 U/L
Females (age 15-60 years) : 20-90 U/L
Children (age 1-12 years) : up to 350 U/L
During the growth spurt of puberty : up to 500 U/L
REFERENCES
LAB/86.3
A guide to diagnostic clinical chemistry By R.N. Walmsley and G.H. White -page 312
TIETZ TEXTBOOK OF Clinical chemistry page 831-832

13
5. ASPARTATE AMINO TRANSFERASE
5.1 INTRODUCTION
Aspartate Amino Transferase & Alanine Amino Transferase
The aminotransferases constitute a group of enzymes that catalyze the interconvertion of
amino acids and α- oxo-acids by transfer of amino groups. Aspartate amino transferase
(AST) and Alanine amino transferase (ALT) are the two enzymes that are of clinical
significance. Distinct isoenzymes of AST are present respectively in cytoplasm and
mytochondria of cells. The α- oxoglutarate/glutamate couple serves as one amino group
acceptor and donor pair in all amino-transfer reactions; the specificity of the individual
enzymes derives from the particular aminoacid that serves as the other donor of an amino
group.
AST
L-Aspartate + 2- Oxaglutarate Oxaloacetate + L –Glutamate
ALT
L-Alanine + 2- Oxaglutarate Pyruate + L –Glutamate
The reactions are reversible, but the equilibria of the AST and ALT reactions favour
formation of aspartate and alanine, respectively. Pyridoxal -5’ phosphate and its amino
analogue, pyridoxamine-5’ –phosphate function as coenzymes in the amino transfer
reactions. Transaminases are widely distributed in tissues. Both AST and ALT are normally
present in human plasma, bile, cerebrospinal fluid and saliva, but none is found in urine
unless a kidney lesion is present.

In viral hepatitis and other forms of liver disease associated with hepatic necrosis, serum
AST and ALT levels are elevated even before the clinical signs and symptoms of the
disease (such has jaundice) appear. Levels for both enzymes may reach values as high as
100 times the upper reference limit, although 20- to 50- fold elevations are most frequently
encountered. Peak values of transaminase activity occur between the 7th and 12th days;
activities then gradually decrease, reaching normal levels by the 3rd to 5th week if recovery is
uneventful. Alcoholic hepatitis has more modest elevations. In infectious hepatitis and
other inflammatory conditions affecting the liver, ALT is characteristically as high as or
higher than AST, and the ALT/AST ratio, which normally and in other conditions is less
than 1, becomes greater than unity. The use of different assay methods for the two
enzymes may alter the activities of the two enzymes relative to each other, and hence the
numerical values of the ratio observed may differ between laboratories. Nevertheless, the
principle that hepatitis is associated with comparable elevations of the two activities
remains valid. The relatively similar elevations of AST and ALT in hepatitis have been
attributed to the release of only the cytoplasmic isoenzyme of AST into the circulation
from reversibly damaged parenchymal cells. When necrosis of the cells occurs,
considerable amounts of mitochondrial AST are also released, depressing the ALT/AST
ratio. The picture in toxic hepatitis is similar to that in infectious hepatitis, with very high
ALT and AST activity being observed in severe cases. Elevations up to 20 times the upper
reference limit may be encountered in infectious mononucleosis with liver involvement and
somewhat lover values in intrahepatic cholestasis. Increased levels may also be observed in
extrahepatic cholestatis, with levels tending to be higher the more chronic the obstruction. The
aminotransferase levels observed in cirrhosis vary with the status of the cirrhotic process;
they range from upper normal to some four to five times normal, with the level of AST
activity higher than that of ALT activity. Elevations probably indicate continuing cellular
necrosis. Five- to 10- fold elevations of both enzymes occur in patients with primary or
metastatic carcinoma of the liver, with AST usually being higher than ALT, but levels are

14
often normal in the early stages of malignant infiltration of the liver. Slight or moderate
elevations of both AST and ALT activities may be observed after the intake of alcohol, in
delirium tremens, and after administration of various drugs, such as opiates, salicylates, or
ampicillin. Although serum levels of both AST and ALT become elevated whenever
disease processes affect liver cell integrity. ALT is the more liver-specific enzyme. Serum
elevations of ALT activity are rarely observed in conditions other than parenchymal liver
disease. Moreover, elevations of ALT activity persist longer than do those of AST activity.
Measurement of both AST and ALT has some value in distinguishing hepatitis from other
parenchymal lesions. After myocardial infarction, increased AST activity appears in serum, as
might be expected from the relatively high AST concentration in heart muscle. On average,
serum levels do not become abnormal, however, until 6 to 8 h has elapsed after the onset
of the chest pain. Abnormal AST levels are observed in more than 97% of cases of
myocardial infarction when correctly timed blood specimens are analyzed. Peak values of
AST activity are reached after 18 to 24 h, and the activity values fall within the normal
range by the fourth of fifth day, provided no new infarct has occurred. The peak values of
AST activity are roughly proportional to the extent of cardiac damage. Average increases
are of the order of four to five times the upper limit of normal; levels of 10 to 15 times
normal are frequently associated with fatal infarct. However, small elevations in serum
levels do not necessarily indicate a favorable prognosis. ALT levels are within normal
limits or are only marginally increased in uncomplicated myocardial infraction, because the
concentration of ALT activity in heart muscle is only a fraction of that of AST activity.
AST (and occasionally ALT) activity levels are increased in progressive muscular dystrophy
and dermatonyositis, reaching levels up to 8 times normal; they are usually normal in other
types of muscle diseases, especially in those of neurogenic origin. Pulmonary emboli can
raise AST levels to two to thee times normal, and slight to moderate elevations (two to five
times normal) are noted in acute pancreatitis, crushed muscle injuries, gangrene, and hemolytic
disease.

Aspartate aminotransferase (AST or SGOT) effects the conversion of alpha keto -
glutarate and aspartate to glutamate and oxaloacetate respectively, by amino group transfer.
The oxaloacetate thus formed is coupled with 2, 4 - dinitrophenylhydrazine to produce a
coloured complex whose absorbance in alkaline solution is measured at 505 nm

3-5 ml clotted blood, avoid haemolysis. Separate serum as soon as possible and perform
the assay or keep in the refrigerator. It is stable up to 24 hours at 40 C. Minimal loss of
activity occurs at 0-4 0C over 1-3 days. Specimen is best stored frozen if they are to be kept
more than 3-4 days
APPARATUS:
Water bath at 37 0C
pH meter
Visible spectrometer wavelength at 505 nm or Colorimeter with green filter Ilford No 604
(520 nm)

15
GLASSWARE:
Volumetric flasks (100 ml and 1 litre volumes)
Measuring cylinders (1 litre)
Automatic micro pipette (100 µl)
Graduated pipettes (1ml, 2 ml, 5ml, 10 ml in 0.1 ml)
Test tubes (150 x16 mm), Beakers (5ml, 10ml, 100ml and 1 litre)
Reagents bottles clear and amber coloured (250 ml and 1 litre)
Polypropylene bottle
CHEMICALS:
Disodium hydrogen ortho-phosphate-anhydrous, analytical grade
Potassium dihydrogen phosphate-anhydrous, analytical grade
Alpha – ketoglutaric acid -analar
DL- aspartic acid -analar, Sodium hydroxide pellets analar
2, 4- dinitrophenylhydrazine-AR; caution: may explode violently when dry,
Hydrochloric acid concentrated-AR (37% w/v), caution: highly corrosive
Sodium pyruvate (analytical grade)
REAGENTS
1. Phosphate buffer pH 7.4: Dissolve 11.9 g of disodium hydrogen phosphate (anhydrous)
and 2.2 g of potassium dihydrogen phosphate (anhydrous) in distilled water and make
up to 1 litre. Check the pH adjust to pH 7.4 if necessary using small amounts of the
appropriate phosphate (e.g. if the pH is more than 7.4 add potassium dihydrogen
phosphate). This solution is stable for about 2 months at 2-8 0C.
2. Sodium hydroxide solution 1 mol/l: Weigh 40 g of sodium hydroxide in beaker, slowly
dissolve in distilled water, transfer into a volumetric flask and make up to 1 litre. Store
in a tightly Stopperd polypropylene bottle. This solution is stable indefinitely at 20 -
250C, which can be achieved by air condition system. However we have observed that
this solution is stable in our room temperature (20-30 0C) for about 2 months.
3. Buffered substrate reagent: Weigh 29.2 mg of alpha- ketoglutaric acid 2.66 g of DL-aspartic
acid into a small beaker. Dissolve in 20 ml of sodium hydroxide solution (1 mol/l)
then adjust to pH 7.4 with more sodium hydroxide solution. Transfer to a 100 ml
volumetric flask and make up to 100 ml with phosphate buffer and mix well. Store the
reagent frozen in screw capped bottles; the volume should be the amount need for a
day’s analysis.
4. Hydrochloric acid 1 mol/l: Dilute 9 ml of hydrochloric acid (concentrated (37% w/v)) to
100 ml with distilled water.
5. Colour reagent 2,4 –dinitrophenylhydrazine 1 mmol/l: Dissolve the equivalent of 19.8 mg of
dry 2,4-dinitrophenylhydrazine in 100 ml of hydrochloric acid ( 1mol/l)and transfer
into a amber colour bottle; Note that the weight of 2, 4 – dinitrophenylehydrazine
must be adjusted to take into account the water content. e.g. If the label reads as 33%
by weight of water is added to ensure safety in transit the calculation is as
follows.100/67 x 19.8 = 29.55 mg. The prepared solution is stable for 2 months at 2 -
8 0C.
6. Sodium hydroxide solution 400 mmol/l: Dissolve 16.0 g of sodium hydroxide in distilled
water in a beaker and make up to 1 litre. Store in a tightly stoppered polypropylene
reagent bottle. This solution is stable indefinitely at 20- 25 0C. We have observed that
this solution is stable at our room temperature at 20-30 0 C for 2 months.
7. Pyruvate standard solution 4 mmol/l: Weigh out 44 mg of sodium pyruvate in a beaker,
transfer into a 100 ml volumetric flask and make up to the mark with phosphate
buffer. Mix well, divide into small portions (about 1ml) and store in the freezer
compartment of the refrigerator. The standard solution is stable for 6 months in the
freezer.

16
5.6 PROCEDURE
1. Two test tubes are required for each serum or control sample( one for the “Test” and
one for a sample blank) one for a reagent blank and one for the standard
2. Transfer 0.5 ml buffered substrate to each tube and pre-incubate in the water bath
(37 0 C) for 5 minutes
3. Add 100 µl of patient’s or control serum to the “Test” tubes or 100 µl distilled
water(reagent blank) or 100 µl pyruvate standard (standard) mix and incubate at 37 0C.
4. After exactly 60 minutes add 0.5 ml colour reagent to each tube, mix and remove from
the water bath.
5. Add 100 µl of patient’s or control serum to the sample blank tubes and mix well.
6. Leave for 20 minutes at room temperature and then add 5.0 ml of sodium hydroxide
solution (400mmol/l) and mix thoroughly.
7. Leave the tubes at room temperature for at least 5 minutes, but not longer than 30
minutes, and then read the absorbance at 505 nm. Set the spectrometer to zero with
the reagent blank.
CALIBRATION
In this method the amount of pyruvate formed is calculated by comparing the absorbance
of the samples (Test- Blank absorbance) with that of the pyruvate standard (4mmol/l).
However alpha - ketoglutarate also contributes to the absorbance and the change in
absorbance is not linearly related to enzyme activity expressed in U/L. The table must
therefore be used to convert the amount of pyruvate formed into U/L.
5.7 CALCULATION
Amount of pyruvate formed = (A Test-A Sample Blank) x 4x1x1000

(µmol/min/litre) A Standard 60
= A Test- A Sample Blank x 66.7
A Standard
With the reagent blank set to zero the spectrophotometer at 505 nm

A = absorbance
Use the table below to convert the amount of pyruvate into U/L (expressed as
µmol/minute/litre at 37 0C)
Calculated pyruvate AST result (U/L at Calculated pyruvate AST result (U/L at
(µmol/min/litre) 37 0C) (µmol/min/litre) 37 0C)
2 4 28 52
4 6 30 56
6 10 32 60
8 12 34 64
10 15 36 69
12 19 38 73
14 23 40 77
16 27 42 81
18 31 44 85
20 35 46 92
22 40 48 98
23 42 50 106
24 44 52 114
26 48 54 125

17
NOTE: When the activity of a sample exceeds 125 U/L the measurement should be
repeated with 10 minute incubation (instead of 60 minutes) and the results multiplied by 6.
When the activity is greater than 750 U/L, the serum should be diluted 1 in 10 with sodium
chloride solution (150mmol/l), an incubation time of 10 minutes should be used and the result
multiplied by 60.
QUALITY CONTROL
of which is unknown to the operator, should be included with each batch of specimens.
Even if single specimens are analysed a control specimen should always be included.

attainable
ROUTINE CONDITIONS VARIANCE: The value should not exceed 16%
REFERENCE VALUES
Approximate reference values: 4 - 42 U/L.
REFERENCES
Reitman, .S. & Frankel. S. (1957) Am. J. Clin.Pathol., 28, 56-63

18
6. ALANINE AMINO TRANSFERASE

Alanine aminotransferase (ALT or SGPT) effects the conversion of alpha-ketoglutarate
and Alanine to pyruvate. Pyruvate formed is coupled with 2, 4-dinitrophenylhydrazine to
produce a coloured complex. The absorbance in alkaline solution is measured at 505 nm.

3-5 ml clotted blood, avoid haemolysis. Separate serum as soon as possible and perform
the assay or keep in the refrigerator. It is stable up to 24 hours at 4 0C. Minimal loss of
activity occurs at 0-4 0C over 1-3 days. Specimen is best stored frozen if they are to be kept
more than 3-4 days

APPARATUS:
Water bath at 37 0C
pH meter
Visible spectrometer wavelength at 505 nm or Colorimeter with green filter Ilford No
604 (520 nm)
GLASSWARE:
Automatic micro pipette (100 µl)
Graduated pipettes (1ml, 2ml, 5ml and 10 ml in 0.1 ml)
Test tubes (150 x16 mm), Beakers (5ml, 50ml, 100ml and 1 litre)
Reagents bottles amber coloured (250 ml and 1 litre)
Polypropylene bottle
CHEMICALS:
Disodium hydrogen phosphate-Anhydrous analytical grade
Potassium dihydrogen phosphate-Anhydrous analytical grade
Alpha ketoglutaric acid AR
DL Alanine AR
Sodium hydroxide pellets analytical grade
2, 4-dinitrophenyl hydrazine AR
Hydrochloric acid AR
Sodium pyruvate analytical grade
REAGENTS
1. Phosphate buffer pH 7.4: Dissolve 11.9 g of disodium hydrogen phosphate (anhydrous)
and 2.2 g of potassium dihydrogen phosphate (anhydrous) in distilled water and make
up to 1 litre. Check the p H and adjust to pH 7.4 if necessary using small amounts of
the appropriate phosphate (e.g. if the pH is more than 7.4 add potassium dihydrogen
phosphate). This solution is stable for about 2 months at 2-8 0C.
2. Sodium hydroxide solution 1 mol/l: Slowly dissolve 40.0 g of sodium hydroxide in distilled
water in a beaker and make up to 1 litre. Store in a tightly stopperd polypropylene
bottle. This solution is stable indefinitely at 20-25 0C. we have observed this solution is
even stable at our room temperature 20-30 0C
3. Buffered substrate reagent: Weigh out 3.56 g of DL Alanine in a beaker and weigh 30 mg
of alpha ketoglutaric acid accurately in another small beaker. Transfer it to same
beaker by dissolving in the phosphate buffer. Add 0.5 ml of sodium hydroxide
(1mol/l) solution. Check the pH. The pH should be at 7.4.Make up to 100 ml with
phosphate buffer and mix. Divide the prepared substrate into small volumes and store
in the freezing compartment or in the freezer. Discard the remaining substrate after
use. Do not freeze the remaining substrate again.

19
4. Hydrochloric acid 1 mol/l: Dilute 9 ml of hydrochloric acid (concentrated (37% w/v))
to 100 ml with distilled water.
5. Colour reagent 2,4 –dinitrophenylhydrazine 1 mmol/l: Dissolve the equivalent of 19.8 mg of
dry 2,4-dinitrophenylhydrazine in 100 ml of hydrochloric acid ( 1mol/l)and transfer
into a amber colour bottle; Note that the weight of 2, 4 –dinitrophenylehydrazine must
be adjusted to take into account the water content. e.g. If the label reads as 33% by
weight of water is added to ensure safety in transit the calculation is as follows.100/67
x 19.8 = 29.55 mg. The prepared solution is stable for 2 months at 2-8 0C.
6. Sodium hydroxide solution 400 mmol/l: Dissolve 16.0 g of sodium hydroxide in distilled
water in a beaker and make up to 1 litre. Store in a tightly stoppered polypropylene
reagent bottle. This solution is stable indefinitely at 20- 25 0C. We have observed that
this solution is stable at our room temperature (20-30 0C) for 2 months.
7. Pyruvate standard solution 4 mmol/l: Weigh out 44 mg of sodium pyruvate in a beaker,
transfer into 100 ml volumetric flask and make up to the mark with phosphate buffer.
Mix well, divide into small portions (about 1ml) and store in the freezer compartment
of the refrigerator. The standard solution is stable for 6 months in the freezer.
6.4 PROCEDURE
1. Two test tubes are required for each serum or control sample( one for the “Test” and
one for a sample blank) one for a reagent blank and one for the standard
2. Transfer 0.5 ml buffered substrate to each tube and pre-incubate in the water bath (37
0C) for 5 minutes
3. Add 100 µl of patient’s or control serum to the “Test” tubes or 100 µl water(reagent
blank) or 100 µl pyruvate standard (standard) mix and incubate at 37 0C
4. After exactly 30 minutes add 0.5 ml colour reagent to each tube, mix and remove from
the water bath.
5. Add 100 µl of patient’s or control serum to the sample blank tubes.
6. Leave for 20 minutes at room temperature and then add 5.0 ml of sodium hydroxide
solution (400mmol/l) and mix thoroughly.
7. Leave the tubes at room temperature for at least 5 minutes, but not longer than 30
minutes, and then read the absorbance at 505 nm. Set the spectrometer to zero with
the reagent blank.

In this method the amount of pyruvate formed is calculated by comparing the absorbance
of the samples (Test- Blank absorbance) with that of the pyruvate standard (4mmol/l).
However alpha - ketoglutarate also contributes to the absorbance and the change in
absorbance is not linearly related to enzyme activity expressed in U/L. The table must
therefore be used to convert the amount of pyruvate formed into U/L.
6.5 CALCULATION
Amount of pyruvate formed = (A Test-A Sample Blank) x 4x1x1000
(µmol/min/litre) A Standard 30
= A Test- A Sample Blank x 133
A Standard
With the reagent blank set to zero the spectrophotometer at 505 nm
A=absorbance

20
Use the table below to convert the amount of pyruvate into U/L (expressed as
µmol/minute/litre at 37 0C)
Calculated pyruvate ALT result (U/L at Calculated pyruvate ALT result (U/L at
(µmol/min/litre) 37 0C) (µmol/min/litre) 37 0C)
2 2 54 42
4 4 56 44
6 4 58 46
8 5 60 47
10 7 62 49
12 7 64 53
14 9 66 55
16 11 68 56
18 13 70 60
20 13 72 62
22 15 74 64
23 15 76 66
24 16 78 67
26 16 80 69
28 18 82 71
30 20 84 73
32 22 86 76
34 24 88 80
36 25 90 84
38 27 92 87
40 29 94 91
42 31 96 95
44 33 98 98
46 35 100 102
48 36 102 109
50 38
52 40
NOTE: When the activity of a sample exceeds 109 U/L the measurement should be repeated
with 10 minute incubation (instead of 30 minutes) and the results multiplied by 3. When the
activity is greater than 750 U/L, the serum should be diluted 1 in 10 with sodium chloride
solution (150mmol/l), an incubation time of 10 minutes should be used and the result
multiplied by 30.
QUALITY CONTROL
of which is unknown to the operator, should be included with each batch of specimens.
Even if single specimens are analysed a control specimen should always be included
REFERENCE VALUES Approximate reference values: up to 2-27 U/L.
REFERENCES
King and wooton- enzymology

21
7. BILIRUBIN
7.1 INTRODUCTION
Haem, released from aged red cells and maturing cells during erythropoiesis, or from
degraded haemoproteins is converted to biliverdin in the reticuloendothelial system.
Biliverdin is reduced to bilirubin which is secreted into the plasma where it is transported
to the liver reversibly bound to albumin. The hepatocytes take the bilirubin up from the
plasma, conjugate it to glucuronic acid and excrete it in the bile. There are six steps in this
process namely; production, transport to the liver, hepatocyte uptake, conjugation, biliary
secretion, gut degradation and excretion.
The total bilirubin produced is around 250-350 mg/day (4 – 6 mmol/day). 15% to 20% is
derived from immature red cell and haemoproteins (early labelled fraction) whilst the
remainder comes from senescent red cells (1g of haemoglobin produces 620µmol of
bilirubin) Haem is converted to bilirubin in the reticuloendothelial system by two enzymes,
haem oxygenase and biliverdin reductase. Haem oxygenase breakes the alpha – CH bridge
of protoporphyrin IX to produce biliverdin IX α, which is then reduced to bilirubin IX α
by biliverdin reductase. Some cleavage occurs at the β, γ and δ bridges, but insignificant
amounts of these isomers are produced. Although bilirubin IX α has two polar propionic
acid side chains it is poorly soluble in water because of intramolecular hydrogen bonding
between the propionic acid residues and other parts of the molecule. This bonding may
also account for the necessity for conjugation with glucuronic acid prior to biliary
excretion. Bilirubin is transported to the liver reversibly bound to albumin. This protein
has one high – affinity binding site and an additional low – affinity site which is activated at
high concentrations of bilirubin. The amount of unbound bilirubin is low; approximately 4
nmol/l at a total plasma concentration of 20 µmol/L. Compounds such as free fatty acids,
sulphonamides, salicylate and ampicillin will displace bilirubin from its binding sites. This
species of bilirubin is called unconjugated bilirubin or indirect reacting bilirubin. The
bilirubin – albumin complex dissociates in the liver and the bilirubin is transported across
the hepatocyte membrane into the cell where it is reversibly bound to cytosolic proteins;
one such protein being ligandin. The function of these proteins appears to be prevention
of efflux of bilirubin from the cell. Bilirubin is conjugated in the endoplasmic reticulum
with glucuronic acid and to a lesser extent with glucose and xylose. The enzyme
responsible is UDP – glucuronosyltransferase which esterifies one or both propionic acid
side chains to produce di – and monoglucuronides. In man the product is predominantly
bilirubin diglucuronide with lesser amounts of the monoglucuronide. The bilirubin
conjugates are secreted into the biliary passages by an active process which is yet to be
clarified. This transport mechanism differs from that responsible for the biliary excretion
of bile acids. In the gut the bacterial flora reduce the bilirubin conjugates to urobilinogens
which are excreted in the faeces as such. Some of the urobilinogens are reabsorbed from
the gut to be re – excreted by the liver (enterohepatic circulation); as these compounds are
water soluble they will also be excreted by the kidney, but this is an unimportant excretory
route. Two points worthy of note are that conjugated bilirubin is more prone to reductive
processes than unconjugated bilirubin and that unconjugated bilirubin can be reabsorbed
from the gut. Thus, if bacterial flora are absent (e.g.: neonate, antibiotic therapy)
deconjugation of bilirubin glucuronides by intestinal mucosal ß – glucuronidase may occur
and reabsorption of the unconjugated compound result in an increase in the plasma
unconjugated bilirubin level. The total plasma bilirubin concentration in the normal subject
is usually less than 20 µmol/L. The clinical sign of jaundice appears when the plasma total
bilirubin rises beyond 40 µmol/L. When normal plasma is evaluated by high performance
liquid chromatography techniques four bilirubin fractions alpha, beta, gamma and delta are
obtained.

22
Alpha fraction
This fraction is unconjugated bilirubin which is water insoluble and bound to albumin. It is
also called indirect reacting bilirubin because the diazo reaction used to measure the plasma
level occurs only after the addition of accelerators. It is the major bilirubin fraction in
normal plasma (> 90%). It does not appear in the urine because of its attachment to
albumin and can only be cleared by the liver.
Beta and gamma fractions

The beta fraction is composed of bilirubin monoglucuronide whilst diglucuronide
constitutes the gamma fraction. These bilirubins (< 10% in normal plasma) are water
soluble and will appear in the urine if present in the blood in excess. They are called direct
reacting bilirubins as they react with the diazo reagent without the addition of accelerators.
Delta fraction
The delta fraction, usually referred to as ‘delta’ bilirubin – not to be confused with bilirubin
IX delta –is an interesting compound with the following characteristics:
 it is direct reacting
 it is tightly bound to albumin
 its plasma level is increased in diseases associated with high plasma levels of
conjugated bilirubin
It appears to be derived from conjugated bilirubin as it can be produced by incubating
bilirubin glucuronides with plasma albumin. It is thought that the bilirubin migrates from
the glucuronic acid residues and becomes covelantly bound to albumin. As noted above
this fraction increases in conditions associated with chronic conjugated
hyperbilirubinaemia (eg: cholestasis) and, as it is tightly bound to albumin, it has a t1/2
comparable to that of protein (20 days) .Thus, after formation delta bilirubin ‘hangs around
‘as it is not cleared by the liver or the kidney. The bile pigments that may be found in the
urine are urobilinogen and conjugated bilirubin. Unconjugated Bilirubin is water insoluble
and bound to albumin, and is thus not available for urinary excretion. The normal subject
excretes 1 – 4 mg (2 – 7 µmol) of urobilinogen daily (derived from the enterohepatic
circulation) Increased values occur in liver disease (inability to excrete the small amounts
reabsorbed from the gut) and in haemolytic disease (increased bilirubin production).
Decreased excretion occurs in bile duct obstruction. (cholestasis) Bilirubin appears in the
urine when the plasma level of conjugated bilirubin rises as in hepatocellular disease and
cholestasis.

The earliest clinical manifestation of hepatobiliary disease is often jaundice, but jaundice
need not necessarily indicate liver pathology (e.g.: haemolysis) and liver pathology can
present without jaundice (e.g.: space-occupying lesions). However, it is convenient to
classify liver disease in terms of jaundice and to this end it is helpful to divide
hyperbilirubinaemia into three categories: Prehepatic: liver disease not present, Hepatic:
hepatocellular disease, Post hepatic: cholestasis (obstruction). Pathological
hyperbiliruminaemia in the neonate if not diagnosed and treated at the early stages may
lead to the development of kernicterus with resultant permanent neurological dysfunction.

Sulfanilic acid is diazotized by the nitrous acid produced from the reaction between
sodium nitrite and hydrochloric acid. Both conjugated and unconjugated bilirubin reacts
with diazotized sulfanilic acid (Diazo reagent) to produce azobilirubin. Caffeine is an
accelerator by splitting the unconjugated Bilirubin protein complex and gives a rapid and
complete conversion to azobilirubin. The pink acid azobilirubin is converted to blue
azobilirubin by an alkaline tartrate reagent and the absorbance of the blue green solution is
measured at 600 nm. Measurement of the azobilirubin in an alkaline medium removes

23
turbidity and increases specificity. There is very little interference by other pigments at
600 nm wavelength. Conjugated Bilirubin is determined by diazotization at an acidic pH,
only the conjugated forms of Bilirubin react with the diazo reagent in the absence of the
accelerator caffeine benzoate. The reaction is stopped by the addition of ascorbic acid
minimizes the effect of haemolysis.

Clear non haemolysed serum, collect 3-5 ml of blood into a clean dry container. A fasting
specimen is preferred to avoid lipaemia. Haemolysis should be avoided because it produces
false values. Specimens should be protected from direct exposure to either artificial light or
sunlight as soon as they drawn because conjugated and unconjugated bilirubin is
photosensitive. The sensitivity to light is temperature dependent. For optimal stability
serum separation should be done as early as possible and assay should be carried out within
2 hours of sample collection. Store the serum in dark and at low temperature (2-8 0C) if
any delay is encountered. By inclusion of a sample blank haemolysed samples can be
assayed for total bilirubin.
APPARATUS AND CHEMICALS
APPARATUS: Spectrophotometer, wavelength at 600 nm or Colorimeter, orange filter,

Ilford 607
GLASSWARE:
Volumetric flasks (100 ml, 500 ml and 1 litre volumes)
Beakers (5ml, 100 ml and 1 litre)
Automatic micro pipettes (50 and 100 µl)
Graduated pipettes (1ml and 10 ml in 0.1 ml)
Test tubes (100 x 13mm) & Rubber bulb
Reagent bottle, clear and amber coloured
CHEMICALS: (ANALYTICAL GRADE)

Bilirubin powder (or commercial Bilirubin standards)
Caffeine
Sodium benzoate
Sodium acetate trihydrate
Sulphanilic acid
Hydrochloric acid, concentrated (37% w/v) caution: highly corrosive
Sodium nitirite
Sodium hydroxide pellets
Potassium sodium tartrate tetra hydrate
Ethylenediamine tetra – acetic acid disodium salt dihydrate
Ascorbic acid
Sodium carbonate anhydrous
REAGENTS
1. Caffeine- benzoate reagent: Dissolve 93 g of sodium acetate trihydrate, 56 g of sodium
benzoate and 1 g of disodium EDTA in approximately 500ml of distilled water. Add
38 g of caffeine. Dissolve and dilute to 1 litre in a volumetric flask. Mix well and filter.
This solution is stable for at least 6 months at 20-25 0C (Room temperature)
2. Sulfaninlic acid reagent: Add 2.5 g of sulfanilic acid to about 200 ml of distilled water in a
500 ml volumetric flask. Using a rubber bulb carefully pipette 7.5 ml of concentrated
hydrochloric acid into the flask. Dissolve and make up to 500 ml. This solution is
stable for up to 6 months at 20-25 0C (Room temperature)

24
3. Sodium nitrite solution: Dissolve 500 mg of sodium nitrite in distilled water and make
up to 100 ml. This solution should be stored at 2-8 0C in an amber coloured bottle and
must be renewed every month. Prepare about 25ml or 50 ml.
4. Diazo reagent: Mix 4 ml of sulfanilic acid reagent with 0.1 ml of sodium nitrite solution,
leave for 2 minutes, then use within 5 hours. Keep at 2-8 0C in an amber coloured
bottle.
5. Alkaline tartrate reagent: Dissolve 75 g of sodium hydroxide and 350 g of potassium
sodium tartrate in about 800 ml of distilled water. Transfer to a volumetric flask and
make up to 1 litre. This reagent is stable for at least 6 months at 20- 25 0C. Store in a
polypropylene bottle.
6. Hydrochloric acid 50 mmol/l: Using a rubber bulb, carefully pipette 4.2 ml of hydrochloric
acid (concentrated) and dilute to 1 litre with distilled water, require only for conjugated
Bilirubin method.
7. Ascorbic acid 40 g/l: Dissolve 0.2 g in 5 ml of water, this solution must be prepared
daily. Required only for the conjugated Bilirubin method.
8. Sodium carbonate 100mmol/l: Weigh out 1.06 g of sodium carbonate anhydrous very
accurately and transfer quantitatively to a 100 ml volumetric flask, containing about 50
ml distilled water. Mix well and make up to 100 ml with distilled water.(Prepare about
50 ml for use)
9. Sodium hydroxide 100 mmol/l: Weigh out rapidly 2.0 g of sodium hydroxide in a beaker,
dissolve and make up to 500 ml with distilled water. ( prepare about 100 ml for use)
10. Tris buffer(0.1 mol/l) pH 7.4 : Tris(hydoxymethyl) amino methane 1.21 g, distilled water
80 ml, adjust pH 7.4 ±0.05, with hydrochloric acid 2 N, make up to 100 ml with
distilled water stable for about 1 month at 4 0C
11. Bovine serum albumin (BSA) diluent 40 g/l: Weigh out 4 g of Bovine albumin powder in a
beaker. Dissolve in the Tris buffer and transfer into 100 ml volumetric flask and make
up to the mark with Tris buffer. This solution is stable for one week at 4 0C
12. Bilirubin standard 342µmol/l: Weigh out 20 mg of Bilirubin powder accurately in a small
beaker or in a weighing bottle. Cover the beaker with aluminium foil or black paper to
protect from sunlight. Add 2 ml of sodium carbonate solution and 1.5 ml of sodium
hydroxide solution and dissolve it .This solution must be red and clear. This procedure
must not be carried out in strong sunlight. Transfer the solution quantitatively to a 100
ml volumetric flask. (This should be protected from direct sunlight by wrapping
around with an aluminium foil or with a black carbon paper). Make up to 100 ml with
BSA diluent mix the solution carefully without forming froth. Divide into small
volumes in clean bottles keep in a box to protect from light and store deep frozen.
(-200C) Do not reuse the standard once it has been used.
7.6 PROCEDURE (TOTAL BILIRUBIN)

1. For the standard, patient specimen, control and their blanks (SB, TB, and CB) pipette
1.0 ml of caffeine benzoate reagent into each of two test tubes.
2. Add 100 µl of standard or patient or control serum to each pair of tubes
3. Add 0.5 ml of diazo reagent to the test, standard, control and 0.5 ml of sulfanilic acid
to the blanks.
4. Mix well and let stand for 10 minutes at room temperature.
5. Add 1.0 ml of alkaline tartrate reagent to each tube and mix thoroughly
6. Read the absorbance at 600 nm (Ilford filter No: 607) immediately, setting the
spectrometer to zero with distilled water.
7.61 PROCEDURE (CONJUGATED BILIRUBIN)

1. For each patient or control specimen label two tubes: one test one blank.
2. Add 100 µl of serum to each tube.
3. Add 1.0 ml of hydrochloric acid (50mmol/l) to each tube.
4. Add 0.5 ml of diazo reagent to the tube marked test and 0.5 ml of sulfanilic acid to the
tube marked blank, mix well.

25
5. After 5 minutes add 50 µl of ascorbic acid solution to each tube.
6. Add 1.0 ml of alkaline tartrate reagent to each tube. Mix well and read the absorbance
of each solution at 600 nm immediately, setting the spectrometer to zero absorbance
with distilled water.

Prepare Bilirubin working standard solutions by diluting the Bilirubin standard solution
(342µmol/l) with BSA solution as shown in the table below. The working standard
solution must be freshly prepared each time a calibration graph is made.
Bilirubin working
(1) (2) (3) (4) (5) (6) (7)
Standard
Bilirubin standard
0 0.5 1 1 2 3 4
Solution 342µmol/l(ml)
Bovine serum Albumin (ml)
4 9.5 9 3 2 1 0
(BSA solution)
Concentration of Bilirubin
Working standard solution 0 17.1 34.2 85.5 171 256.5 342
(µmol/l)
A calibration graph is prepared from the Bilirubin working standards using the volumes of
standard and reagents described in the table below:
Tube No 1 (Blank) 2 3 4 5 6 7
Caffeine
benzoate 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Reagent (ml)
Bilirubin
working 100 100 100 100 100 100 100
Standard (µl)
Mix well, protect the tubes from light then add
Diazo reagent
- 0.5 0.5 0.5 0.5 0.5 0.5
(ml)
Mix well and allow the solutions to stand at room temperature for 10 minutes. Protect the tubes from light.
Sulfanilic acid
0.5 - - - - - -
Reagent(ml)
Mix well
Alkaline
tartrate 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Reagent (ml)
Mix well. Read the absorbance of each tube at 600 nm after setting the spectrometer to zero with the blank
(Tube No 1)
Plot the absorbance of the each tube on the vertical axis against the concentrations in
µmol/l of working standards on the horizontal axis
o The calibration graph should be prepared whenever the new batch of reagents are
prepared or any changes made in the spectrophotometers
o Freshly prepare all reagents and use clean glassware
o Measure the standards (each concentration) in duplicate
o Check the calibration graph by measuring a quality control serum

26
NOTE:
 All the tubes or racks used for the assay should be covered with black papers to
protect from direct light
 Icteric serum can be diluted 1 in 2 or highly icteric serum can be diluted 1 in 4 with
normal saline and multiply the result by the diluting factor
 Carryover is minimized by measuring the absorbance of all the serum blank solutions
first, followed by all the test solutions. The colour of the azobilirubin is stable for
about 30 minutes. After 30 minutes turbidity may occur and the absorbance of the
serum blank increases
7.7 CALCULATION
If the calibration graph is linear, calculate the results using the following formula:
Concentration of Bilirubin (µmol/l) = T – TB x 342
S – SB
Where:
T =Absorbance reading of sample or control
TB =Absorbance reading of control or patient sample blank
S =Absorbance reading of Bilirubin standard (342 µmol/l)
SB =Absorbance reading of standard blank
If the calibration graph is not linear, then results should be read from a calibration curve
prepared using working standards 1, 2, 3, 5 and 6.
QUALITY CONTROL
At least two serum control specimens having stated values in the range 20 – 200 µmol/l,
one of which should be unknown to the operator, should be included with each batch of
specimens. If single specimens are analysed a control specimen should always be included.

attainable.
ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed
12%
REFERENCE VALUES
Total bilirubin in adults: 3 – 21 µmol /l
Conversion from SI units into ‘old’ units: µmol/l x 0.0585=mg/dl
REFERENCES
WHO manual LAB /86.3

27
8. CALCIUM
8.1 INTRODUCTION
Calcium is the fifth most common element and the most prevalent cation found in the
body. An average human body contains approximately 1kg (24.95 mol) of calcium.
Calcium is found in three main compartments: the skeleton, soft tissues, and the extra
cellular fluid. The skeleton contains 99% of the body’s calcium, predominantly as extra
cellular crystals of unknown structure with a composition approaching that of
hydroxyapatite. Soft tissues and extra cellular fluid contain about 1% of the body’s calcium.
In blood, virtually all of the calcium is in serum or plasma, which has a mean normal
calcium concentration of approximately 9.5mg/dL (2.37 mmol/L). Calcium exists in three
physiochemical states in plasma, of which approximately 50% is free or ionised. Another
40%is bound to plasma proteins, chiefly albumin. Because calcium binds to negatively
charged or anionic sties on albumin, it’s binding is pH-dependant. Alkalosis leads to an
increase in binding and a decrease in free calcium; conversely, acidosis leads to a decrease
in binding and an increase in free calcium. For each 0.1-unit change in pH, approximately
0.2-mg/dL (0.05mmol/L) of inverse change occurs in the serum free calcium
concentration. Approximately 20% of protein-bound calcium in serum is associated with
globulins. In some patients with multiple myeloma, the high concentrations of serum
globulin may bind sufficient calcium, about 10%, is complexed with small diffusible anions
including bicarbonate, lactate, phosphate and citrate. Calcium can be redistributed among
the three plasma pools, acutely of chronically, independently affecting the quantities of free
calcium and total calcium in the serum. The skeleton is a major storehouse for providing
calcium for the extra cellular and intracellular pool. Approximately 5g of calcium is rapidly
available from the skeletal exchangeable pool, which is accessible for maintaining normal
physiological functions. Intracellular calcium has many important physiological functions
within the cells, including muscle contraction, hormone secretion, glycogen metabolism
and cell division. Extra cellular calcium provides a source for maintenance of intracellular
calcium. In addition, it has an important role in providing calcium ions for bone
mineralization, coagulation cascade and maintaining plasma membrane potential. Calcium
stabilises the plasma membranes and influences permeability and excitability. A decrease in
serum free calcium concentration increases neuromuscular excitability and tetany. An
elevated free calcium concentration results in reduced neuromuscular excitability.

Hypercalcaemia is found commonly in clinical practice. It may be uncovered as a
biochemical abnormality in an otherwise asymptomatic patient or in association with
severe illness. Hypercalcaemia occurs when the flux of calcium into the extra cellular fluid
is greater than the efflux of calcium out of this compartment. For example, when excessive
resporption of bone mineral occurs in malignancy, hypercalciuria develops. When the
capacity of the kidneys to excrete filtered calcium is exceeded, hypercalcaemia develops.
Hypercalcaemia can be due to increased intestinal absorption of calcium (vitamin D
intoxication), enhanced renal retention of calcium (thiazide diuretics), increased skeletal
resorption (immobilization), or a combination of these mechanisms (primary hyper
parathyroidism). The pathogenesis, clinical presentation, and differential diagnosis
therefore vary widely. Hypocalcaemia is due to the reduction in either the albumin-bound
or free fraction. Hypoalbuminemia is probably the most common cause of reduction in the
concentration of total serum calcium. Common clinical conditions associated with low
serum albumin concentrations include chronic liver disease, nephritic syndrome,
congestive heart failure, and malnutrition. Chronic renal failure is also frequently associated
with hypocalcaemia. Contributing reasons for the low calcium values are
hyperphosphatemia, impaired synthesis of 1, 25(OH)2D due to inadequate renal mass, and
skeletal resistance to the action of Parathyroid hormone. Magnesium deficiency is the other
common clinical cause of hypocalcemia. Magnesium deficiency impairs PTH secretion as

28
well as the action of PTH on bone and kidneys. Acute symptomatic hypocalcaemia may
be noted in hospitalized patients for various reasons. Patients undergoing surgical
treatment for hyperthyroidism or primary hyperparathyroidism of receiving therapy for
haematological malignancies may have rapid remineralisation of bone (hungry bone
syndrome) causing a drop in serum calcium. Acute hemorrhagic and edematous pancreaitis
is frequently complicated by hypocalcaemia. Ostomalacia or rickets secondary to vitamin D
deficiency may also be associated with hypocalcaemia. The hypocalcaemia may be due in
part to impaired intestinal absorption of calcium. In addition, vitamin D deficiency renders
the skeleton resistant to PTH and thereby limits calcium resorption from bone.

Serum or plasma calcium is measured with o-cresolphthalein complexone reagent
containing ethanediol which maintains a clear solution in the presence of proteins and
suppresses the ionization of o-cresolphthalein complexone in the reagent. Interference by
magnesium is eliminated by the inclusion of 8-hydroxyquinoline.

2-3 ml clotted blood collected into acid washed container. Do not apply the tourniquet,
avoid haemolysis. Fasting specimen is preferable. Separate the serum from red cells as early
as possible. Pyrex vials with Teflon lined screw caps are recommended for specimen
storage. Specimen may stored at -20 0C for several weeks or months

APPARATUS: Spectrophotometer with wavelength 575 nm or colorimeter with yellow
filter-Ilford 606(580nm), Safety bulb
GLASSWARE:
Volumetric flask class A (100ml, 500ml and 1 litre volumes)
Automatic micro pipettes (50 µl)
Volumetric pipettes (5 ml, 10ml)
Graduated pipettes (1 ml, 2 ml, 5ml and 10 ml in 0.1ml)
Beaker (5ml, 250 ml)
Measuring cylinder (50ml), Test tubes (125 x 16 mm)
CHEMICALS:
Hydrochloric acid concentrated (37% w/v); caution: highly corrosive
O-cresolphthalein complexone-AR
Ethanediol-AR
2-amino-2-methyl-1-propanol-AR
8-hydroxyquinoline-AR
Calcium carbonate-AR
NOTES: All glassware must be thoroughly cleaned then soaked overnight in hydrochloric
acid (0.5mol/l) to remove traces of calcium then thoroughly rinsed with distilled or
deionised water and finally dried before use.
REAGENTS
1. Hydrochloric acid 0.5 mol/l: Adding the acid to the distilled water, dilute about 45 ml of
hydrochloric acid (concentrated) to 1 litre with distilled water. Use this solution for
soaking glassware as described above.
2. Stock CPC reagent: Add 38 ml ethanediol and 13 ml of 2- amino-2-methyl-1-propanol to
a 500 ml volumetic flask containing about 400 ml of distilled water. Weigh out 15 mg
of o-cresolphthalien complexone and add it to the volumetric flask, mix until the all
the chemicals are completely dissolved, make up to the mark with distilled water and

29
transfer the reagent into a clean brown bottle. This solution is stable for 3 weeks at 4
-6 0C
3. Working CPC reagent: Weigh out 100 mg of 8-hydroxyquinoline and transfer into a 100
ml volumetric flask using small volumes of stock CPC solution. Add about 80 ml of
stock CPC solution mix until the chemical is completely dissolved and make up to the
volume to 100ml using stock CPC reagent. The 8-hydroxyquinoline dissolves quite
slowly. This solution is stable for 1 week at 4-6 0C and should have and absorbance at
575 nm of about 0.2 when measured with the spectrometer set to zero with distilled
water. An absorbance higher than 0.2 indicates either that the reagent has deteriorated
or that it is contaminated with calcium (use of dirty container or cuvettes)
4. Calcium stock standard 25mmol/l: Dry about 300 mg of calcium carbonate in a dry
container in an oven for 4 hours at 80-100 0C. Remove the container from the oven
and immediately close it with a lid. When it has attained to room temperature weigh
out exactly 250 mg and transfer to a 100 ml volumetric flask. Dissolve the calcium
carbonate in a minimum volume of hydrochloric acid (concentrated) approximately
0.5 ml is required, and then make up to100 ml with distilled water.
5. Calcium working standard 2.5mmol/l: Using a volumetric pipette transfer 10.0 ml of the
stock standard to a 100 ml volumetric flask. Make up to 100 ml with distilled water.
8.6 PROCEDURE
1. The analysis must be performed in duplicate. Transfer 50 µl of serum or plasma to
each of two clean tubes, add 5.0 ml working CPC reagent to each tube using a 5 ml
volumetric pipette and mix well.
2. Transfer 50 µl of working calcium standard (2.5mmol/l) to each of 2 clean tubes; add
5.0 ml working CPC reagent to each tube using 5 ml volumetric pipette and mix well.
3. Transfer 50 µl of distilled water to a clean tube, add 5.0 ml working CPC reagent, Mix
well. This is the reagent blank.
4. Set the spectrometer to zero at 575 nm with distilled water and measure the
absorbance of the reagent blank which should be about 0.2.
5. Measure the absorbance of the standards (2.5mmol/l) and serum sample
If the absorbance of duplicate readings varies by more than 0.015 then precision is
unsatisfactory. Check that the test tubes and pipettes are clean. Check the precision of the
50 µl volumetric pipette. Use a 5.0 ml volumetric pipette for the working CPC reagent.
Check that the spectrometer cuvettes (cells) are clean.

The calibration graph must be prepared in order to confirm the linearity of the method
and should be checked monthly. The calibration graph should not be used for calculating
patients’ results. Prepare the calibration graph standards from the calcium stock standard
as described in the table below in clean test tubes as accurately as possible using 1, 2 and 10
ml graduated pipettes
Tube Number 1 2 3 4 5
Calcium stock standard

0.2 0.7 1.0 1.2 1.5
( 25mmol/l) ml
Distilled water (ml) 9.8 9.3 9.0 8.8 8.5
Calcium concentration (mmol/l) 0.5 1.75 2.5 3.0 3.75

30
Transfer 50 µl of distilled water into a clean test tube for the reagent blank and transfer
50 µl of each calibration graph standard to 5 other test tubes. Add 5.0 ml of working CPC
reagent to each tube using a 5 ml volumetric pipette and safety bulb. Mix and measure the
absorbance of each tube at 575 nm setting the spectrometer to zero with distilled water.
Plot the absorbance of each tube on the vertical axis against the calcium concentration of
the calibration graph standards in mmol/l on the horizontal axis.
The purpose of preparing the calibration graph is to confirm the linearity of the method. If
this is not linear beyond 3.0 mmol/l, then patients’ samples with calcium concentrations
greater than 3.0 mmol/l should be diluted two-fold with distilled water before analysis. If
the graph is linear up to 3.75 mmol/l then samples with calcium concentrations greater
than 3.75 mmol/l should be diluted two-fold with distilled water before analysis.
8.7 CALCULATION
Calculate the results using the following formula:
Concentration of calcium (mmol/l) = T-B x 2.5
S-B
Where:
T = Absorbance reading of sample or control
S = Absorbance reading of working calcium standard (2.5 mmol/l)
B = Absorbance reading of reagent blank
If the sample or control result is above the linearity of the method then repeat the analysis
after accurately diluting 200 µl of sample with 200 µl of distilled water in a clean tube. Use
50 µl of the diluted sample for the analysis. Remember to multiply the result by 2 to obtain
the calcium concentration of the sample.
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 2.00-2.90 mmol/l
one of which is unknown to the operator, should be included with each batch of
specimens. If single specimens are analysed a control specimen should always be included.
OPTIMAL CONDITIONS VARIANCE : A coefficient of variation of around 1.5%

ROUTINE CONDITIONS VARIANCE : The value obtained for the RCV should not
exceed 4%
REFERENCE VALUES
The reference interval for healthy ambulant adults is 2.25-2.60 mmol/l. Conversion of SI
units into “old” units: mmol/l x 4 = mg/dl.
The reference values are only appropriate if the patient has a normal serum albumin
concentration. If the patient has a low serum albumin then it may be helpful to report the
albumin corrected calcium as well as the measured calcium. The albumin corrected calcium
is calculated in this way:
(40 – Patient’s albumin) + measured calcium = albumin corrected calcium
40
For example if the patient has an albumin of 20 g/l and a measured calcium of 1.90
mmol/l, then the albumin corrected calcium is 2.40 mmol/l
(40-20) + 1.90 = 2.40 mmol/l
40
The albumin corrected calcium will be approximately 2.20-2.60 mmol/l if low measured
calcium is a consequence of a low serum albumin.

31
NOTE: The estimation of calcium is difficult, particularly because of the possibility of
contamination by calcium. It is essential that high quality chemicals are used and that the
recommendations regarding cleaning of glassware are strictly followed. It may be found
helpful to keep separate test tubes, pipettes etc, only for the analysis of calcium When
preparing the reagents, be careful not to contaminate other chemicals or glassware with
calcium carbonate.
PRECAUTIONS
 Use dry glass container which must be chemically cleaned and acid washed
 Avoid use of plastic containers and use of rubber stoppers
 Avoid use of acid etched glassware. It may lead either to calcium loss because
adsorption of calcium on to the damage surface or to contamination with calcium
because the etched areas cannot be cleaned thoroughly.
REFERENCES
LAB/86.3
CALCIUM IN URINE
SPECIMENS: a 24 hour urine collection in an acid washed 2.5 L bottle; 10 ml of 1 N HCl
is added as the preservative. Acid washed beaker and a funnel should be issued from the
laboratory along with collection bottle.
PROCEDURE:
 Mix the 24 hour urine collection and measure the total urine volume
 Follow the procedure as for serum calcium analysis
Urine samples with calcium concentration greater than 3.75 mmol/l should be diluted with
distilled water before analysis. Multiply the results by the dilution factor
CALCULATION:
Concentration of calcium (mmol/l) = T-B x 2.5
S-B
Concentration of calcium in 24 hour urine = T-B x 2.5 x (TV in litre) mmol/24 hour
S-B
Where:
T = Absorbance reading of sample or control
S = Absorbance reading of working calcium standard (2.5 mmol/l)
B = Absorbance reading of reagent blank
TV =24 hour urine total volume in litre
In children concentration of urinary calcium= T-B x 2.5 x TV in litre x 1000 µmol/kg/24 hours
S-B Body weight in kgs
REFERENCE VALUES:
New Born : 0-17.5 µmol/kg/24 hours
Infants : up to 1000 µmol/kg/24 hours
Older Children : up to100 µmol/kg/24 hours or 750-3750 µmol/24hours
Adults
Calcium in diet
Calcium free : 0.13-1.0 mmol/24 hours
Low –average : 1.25-3.75 mmol/24 hours
Average (800mg/day) : 2.5-7.5 mmol/24 hours

32
9. CREATININE
9.1 INTRODUCTION
Creatine is synthesized in the kidneys, liver and pancreas by two enzymatically mediated
reactions. In the first, transamidination of arginine and glycine forms guanidinoacetic acid:
the second methylation of guanidinoacetic acid occurs with S- adenosylmethionine as
methyl donor. Creatine is then transported in blood to other organs such as muscle and
brain, where it is phosphorylated to phosphocreatine a high energy compound.
Interconversion of phosphocreatine and creatine is a particular feature of metabolic
processes of muscle contration; some of the free creatine in muscle spontaneously converts
to creatinine, its anhydride. Between 1 and 2% of muscle creatine is converted to creatinine
daily. Because the amount of endogenous creatinine produced is propotional to muscle
mass, the production varies with age and sex; non obese men excrete about 1.5 g/day,
women 1.2 g/day. Daily excretion of creatinine can be 10% to 30% greater as a result of
dietary intake of creatine and creatinine in meats. On the whole however, dietary
fluctuations of creatinine intake cause only minor variation in daily creatinine excretion on
the same individual. The excretion rate in one individual, in the absence of renal disease, is
relatively constant and parallels endogenous production. Most of the interindividual
variations of creatinine excretion in healthy persons are attributable in the main to age, sex,
and lean body mass and intraindividual variation tends to be less than 15% from day to
day.
As creatinine is endogenously produced and released into body fluids at a constant rate and
its plasma levels maintained within narrow limits, its clearance may be measured as an
indicator of glomerular filtration rate. However, a small quantity of creatinine is reabsorbed
by the tubules and a small quantity of creatinine appearing in the urine(7-10%)is due to
tubular secretion. As a result creatinine clearance (if creatinine is measured with an accurate
method) is approximately 7% greater than inulin clearance. Some methods for creatinine
used in clinical laboratories are nonspecific, however, and thus this difference is often
smaller. The creatinine clearance is performed by obtaining a 4-, 12- or 24-h urine
specimen and also a blood specimen within the period of urine collection. The volume of
the urine is measured, urine flow rate is calculated (millimetres per minute), and the assay
for creatinine is performed on plasma and urine to obtain the concentration in milligrams
per decilitre or per millilitre. Two factors influence measurement of creatinine clearance
and thus its interpretation. First, the most common methods for measuring creatinine use
the nonspecific alkaline picarate reaction, and thus noncreatinine chromogens in plasma
increase the apparent plasma concentration by as much as 30% if serum values are less
than 1.0mg/dL, and by approximately 10% is values exceed 1.0mg/dL. The percent
increase is progressively less with higher creatinine concentrations. (Urine contains
considerably fewer noncreatinine chromogens.) This overestimation of plasma creatinine
concentration results in underestimation of creatinine clearance and partially offsets the
apparent high clearance of creatinine that is due to tubular secretion. As a result, the
endogenous creatinine clearance agrees closely with the inulin clearance throughout a
substantial range of clearances. However, if accurate methods are used for assay of plasma
creatinine, the GFR estimated by creatinine clearance may not correlate with the GFR
estimated by inulin clearance. Secondly, GFR measured by creatinine clearance and GFR
measured by inulin clearance in the same patient progressively diverge as renal failure
progresses and plasma creatinine level rises. The greater apparent GFR found by creatinine
clearance may be due to an increase in tubular secretory activity for creatinine when plasma
levels increase much above normal and to the relatively smaller contribution of
noncreatinine chromogens in the nonspecific assay of plasma creatinine. In clinical
practice, it is now accepted that, by the time patients have lose one half to two thirds their
normal renal function, as demonstrated by creatinine clearance, it is more reliable and

33
prudent to monitor their subsequent renal function and response to therapeutic
initiatives by using radioisotopic markers of glomerular filtration and renal plasma flow.
Protein free filtrate is mixed with an alkaline picrate solution which forms a yellow –red
complex with Creatinine. The absorbance of the complex is measured at 500 nm.
Non haemolysed serum; collect about 4 - 5 ml blood into a clean dry bottle. Avoid
haemolysis. Separate the serum as early as possible from the cells within 12 hours of
collection. Serum is stable at 2-8 0C up to 24 hours
Referral: Send about 1.0 ml serum kept cool to reach destinations within 18 hours
APPARATUS:
Spectrophotometer at wavelength 500 nm or Colorimeter with blue green filter, Ilford 603)
Volumetric flask (100 ml, 500 ml Sodium hydroxide pellets
and 1litre volumes) Picric acid; Note: water is added to
Automatic micro pipettes (50µl, picric acid to ensure safety in transit
100µl, 500 µl and 1000µl-5000µl) Creatinine anhydrous -AR
Graduated pipettes (5ml, 10 ml in Hydrochloric acid concentrated
0.1 ml) (37% w/v) caution: highly corrosive
Beakers (100ml, 500 ml) Sulphuric acid-AR
Test tubes (125 mm x 16 mm) Sodium tungstate dihydrate
Conical centrifuge tubes 15 ml Polyvinyl alcohol
Reagent bottles, clear and amber
coloured (500 ml), Rubber bulb
REAGENTS
1. Saturated picric acid solution: Picric acid is supplied as a moist chemical. Weigh out the
equivalent of 7 g of picric acid i.e. mix well the bottle and weigh out about 10.5 – 11 g
if your picric acid container states that 50 % by weight of water has been added. Add
11 g of moist picric acid to 500 ml distilled water, stir for several hours to ensure that a
saturated solution is produced. Transfer in to a brown bottle. This solution is stable
indefinitely at room temperature.
Note: the amount of picric acid weighed out will depend on the water content of the
chemical
2. Picric acid 0.036 mol/l: Measure 705 ml of saturated picric acid solution in to volumetric
flask and make up to 1 litre with distilled water store in a brown bottle. This solution is
stable indefinitely
3. Acid tungstate solution: Weigh out 11.1 g of sodium tungstate dihydrate (9.8 g anhydrous
salt) and dissolve in about 300 ml of distilled water in a 1 litre volumetric flask.
Dissolve 1 g of polyvinyl alcohol in about 100 ml of distilled water with heating (do
not boil) Allow to cool to room temperature then transfer into the volumetric flask
containing sodium tungstate. Measure 2.1 ml of concentrated sulphuric acid in to 300
ml of distilled water in a beaker mix well. Add this solution also-in to the same
volumetric flask containing tungstate solution. Dilute to 1 litre with distilled water
when the solution is cool to room temperature. Store in a brown bottle.
4. Sodium hydroxide solution 1.4mol/l: Dissolve 56 g of sodium hydroxide in distilled water
and dilute to 1 litre, store in a polypropylene bottle.

34
5. Hydrochloric acid 0.1mol/l: Carefully pipette 9.0 ml hydrochloric acid concentrated in
to a volumetric flask containing distilled water, dilute to one litre with distilled water.
6. Creatinine standard 1.32mmol/l: Keep about 200 mg of Creatinine for overnight in a
desiccator. Weigh out 149 mg of Creatinine anhydrous and dissolve in a small volume
of hydrochloric acid (0.1mol/l) in a small beaker, transfer quantitatively to a one litre
volumetric flask and make to 1 litre with hydrochloric acid. Store in a brown bottle
9.6 PROCEDURE
1. Label two conical centrifuge tubes one for the quality control serum and one for the
patient’s sample. Pipette 4.0 ml of Acid tungstate reagent into each tube.
2. Pipette 500 µl of quality control serum or patients’ serum to the appropriate tubes.
Mix vigorously for about 10 seconds then centrifuge for about 10 minutes to obtain a
clear supernatant.
3. Transfer 3.0 ml of clear supernatant in to test tubes for each quality control serum or
patient’s serum. For blank 3.0 ml of distilled water, for the standard pipette 3.0 ml of
distilled water and 50 µl of Creatinine standard solution.
4. To each tube add 1.0 ml of picric acid solution (0.036 mol/l) mix well
5. Add 0.5 ml sodium hydroxide solutions (1.4mol/l) to the first tube, mix well at 30
second intervals add sodium hydroxide solution to remaining tubes.
6. Exactly 15 minutes after addition of sodium hydroxide read the absorbance against the
reagent blank at 500 nm. Read absorbance of the tubes in sequence maintaining 30
seconds intervals between readings
9.7 CALCULATION
Dilution of standard in the assay : 0.05ml STD + 3.0 ml of distilled water
= 3/0.05 = 60
Concentration of the stock standard = 1.32 mmol/l (1320 µmol/l)
In the assay condition =1.32/60 =0.022 mmol/l
Dilution of serum in the assay =1 in 9
Serum Creatinine concentration =Abs of Test x Con of Std x Dilution of serum
Abs of Std
=T/S x 0.022 x 9
=T/S x 0.198
=T/S x 200 µmol/l
QUALITY CONTROL
attainable.
ROUTINE CONDITIONS VARIANCE: This value should not exceed 8%
REFERENCE VALUES: For an adult 71-133 µmol/l

Children under 12 years 20-80 µmol/l
9.8 LIMITATIONS
Proteins give positive Jaffe reaction therefore
 The supernatant fluid should be clear
 Pipetting of 3 ml of supernatant should be done carefully without disturbing the
precipitate.
 Sodium hydroxide solution should be added in timed intervals
 Absorbance reading should be measured exactly 15 minutes after addition of Sodium
hydroxide
 Interfering substances reacting like Creatinine are acetone, acetoacetate, pyruvate and
some cephalosporin antibiotics contribute to total colour production
 Blood constituents such as glucose, ascorbate histidine and adrenaline may cause
disturbances to the colour development.

35
10. URINE CREATININE
24 hours collection of urine: empty the bladder completely and discard the sample, note
the time, from that time onward collect the sample into a beaker and transfer (use a funnel)
into an amber colour bottle (2.5 L) with preservative till 24th hour, keep the bottle in the
refrigerator during collection period. A sample of blood (3-4 ml) should be drawn during
the collection period of urine. If the bottle is inadequate, collect the excess urine into a
chemically clean bottle and store in the refrigerator until it is dispatched to the laboratory.
After dilution, the urine is mixed with picric acid and sodium hydroxide solution which
forms a yellow red complex with Creatinine. The absorbance of the complex is measured
at 500 nm.
Same as for serum Creatinine estimation.
PRESERVATIVE
1 Normal hydrochloric acid (1 N)-10 ml or Thymol in Isopropanol (100g/l)-5 ml
10.4 PROCEDURES
1. Mix the 24 hours urine collection. Measure the total urine volume.
2. Pipette 1 ml of urine into a 100 ml volumetric flask. Make up to 100 ml with distilled
water. Mix well (1:100 dilution)
3. Three test tubes are required. One for the Test, one for a reagent blank, and one for
the standard
4. Transfer 3.0 ml of diluted urine into the Test. 3.0 ml of distilled water into the reagent
blank. 3 ml of distilled water and 0.1 ml of standard solution into the standard.
5. To each tube add 1.0 ml picric acid solution (0.036mol/l). Mix well.
6. Add 0.5 ml sodium hydroxide solution (1.4mol/l) to the first tube, mix well at 30
seconds intervals and add sodium hydroxide solution to the remaining tubes.
7. Exactly 15 minutes after adding sodium hydroxide read the absorbance against the
reagent blank at 500 nm. Read the tubes in sequence maintaining 30 seconds
intervals between readings.
10.5 CALCULATION
Same method as serum Creatinine but 3 ml of diluted urine (1:100 with distilled water) is
used instead of filtrate
No protein precipitation is needed
 Concentration of standard = 1.32 mmol/l (1320 µmol/l)
 Standard dilution is = 1 in 30 (i.e. 0.1 ml standard diluted with 3.0
ml of distilled water)
 ∴standard concentration = 1.32 mmol/l = 0.044 mmol/l (in assay condition)
30
 urine dilution is = 1: 100
 ∴ urine Creatinine = Test x 0.044 x 100 mmol/l
Standard
 24 hour Urine Creatinine = Test(T) x 4.4 x Total volume in litre
(mmol/24 hour) Standard(S)
CHILDREN
 Urine Creatinine = T x 4.4 x Total volume in litre x 1000
(µmol/kg/24 hours) S x Body weight in kgs
REFERENCE VALUES: For an adult 8.84 to 17.6 mmol/24 hours

36
Children under 12 years 44 -354 µmol/kg/24 hours
CREATININE CLEARANCE
 The following details of the patients are required for calculation of Creatinine
Clearance
 Height in cm
 Body weight in kg(BW)
 Age
 Total volume of urine collection over timed period ( 24 hours)
 Blood sample should be collected during the 24 hours urine collection
 Serum Creatinine and 24 hours urine Creatinine should be performed for calculation
of Creatinine clearance
 Creatinine Clearance =UV/P
 U-Urine Creatinine in mmol/l
 V-Rate of urine flow in ml/minute=24 hours urine volume in ml
24 x 60
 P-Serum Creatinine in mmol/l
Conversion of urinary Creatinine excretion in mmol/24 hours to mmol/l

Concentration of urine Creatinine in mmol/l=Concentration of urinary Creatinine mmol/24 hours
Total volume in litre
Creatinine Clearance=Concentration of urine Creatinine in mmol/l x 24 hour urine volume in ml

(ml/minute) Concentration of serum Creatinine in mmol/l 24 x 60
Clearance varies with body size and is proportional to the body area (A) where this varies
much from the normal in adults and in all cases in children.
The determined clearance is corrected to a standard surface area of 1.73 m2 by multiplying
1.73/A. The A [Body surface area (in m2) can be calculated using a nomogram. From
height (in cm) and weight (in kg)]
Corrected Creatinine Clearance= Creatinine clearance x 1.73 ml/minute/1.73 m2

A
REFERENCE VALUES: Refer appendix 3
REFERENCES
WHO manual
Varley’s practical clinical Biochemistry sixth edition

37
11. CHOLESTEROL
11.1 INTRODUCTION
Although every living organism examined has been found to contain sterols, cholesterol is
found almost exclusively in animals and humans, in which it is also the main
sterol.Virtually all cells and body fluids contain some cholesterol.Like other
sterols,cholesterol is a solid alcohol of high molecular weight and possesses the tetracyclic
perhydrocyclopentanophenanthrene skeleton. The molecule contains 27 carbon
atoms.Cholesterol is the initial starting point in many metabolic pathways. These include
vitamin D synthesis, steroid hormone synthesis, and bile acid metabolism.
Cholesterol is presented to the intestinal wall from three sources: the diet, bile and
intestinal secretions and cells. Animal products, especially meat, egg yolk, seafood, and
whole-fat dairy products, provide the bulk of dietary cholesterol. Cholesterol intake varies
considerably according to the dietary intake of animal products. A similar amount of
cholesterol is present in the gut from biliary secretion and the turnover of mucosal cells.
Practically all cholesterol in the intestine is present in the unesterified (free) form.
Esterified cholesterol in the diet is rapidly hydrolyzed in the intestine to free cholesterol
and free fatty acids (FFA) by cholesterol esterase in pancreatic and small intestinal
secretions.
Although portion of the body’s cholesterol is derived from dietary intake, most tissue and
plasma cholesterol is synthesized endogenously by the liver and other tissues from simpler
molecules, particularly acetate.
Once synthesized cholesterol is released into the circulation for transport in combination
with specific apoproteins, the apolipoproteins, in complexes known as lipoproteins.
Minimal cholesterol esterification occurs within the liver before its release, and cholesterol
is mainly esterified within the vascular compartment. Esterification is important because it
serves to enhance the lipid carrying capacity of the lipoproteins. The reaction is catalyzed
by the enzymes lecithin-cholesterol acyltransferase (LCAT) in the plasma and acyl-
cholesterol acyltransferase (ACAT) intracellularly.The intracellular ACAT pathway is the
major pathway in the liver, intestine, adrenal cortex, and probably in the arterial wall.
Once cholesterol enters the cell, the esters are hydrolyzed by the action of specific esterases
and enters into specific metabolic pathways.
Cholesterol reaching the liver is either secreted unchanged into bile or metabolized to bile
acids. Approximately one third of the daily production of cholesterol is catabolized into
bile acids. The first step in the bile acid synthesis involves the rate limiting step, 7 alpha
hydroxylation. Two bile acids, cholic and chenodeoxycholic, constitute the primary bile
acids.They are conjugated with either glycine or taurine and enter the bile canaliculi. Some
of the bile acids are deconjugated and converted by bacteria in the intestine to secondary
bile acids. Cholic acid is converted to deoxycholic acid, and chenodeoxycholic acid is
metabolized to lithocholic acid.Virtually all bile acids except lithocholic are reabsorbed in
the lower third of the ileum and returned to the liver via the portal vein, thus completing
the enterohepatic circulation.

High prevalence of atherosclerosis and ischaemic heart disease is seen where dietary fat
intake is relatively high. High plasma levels of LDL, IDL and possibly VLDL are
associated with an increased risk of premature atherosclerosis and ischaemic heart disease.
This relationship appears to be a continuous (curvilinear) one, i.e. there is no threshold
above which risk abruptly appears. Plasma HDL cholesterol concentration is a negative

38
risk factor, so that high levels appear to protect against ischaemic heart disease and low
levels are associated with an increased risk of ischaemic heart disease.

The cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator
quinoneimine is formed from hydrogen peroxide and 4 – aminophenazone in the presence
of phenol and peroxidase.
11.4 PATIENT PREPARATION

 No change in dietary habits for at least 3 weeks
 12 hours fasting is preferred but not an absolute requirement
BLOOD DRAWING TECHNIQUE

 Patient should be seated.
 Blood is drawn preferably with out a tourniquet.
 Patient should be subjected to minimum stress during blood drawing.

12 hours fasting; 2-3 ml clotted blood; blood drawn with out a tourniquet; avoid
haemolysis. Serum should be separated from cells as early as possible. Specimen preferably
be analysed on the day of collection. Serum is stable for 4 days at 4 0C, for 3 months at
-20 0C and for many years at -70 0C. As referral sample send about 0.5 ml clear serum, kept
cool, to reach destination within 24 hours.
APPARATUS:
Spectrophotometer at wavelength 500 nm
Water bath at 37 0C
Vortex mixer
Automatic micropipette 10 µl
Automatic pipette 1000µl
GLASSWARE:
Test tubes 100mm x 13mm
Semi micro cuvette (capacity =1ml)
REAGENTS:
There are various brands of commercially available reagents and standards for cholesterol
estimation. Evaluate the kits using quality control samples (low, high and normal values)
and consider the following to select the commercially available kits
1. Performance of the test
2. Reagent stability
3. Expiry date
4. Feasibility of test procedure
5. Interfering substances
6. cost
11.7 PROCEDURE
Following procedure is based on commercially available reagent kits. Test procedure may differ among
various products. Therefore strict to the test procedure provided with reagent kits
1. label tubes for Blank, Standard, Quality control and test
2. Add 10 µl of distilled water to Blank , 10 µl of standard solution to Standard, 10µl of
QC sample to Quality control and 10µl of patients serum to test

39
3. Add 1000 µl of cholesterol reagent to all the tubes
4. Mix well and incubate all the tubes at 37 0C for 5 minutes in water bath
5. Mix well, zero the spectrophotometer with reagent blank and read the absorbance at
500 nm
11.8 CALCULATION
Concentration = Absorbance of test x Standard concentration (mmol/l)
Absorbance of standard
QUALITY CONTROL
Include Quality Control sample for every batch of tests.
OPTIMAL CONDITIONS VARIANCE : A coefficient of variation of around 7%

ROUTINE CONDITIONS VARIANCE : This value should not exceed 14%
REFERENCE VALUES: Refer appendix 3
11.9 LIMITATION
 The test is linear up to a cholesterol concentration of 750 mg/dl (19.3mmol/l). Dilute
samples with a higher cholesterol concentration 1+2 with physiological saline (0.9 %)
and repeat the determination. Multiply the result by 3.
 Haemoglobin up to 200 mg/dl does not interfere with the test
 Bilirubin >5mg/dl and ascorbic acid>10 mg/dl interfere the test
 This limitation varies with kits.
PRECAUTIONS
 Allow the samples, standard, QC and reagents to attain room temperature
 Mix the thawed sample well.
 Do not include more than 20 samples for a batch to maintain good quality
 Verify the temperature of the water bath (at 37 0C) before the commencement of the
test.
 As the final volume would be 1 ml, use appropriate tubes. (You may encounter
difficulties in pipetting with long tubes.)
 Wipe outside of the pipette tip using a piece of gauze
 Add serum to the bottom of the tube
 Mix well in each step
 Any colour change of the blank should be compared with previous blank readings
REFERENCES
Leaflets of commercially available kits
OTHER METHODS
Liebermann –Burchard method
Specific enzymatic methods (as described above) have replaced the chemical methods based on Libermann- Burchard and
Salkowski reactions.

40
12. GLUCOSE
12.1 INTRODUCTION
Glucose is the primary energy source for the human body. It is derived from the
breakdown of carbohydrates in the diet (grains, starchy vegetables and legumes) and in the
body stores (glycogen), as well as by endogenous synthesis from protein or the glycerol
moiety of triglycerides. When energy intake exceeds expenditure, the excess is converted to
fat and glycogen for storage in adipose tissue and liver or muscle respectively. When energy
expenditure exceeds caloric intake, endogenous glucose formation occurs from the
breakdown of carbohydrate stores and from non carbohydrate sources. (Amino acids,
lactate, and glycerol)
The glucose level in blood is maintained within a fairly narrow range under diverse
conditions (feeding, prolonged fasting or severe exercise) by regulatory hormones. These
include insulin, which decreases blood glucose, and the counter regulatory hormones
(glucagon, cortisol, noradrenaline and growth hormone) which increase blood glucose
levels.

Diabetes mellitus is a group of metabolic disorders of carbohydrate metabolism in which
glucose is underutilized, producing hyperglycemia. Some patients may develop acute life
threatening hyperglycemic episodes, such as ketoacidosis or hyperosmolar coma. As the
disease progresses the patients are at increased risk of developing specific complications
including retinopathy leading to blindness, renal failure, neuropathy(nerve damage),and
atherosclerosis.The last may result in stroke, gangrene or coronary disease.
(Please refer the article in annexure for current World Health Organization recommended
criteria for diagnosis of Diabetes Mellitus.)
Hypoglycemia is a blood glucose concentration below the fasting range, but it is difficult to
define specific limits. No symptoms are specific for hypoglycemia. A rapid decrease in
plasma glucose to hypoglycemic levels usually triggers a sympathetic response, with the
release of nor adrenaline, which produces classical signs and symptoms of hypoglycemia:
weakness. Sweating, nausea, rapid pulse, lightheadedness and hunger. The brain is totally
dependent on blood glucose, and very low levels of plasma glucose(less than 20 or 30 mg
/dl cause severe central nervous system dysfunction.
Neonatal blood glucose concentrations are much lower than adults and decline shortly
after birth when live glycogen stores are depleted. Glucose levels as low as 30 mg/dl in a
term infant and 20mg/dl in a premature infant may occur without any clinical evidence of
hypoglycemia. The more common causes in the neonatal period include prematurity ,
maternal diabetes and maternal toxemia. These are usually transient. Hypoglycemia with
onset in early infancy is usually less transitory and may be due to inborn errors of
metabolism or ketotic hypoglycemia, which usually develop after fasting or febrile illness.

The aldehyde group of β - D – Glucose present in the plasma is oxidized by the enzyme
Glucose oxidase to gluconic acid with liberation of hydrogen peroxide. The hydrogen
peroxide is converted to water and molecular oxygen by the enzyme peroxidase. In the
presence of an oxygen acceptor or 4 - aminophenazone together with phenol, a pink
colour is formed which is measured at 510 nm.
SPECIMEN CONTAINERS

41
 Blood containers should be leak proof and be easy to close and open without
contaminating the fingers.
 Screw capped bottle with a rubber liner (Bijou bottles) are satisfactory.
 Bottles should be washed with a detergent, rinsed in several changes of clean water,
rinsed in distilled water and dried well.
ANTICOAGULANT AND PRESERVATIVE

 4 mg of a mixture of potassium oxalate and sodium fluoride in the ratio of 3:1 is
sufficient to collect 1 ml of blood.
 A solution can be prepared so that 0.1 ml contains 3 mg of potassium oxalate and 1mg
of sodium fluoride.
PREPARATION OF BLOOD SUGAR BOTTLES

 Weigh out 3 g of potassium oxalate and 1 g of sodium fluoride separately into beakers
 Dissolve the chemicals well and transfer into a 100 ml volumetric flask, mix well and
make up to 100ml with distilled water.
 Store the solution in a bottle at room temperature
 To collect 1 ml of blood add 0.1 ml of the prepared solution into bijou bottle and dry
in the oven at 60 0C
 Allow to cool; stopper and label the bottles, the amount of blood is to be collected
should be mentioned

 1 ml blood collected into blood sugar bottle
 Haemolysis free plasma
FASTING SPECIMEN
 For adults the fasting time is usually 10-12 hours.
 For children the fasting time is 6 hours unless longer time is indicated.
POST PRANDIAL SPECIMEN

 Blood collected 2 hours after a meal
RANDOM SPECIMEN
 Blood sample collected at any time regardless of food intake
STABILITY
 Glucose stabilized up to 24 hour at room temperature when collected in an oxalate
and fluoride mixture.
 Plasma should be separated soon after collection preferably within 1 hour
 Separated plasma should not contain RBC or Leucocytes
BLOOD COLLECTION
VENOUS BLOOD
 Avoid an intravenous (IV) infusion arm
 Do not shake the blood but gently mix with the anticoagulant.(to prevent haemolysis)
 Exact amount of an anticoagulant and blood should be mixed since sodium fluoride
inhibits the action of glucose oxidase and peroxidase in the assay.

APPARATUS:
Analytical balance accurately calibrated
Oven, temperature at 100 0C
Water bath, temperature at 37 0C

42
Visible Spectrophotometer with 510 nm
Volumetric flask (100 ml A grade Benzoic acid
and 500 ml volumes) 4 - amino phenazone/4 - amino anti
Graduated pipettes (0.5ml, 1 ml, 2 pyrine-AR
ml in 0.1 ml) Glucose oxidase lyophilized powder
Petri dish, watch glass or beaker from Aspergillus
Pasteur pipette Peroxidase lyophilized powder
Test tubes (100 mm x 13mm) Phenol crystals-AR
Reagent bottles clear and amber Tween 20
coloured D-Glucose anhydrous-AR
Rubber bulb Disodium hydrogen phosphate
dihydrate – AR (Na2HPO4.2H2O)
Potassium dihydrogen phosphate-
AR (KH2PO4)
Sodium azide AR
REAGENTS
1. Benzoic acid solution 1g/l: Weigh 1g of benzoic acid and transfer it to a 1 litre volumetric
flask. Add about 800 ml of distilled water and mix to dissolve the chemical completely.
Make up to 1 litre mark with distilled water and mix well. Transfer to a clean bottle,
label the bottle and store at room temperature. The solution is stable indefinitely.
Benzoic acid does not dissolve easily (Distilled water at 50 – 70 0C can be used –
Monica)
2. Stock glucose standard solution 1 g %( 55.55 mmol/l)
 Use dry and clean glassware
 Weigh 1.3 g of D-Glucose anhydrous (analytical grade) into a watch glass or Petri
dish or into a beaker. Spread the chemical over the bottom of the container and
keep in an oven at 60- 80 0C for 4 hours.
 Allow to cool in a dessiccator and weigh out 1 g of dried glucose accurately
 Transfer the chemical from the weighing container to a volumetric flask using a
funnel. Wash any chemical remaining in the container into the volumetric flask
with the benzoic acid solution (1 g/l). Always use a funnel to transfer the chemical
or solutions from any container to a flask
 Half fill the volumetric flask with benzoic acid solution and mix until the chemical
is completely dissolved. Make the solution up to 100 ml with benzoic acid
solution. Make sure the bottom of the meniscus of the fluid is on the graduation
mark when viewed at eye level
 Use a Pasteur pipette to add the final volume of the benzoic acid solution to the
flask.
 Mix the solution well by inverting the flask for several times. Rinse the bottle with
small quantity of the standard solution, transfer in to the bottle and put the date of
the preparation on the label
 The standard is stable for three months at 2- 8 0C
NOTE: The glucose standard solution should be kept at room temperature for 24
hours to α - β forms to reach in equilibrium after preparation (H&W- 6ht edition)
3. Working glucose standard 5.55 mmol/l:
 Allow the stock glucose solution to attain room temperature.
 Pipette accurately 10 ml of stock glucose solution using a volumetric pipette A
grade ( bulb pipette)
 Carefully dispense into a 100 ml volumetric flask A grade

43
 Make up to the 100 ml mark with benzoic acid solution (1 g/l) use a Pasteur
pipette to add the final volume of the benzoic acid solution to the flask. Make sure
the bottom of the meniscus of the fluid is on the graduation mark when viewed at
eye level. Stopper and mix the solution well by inverting the flask several times.
 Rinse a clean dry bottle with small quantity of standard solution and transfer the
solution into the bottle. Store in the refrigerator at 2- 80 C. This solution is stable
for three months at 2- 80 C
4. Phosphate buffer 100 mmol/l pH 7.0 :
Disodium hydrogen phosphate dihydrate [Na2HPO4.2H2O] 12.95 g
Anhydrous Potassium dihydrogen phosphate [KH2PO4 ] 4.95 g
Sodium azide [NaN3 ] 0.50 g
Distilled water to 1 litre
 Measure about 800 ml of distilled water into a 1 litre volumetric flask
 Weigh out chemicals and add one by one in the order into the flask. Mix to
dissolve the chemicals
 Check that the pH is 7.0 ± 0.05 with a pH meter, make up to 1 litre mark with
distilled water and mix well.
 Transfer the reagent to a clean bottle and label. The reagent is stable for 3-4
months at 2-8 0C
5. Colour reagent(100ml)
4 – Amino phenazone 16 mg
Glucose oxidase 1800 units
Peroxidase 100 units
Phenol 105 mg
Tween 20 50 µl
Phosphate buffer to 100 ml
To prepare 500 ml of colour reagent:

i. Glucose oxidase (GOD): Available as lyophilised powder and as liquid form.
Different products are found in different definitions for units of activity of
glucose oxidase. Read the label for the activity. E.g. 250 units/mg
Therefore the amount of GOD powder to be weighed to contain 1800 units of
GOD is 1800/250=7.2 mg
To prepare 500 ml of colour reagent 7.2 x 5 =36 mg is required, weigh out 36 mg
of GOD lyophilized powder accurately in a small beaker and dissolve in 10 ml of
phosphate buffer carefully.
ii. Peroxidase (POD): Read the label for the activity. E.g. 63 units/mg
Therefore the amount of peroxidase powder to be weighed to contain 100 units
of POD is 100/63 = 1.58 mg
To prepare 500 ml colour reagent 1.58 x 5 = 7.9 mg is required; weigh out 7.9 mg
of POD in a small beaker and dissolve in about 10 ml of phosphate buffer.
iii. Transfer about 400 ml of phosphate buffer into a 500 ml volumetric flask
iv. Add the glucose oxidase solution into the flask using a funnel. Rinse out the
beaker into the flask with a little of the phosphate buffer to make sure all the
GOD is transferred to the flask
v. Add the POD solution into the flask described as above ( iv)
vi. Weigh out 80 mg of 4 – aminophenazone in a small beaker and transfer into the
flask with rinsing with the phosphate buffer.
vii. Weigh rapidly 525 mg of crystalline phenol in a beaker. Transfer into the flask
carefully using the funnel. Rinse the beaker into the flask with phosphate buffer
and mix well.
NOTE: Phenol is highly corrosive, toxic and hygroscopic chemical. Therefore
handle it with great care. To avoid damaging the balance pan, always remove the
beaker when adding or subtracting the chemical. Make sure the stock bottle of
phenol is tightly stoppered after use.

44
viii. Measure 250 µl (0.25 ml) of Tween 20 and add into the flask. Make up to the
500 ml of mark with phosphate buffer, stopper and mix well.
ix. Transfer to a clean brown bottle, label and store at 2-8 C. The reagent is stable for
about one month.
12.6 PROCEDURE
1. Label sufficient test tubes for the batch including standard (S) Quality controls (C1,C2)
and patients samples (1,2,3, etc)
2. pipette into the appropriately labelled 13 x 100 mm tubes as follows
S1 S2 C1, C2 1, 2, 3
Distilled water (ml) 1.9 1.8 1.9 1.9
Glucose standard 5.5 mmol/l (µl) 100 200 - -
Quality control/Patient’s plasma (µl) - - 100 100
Mix well
3. Label a second set of tubes including reagent blank (B), standard (S1, S2), Quality
controls (C1, C2 ) and patient’s samples (1, 2, 3 )
4. Pipette into the tubes as follows.
Blank S1 S2 C1, C2 1, 2, 3
Distilled water (µl) 100 - - - -
Diluted standards (µl) - 100 100 - -
Diluted patient’s sample(µl) - - - 100 100
Colour reagents(ml) 1.2 1.2 1.2 1.2 1.2
5. Mix all tubes well, incubate at 37 in a water bath for 15 minutes.
0C
6. Shake tubes two or three times during this period to ensure adequate aeration
7. Remove from the water bath, cool to room temperature and read the absorbance in a
spectrophotometer at 510 nm. Set the instrument to zero with the reagent blank (B).
8. Perform the standards in duplicate for greater accuracy and precision.
9. Calculate the results in mmol/l and check the quality control results.
NOTE: This procedure is designed for spectrophotometers that require a minimum
volume of reaction mixture in the cuvette of 1 ml. Economical use of reagents is possible
with this protocol, thus the cost per test can be kept to the minimum. However if the
laboratory employs a spectrophotometer requiring a large volume of the reaction mixture
for measurement, viz 5 ml, it is advisable to increase the volume of all reagents mentioned
under step 4 proportionately.

The calibration graph must be prepared in order to confirm the linearity of the method
and should be checked whenever a new batch of reagents are introduced or any change in
the spectrophotometer is being made.
Prepare the calibration graph standards from the Glucose working standard 5.55 mmol/l
as described in the table below in clean tubes (13 x 100 mm) as accurately as possible.
S1 S2 S3 S4 S5 S6
Glucose working standard 5.55mmol/l (µl) 50 100 200 300 400 500
Distilled water (ml) 1.95 1.9 1.8 1.7 1.6 1.5
Glucose concentration mmol/l 2.77 5.55 11.1 16.6 22.2 27.7
Mix well
Follow the procedure 12.6 and draw a calibration graph by plotting the absorbance values
of standards against the concentration of standards.
The points should be linear and the graph should pass through the origin
Since the procedure for standard tube S2 and test is identical, the standard S2 will represent
a concentration of 5.55 mmol/l. the glucose concentration represent by other standard
tubes are S1=2.77, S3=11.1, S4=16.6, S5=22.2, S6=27.7 mmol/l

45
12.7 CALCULATION
When the calibration graph is linear one of the standards used to prepare the calibration
graph should be included in each batch of tests.
The Beer & Lambert formula can be used to calculate the concentration of unknown
samples.
Concentration of Glucose in mmol/l = Test /Standard x Concentration
QUALITY CONTROL
Include QC sample for each batch of tests
OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 3 % should be

attainable.
ROUTINE CONDITIONS VARIANCE: This value should not exceed 6 %
REFERENCE VALUES
Random plasma Glucose level ≤7.8 mmol/l
Fasting plasma Glucose level 3.3-6.1 mmol/l
12.8 LIMITATION
 Any sample that gives a glucose value >25 mmol/l should be diluted 1:2 with 0.9 %
Sodium chloride solution and the correct value obtained by multiplying the result by 3.
 At high plasma levels of uric acid, glutathione and Bilirubin may interfere with the
assay by causing a decrease in glucose values. Ascorbic acid will decrease glucose
values by retarding colour development. Do not report results from specimens with
suspected interference. Inform the requesting physician of the problem.
PROBLEMS AND PRECAUTIONS

 In preparation of sugar bottle add correct volume of anticoagulant/fluoride mixture as
excess NaF may lead to falsely low glucose levels.
 As this is an enzymatic method the factors that control enzymatic reactions should be
kept under control. ( pH of buffer at 7.0 , temperature at 37 0C and the period of
incubation)
 No colour development or low colour development may be due to
o Expired colour reagent
o Unsuitable or expired glucose oxidase or any other chemicals
o Check the incubation time and temperature of the water bath
o Pipetting errors
 As the final volume is 1.3 ml, use a semi micro cuvette (1 ml). Do not use macro
cuvettes (2 ml) as it will cause errors in absorbance readings.
12.9 OTHER METHODS

1. O-Toluidine
COPPER REDUCTION (NOT RECOMMENDED DUE TO NON SPECIFICITY)
2. Phosphomolybdate (Folin wu)
3. Arsenomolybdate (Somogyi-Nelson)
4. Neocuproine
5. Alkaline fericyanide method
ENZYMATIC
6. Hexo kinase (HK)
7. Glucose oxidase (GOD)-oxygen consumption
8. Glucose dehydrogenase
REFERENCES
Trinder,p. (1969).Annals of Clin.Biochem.6:24-27
Barham D and Trinder P. (1972). Analyst 97:142-145

46
13. INORGANIC PHOSPHATE
13.1 INTRODUCTION
Phosphorus in the form of inorganic or organic phosphate is an important and widely
distributed element in the human body. An adult human has approximately 600g (19.4
mol) of phosphate expressed as phosphorus, of which about 85% is in the skeleton and
the rest principally in soft tissues. In the soft tissues most phosphate is cellular. Although
both inorganic and organic phosphate are present in cells, most is organic and
incorporated into nucleic acids, phospholipids and high energy compounds involved in
cellular integrity and metabolism.
Serum phosphate has a diurnal variation. It is higher in the afternoon and evening. The
serum phosphate level is dependent on meals and variation in the secretion of hormones
such as parathyroid hormone. Serum calcium and phosphate levels are regulated by the
kidneys.
The most important intracellular function of phosphate is the high energy bond in
adenosine triphosphate. These energy sources maintain many physiological functions such
as muscle contractility, neurological functions and electrolyte transport. Phosphate is a
constituent of cyclic adenine and guanine nucleotides as well as nicotinamide adenine
dinucleotide phosphate, which is important in many enzyme systems. It is an element in
phospholipid cell membranes, nucleic acids and phosphoproteins. It is also involved in the
regulation of intermediary metabolism of proteins, fats and carbohydrates, as well as in
gene transcription and cell growth.
Extra cellular phosphate maintains the critical intracellular concentration and provides
substrate for bone mineralization.
The skeleton serves as a store house for phosphate. The cellular demands for metabolic
function in bone cells are similar to those in other cells.

Hypophosphataemia is defined as the concentration of inorganic phosphate in the serum
below the normal reference interval. Hypophosphatemia does not necessarily imply
intracellular phosphate depletion. Hypophosphataemia may be present when cellular levels
are normal, and cellular phosphate depletion may exist when serum concentrations are
normal or even high. Phosphate depletion may have four general causes
 Intra cellular shift (A high carbohydrate diet stimulates insulin secretion, increasing the
transport of glucose and phosphate into the cell.)
 Renal Phosphate wasting (Any cause of excessive parathyroid hormone secretion may
result in hypophosphataemia due to phosphaturia
 Decreased intestinal phosphate absorption (Increased loss due to vomiting, diarrhoea
and phosphate binding antacids: Decreased absorption in malabsorption syndromes.)
 Cellular phosphate loss (Acidosis results in catabolism of organic compounds within
the cell so that inorganic phosphate shifts into the plasma and excreted in the urine.
Hyperphosphatemia is usually due to acute or chronic renal failure because the kidneys fail
to excrete the amount taken in the diet. Lack of Parathyroid hormone and increased
growth hormone causes increased tubular reabsorption of phosphate resulting in increased
phosphate levels in blood. Increased phosphate intake, increased extra cellular phosphate
load in acidosis and any cause leading to cell lysis causes hyperphostaemia

47
The filtrate obtained after removing proteins by means of trichloro acetic acid is treated
with an acid molybdate reagent which reacts with inorganic phosphate to form
phosphomolybdic acid. The molybdenum of the phosphomolybdic acid is reduced by
means of 1-amino-2- naphthol-4-sulphonic acid to give a blue compound which is
measured colorimetrically at 700 nm.

Clear, non haemolysed serum is suitable, collect about 4-5 ml blood without haemolysis
into a clean container. Ideally specimens should be obtained without the tourniquet from a
recumbent fasting patient. Serum should be separated from erythrocytes as soon as
possible within 1 hour of collection, as organic phosphate present in erythrocytes are
hydrolysed with formation of inorganic phosphate causing the serum concentration to rise.
Hydrolysis proceeds more rapidly at room temperature and at 37 0C. Haemolysed samples
are unsuitable because erythrocyte contain high concentration of organic phosphates which
can be hydrolysed to inorganic phosphate during storage.
APPARATUS:
Centrifuge
Spectrophotometer
Automatic pipette (500 µl, 1000 µl)
Volumetric flasks (100 ml, 200ml, Ammonium molybdate-AR
500 ml) 1-Amino-2-napthol-4-sulphonic acid
Beakers (5ml, 200 ml, 500ml) Perchloric acid AR
Graduated pipette (0.5 ml, 1ml, 5ml, Sodium metabisulphite-AR (store in
10 ml) refrigerator)
Conical centrifuge tubes 15 ml Sodium sulphite-AR (store in
Test tubes (16 x 150 mm) refrigerator)
Potassium dihydrogen phosphate
KH2 PO4 - AR
Trichloro acetic acid - AR
REAGENTS
1. Reducing agent: Dissolve 12 g of sodium Meta bisulphite and 2.4 g of sodium sulphite in
about 80 ml of distilled water. Add 0.2 g of 1-amino-2-naphthol-4-sulphonic acid.
Dissolve and dilute to 100 ml in volumetric flask. Store in the refrigerator in a brown
bottle. The solution keeps up to 4 weeks. It is better prepare fresh reagent ( about 10
ml)
2. Trichloroacetic acid 10 %: Dissolve 10 g of Trichloroacetic acid in distilled water and
make up to 100 ml with distilled water. Store in the refrigerator.
3. Perchloric acid AR
4. Ammonium Molybdate 5%: Dissolve 5 g of Ammonium molybdate in distilled water and
make up to 100 ml with distilled water. Store at room temperature. Discard the
solution when a precipitate appears.
5. Stock phosphate standard solution 32mmol/l: Keep about 2.5 g of pure potassium
dihydrogen phosphate in a dessicator to dry, weigh exactly 2.194 g of potassium
dihydrogen phosphate and transfer into a 500 ml flask. Dissolve in distilled water and
make up to 500 ml with distilled water. Store at room temperature.
6. Working phosphate standard solution 0.128 mmol/l: Dilute 2 ml of stock phosphate
standard to 500 ml with distilled water. Store at room temperature

48
13.6 PROCEDURE
1. Label sufficient centrifuge tubes for quality control (C) and patient’s samples (1, 2, 3)
2. Pipette into tubes as follows
C 1, 2, 3
Trichloroacetic acid 10 % (ml) 9.0 9.0
Quality control serum (ml) 1.0 -
Patient’s sample (ml) - 1.0
3. Mix well and leave for about 10 minutes and centrifuge ( 2500-3000 rpm) for
about 10 minutes
4. Label a set of test tubes including reagent blank (B) Standard (S) Quality control (C1,
C2) and patient’s samples (1, 2, 3 …)
5. Pipette into the tubes as follows
B S C 1, 2, 3
Trichloro acetic acid 10% (ml) 5.0 - - -
Working standard solution (ml) - 5.0 - -
Supernatants from tubes above(ml) - - 5.0 5.0
Perchloric acid (ml) 0.4 0.4 0.4 0.4
Ammonium Molybdate 5% (ml) 0.4 0.4 0.4 0.4
Reducing reagent (ml) 0.2 0.2 0.2 0.2
6. Mix the tubes after each addition of reagents.

7. Leave at room temperature for 10 minutes
8. Read the absorbance at 700 nm , set the instrument to zero with tube B
NOTE: For inadequate specimen use appropriate volumes of serum and reagents.
The supernatant should be clear.
13.7 CALCULATION
Concentration of standard is 0.128 mmol/l, serum dilution is 1:10
∴Concentration of phosphorus = T x 0.128 x 10
S
= T x 1.28 mmol/l
S
REFERENCE VALUES : For an adult 0.80 -1.44 mmol/l
13.8 LIMITATION
Glucose phosphate, CPK and other organic phosphates may also be hydrolyzed by assay
conditions, resulting in overestimation of inorganic specimens.
PRECAUTIONS
 Glass ware should be properly cleaned and rinsed because phosphate is a common
component of many detergents
 Discard Ammonium Molybdate solution when a precipitation formed at the bottom
of the container
 Recommend to prepare only 10 ml of reducing agent at a time as the stability of the
reagent is one month.

49
14. INORGANIC PHOSPHATE IN URINE
14.1 INTRODUCTION
Urinary phosphate excretion varies with age,muscle mass,renal function,parathyroid
hormone level,the time of the day and diet.

Urine is diluted with distilled water and mixed with an acid molybdate reagent which reacts
with inorganic phosphate to form phosphomolybdic acid. The molybdenum of the
phosphomolybdic acid is reduced by means of 1-amino-2-naphthol-4-sulphonic acid to
give a blue coloured complex which is measured at 700nm spectrophotometrically.

A 24 hours collection of urine is need. 10 ml of 1 N HCl is added as the preservative
APPARATUS, CHEMICALS, GLASSWARE AND REAGENTS ARE AS FOR SERUM

PHOSPHATE ESTIMATION
14.4 PROCEDURE
1. Mix the 24 hours urine collection
2. Measure the total volume of urine
3. Pipette 1 ml of urine into a 100 ml volumetric flask. Make up to 100 ml with distilled
water.
4. Label 3 test tubes for Blank (B), Standard (S) and Test (T)
5. Pipette 5 ml of distilled water into the tube B, 5 ml of working standard into the tube S
and 5 ml of diluted urine into the tube T
6. Add 0.4 ml of Perchloric acid into each tube, Mix well
7. Then add 0.4 ml of Ammonium Molybdate 5% solution to each tube and mix well
8. Add 0.2 ml of reducing agent to each tube, Mix well and leave for 10 minutes at room
temperature
9. Read the absorbance at 700 nm, setting the spectrophotometer to zero with blank
solution
14.5 CALCULATION
Concentration of the Standard = 0.128 mmol/l
Urine phosphate = T x 0.128 x 100 mmol/l
S
= T x 12.8 mmol/l
S
24 hour urine phosphate = T x 12.8 x V (24 hour urine volume in litre) mmol/24 hrs
S
In children urinary phosphate excretion=T x 12.8 x Total volume in litre

(mmol/kg/24hrs) S x Body weight in kg
REFERENCES VALUES:
For an adult : 16-48 mmol/24 hours
For children : Refer appendix 3

50
15. TOTAL PROTEIN

15.1 INTRODUCTION
The term ‘ plasma proteins’ describes the very large number of complex molecules that
share a common primary structure ; but have an enormous diversity of function .Many of
the plasma proteins are classified according to function, e.g. enzymes, clotting factors,
acute phase proteins, immunoglobulins, complement components, protease inhibitors,
apolipoproteins and transport proteins. Normal adult plasma contains 60 -80 g/L of total
protein,the major contributor being albumin and the rest due to globulins.
GLOBULINS
Electrophoresis of normal serum performed on a carrier such as cellulose acetate reveals
four major non-albumin bands staining for protein/lipoprotein, designated by mobility as
α1, α2 β, γ
Band Concentration (g/L) Major components
α1-globulin 2-5 α1- antitrypsin, Apolipoprotein
α2 –globulins 4-10 Caeruloplasmin, α2 macroglobulins,
Haptoglobins
β-globulin 6-12 Transferrin β-lipoproteins
γ-globulins 8-16 Immunoglobulins

The two general causes of alterations of serum total protein are a change in the
volume of plasma water and a change in the concentration of one or more of the
specific proteins in the plasma. Decrease in the volume of plasma
water(haemoconcentration) is reflected as relative hyperproteinemia: concentrations
of all the individual plasma proteins are increased to the same degree.
Hyperproteinemia is noted in dehydration due to inadequate water intake or to
excessive water loss as in severe vomiting, diarrhoea or diabetic acidosis.
Hemodilution(increase in plasma water volume) is reflected as relative
hypoproteinemia; concentrations of all the individual plasma proteins are decreased
to the same degree. Hemodilution occurs with water intoxication or salt retention
syndromes, during massive intravenous infusions and physiologically a recumbent
position is assumed. A recumbent position decreases total protein concentration by
0.3 to 0.5 g/dL.
Of the individual serum proteins, albumin is present in such high concentrations
that low levels of this protein alone may cause hypoproteinemia. Such
hypoproteinemia is common and has many causes as described earlier. Mild
hyperproteinemia may be caused by an increase in the concentration of specific
proteins normally present in relatively low concentrations as for example in acute
phase proteins and polyclonal immunoglobulins as a result of infection.Marked
hyperproteinemia may be caused by high levels of the monoclonal
immunoglobulins produced in multiple myeloma and other malignant
paraproteinemias.

51

Serum proteins form a violet- blue complex with copper ions in alkaline solution. The
absorbance of the complex is measured at 540 nm. A method using a sample blank is
recommended in order to avoid errors due to turbidity.

3 ml clotted blood collected into dry clean glass bottle without applying a tourniquet, avoid
haemolysis, fasting specimen is desired to decrease lipaemia. Separate the serum from cells
as early as possible. Serum is stable at 4 0C
APPARATUS:
Spectrophotometer at wavelength 540 nm or colorimeter with yellow-green filter, Ilford
605(550 nm)
GLASSWARE:
Graduated pipette (5 ml in 0.1 ml)
Measuring cylinders (100ml and 500 ml)
Test tubes (150 mm x16 mm)
Beaker (250 ml)
Polyethylene reagent bottles (I litre)
CHEMICALS:
Sodium chloride-AR
Sodium azide-AR caution: handle with care
Sodium hydroxide pellets-AR
Copper sulphate pentahydrate-AR
Potassium sodium tartrate tetrahydrate-AR
Potassium iodide-AR
Albumin bovine, a fraction v powder is suitable
REAGENTS
1. Sodium hydroxide solution 6 mol/l: Dissolve 120 g of sodium hydroxide little by little in
about 400 ml of distilled water. After cooling dilute to 500 ml. Store in a tightly- closed
polyethylene bottle. This solution is stable indefinitely at 20 -25 0C(room temperature)
2. Biuret reagent: Dissolve 3.0 g of copper sulphate in about 500 ml of distilled water. Add
9.0 g of potassium sodium tartrate and 5.0 g of potassium iodide. When they have
completely dissolved, add 100 ml of sodium hydroxide solution (6 mol/l) and make up
to 1 litre. The solution is stable indefinitely at 20 -25 0C(room temperature) store in a
tightly closed polyethylene bottle
3. Blank Biuret reagent: Dissolve 9.0 g of potassium sodium tartrate and 5.0 g of potassium
iodide in distilled water. Add 100 ml of sodium hydroxide solution (6mol/l) and make
up to 1 litre with distilled water. When kept in a tightly-stoppered polyethylene bottle
this solution is stable indefinitely at 20 -25 0C(room temperature)
4. Sodium chloride/Sodium azide solution: Weigh out 9.0 g of sodium chloride and 1.0 g of
sodium azide, dissolve and make up to 1 litre with distilled water. This solution is
stable indefinitely at 20 -25 0C (room temperature)
5. Protein standard 80 g/l: Weigh out about 4.3 g of bovine albumin powder and dry it in
the oven at about 60 C for 8-10 hours. After drying weigh out exactly 4.0 g of dried

52
bovine albumin powder. Float the powder on the surface of about 30 ml of sodium
chloride/sodium azide solution in a small beaker. When the albumin has dissolved
transfer the solution into a 50 ml volumetric flask (pour slowly down the side of the
flask to avoid frothing). Rinse the beaker with small volumes of sodium
chloride/sodium azide solution and make the final volume to 50 ml. This solution is
stable for 6 months at 2-8 0C. Store in a clean sterile bottle.
15.6 PROCEDURE
1. For the standard and each patient or control sample, pipette into test tubes 2.5 ml of
Biuret reagent (standard and test) and 2.5 ml of blank Biuret reagent (standard blank
and test blank)
2. Add 50 µl of standard (80 g/l) or samples to each pair of tubes.
3. A reagent blank is set up for each batch and contains 2.5 ml of Biuret reagent and 50
µl of water.
4. Mix each tube and allow them to stand at room temperature for 30 minutes or at
370 C for 10 minutes.
NOTE: the same temperature must be used for standards and samples.
5. Measure the absorbance at 540 nm (yellow-green filter, Ilford No 605) setting the
spectrometer to zero with blank Biuret reagent. First read the absorbance of the
sample blanks, then the reagent blank, and then the tests.
6. If results are greater than 100 g/l then repeat the analysis using 20 µl of sample.
Multiply the result by 2.5 to obtain the protein concentration.
CALIBRATION
Although the measurement of total protein is simple, results of external quality assessment
programmes indicate that many laboratories have difficulty in producing accurate without
sample blank correction.
The recommended method proposes the use of a bovine albumin solution as a calibrator.
One alternative is to use lyophyilised serum calibrators. However many of these show
significant turbidity when they are reconstituted and sometimes it is difficult to know
whether the contribution that the turbidity makes to the absorbance at 540 nm has been
subtracted in the calculation of the total protein value. When possible use a calibrator with
a value assigned by a total protein method that incorporates a sample blank. A sample
blank for the standard (SB) is not necessary if a solution of bovine albumin is used.
A second alternative is to use an out – of-date of human serum albumin from a blood
transfusion or pharmacy department. Use the stated concentration of albumin for the total
protein value. One bottle will last for many months at 4-6 0C if small volumes (5-10 ml) are
withdrawn as required using a sterile syringe.
A calibration curve must be prepared as described below to check the linearity of the
spectrometer. If it is linear, then a single standard can be used routinely as described under
“Technique” .The calibration curve should be repeated at least once a month.
PREPARATION OF CALIBRATION GRAPH USING BOVINE ALBUMIN

Prepare a calibration graph to confirm that the method is linear with your spectrometer to
at least 80g/l. Provided that it is linear, a single standard (80 g/l) can then be used with
each batch of patients’ samples.
Working standard No 1 2 3 4
Protein standard 80 g/l (ml) 0.25 0.50 0.75 1.0
Sodium chloride/sodium azide solution (ml) 0.75 0.50 0.25 0

53
Concentration of working standard solution (g/l) 20 40 60 80
Label five test tubes as follows: reagent blank (RB),

Working standard: No.1 (20 g/l);
No.2 (40 g/l);
No.3 (60g/l);
No.4 (80g/l);
Pipette 2.5 ml of Biuret reagent into each tube; add 50 µl of distilled water to the reagent
blank, and 50 µl of each working standard solution into the corresponding tubes. Mix and
leave to stand at room temperature for 30 minutes or at 37 0C for 10 minutes.
Read the absorbance at 540 nm after setting the instrument to zero with the reagent blank.
Prepare the calibration graph by plotting the absorbance against the protein concentration
for each tube.
15.7 CALCULATION
S= A standard - A standard blank – A reagent blank
T= A sample - A sample blank- A reagent blank
The serum protein concentration of sample=T/S x 80 g/l

Where 80 is the concentration of the standard (g/l)
Conversion from SI to “old” units g/l x 0.1=g/100 ml
QUALITY CONTROL
At least two quality control specimens, having stated values in the range 40-80 g/l, one of
which is unknown to the operator, should be included with each batch of specimens. If
single specimens are analysed a control specimen should always be included.

attainable.
4%
REFERENCE VALUES
Approximate reference values for an adult 60-80 g/L
REFERENCES
Doumas, B.T. (1975) Clin. Chem, 21, 1159-1166.

54
16. UREA
DIACETYLE MONOXIME METHOD
16.1 INTRODUCTION
Urea is the major nitrogen containing metabolic product of protein catabolism in humans,
accounting for more than 75% of the non protein nitrogen eventually excreted. The
biosynthesis of urea from amino nitrogen-derived ammonia is carried out exclusively by
hepatic enzymes of the urea cycle.
More than 90% of urea is excreted through the kidneys, with losses through the
gastrointestinal tract and skin accounting for most of the remaining minor fraction. Urea is
neither actively reabsorbed nor secreted by the tubules but is filtered freely by the
glomeruli. In a normal kidney, 40 to 70% of the highly diffusible urea moves passively out
of the renal tubule and into the interstitium, ultimately to re-enter plasma. The back
diffusion of urea is also dependent on urine flow rate; with more entering the interstitium
in slow-flow states. Urea production is dependent on several non renal variables such as
diet and hepatic synthesis.

A wide variety of renal diseases with different permutations of glomerular, tubular,
interstitial or vascular damage can cause an increase in plasma urea concentration. The
usefulness of urea as an independent indicator of renal function is limited by the variability
of its blood levels as a result of non renal factors. Mild dehydration, high protein diet, the
increased protein catabolism, muscle wasting as in starvation, reabsorption of blood
proteins after a gastrointestinal haemorrhage, treatment with cortisol, and decreased
perfusion of kidneys may cause increased blood urea that is called pre renal ureamia.
Impaired perfusion may be due to decreased cardiac output or shock secondary to blood
loss or other causes. Urea values above 20 mmol/L always indicate intrinsic renal disease.
However the plasma urea usually does not rise until the glomerular filtration rate falls to
less than 50%.

Proteins in whole blood, plasma or serum are precipitated with trichloroacetic acid. The
urea in the supernatant reacts with diacetyle monoxime in the presence of
thiosemicarbazide and cadmium ions under acid conditions. The absorbance of the
resulting rose-purple solution is measured at 530 nm.

3 ml of clotted blood collected into clean dry bottle; avoid haemolysis. Separate serum
from cells as early as possible. Serum urea is stable for 24 hours at room temperature
(250C), for 7 days at 2-60C, for longer duration (2-3 months) when frozen. As a referral
sample send about 0.5 ml clear serum, kept cool, to reach correct destination within 24
hours.
APPARATUS:
Water bath or heating block (temperature range 10- 110 0C)
Spectrophotometer wavelength 530 nm
Colorimeter green filter, Ilford 604 (520nm)
Rack for boiling tubes

55
Automatic micro pipettes (50 and 100 µl)
GLASSWARE:
Amber coloured reagent bottles (500 ml volume)
Graduated pipettes (5 and 10 ml in 0.1 ml)
Graduated cylinders (50 ml, 100 ml and 500 ml)
Volumetric flasks (50 and 100 ml and 500 ml volumes)
Glass stoppered boiling tubes (160 x 18 mm)
Conical centrifuge tubes 15ml
CHEMICALS:
Benzoic acid-AR
Cadmium sulphate (as 3CdSO4.8H2O)-AR
Diacetyle monoxime-AR
Orthophosphoric acid (85% w/v)-AR caution: corrosive, handle with care
Sulphuric acid, concentrated (95 – 97 % w/v)-AR caution: corrosive, handle with care
Thiosemicarbazide-AR
Trichloroacetic acid-AR caution: corrosive, handle with care
Urea-AR
REAGENTS
1. Acid reagent : Add about 200 ml of distilled water to a 500 ml volumetric flask. Keep
the flask in basin containing water and then add slowly 44 ml of sulphuric
(concentrated) acid and 66 ml of orthophosphoric acid. Cool the solution to room
temperature but do not use ice water as a cooling bath. Add 50 mg thiosemicarbazide
and dissolve, then add 1.6 g cadmium sulphate and dissolve, add 1.5 ml of the urea
working standard solution (2.5mmol/l). Make up to 500 ml with distilled water.
Transfer to an amber coloured bottle. This reagent is stable for at least six months at
2- 8 0C.
NOTE: The presence of a small amount of urea in the reagent improves the linearity
of the calibration curve. The cadmium sulphate improves the stability of the final
coloured product.
2. Diacetyl monoxime reagent: Weigh out 2.0 g of diacetyl monoxime, dissolve in
distilled water and dilute to 500 ml with distilled water in a volumetric flask. This
solution is stable for at least six months at 2-8 0C
3. Colour reagent: Use a graduated cylinder and mix 50 ml of acid reagent with 50 ml of
diacetyl monoxime reagent in a small bottle. This amount is sufficient for 33 reactions.
This reagent must be prepared daily, therefore, the volume made up should depend on
the number of reactions anticipated; 3 ml is required for each reaction.
4. Benzoic acid solution 1 g/l: Weigh out 0.5 g of benzoic acid and transfer to a 500 ml
volumetric flask. Add distilled water, mix well to dissolve and make up to 500 ml with
distilled water. This solution is stable for several months at 20-250C (room
temperature)
5. Trichloroacetic acid solution 50 g/l: Weigh out 25 g of trichloroacetic acid in a beaker,
dissolve, transfer into 500 ml volumetric flask and make up to 500 ml with distilled
water. This solution is stable for several months at 20-25 0C. We recommend storing
the solution in the refrigerator.
6. Stock urea solution 125 mmol/l: Weigh out 1g of urea –AR in a beaker and keep in
the dessicator overnight. Weigh out 750 mg urea from the dessicator and transfer into
a 100 ml volumetric flask. Add 50 ml of benzoic acid solution; dissolve and dilute to
100 ml with benzoic acid solution. This reagent is stable for several months at 2-80 C
7. Working urea standards: Prepare working urea standards in 50 ml volumetric flasks
according to the table below;

56
Working standard No 1 2 3 4 5 6
Stock urea standard (ml) 1 2 3 4 6 8
Benzoic acid solution Up to 50 ml for each
Concentration of working standard 2.5 5.0 7.5 10 15 20
Solution (mmol/l)
These standards are stable for several months at 2-80 C

In this method the formation of the coloured product depends on the composition of the
colour reagent and the period of heating at 100 0C. Small variations may occur from day to
day and it is therefore essential to check the calibration each time that patients’ samples are
analysed. When you are familiar with the shape of the calibration graph on your
spectrometer you may find that you can omit some of the standards, e.g. prepare your daily
calibration using for example the 5, 10 and 20 mmol/l standards, or use the 10 mmol/l
standards should be prepared when the acid reagent and diacetyl monoxime reagent are
renewed to check that the reagents are correct.
Follow the procedure described under “Procedure”. Plot the absorbance of each tube on
the vertical axis against the concentration of the working urea standard solutions in mmol
on the horizontal axis.
16.6 PROCEDURE
1. Pipette 0.5 ml of trichloroacetic acid solution using a rubber bulb into centrifuge tubes
for each standard, control serum and patient’s sample. Add 50 µl of standard, control
serum or patient’s sample to the appropriate tube, mix and leave at room temperature
for 5 minutes, then centrifuge to obtain a clear supernatant.
2. Label another set of tubes (18 x 160mm) and pipette 3.0 ml of colour reagent into test
tubes for each standard, blank, control serum and sample.
3. Add 100 µl of trichloroacetic acid solution to the blank tube, and 100 µl of supernatant
form the standard, control or samples to the appropriate tube.
4. Mix well and heat at 100 0C for 15 minutes exactly.
5. Cool the tubes to room temperature in a bowl of water (about 5 min) mix and read the
absorbance at 530 nm (green filter, Ilford No 604) first read the absorbance of the
blank against distilled water and note down the reading, then set to zero with the blank
and read the standards and unknowns. The absorbance measurements should be made
as soon as possible and not more than 30 minutes after the end of step 4.
16.7 CALCULATION
Read the results from the calibration curve or use the following formula if your calibration
graph is linear.
Concentration of urea in mmol/l = T x C
S
Where:
T = Absorbance reading of patient’s test
S = Absorbance reading working standard
C = Concentration of working standard (10 mmol/l)
If the result is greater than 20mmol/l, dilute 50 µl of supernatant from that sample with
100 µl of trichloroacetic acid solution. Mix well. Repeat the analysis using 3 ml of colour
reagent and 100 µl of the diluted supernatant. Remember to multiply the result by 3 to
obtain the urea concentration in the patient’s sample.

57
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 3-20 mmol/l, one
of which is unknown to the operator, should be included with each batch of specimens. If
single specimens are analysed, a control specimen should always be included.

attainable.
6%
REFERENCE VALUES
Approximate reference intervals for “healthy” ambulant adults: 2.5-6.6 mmol/l
Conversion of SI units into “old” units: mmol/l x 6 = mg/100 ml
Blood urea reference values are method dependable. Blood urea estimation by urease
method usually gives higher values
NOTE: The method recommended uses the reagents described in the above
references. However a protein precipitant has been included because the direct method
(No protein precipitation) gave results on patients’ samples which were significantly
higher than other methods.
16.8 LIMITATION
Cadmium sulphate has been included as recommended by Wybenga et al. In the absence
of cadmium ions, the absorbance decreased by about 7 % in 30 minutes; when the colour
reagent is prepared as described, then the absorbance decreased by about 4% in 30
minutes. Provided that the batch size is kept small, so that the absorbance readings can be
made over a period of a few minutes, the presence or absence of cadmium ions has little, if
any effect on the results.
REFERENCES
WHO Manual LAB/86.3

58
17. URIC ACID
PHOSPHOTUNGSTATE REDUCTION –CARAWAY METHOD
17.1 INTRODUCTION
Uric acid is the end product of purine metabolism (adenine,guanine) in the human. On a normal
diet some 5-6 mmol of urate is produced daily.Of this amount about 3-4 mmol is produced from
purines synthesized in the body (de novo synthesis), whilst the remaining 1-2 mmol are contributed
by dietary purine.Urate, the end product of purine base degradation, is formed from xanthine by the
enzyme xanthine oxidase.
Around 25-30% of the 5-6mmol of urate produced daily is eliminated via the gastrointestinal tract,
where it is degraded by bacterial uricases. The remainder is excreted by the kidney. In renal failure
the intestinal excretion can be markedly increased.
Some 98% of the filtered urate is reabsorbed in the proximal tubule. The 2% that escapes
reabsorption contributes around 20% of the total excreted, proximal tubular secretion accounting
for the remainder. Minor reabsorption also occurs in the distal nephron Renal clearance is
influenced by various drugs and metabolic products.

Hyperuricemia is most commonly defined by serum or plasma uric acid concentrations greater than
7.0 mg/dL (0.42 mmol/L) in men or greater than 6.0 mg/dl (0.36 mmol/l) in women (if specific
methods are used to measure the uric acid). Asymptomatic hyperuricemia is frequently detected
through biochemical screening; Long term follow up of asymptomatic hyperuricemic patients is
undertaken because many are at risk for renal disease that may develop as a result of hyperuricemia
or hyperuricosuria; few of these patients ever develop the clinical syndrome of gout.
Gout occurs when monosodium urate precipitates from supersaturated body fluids;the deposits of
urate are responsible for the clinical signs and symptoms.Gouty arthritis may be associated with
urate crystals in joint fluid as well as with deposits of crystals(tophi) in tissues surrounding the joint.
The deposits may occur in soft tissues as well , and wherever they occur they elicit an intense
inflammatory response consisting of polymorphonuclear leukocytes and macrophages.Renal disease
associated with hyperuricemia may take one or more of several forms: (1)gouty nephropathy with
urate deposition in renal parenchyma,(2)acute intratubular deposition of urate crystals, and (3) urate
nephrolithiasis Hyperuricemia is also attributable to primary defects of enzymes in the pathways of
purine metabolism.

Serum proteins are precipitated with acid tungstate and a clear supernatant is obtained after
centrifugation. A portion of the supernatant is added to alkaline phosphotungstate.
Phosphotungstate reagent oxidizes the urate to allantoin and itself reduced to tungsten blue which is
measured by its absorbance at 700 nm

4ml of clotted blood, collected into clean dry bottle, avoid haemolysis. Separate the serum from cells
as early as possible. Uric acid in serum is stable for 48-72 hours at room temperature (250C), for 3-7
days at 4-6 0C and for 6-7 months when frozen. As a referral sample send about 1 ml of clear serum,
kept cool, to reach the correct destination within 24 hours
APPARATUS:
Water bath at 25 0C or Basin with water temperature maintained at 250C
Automatic micro pipette 500 µl
Quick fit complete assembly (round bottom flask and condenser) for refluxing

59
GLASSWARE:
Conical centrifuge tubes (15ml)
Test tubes 16 x 150 mm
Volumetric flask 100 ml, 200 ml and 1 L
Graduated pipettes 1 ml, 5 ml and 10 ml
CHEMICALS:
Molybdate free sodium tungstate dihydrate-AR
Orthophosphoric acid-AR
Sodium carbonate-AR
Sulphuric acid-AR
Uric acid –AR
Formaldehyde 40% solution
REAGENTS
1. Stock Phosphotungstic acid reagent: Weigh out 50 g of molybdate free sodium tungstate dihydrate or
44.5 g anhydrous salt and dissolve in about 400 ml of distilled water in refluxing flask. Add 40
ml of Orthophosphoric acid (concentrated). Mix well and reflux gently for 2 hours. Allow the
solution to cool. Then transfer with rinsing to a 500 ml volumetric flask. Make up to 500 ml
with distilled water. Mix well. Transfer this solution into a clean dry brown bottle. This solution
is stable in the refrigerator for about one year.
2. Working Phosphotungstic acid reagent: Dilute 10 ml of stock phosphotungstic acid reagent to 100 ml
with distilled water. Transfer in to a clean, dry brown bottle. Stable in the Refrigerator for two
weeks.
3. Sodium carbonate 10g/dl: Weigh 10 g of Sodium carbonate anhydrous, dissolve in distilled water
and make up to 100 ml with distilled water. Store in a poly propylene bottle.
4. Sodium tungstate 10g/dl: Weigh out 10 g Sodium tungstate dihydrate, transfer into a 100ml
volumetric flask, dissolve and make up to 100 ml with distilled water. Store in a brown bottle.
5. Sulphuric acid (2/3 N) 0.34mol/l: Add slowly 3.7 ml of sulphuric acid concentrated in to 150 ml
distilled water in a beaker. Cool and stir well. Transfer in to a 200 ml volumetric flask and make
up to 200 ml distilled water. Transfer in to a brown bottle.
6. Acid tungstate reagent: To 80 ml of distilled water while mixing add 5 ml of sodium tungstate
solution, 0.05 ml of phosphoric acid (concentrated), 5 ml of 2/3 N sulphuric acid solution.
Transfer in to a brown bottle. Keep at room temperature.
7. Stock Uric acid standard solution 1.5mmol/l: Keep about 300 mg of uric acid analytical grade chemical
in a desiccator overnight. Weigh out 252.2 mg of uric acid and transfer in to a one litre
volumetric flask. Weigh out 150 mg of Lithium carbonate; dissolve in about 50 ml of distilled
water. Filter, heat the filtrate to 60 0C. Add this warm solution to the volumetric flask
containing uric acid. Mix until the uric acid completely dissolved. Allow the flask to cool. Then
add 20 ml formaldehyde 40 % solution. Add 500 ml of distilled water .Then add slowly 25 ml
of sulphuric acid solution. (Prepared by adding 1 ml of sulphuric acid (concentrated) to 35 ml
of water) Make up to one litre with distilled water. Mix and store in a brown bottle. This
solution is stable for about one year at 4- 6 0C.
8. Working uric acid standard: Allow the stock uric acid standard solution to attain the room
temperature. Pipette 2 ml of stock standard solution in to 100 ml volumetric flask. Make up to
100 ml with distilled water. This standard is equivalent to 300 µmol/l under the assay conditions
employed. Solution is stable for one week at 4-6 0C.
9. Working Uric acid standard series for calibration: Prepare working standard series by quantitative
transfer of 1ml, 2ml, 3ml and 4 ml of stock solution to each of four 100 ml volumetric flasks.
Dilute to 100 ml with distilled water. Mix well. These standards are equivalent to 150µmol/l,
300µmol/l, 450µmol/l and 600µmol/l under the employed assay condition.

60
17.6 PROCEDURE
1. Pipette 4.5 ml of acid tungstate regent in to centrifuge tubes for each quality control sample and
patient’s sample.
2. Pipette 0.5 ml of quality control serum or patients sample to the appropriate tube with mixing.
3. Leave at room temperature for 10 minutes and centrifuge for 10 minutes at 3000 rpm to obtain
a clear supernatant.
4. Label a set of test tubes for each blank, standard, quality control sample and patient’s samples.
5. Pipette 2.5ml of distilled water to the tube marked blank, 2.5ml of working standard to the tube
marked Standard, and 2.5 ml supernatants from the quality control sample or patients’ samples
to the appropriate tube. Keep all the tubes in a water bath at 250C.
6. Add 0.5 ml of Sodium carbonate solution to all tubes, mix well and allow standing for 10 min at
25 0C.
7. Then add 0.5 ml of working phosphotungstic acid reagent to all tubes. Mix immediately. Allow
to stand at 250C for 30 min.
8. Measure the absorbance at 700 nm, setting the spectrophotometer to zero with blank solution.

Run the uric acid working standards series together with the regent blank exactly in the same
manner as describe under technique. Plot the absorbance values of standards against the
concentration of standards. The points should be linear and the graph should pass through the
origin.
17.7 CALCULATION
Calculate the uric acid concentration in the patient’s specimens from the absorbance of the standard
as follows
Concentration of Uric acid in µmol/l= T/S x C=T/S x 30 x 10=T/S x 300 µmol/l

Where
T= Absorbance of patient’s test
S=Absorbance of working standard
C=Concentration of working standard (30µmol/l)
Serum dilution 1:10
QUALITY CONTROL
Include Quality control sera for each batch of test
REFERENCE VALUES : For an adult 120 – 360 µmol/l
17.8 LIMITATION
Haemolysed sera interfere with the measurement of uric acid.
Supernatants should be clear as turbidity may interfere with colour development

61
18. URINE URIC ACID

Method similar to used for serum uric acid can be applied to urine uric acid estimation

Collect 24 hour urine into a clean, dry, sterile 2.5 L bottle and keep in the refrigerator during the
collection.
18.2 PROCEDURE
1. Mix the urine collection well. Measure the total volume. Warm an aliquot of urine for a few
minutes to 600C to dissolve any urates in the deposit. Add 1ml of urine in to a 100ml
volumetric flask. Make up to 100ml with distilled water. Mix well. (Dilution 1 in 100)
2. Pipette 2.5 ml of distilled water to the tube marked B for blank , 2.5 ml of working standard to
the tube marked S as standard and pipette 2.5 ml of diluted urine in to the tube marked T for
test. Keep all the tubes in a water bath at 250C
3. Carry out the same procedure as for serum uric acid estimation from step 6.
18.3 CALCULATION
Calculate urine uric acid concentration in the sample using the absorbance of the standard as
follows.
Urine uric acid concentration µmol/l = T/S x C

Where T = Absorbance of patients test
S = Absorbance of working standard
C = Concentration of working standard
Urine dilution 1:100
Urine Uric acid µmol/l = T/S x 30 x 100 (dilution factor=100)

Urine Uric acid (mmol/l) = T/S x 30 x 100
1000
24 hour urine uric acid =T/S x 30 x100 x 24 hour urine volume in litre (mmol/24 hour)
1000
REFERENCE VALUES : 1.48 - 4.43 mmol/24hours

62
19. ELECTROLYTES
DETERMINATION OF SERUM SODIUM AND POTASSIUM BY FLAME PHOTOMETRY [FLAME
EMISSION SPECTROSCOPY]
19.1 INTRODUCTION
SODIUM
In a normal adult the total body sodium is about 55mmol/kg of body weight; about 30% is tightly
bound in the crystalline structure of bone and thus is nonexchangeable. Thus only 40 mEq/kg is
exchangeable among the various compartments and accessible to measurements. The exchangeable
sodium is distributed primarily in the extra cellular space. About 97% to 98% of the exchangeable
sodium is found in the extra cellular water space and only 2% to 3% in the intracellular water space.
Approximately 16% of exchangeable sodium is in plasma, 41% is in interstitial fluid (ISF) that is
readily accessible to the plasma compartment, 17% is in ISF of dense connective tissue and
cartilage, 20% is in ISF of bone and 3% to 4% in the transcellular water compartment. Total bone
sodium (exchangeable and nonexchangeable) accounts for the 40% to 45% of the total body
sodium.
The amount of sodium in the body is a reflection of the balance between sodium intake and output.
Sodium intake depends on the quantity and type of food intake. Under normal conditions, the
average adults takes in about 50 – 200 mmol of sodium/ day. Sodium output occurs through three
primary routes; the gastrointestinal tract, the skin and the urine.
Under normal circumstances loss of sodium through the GIT is very small. Faecal water excretion is
only 100- 200 ml/day for a normal adult and faecal sodium excretion only 1 to 2 mmol/ day.
However, one should bear in mind that although losses of water and electrolytes are normally small,
the total volume of GIT fluid secreted is large, averaging about 8 L/day. Almost all this volume is
normally reabsorbed. However, with impaired GIT reabsorption, loss of water and electrolytes are
large. Thus with severe diarrhoea or with gastric or intestinal drainage tubes, sodium loss via the
GIT may exceed 100mmol/ day.
The sodium content of sweat averages about 50 mmol/ L but is somewhat variable. The sweat
sodium concentration is decreased by aldosterone and increased in cystic fibrosis. The rate of sweat
production is highly variable, increasing in hot environments, during exercise, and with fever. Under
extreme conditions sweat production can exceed 5 L/ day, accounting for a loss of more than 250
mmol of sodium .Under normal conditions, in a cool environment; sodium losses from the skin are
small. With extensive burns or exudative skin lesions there is great loss of sodium and water.
Major route of sodium excretion is through the kidney. Furthermore, urinary excretion of sodium is
carefully regulated to maintain body sodium homeostasis, which in turn is critical to control of extra
cellular volume.
Sodium is freely filtered by the glomerulus. Approximately 70% of the filtered sodium is reabsorbed
by the proximal tubule, about 15% by the loop of henle, 5% by the distal tubule, 5% by the cortical
collecting tubule, and another 5% by the medullary collecting duct; thus normally less than 1% of
the filtered sodium is excreted.
POTASSIUM
Approximately 98% of the total body potassium is found in the intracellular water space
(ICW),reaching a concentration there of about 150 to 160 mmol/ l .In the extra cellular water space
,the concentration of potassium is only 3.5 to 5 mmol/l . Total body potassium in an adult male is
about 50 mmol/kg of body weight and is influenced by age, sex, and very importantly muscle mass,
since most of the body’s potassium is contained in muscleThe amount of potassium in the body is a
reflection of the balance between potassium intake and output. Potassium intake depends on the
quantity and type of food intake. Under normal conditions the average adult takes in about 50 to
100 mmol/ day, about the same amount as sodium. Potassium output occurs through three primary
routes; the gastrointestinal tract, the skin and the urine.

63
Under normal circumstances loss of potassium through the GIT is very small, amounting to less
than 5mmol/day for an adult. The concentration of potassium in the sweat is less than that of
sodium, and so potassium losses via the skin are usually small.
The major means of potassium excretion is by the kidney. The kidney is capable of regulating the
excretion of potassium to maintain body potassium homeostasis.

SODIUM
Hyponatremia occurs when there is a greater excess of extra cellular water than of sodium or a
greater deficit of sodium than water.
The symptoms of hyponatremia depend on the cause, magnitude, and rate of fall in serum sodium.
With acute, pronounced hyponatremia caused by water intoxication, nausea, vomiting, seizures, and
coma may occur. Symptoms are less fulminant with chronic hyponatremia caused by salt depletion.
With progressively severe degrees of chronic hyponatremia, constant thirst, muscle cramps, nausea,
vomiting, weakness, lethargy, and finally delirium and impaired consciousness may occur.
Hypernatremia occurs when there is greater deficit of extra cellular water than of sodium. Greater
excess of sodium than of water rarely occurs.
Hypernatremia usually occurs as a chronic process secondary to loss of water in excess of sodium.
Symptoms are therefore those of dehydration.
POTASSIUM
POTASSIUM EXCESS
Potassium accumulates in the body when the intake of potassium exceeds output because of some
abnormality of the potassium homeostatic mechanism. It should be noted that under most
conditions the normal kidney is capable of excreting a great deal of potassium, and a high potassium
intake leads to potassium retention only when kidney function is compromised.
POTASSIUM DEPLETION
This occurs when potassium output exceeds intake. Only small amount of potassium is loss in the
faeces under normal conditions. GIT loss of potassium during diarrhoea and drainage of GIT
secretions can be large.
Alkalosis results in the total body potassium depletion. With alkalosis, potassium moves from the
extra cellular compartment to the intra cellular space. In the cells of the distal nephrone of the
kidney, this increase in intra cellular potassium stimulates potassium secretion and therefore
increases renal excretion of potassium.

Using compressed air diluted serum is sprayed as a fine mist of droplets in to a non luminous flame.
In the flame the elements in the compound are converted in to the atomic state. As the temperature
rises due to the thermal energy of the flame, a small portion of these atoms excited and the
electrons moves to higher energy level. When atoms excited they emit light in the form of a fixed
wavelength to produce a spectrum. Light emitted from the thermally excited ions is directed to
photo sensitive detector system. The amount of light emitted depends on the concentration of
metallic ions present. The response compared with those obtained from standards.

Haemolysis free serum is suitable. Blood collected into clean, dry bottle or commercially available
evacuated tubes or capillary blood collected in either micro tube or capillary tubes.
Blood should not be collected from the arm receiving an electrolyte or intravenous infusion. Avoid
muscle activity (clenching the fist) when collecting the blood sample as this can artificially increase
the potassium values. Blood specimens should not chilled before separation of serum, false increase
in potassium level occurs as a result of K+ leakage from erythrocytes and other cells.

64
Serum should be separated from cells immediately within 1 hour of collection at room
temperature. Blood samples should not be centrifuged for longer periods. Grossly lipaemic serum
samples are unsuitable for electrolyte estimation. Serum samples should be stored at 20C to 40C or
frozen for delayed analysis.
APPARATUS: Disposable sample cups (plastic)

Analytical balance
Flame photometer CHEMICALS:
Automatic micro pipette 100µl Sodium chloride (Analytical grade)
Potassium chloride (Analytical grade)
GLASSWARE: Corning diluent concentrated
Conical flasks 50 ml Deproteinising solution (Corning)
Graduated pipette 25 ml LP gas
REQUIREMENTS (ENVIRONMENTAL CONDITIONS)

TEMPERATURE : Operating 100C to 35 0C
HUMIDITY : Operating 85% maximum at 350 C
FUEL : High grade propane should be free of heavy hydrocarbon deposits and regulated
at the cylinder to approximately 2.1 kg/cm2 (30 psig)
AIR : A supply of clean air at a minimum pressure of 1 kg/cm2 (14 psig) at 6 litres
/minute, as supplied by a Corning 850 Air Compressor. Maximum inlet pressure
2.1 kg/cm2 (30 psig).If condensation problems arises a ‘Corning 856 Air
Compressor’ should be used, which has a water separator fitted.
VOLTAGE : 90V to 127V or 198V to 264 V, 50/60Hz
POWER : 20VA, 410 only
REAGENTS
All glassware used to prepare standard solutions should be chemically cleaned and
finally rinsed with distilled water.
Weigh out separately into two watch glasses or into two Petri dishes about 15 g of analytical grade
Sodium chloride and about 1 g of Potassium chloride.
Dry for 6 hours at 100 0C in an oven and allowed to cool to room temperature in a desiccator.
1. Stock Sodium solution 1000 mmol/l: To prepare 200ml, weigh accurately 11.7 g of dried
Sodium chloride in a weighing bottle or in a beaker. Transfer it in to an ‘A grade’ 200 ml
volumetric flask using a funnel. Wash in any chemical remaining in the weighing bottle or in the
beaker in to the flask with a little amount of distilled water (glass distilled water) or deionised
water. Dissolve in about 150 ml of distilled water and make up to 200 ml with distilled water.
Use a pasture pipette or a wash bottle to add the final volume of distilled water to the flask. Mix
the solution well by inverting the flask for several times.
2. Stock Potassium solution 100 mmol/l: Weigh accurately 0.746 g of dried Potassium chloride
in a weighing bottle or beaker. Transfer it in to an ‘A grade’ 100 ml volumetric flask using a
funnel. Wash any remaining in the weighing container in to the flask with distilled water.
Dissolve in about 80 ml of distilled water. Make up to 100 ml with distilled water. Use a Pasture
pipette or a wash bottle to add the final volume of distilled water to the flask. Mix the solution
well by inverting the flask for several times.
3. Flame photometry deproteinising solution: The pack of deproteinising solution contains
deproteinising base solutions and Sachet of catalyst. For use add the catalyst into base solution
and mix thoroughly. This solution is stable for 4 weeks at 2-80 C.
4. Working Standard solution (Sodium-140 mmol/l, Potassium-5.0 mmol/l): Into a clean
500 ml ‘A grade’ volumetric flask, add 70 ml of stock solution-1(Stock sodium solution) and 25
ml of stock solution-2 (Stock potassium solution) Make up to 500 ml with good quality distilled
water or deionised water. Use a pasture pipette or a wash bottle to add the final volume of
distilled water to the flask. Mix the solution well by inverting the flask for several times. Rinse a

65
polypropylene bottle with little of the prepared solution. Then transfer the standard solution
into the bottle. Label and keep at room temperature. (Contamination with sodium may occur if
a glass bottle is used) Stopper the bottle tightly to avoid evaporation.
5. Working diluent concentrate: Diluent concentrate recommended by the manufacturer should
be used. For ‘Corning 410 C’ Corning diluent concentrate is used. Pipette 0.5 ml diluents
concentrate in to clean 500 ml volumetric flask. Dilute up to 500 ml with good quality distilled
water or deionised water. Store the working diluent concentrate in a polypropylene bottle. This
solution is stable for 5 days at room temperature.
19.6 PROCEDURE
The details of the operation vary from one instrument to another. Following steps are related to
‘Corning 410’ clinical model flame photometer.
1. Sample dilution: Dilute each serum, quality control sample and working standard solution
1:200 with working diluent concentrate. Into 50 ml conical flasks pipette 19.9 ml of working
diluent concentrate and 0.1 ml of working standard solution or quality control sample or
patient’s serum and mix well.
2. Turn on the fuel supply at source
3. Depress the ‘power’ switch to switch on the instrument 410. The ‘power on’ LED will be
illuminated, the air compressor will start an ignition cycle will commence.
4. If the flame on LED is not illuminated at the end of the ignition cycle, (Refer the operator’s
manual available with the instrument) check that the air pressure gauge indicates a reading
between 11 and 13 psig, if it does not, lower the air regulator locking ring and adjust the
regulator for a reading of 12 psig on the air pressure gauge. Raise the locking ring to lock the air
regulator adjuster.
5. Set the filter selector to the required position. Non luminous blue flame with distinct cones can
be seen, if does not; adjust the fuel to get distinct blue cone flame.
6. Insert the Nebulizer inlet tube in a beaker containing approximately 100 ml of diluent and allow
15 minutes for the operating temperature to stabilize. This will ensure a stable burner
temperature when solutions are aspirated, after the warm up period.
7. While aspirating the diluent, adjust the ‘blank’ control so that the display reads zero
8. Aspirate a pre diluted standard solution
9. Allow 20 seconds for a stable reading and then adjust ‘coarse’ and ‘fine’ controls for a
convenient reading (if a 140 mmol/l Sodium standard is aspirated, set the display to 140)
10. Carefully adjust the ‘fuel’ control for a maximum reading on the display, ensuring that only
small adjustments are made, with a pause of several seconds between adjustments.
11. Remove the standard solution, wait 10 seconds, then aspirate a blank solution of diluent for 20
seconds. Adjust the ‘blank’ control for a zero reading. Remove the blank solution and wait 10
seconds.
12. Repeat paragraphs 8, 9 and 11 until the blank reading is zero (within ± 0.2) and the calibration
reading is within ± 1%.
13. Aspirate each of the unknown solutions for 20 seconds, then note the reading in mmol/l
14. Check the calibration frequently
15. When analyzing large batches of samples, recheck instrument calibration every 10 samples with
a single standard solution.
SHUT DOWN PROCEDURE

1. Aspirate deproteinising solution diluted 1 in 100 with deionised water for one minute
2. Aspirate diluent for 2 minutes
3. Turn off the fuel supply at source when the “flame on” LED is extinguished; switch off the
“power” switch.
NOTE:
 Always use same batch of diluent for the blank, dilution of samples and standard, alternatively
the corning dilutor can be used.

66
 In any difficulty of obtaining a maximum sodium reading proceed as follows: Open the
inspection flap and adjust the ‘fuel’ control until the flame just starts to lift off the burner. Then
turn the ‘fuel’ control back, counter clockwise, until the cones of the flame are on the burner.
Close the flap and proceed with paragraph 11.
PRECAUTIONS
A diluent recommended by the manufacturer of instrument should be used. Deionised or high
quality distilled water should be used to prepare the diluent. Deionised or distilled water must be
free from contaminative elements (bacteria or moulds can cause inaccuracies by interrupting or
blocking the flow of sample through the Nebulizer always use the same batch of diluent for the
blank and the dilution of samples and standards.
 Anticoagulants containing sodium or potassium salts must not be used. Use serum for
measurement of sodium and potassium
 Dilute the sera with care. Good quality calibrated pipette or a sensitive diluter must be used.
Use the same pipette or dilution equipment for both standard and sample
 Accuracy of the results depends on the accuracy and purity of the calibration standard. Always
use accurately prepared standards.
 Both the accuracy and precision of results depends on maintenance and adherence to operating
instructions provided by the manufacturer. Careful cleaning of the atomizer-burner, cleanliness
of sample containers, the aspirating systems, proper adjustment of flame size, (blue flame with
distinct cone) aspiration rate, and geometry of the flame and uniform entry of atomized, diluted
sample into the flame are also critical for accuracy and precision. Thermal equilibrium must be
established before analysis of unknown samples. Warm up period is necessary because the
initial evaporation of water in the flame decreases the temperature of the burner and the entire
burner chamber.
 Safety: Propane is highly inflammable and potentially explosive and commonly supplied as a
liquid under pressure in a cylinder for use with the 410. Cylinder should never be subjected to
heat or mechanical shock. Leakage of propane from tank, instrument fittings or from valves
may be detected with the aid of soap solutions.
 Site conditions:
 Never install the flame photometer beneath overhanging cupboards. There must be at least
1 metre of clear space above the 410 chimney
 The environment must be clean and free from dust
 The instrument must be placed on a strong, level work stop, free from vibration
 Avoid the instrument to direct sunlight or draughts
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 120-150 mmol/l for
sodium, 3.0-6.0 mmol/l for potassium, and one of which is unknown to the operator should be
included with each batch of specimens. If single specimens are analysed a control specimen should
always be included
OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 1% for sodium and
1.5% potassium should be attainable
ROUTINE CONDITIONS VARIANCE: The value should not exceed 2% for sodium and 3% for
potassium
REFERENCE VALUES
Serum Sodium : 136mmol/l - 146mmol/l
Serum Potassium: 3.5mmol/l - 5.6mmol/l

67
19.7 LIMITATION
Haemolysed sera interfere with the measurement of Electrolytes which causes liberation of
potassium from the red blood cells.
REFERENCES
Operator’s Manual Corning 410
LAB/86.3
20. URINE SODIUM AND POTASSIUM
Same as for serum sodium and potassium

Collect 24 hours urine (timed collection) into a dry, sterile, 2.5 litres brown bottle without addition
of any preservatives
20.1 PROCEDURE
 Mix the 24 hour urine collection and measure the total volume using a clean measuring cylinder
 Perform the test as for serum electrolytes
 If the sodium level is low, dilute the neat sample 1:50 or 1:100 and divide the result by 4 or 2
respectively; dilution is depend on the level of the sodium
 If the potassium level is higher than 10 mmol/l then dilute the urine 1:500 or 1:1000 and
multiply the result by 2.5 or 5 respectively. Dilution depends on the level of potassium
20.3 CALCULATION
Sodium (mmol/24 hour) = mmol/l x 24 hour urine volume in litre
Potassium (mmol/24 hour) = mmol/l x 24 hour urine volume in litre
REFERENCE VALUES
Urine Sodium : 40-220 mmol/24 hour
Urine Potassium : 25-150 mmol/24 hour
REFERENCES
Operator’s Manual Corning 410
LAB/86.3

68
21. APPENDIX 1 - SAMPLE COLLECTION AND TRANSPORTATION

SPECIMEN COLLECTION
Collection of Blood
Blood for analysis may be obtained from veins, arteries or capillaries. Venous blood is the
specimen of choice and venipuncture is the method of for obtaining this specimen. Prior to
the drawing of blood the phlebotomist should ensure the identity of the patient. The
appropriate containers and required volumes should be estimated. All the containers should
be labelled prior to the collection of blood.
VENIPUNCTURE
The phlebotomist should verify that the patient is fasting, if fasting be necessary to ensure
medically useful results. The patient should be comfortably seated or supine (if sitting is not
feasible.) and should be in that position for 20 minutes. The patients arm should be in a
straight line from the shoulder to the wrist. An with an inserted intravenous line should be
avoided,as should an arm with extensive scarring or haematoma at the intended collection
site .If a woman has had a mastectomy arm veins on that side of the body should not be
used since the surgery may have caused lymphostasis affecting the blood composition. The
median cubital vein in the antecubital fossa is the preferred site for collecting venous blood
in the adults. The area should be cleaned with alcohol swab.(Use benzylkonium chloride as
the cleaning during determinations of plasma alcohol.)When the skin is cleaned a tourniquet
is applied 10 tp 15 cms to obstruct the return of venous blood to the heart and to distend
the veins.(A tourniquet should be 2.5 cms wide and 38-40 cms in length. The appropriate
needle must also be selected. The larger the gauge size the smaller the bore. The usual
choice for an adult with normal veins is gauge 21 and they should be sharp, sterile and
without barbs. If blood is drawn for trace metals the needle should be stainless steel and
free from contaminants.
 In children butterfly needle with attached tubing is preferred. Appropriate volumes

should be carefully estimated and the analytical methods at the laboratory should be
able to accommodate small volumes of specimens.
 Application of a tourniquet is not recommended when blood is drawn for serum
proteins, calcium and lipid profile. When the flow of blood is obstructed by the
tourniquet, the filtration pressure across the capillary walls is increased which causes
fluid and low molecular weight compounds to pass through the capillary wall causing a
relative hemoconcentration. The first drawn sample is the most representative of the
composition of circulating blood. Therefore the first drawn blood specimen should be
used for those tests pertinent to critical medical decisions.
 Vigorous suction on a syringe during collection or forceful transfer from the syringe to
the receiving vessel may cause haemolysis of blood.
 Evacuated tubes are preferred to syringes because they are easy to use and less
likelihood of contamination of their outside with blood. There are two main classes of
blood tubes, those containing a serum separating material and without. Tubes with
siliconised stoppers are preferred as there is less interference by silicone with test
procedures. Tubes containing glycerin should not be used for lipid measurements.
SKIN PUNCTURE
If only a small volume of blood is required for a test skin puncture is performed and
capillary blood is obtained. In an adult or grown child, blood may be obtained by
puncturing the tip of a finger. When the skin is dry after cleaning with alcohol the tip of the
finger is punctured by a sharp stab with a lancet.

69
 To improve circulation of the blood, the finger may be warmed by the application of
warm, wet wash cloth for 3 minutes prior to pricking.
 The first drop is wiped off and subsequent drops are transferred to the appropriate
tubes. The blood may be collected into the capillary tubes by capillary attraction.
 In an infant the lateral and medial planter surface of the foot should be used for skin
puncture. Blood collection on ambulatory patients from anywhere on the foot is
avoided.
 For collection of blood specimens on filter paper for neonatal screening the skin is
cleaned and punctured as described earlier. Then the filter paper is gently touched
against a large drop of blood which is allowed to soak into the paper to fill the marked
cycle. The filter paper should be air dried.
ARTERIAL PUNCTURE
Arterial punctures require considerable skill and are usually performed by specially trained
medical officers. Femoral artery is the preferred site. The needle and syringe should be
flushed out with a heparin solution to ensure anticoagulation and to expel trapped air. If the
collected specimen is intended for blood gas analysis the nozzle of the syringe containing
the blood should be sealed and the syringe placed in ice water (melting ice) for immediate
transport to the laboratory. Analysis should be performed within 15 minutes.
Anticoagulants and Preservatives for blood

 Heparin is the most widely used anticoagulant for clinical chemical analysis. It is
available as sodium, potassium, lithium and ammonium salts. It acts as an
antithrombin to prevent the transformation of prothrombin into thrombin and thus
the formation of fibrin from fibrinogen.
 The chelating agent ethylenediamenetetraacetic acid is useful for haematological
examinations as it preserves the cellular components of blood. EDTA prevents
coagulation by binding calcium which is essential for the clotting mechanism.
 Sodium fluoride is usually considered as a preservative for blood glucose. It inhibits
the action of enzyme enolase in the glycolytic pathway. The sodium fluoride and
potassium oxalate should be in the 1: 3 concentrations. Excess of the preservative will
cause inhibition of the glucose oxidase reagent resulting in low glucose values.
 Oxalates of sodium, potassium, ammonium and lithium inhibit blood coagulation by
forming insoluble complexes with calcium ions. During the preparation of
tubes/bottles with oxalates the temperature of the drying oven should be carefully
controlled to below 1000 C in order to avoid decomposition to carbonate which has
no anticoagulant activity.
URINE COLLECTION
The type of urine sample to be collected is dictated by the tests to be performed.
 A clean early morning fasting specimen is generally the most concentrated and thus is
preferred for microscopic examination and for the detection of abnormal amounts of
constituents such as proteins, amino acids and β chorionic gonadotrophins.
 A double voided specimen is the urine excreted during a timed period following the
complete emptying of the bladder. It is used for example to assess the glucose
excretion during a glucose tolerance test.
 Timed urine specimens are used to minimize the influence of short term biological
variations. When specimens are to be collected over a specified period of time the
patient’s close adherence to instructions is important. The bladder should be emptied
at the time the collection is to begin, and this urine is discarded. Thereafter all urine
should be collected until the end of the scheduled time. The urine should be collected
in to a chemically clean container and should be transferred to the bottle using a
funnel. Any excess urine should be collected into a separate refrigerated container. At
the end of the collection period the last urine sample should be added into the bottle
and dispatched to the laboratory with out delay. Other relevant information (weight

70
and height of the patient) and a blood sample collected during the period of urine
collection should be provided. Acid washed beaker and a funnel should be provided
to the patient in urinary calcium estimations.
Urine preservatives
One of the most satisfactory forms of preservation of urine specimens is refrigeration; it is
even more successful when combined with chemical preservation.
HANDLING OF SPECIMENS FOR TESTING

 Every specimen should be clearly labelled for proper identification. The minimum
information on a label should include the patient’s name, location and identifying
number, date and time of sample collection.
 The specimen must be properly treated during the transport and from the time serum
has been separated until it is analysed.
 Plasma and serum should be separated from cells as soon as possible and certainly
within 2 hours. If the specimens cannot be analysed at once the separated serum
should be stored in the refrigerator in capped tubes both to maintain stability and
prevent evaporation. If the analytes are unstable in the refrigerator, freeze the serum
at -200 C or - 70 0C. At the time of analysis the specimen should be brought to the
room temperature.
 During the transportation of specimens to the referral laboratory extreme precautions
should be made to ensure that the specimen reaches the destination in leak proof
containers. The tube used for the holding of the specimen (primary tube) should be
so constructed that the contents do not escape if the container is exposed to extremes
of heat, cold and sunlight. The secondary container used to hold one or more
specimen tubes must be constructed to prevent the tubes from knocking each other.
For transportation of refrigerated specimens solid carbon dioxide may be used.
 Various laws and regulations apply to the shipment of biological specimens.

71
22. APPENDIX 2 – DIABETES MELLITUS
Definition: A group of diseases characterized by elevated blood glucose level
(hyperglycaemia) resulting from defects in insulin secretion, in insulin action or both.
Classification
 Type 1 Diabetes mellitus
(Insulin dependent, juvenile D.M.)
Immune mediated
Autoimmune destruction of β cells of the pancreas
Age of onset: childhood and adolescence; any age
Antibodies against islet cells
Idiopathic
Asian /African: permanent low insulin
 Type 2 Diabetes mellitus

Maturity onset; non insulin dependent
Due to insulin insensitivity hyper secretion of insulin
Relative insulin deficiency
characteristics Type 1 Type 2

Age of onset <35 years >35years
Genetic predisposition low high
Antibodies to β cells yes No
Body habitus Normal/wasted Obese
Plasma insulin Low /absent High
C -peptide
Metabolic feature Insulin deficiency Insulin insensitivity
 Specific types of Diabetes

Genetic defects of islet cell function
Endocrinopathies
Drug induced
 Gestational D.M
Any degree of clinical glucose intolerance with onset or first recognition during
pregnancy.
Symptoms
Polyuria
Polydypsia
Blurring of vision
Weight loss
Diagnostic Strategy for Diabetes (Refer annexure 1) W.H.O 2002 – pg 17

Corrections – Fasting plasma glucose and random plasma glucose
Fasting plasma glucose without symptoms
Fasting plasma glucose on 2 occasions

Normal fasting plasma glucose : 3.3 - 6.1 mmol/L
Impaired fasting glycaemia :> 6.1 <6.9 mmol/L
Diabetes :> 7.0 mmol/L
FPG = 7.0 mmol/l →repeat →7.0 mmol/L→DM
Oral Glucose Tolerance Test
Provide information on latent DM

72
Is more sensitive than FPG
Preparation of the patient

Thee days carbohydrate rich diet and activity
No medication on the day of the test
12 hour fast
No smoking
Glucose load: adults : 75g in 300-400 ml of water
Children : 1.75 g/Kg up to 75g of glucose
Plasma glucose sampling : 10 min before glucose load
120 min (2 hours) post glucose
Urine glucose corresponding to the samples
Evolution;
Fasting plasma 120min glucose

Glucose
IFG 6.1 -6.9 mmol/L
(110 – 125mg/dl)
IGT < 7.0 mmol/L 7.8 – 11.1m.mol/L

(< 126mg/dl) (140- 199mg/dl)
Diabetes >7.0m.mol/L >11.1 mmol/L

(>126 mg/dl) > 200mg/dl
OGTT is affected by
 Metabolic stress( ↑ glucose secretion)
Major surgery, M.I, drugs (steroids)
 Malabsorption
 Vomiting
Gestational Diabetes
Diagnosis;
Fasting plasma glucose >7.0 mmol/L (126mg/dl)
Random plasma glucose >11.1 mmol/L (200mg/dl)
Lab diagnosis
One step approach 75g glucose-OGTT
Two step approach

 First OGTT with 50g glucose; cut off value after 1 hour plasma glucose > 7.8
mmol/L (140 mg/dl)
 Second OGTT with 75g of glucose load and evaluation as the standard OGTT
Monitoring of Disease
Maintain Plasma glucose level as close as possible to normal levels during the day.
 Use of the glucometer – only for monitoring not for the diagnosis
 CALIBRATED glucometer

73
23 APPENDIX 3 - REFERENCE RANGES

Reference Ranges
Analytes Sample Reference range Units Some common indication
ACE 5 ml clotted blood / Adult 30-100 U/L Sarcoid
(Angiotensin converting enzyme) Lithium Heparin
1 ml Blood Children 6m-4y 35-75
Children 4-9y 42-90
Children 9-13y 49-105
Boys 13-18y 45-98
Girls 13-18y 35-75
ACTH 4 - 5 ml of blood into EDTA Cord 50-570 ng/L Pituitary function

(Adrenocorticotropic Hormone ) bottle ( Separated and frozen Newborn 10-185 Adrenal function
within 30 min) Avoid glass Adult
tubes as they adsorb ACTH 8 h unrestricted activity 8-79
Use pre-chilled polystyrene 16 h supine 7-30
tubes
AFP Maternal 3-5 ml clotted blood 14 wks 46 µg/l Neural tube defects
15 wks 58 Downs syndrome
16 wks 64
17 wks 72
18 wks 84
19 wks 94
20 wks 108
21 wks 118
22 wks 120
AFP 3-5 ml Clotted blood Adult < 10 µg/L Hepatoma

(Alpha Feto protein) Newborns <55000 U/ml Testicular teratoma
Contact Department of (may be higher if premature)
Biochemistry (Endocrine) for Infants at 8 wk <3100 U/ml
daily/weekly Paediatric reference Infants at 20 wk <40 U/ml
ranges Children <15 U/ml

74
Albumin 2 ml of Clotted Blood New borne 25-50 g/L Malabsorption and

1 Year 35-50 malnutrition
2-3 Year 36-50 Protein losing states
4th Year and after 37-50 Chronic liver disease
Adult 30-45
Aldosterone 4 -5 ml EDTA / Heparin or Newborn 0.14-1.66 nmol/L Adreno cortical function

Clotted Blood 1wk -1 y 0.03-4.43
1–3y 0.14-1.66
3-5 y <0.14-2.22
5-7 y <0.14-1.39
7-11 y 0.14-1.94
11-15 y <0.14-1.39
Adult, average Sodium diet (100-200 nmol/day)

Supine 0.08-0.27
Upright (At least 2 hours)

Female 0.14-0.83
Male 0.166-0.609
Alkaline Phosphatase Isoenzymes 5 ml clotted blood Reference range available in text books Qualitative by Differentiation of increased
(Only analysed if patient has alkaline electrophoresis ALP
phosphatase >250 U/L) (Liver , Bone disorders)
ALP 3 ml clotted blood Males (age 20-60 Years) 20-90 IU/L 37C Bone and Liver diseases
( Alkaline Phosphatase) Females (age 15-60 Years) 20-90
Children (age 1-12 Years) up to 350
During the growth spurt of puberty up to 500
Alpha-1 Antitrypsin 4 -5ml Lithium Heparin or Newborn 1.45-2.7 g/L α 1- antitrypsin deficiency
(Phenotyping performed if <1.4 5 ml clotted blood Adult 0.78-2.00
g/L) >60 years 1.15-2.00

75
ALT 3 ml clotted blood Adults 2-27 IU/L 37C Liver disease

(Alanin Aminotransferase) Infants 10-80
Children 10-40
Amino Acids (Plasma) 4.ml Lithium Heparin or Reference ranges are available in text books µmol/L Metabolic diseases
5 ml clotted blood
Amino Acids (Urine) Early morning first urine This is a special test, contact the reference laboratory Metabolic diseases
sample. Sample should be
frozen and a control sample
should be provided
Ammonia 4 ml EDTA in pre chilled Adult <40 µmol/L Acute hepatic failure
tubes. Must reach the lab Newborn 53-88 Urea cycle defects
within 20 minutes. A control Infants and older children 21-47
sample should be provided.
Contamination from
environment, smoking,
contamination of the glass
ware should be avoided. For
specific instructions contact
the Reference laboratory
Amylase 3 ml Clotted blood 70 – 340 IU/L 37C Acute Pancreatitis
Androstenedione (plasma) 3 ml clotted blood Pre pubertal 0.3-1.7 nmol/L Congenital adrenal cortical
Puberty (10-17 y) 0.3-8.4 disease
Adult
Male 2.6-7.2
Female 3-9.6
Apo- Lipoprotein B 3 ml clotted blood Male 0.63-1.33 g/L Investigation of lipid disorders
Female 0.60-1.26
Apo-Lipoprotein A-1 3ml clotted blood Male 0.94-1.78 g/L Investigation of lipid disorders
Female 1.01-1.99
AST 3 ml Clotted blood, avoid Newborn 10-75 IU/L 37C Hepatocellular disease
(Aspartate aminotransferase) haemolysis Children 10-45 Cardiac disease
Adult 4-42

76
Bicarbonate 5 ml clotted blood Newborn 18-23 mmol/L Acid base disorders

Adult 23-31
Bilirubin (Urine) Random Urine Not normally detectable Qualitative Liver disease
Bilirubin (Total) 3 ml clotted blood Cord blood < 50 µmol/L Liver disease
Protect from light Cord blood premature infants < 58
First 24 h <103
2-5 days <205
>1 month 1.7-26
Adult 3-21
Bilirubin-Direct 3 ml clotted blood Adults and Children 0-3.4 µmol/L Neonatal jaundice
Protect from light Liver disease
CA 125 5 ml clotted blood < 35 kU/L Ovarian cancer
(Refer the analytical method)
CA 15-3 5 ml clotted blood Non pregnant < 28 kU/L Raised in cirrhosis
Pregnant Breast cancer
1 & 2 Trimester < 50
CA 19-9 5 ml clotted blood < 37 kU/L Pancreatic cancers
Calcium (Urine) 24 hours Urine Collected into New Born 0-17.5 µmol/kg/24 hours Disorders of calcium
acid washed bottle Infants up to 1000 µmol/kg/24 hours metabolism
Older Children up to100 µmol/kg/24 hours
Or 750-3750 µmol/24 hours
Adults
Calcium in diet
Calcium free 0.13-1.0 mmol/24 hours
Low –average 1.25-3.75 mmol/24 hours
Average( 800mg/day) 2.5-7.5 mmol/24 hours

77
Calcium ( Ionised) 3 ml Heparinised blood Cord blood 1.25-1.5 mmol/L Disorders of calcium
ISE (Ion Selective Electrode) Newborn 3-24 h 1.07-1.27 metabolism
24-48 h 1.00-1.17
Thereafter 1.12-1.23
Calcium ( Total) 3 ml clotted blood Collected Cord Blood 2.33-3.05 mmol/L Disorders of calcium
into acid washed bottle without metabolism
tourniquet New Born ( 1st week)
Bottle fed 1.85-2.75
Breast fed 2.05-3.05
Thereafter up to 12 years 2.20-2.75
Adults 2.25-2.60
CEA 5 ml clotted blood Non smokers <2.5 µg/L Tumour marker, especially of
(Cacino Embryonic Antigen) Smokers 2.6-5.0 colorectal, lung, breast and
pancreas
Ceruloplasmin 5 ml clotted blood 1 day- 3 months 5-18 mg/dl Wilson’s disease
6/12 months-1 year 33-43
1 year-7 years 24-56
>7 years , Adult 18-45
Chloride 5 ml clotted blood Cord blood 96-104 mmol/L Acid base disturbances
Newborn 0-30 days 98-113
After 1 month 98-107
CSF Infant 110-130 mmol/L

Adult 118-132
Urine (24 hours sample) Infant 2-10 mmol/day

Child 15-40
Adult 110-250
(Varies greatly with Cl intake)
Sweat Normal 5-35 mmol/L Cystic fibrosis

Marginal 30-70
Cystic fibrosis 60-200

78
Cholesterol 3 ml Clotted blood Cord Blood 0.60-3.50 mmol/L Lipid disorders

1-6 week 2.40-5.60
up to 1 year 3.50-6.80
1-3 years 1.15-4.70
4-6 years 2.80-4.80
Male Female
6 - 9 years 3.26-4.94 3.16-5.41 mmol/L
10-14 years 3.36-5.28 3.21-5.61
15-19 years 2.95-5.12 3.23-5.48
22-24 years 3.21-5.64 3.16-5.59
25-29 years 3.44-6.32 3.33-5.75
30-34 years 3.57-6.58 3.37-5.96
35-39 years 3.78-6.99 3.63-6.27
40-44 years 3.91-6.94 3.81-6.53
45-49 years 4.09-7.15 3.94-6.86
50-54 years 4.09-7.17 4.20-7.38

55-59 years 4.04-7.15 4.45-7.77
60-64 years 4.12-7.15 4.45-7.69
65-69 years 4.09-7.10 4.43-7.85
> 70 years 3.73-6.86 4.48-7.25
Cholinesterase phenotype 5 ml clotted blood of family Dibucaine 77-83 % Scoline apnoea

(Pseudo cholinesterase) members of patient and Fluoride 56-64 Organophosphorus pesticide
control sample exposure
Cholinesterase screen 5 ml clotted blood of family 0.6-1.4 kU/L at 25 C Scoline apnoea
(Pseudo cholinesterase) members of patient and 1.08-2.4 kU/L at 37 C Organophosphorus pesticide
control sample exposure
CK (Total) 3 ml clotted blood New Born <300 IU/L 37 C MI
(Creatine kinase) Children <200 Skeletal muscle disease
Male 38-174
Female 26-140
CK-MB 3 ml clotted blood < 12 or < 2.8 % of Total CK IU/L 37C Myocardial infarction

79
Refer the procedure and reference ranges in the kit

Copper 5 ml clotted blood collected Birth – 6 month 3.14-10.99 µmol/L Wilson’s disease
into acid washed bottle 6y 14.13-29.83
12 y 12.56-25.12
Adult
Male 10.99-21.98
Female 12.56-24.34
Cortisol 5 ml clotted blood At Birth 94-610 Nmol/L Adrenocortical function

12 hrs 440
24 hrs 193
Older children 0800 hrs 200-720

2200 hrs <205
Adult
0800 hrs 138-635
1600 hrs 83-441
2000 hrs fraction of < 50% of 0800 hrs
Cortisol-free (Urine) 24 hours urine collected into a Child 5.5-74.5 nmol/day Adrenocortical function
container with 10 g of Boric Adolescent 13.8-152
acid. Sample should be Adult 27.6-276
refrigerated during the
collection
C-Peptide (RIA) 3 ml clotted blood 0.26-0.62 nmol/L Insulinoma

Refer the procedure and reference range of the Pancreatic β-cell function
kit/method Insulin overdose
C-Reactive Protein 3 ml clotted blood < 6 months < 3.6 mg/L Acute phase protein
>12 months <6
Creatinine 4 ml clotted blood Under 12 years 20-80 µmol/L Renal function

Adults 71-133 µmol/L
Refer the procedure and reference range of the
kit/method

80
Creatinine (Urine) 24 hours urine Under 12 years 44-354 µmol/kg/24 hours Marker of renal function
Adults 8.84-17.6 mmol/24 hours
Creatinine Clearance 24 hour urine and blood Newborn (up to 1 month) 40-65 ml/min/1.73 m2 Renal function
sample Male Female
( taken during the collection of Under 12 years 98-150 95-123 ml/min/1.73 m2
24 hour urine sample) 20-30 years 88-146 81-134
30-40 years 82-140 75-128
40-50 years 75-133 69-112
50-60 years 68-126 64-116
60-70 years 61-120 58-110
70-80 years 55-113 52-105
DHEA-Sulphate 3ml clotted blood Pre pubertal 0.25-1.00 µg/ml Adrenocortical function
Tanner Age Male

1 <9.8 y 0.13-0.83
2 9.8 -14.5 y 0.42-1.09
3 10.7- 15.4 y 0.48-2.00
4 11.8-16.2 y 1.02-3.85
5 12.8-17.3 y 1.20-3.70
Adult 1.80-4.50
Tanner Age Female

1 <9.2 y 0.19-1.14
2 9.2-13.7 y 0.34-1.29
3 10-14.4 y 0.32-3.26
4 10.7-15.6 y 0.58-2.60
5 11.8-18.6 y 0.44-2.48
Adult 0.60-2.55
Ethanol 3 ml clotted blood. Avoid Impairment 11-22 mmol/L Ethanol level

alcohol swabs to clean the Depression of CNS >21.7
venepuncture site aqueous Fatalities reported >86.8
benzalkonium chloride

81
preferred
Faecal Fat Minimum 3 day collection- Infant breast fed <1 g/day Gastrointestinal malabsorption
patient must be on a normal 0-6 years <2
diet(Collected between two Adult <7
markers) Adult (fat free diet) <4
Faecal Reducing substances Fresh faeces (send to lab Undetectable Qualitative test Gut sugar malabsorption
within 20 minutes or freeze)
Ferritin 5 ml clotted blood Newborn 25-200 µg/L Iron status

1 month 200-600
2- 5 month 50-200
6 month -15 years 7-140
Adult
Male 20-250
Female 10-120
Folate 5 ml clotted blood 2-16 y 11-48 nmol/L Megaloblastic anaemia

>16 7-36
Fructosamine 3 ml clotted blood Adult 205-285 µmol/L Glycaemic control

Child 5% below adult level
FSH 3 ml clotted blood Pre pubertal Pituitary-Gonadal axis

0-6 months <1-4 mIU/ml
6 months – 1 year <1-13
Children <10 years <1-3
Tanner Age Male
1 <9.8 y 0.26-3.0 mIU/ml

2 9.8 -14.5 y 1.8-3.2
3 10.7- 15.4 y 1.2-5.8
4 11.8-16.2 y 2.0-9.2

82
5 12.8-17.3 y 2.6-11.0
Adult 2.0-9.2
Tanner Age Female

1 <9.2 y 1.0-4.2
2 9.2-13.7 y 1.0-10.8
3 10-14.4 y 1.5-12.8
4 10.7-15.6 y 1.5-11.7
5 11.8-18.6 y 1.0-9.2
Adult
Follicular 1.8-11.2
Mid cycle 6.0-35.0
Luteal 1.8-11.2
Post menopausal 30-120
Gamma GT 3 ml clotted blood Newborn <200 IU/L 37 C Liver function

(Gamma Glutamyl Transferase) Infants <120 Alcohol abuse
Children <35
Adult
Male ≤50
Female ≤30
Gastrin 3 clotted blood. (12 hour Child < 10-125 ng/L Zollinger-Ellison syndrome
fasting) Serum should be Adult 16-60 y Male <100
centrifuged, separated & Female <75
frozen at -20 C without delay.
Samples must not be >60 y <100
haemolysed and lipaemia
should be avoided
Glucose 2 ml Blood collected into Plasma Glucose level

83
sodium fluoride and potassium Cord blood 2.5-5.3 mmol/L

oxalate in 1:3 ratio (Refer the Premature 1.1-3.3
volume of blood to be Neonate 1.7-3.3
included in the sugar bottle Newborn
from the local laboratory) 1 day 2.20-3.30
>1 day 2.80-5.00
Child 3.30-5.50
Glucose Fasting Plasma Glucose level mmol/L Diagnosis of Diabetes Mellitus
(10-12 hours fasting) Adult 3.3-6.1

Random Plasma Glucose level ≤7.8
Post Prandial Plasma Glucose level ≤11.1
Oral Glucose Tolerance Test

Fasting < 6.1
After 2 hours < 7.8
(Refer recent WHO criteria for diagnosis of diabetes
mellitus)
Glucose (CSF) 1 ml of CSF into Fluoride/ 70 % of plasma glucose mmol/L Meningitis (Bacterial/Viral)
Oxalate bottle (accompanied
with the blood sample into
fluoride oxalate)
Growth Hormone (serum) 3 ml clotted blood Newborn 1st day 5-53 ng/ml Pituitary function
1 week 5-27
1 month – 1 year 2-10
Child fasting at rest 0.7-6.0
Adult 0.7-6.0

84
HBA1c 5 ml blood in EDTA bottle 1-5 years 2.1-7.7 % Glycaemic control

(Glycated haemoglobin A 1c) 5-16 years 3.0-6.2
Adult
(Column chromatography, Cation exchange )
4.5-8.5
HCG 5 ml clotted blood Male and non pregnant female <5 IU/L Pregnancy
(Human Chorionic Gonadotrohpin) Female Germ cell tumours
After After LMP
fertilization
2 wk 4 wk 5-100
3 wk 5 wk 200-3000
4 wk 6 wk 10000-80000
5-12 wk 7-14 wk 90000-500000
13-24 wk 15-26 wk 5000-80000
26-38 wk 27-40 wk 3000-15000
Trophoblastic disease >100000
HDL Cholesterol 4 ml of clotted blood colleted Age Male Female mmol/L Lipid disorders
without tourniquet Cord blood 0.16-1.37 0.34-1.45
5-9 y 0.98-1.94 0.93-1.89
10-14 y 0.96-1.91 0.96-1.81
15-19 y 0.78-1.63 0.91-1.91
20-24 y 0.78-1.63 0.85-2.04
25-29 y 0.80-1.63 0.96-2.15
30-34 y 0.72-1.63 0.93-1.99
35-39 y 0.75-1.60 0.88-2.12
40-44 y 0.70-1.73 0.88-2.28
45-49 y 0.78-1.66 0.88- 2.25
50-54 y 0.72-1.63 0.96-2.38
55-59 y 0.72-1.84 0.96-2.35
60-64 y 0.78-1.91 0.98-2.38
65-69 y 0.78 -1.94 0.91-2.48
>70 y 0.80-1.94 0.85-2.38
Homocystine Plasma <15 mmol/L Cardiac risk

Refer the procedure and reference range of the
kit/method

85
Hydroxy progesterone(17 OHP) Serum 3 ml clotted Blood Male, Puberty stage -1 0.1-2.7 nmol/L Adrenal status
Adult 1.5-7.5
Female, Puberty stage -1 0.1-2.5
Follicular 0.6-3.0
Luteal 3.0-15.5
Postmenopausal ≤2.1
Congenital Adrenal
Neonates <30
Hyperplasia
>1 month 6
Hydroxyindoleacetic Acid 24 hours urine Adult 10.4-31.2 µmol/24 hours Carcinoid syndrome
(5-HIAA) Children 7-70
Insulin (12 hour fasting ) 5 ml clotted serum should be Newborn 21-139 pmol/L Insulinoma
centrifuged , separated & Adult 13-174 Insulin overdose
frozen at -20 C within 2 hours >60 years 42-243
Iron 5 ml clotted blood collected New Born up to 1 month 17.9-44.75 µmol/L Iron status
into acid washed bottle Infant (1month-1 year) 7.16-17.9
Child (1 year-12 year) 8.95-21.48
Adult
Male 11.64-30.43
Female 8.95-30.43
(Strongly method dependent)
Iron Binding Capacity , Total 5 ml clotted blood collected Infant 17.9-71.6 µmol/L Iron status
(TIBC) into acid washed bottle Thereafter 44.5-80.55 µmol/L
Lactate 4 ml of blood .Patient should Venous 0.5-13 mmol/L Investigation of metabolic

be complete rest for 2 hour Arterial 0.5-1.6 disorders
and fasting & preferably Lactic acidosis
without tourniquet. (Should be
collected into container
containing 10 mg of NaF & 2
mg of potassium oxalate per 1
ml of blood. Specimen should

86
be cooled immediately & cells

separated within 15 minutes)
LDH (Lactate dehydrogenase) 5 ml clotted blood 0-4 days 290-775 IU/L 37 C Haematological abnormalities
Total(L P) 4-10 days 545-2000 Liver disease
10days -2 years 180-430
2 years -12 years 110-295
12 -60 years 100-190
>60 years 110-210
LDL Cholesterol Male Female Lipid status
(Calculated) Cord blood 0.5-1.45 0.54-1.50 mmol/L
5-9 y 1.63-3.34 1.76-3.63
10-14 y 1.66-3.44 1.76-3.52
15-19 y 1.61-3.37 1.53-3.55
20-24 y 1.71-3.81 1.48-4.12
25-29 y 1.81-4.27 1.84-4.25
30-34 y 2.02-4.79 1.81-4.04
35-39 y 2.10-4.90 1.94-4.45
40-44 y 2.25-4.82 1.92-4.51
45-49 y 2.51-5.23 2.05-4.82
50-54 y 2.31-5.10 2.28-5.21
55-59 y 2.28-5.26 2.31-5.44
60-64 y 2.15-5.44 2.59-5.80
65-69 y 2.54-5.44 2.38-5.72
>70 y 2.28-4.82 2.49-5.34
LH 5 ml clotted blood Pre pubertal mIU/L Pituitary- gonadal axis
(Luteinizing Hormone) 0-6 month 1-18
6 month -10 year <1-5
Tanner Age Male

1 <9.8 y 0.02-0.3
2 9.8-14.5 y 0.2-4.9
3 10.7-15.4 y 0.2-5.0
4-5 11.8-17.3 y 0.4-7.0
Adult 1.5-9.0
Tanner Age Female

87
1 <9.2 y 0.02-0.18
2 9.2-13.7 y 0.02-4.7
3 10-14.4 y 1.0-12.0
4-5 10.7-15.6 y 0.4-11.7
Adult
Follicular 29
Mid cycle 18-49
Luteal 2-11
Lipoprotein (a) 3 ml clotted blood 20-570 mg/L Lipid disorders

(refer the method for reference range)
Lithium 5 ml clotted blood Therapeutic 0.6-1.2 mmol/L Therapeutic dose monitoring
Toxic >2 Overdose
Magnesium 3 ml clotted bottle collected Newborn 2-4day 0.6-0.9 mmol/L Electrolyte status
into acid washed bottle 5 months – 6year 0.70-0.95
(preferably without tourniquet) 6-12 years 0.70-0.86
12-20 years 0.70-0.91
Adult 0.66-1.07
Mucopolysaccharide screening Random Urine, fresh Negative Qualitative Mucopolysaccharidosis

Myoglobin Random Urine Any Myoglobin detected is clinically significant Qualitative Acute tubular necrosis
Myoglobin (Serum) 4 ml of clotted blood Male 19-92 µg/L Acute myocardial infarction
Female 12-76
Increases slightly with age
Occult Blood Samples from 3 consecutive Not normally detected Qualitative GI bleeding
days Colonic cancer
Oestradiol (17-β Oestradiol) 5 ml Clotted blood Children <60 pg/ml Gonadal function
Male <40
Female
Follicular phase 30-120
Ovulatory peak 150-400
Luteal phase 70-200
Menopause <60

88
Osmolality Random Urine 50-1200 mOsmol/kg Urine concentrating ability

Depending on the fluid intake
Oxalate 24 urine collected into bottle Male 0.23-0.68 mmol/24 hours Hyperoxaluric stone forming
containing 10 ml of 1 N HCL Female 0.23-0.63
Children 0.10-3.0
Excessive Vitamin C in Urine affects assay results
Para Thyroid Hormone (PTH) 5 ml clotted blood Cord blood ≤0.32 pmol/L Parathyroid tumour
Intact (IRMA) Hypercalcaemic states
2 y – Adult 0.95-6.8
Phosphate -inorganic 5 ml Clotted blood Cord Blood 1.03-2.45 mmol/L Bone function
(Lower values found in breast fed infants) Renal function
New Born ( 1st week)
1st week 1.87-2.91
nd
2 week 1.58-2.87
up to 1 year 1.30-2.10
Thereafter up to 12 years 1.16-1.91
Adults 0.80-1.44
Phosphate-inorganic (Urine) 24 hours Urine Older children 0.49-0.65 mmol/kg/24/hour Parathyroid , renal and bone
Infants 6.5 mmol/kg/24 hour disorders
Adults 16-48 mmol/24 hours
Potassium 3 ml clotted blood (avoid <2 month 3-7 mmol/L Electrolyte status
haemolysis & do not 2-12 month 3.5-6.0
refrigerate) > 12 month 3.5-5.0
Adult 3.5-5.6
Pregnancy Test(Urine) Random Urine Qualitative Not applicable Pregnancy
Progesterone 5 ml clotted Pre pubertal child (1-10 y) 0.2-1.7 nmol/L Ovulatory status
Puberty
Tanner Male Female
1 <0.3-1.0 <0.3-1.0
2 <0.3-1.0 <0.3-1.7
3 <0.3-1.5 .3-14.3
4 <0.3-3.4 <0.3-41.3
5 0.7-2.6 0.3-30.2

89
Adult
Male 0.4-3.1
Female
Follicular 0.5-2.2
Luteal 6.4-79.5
Pregnancy 7-13 wk 32.6-139.9
4-37 wk 62-262.4
30-42 wk 206.7-728.2
Prolactin 5 ml of clotted blood Cord blood 45-539 µg/L Pituitary function

Newborn 5-3 d 30-495
Children
Tanner Male Female
1 <10 3.6-12
2-3 <6.1 2.6-18
4-5 2.8-11 3.2-20
Adult
Male 3-14.7
Female 3.8-23.2
Pregnancy 3rd trimester 95-473
Prostate Specific Antigen (PSA) 5 ml clotted blood <4 µg/L Prostate cancer
Protein (CSF), Lumbar CSF in plain container Premature 15-130 mg/dl Inflammatory conditions of the
Full term newborn 40-120 meninges
<1 month 20-80
Thereafter 15-40
Protein(Total) 3 ml clotted blood New borne 46-77 g/L Malnutrition , liver disease and
1 Year 56-73 protein losing conditions
2-3 Year 58-76
4th Year and after 60-80

90
Adult 60-80
Body Fluid (Transudates) <3
(Exudates) >3
Renin 5 ml blood taken into a bottle 0-3 y <16.6 µg/L/hr Hypertensive states
containing EDTA ,must be 3-6 y <6.7
separated at room temperature 6-9 y <4.4
and frozen at -20 C or 9-12 y <5.9
lower(Contact the reference lab 12-15 y <4.2
before collection of blood) 15-18 y 4.3
Normal sodium diet
Supine 0.2-1.6
Upright (4 hours) 0.7-3.3
Low sodium diet
Supine 1.0-5.4
Upright (4 hours) 0-19.0
SHBG 5 ml clotted blood Male 10-50 nmol/L Gonadal function

(Sex Hormone Binding Globulin) Female 30-90
Pre pubertal 55-120
Sodium 3 ml clotted blood Newborn 134-146 mmol/L Electrolyte status
Infant 139-146
Child 138-145
Thereafter 136-146
Somatomedin C ( IGF-1) 4 ml blood collected into 400-2000 IU/L Growth Hormone disorder
EDTA bottle investigation
Stone analysis Renal Calculus Not applicable Qualitative Investigation of Nephrolithiasis
Quantitative
Sugar Chromatography (Urine) Random urine, Fresh early Not detectable Qualitative Gut sugar malabsorption
morning sample preferable
T3 (Free) 3 ml clotted blood Child 2.6-4.8 pg/ml Thyroid function
Adult 2.08-6.74
T4(Free) 3 ml clotted blood Premature infant ng/dl Thyroid function
(Gestational age in week)
25-27 0.6-2.2
28-30 0.6-3.4

91
31-33 1.0-3.8
34-36 1.2-4.4
Infants, Children and Adults

1-4 days 2.2-5.3
2-20 weeks 0.9-2.3
5-24 months 0.8-1.8
2-7 years 1.0-2.0
8-20 years 0.8-1.9
21-45 years 0.9-2.5
Adults >45 years 0.8-2.3
Testosterone (Free) 5 ml Clotted blood Age Male Female pg/ml Gonadal function
Cord 5-22 4-16
Newborn
1-15 d 1.5-31.0 0.5-2.5
1-3 month 3.3-18.0 0.1-1.3
3-5 month 0.7-14.0 0.3-1.1
5-7 month 0.4-4.8 0.2-0.6
Pre pubertal
1-10 y 0.15-0.6 0.15- 0.6
Adult 52-280 1.6-6.3
Testosterone (Total) 5 ml Clotted blood Pre pubertal Child 1-10 y ng/ml
Male <0.03-0.1
Female <0.03-0.1
Puberty Male
Tanner Age
1 <9.8 y <0.03-0.1
2 9.8-14.5 y 0.18-1.5
3 10.7-15.4 y 1.0-3.2
4 11.8-16.2 y 2.2-6.2
5 12.8-17.3 y 3.5-9.7
Adult 3.5-103
Puberty Female
Tanner Age
1 <9.2 y 0.03-0.1
2 9.2-13.7 y 0.07-0.28

92
3 10-14.4 y 0.15-0.35
4 10.7 -15.6 y 0.13-0.32
5 11.8 -18.6 y 0.20-0.38
Adult 0.1-0.5
Thyroglobulin 5ml Clotted blood 3.0- 42 µg/L Carcinoma of thyroid
Total Protein (Urine) 24 hours urine 20-150 mg/24hours Renal function

preservative:1ml of 10% Protein losing status
Thymol in isopropanol
solution
Transferrin saturation (calculated) Male 20-55 %
Female 15-54 %
Triglycerides 3 ml of clotted blood. 14 hours Male Female Lipid disorders

fasting is necessary Cord Blood 0.15-1.07 0.12-0.86 mmol/L
0-9 years 0.34-1.13 0.40-1.24
10-14 y 0.36-1.41 0.42-1.48
15-19 y 0.42-1.67 0.44-1.40
20-29 y 0.50-2.81 0.41-1.63
30-39y 0.56-3.62 0.44-1.99
40-49 y 0.62-3.70 0.51-2.42
50-59 y 0.65-3.23 0.59-2.62
Troponin I 2 ml lithium Normal < 0.5 µg/L Myocardial infarction

Heparin or 5 ml clotted MI >3 Unstable angina
(Refer the method for reference range)
Troponin T 2 ml lithium Refer the method for reference range Myocardial infarction
Heparin or 5 ml clotted Unstable angina
TSH 5m Clotted blood Premature infants mIU/L Thyroid function
(Thyroid Stimulating Hormone) (Gestational age in weeks)
25-27 0.2-30.3
28-30 0.2-30.6
31-33 0.7-27.9
34-36 1.1-21.6

93
Infants , children and adults

1-4 days 1-39
2-20 wk 1.7-9.1
5-24 months 0.8-8.1
2-7 years 0.7-5.7
8-20 years 0.7-5.7
21-45 years 0.4-4.2
Adult >45 years 0.3-5.0
Urea 3 ml clotted blood Cord blood 7.5-14.3 mmol/L Renal function

Premature 1 wk 1.1-8.9 Dehydration
Newborn 1.4-4.3
Infant/ Child 1.8-6.4
Adult 2.5-6.6
>60 y 2.9-7.5
Uric Acid 4 ml clotted blood 1-5 y 100-350 µmol/L Gout

6-11 y 130-390 Tumour lysis
Pre eclampsia
12-19 y
Male 180-460
Female 160-340
Adult 120-360
Uric Acid (Urine) 24 hours urine collected into 1.48-4.43 mmol/24hours

sterile 2.5L bottle (Should be
refrigerated during the
collection)
Urine Analysis Random Urine Not applicable Qualitative Renal disease
(with Microscopy)
Vitamin A 5ml Clotted blood 1-6 y 0.7-1.5 µmol/L Nutritional status
(fasting) 7-12 y 0.91-1.71
Protect from light separate the 13-19 y 0.91-2.51
serum immediately. Adult 1.05-2.8
Store at -20 0 C

94
Vitamin B 12 5ml Clotted blood Newborn 125-590 pmol/L Megaloblastic anaemia

Thereafter 103-157
>60 years 81-590
Vitamin D (25 OHD3) 5ml Clotted blood Children Vitamin D metabolism and
(25 Hydroxy cholecalciferol) 1-30 d 1.9-33.4 ng/ml Calcium metabolism
31d - 1 y 7.4-53.3
Adult 14-60
Vitamin D(1,25 OHD3) 5ml Clotted blood 43-154 pmol/L Vitamin D metabolism and
(1,25-dihydroxy cholecalciferol) Calcium metabolism
Vitamin E 5ml Clotted blood 11.6-46.4 µmol/L Nutritional status
REFERENCES:
1. Teitz text book of clinical chemistry by Carl A.Burtis, Edward R.Ashwood; 2nd and 3rd Edition
2. Clinical chemistry – theory , analysis, correlation by Lawrence A.Kaplan, Amadeo J. Pesce; 3rd Edition 1996
3. WHO Guidelines on standard operating procedures for clinical chemistry, Sep 2000
4. Nelson text book of paediatrics by Behrman, Kliegman, Jenson; 16th Edition 2000
5. Forfar and Arneil’s Text book of paediatrics
6. Biochemical basis of paediatric disease by Steven J Soldin,Nader Rifai,Jocelyn M. Hicks ;3rd Edition 1998

95
WE SINCERELY THANK THE FOLLOWING COLLEAGUES / MEMBERS OF MRI AND WHO COUNTRY OFFICE
Administrative staff
Ms. Kumuduni Ragel
Secretary WHO country office Sri Lanka
Mrs. G. Subramanium
Accountant MRI
Mr. A. Ravichandran
Financial staff
Mrs. Margret Prera

Planning unit Ministry of Health
Miss. Nilakshi Devindi Gunatillaka
Members of staff and NEQAS team
Ms. Manjula Subashini

Mr. K. S. T. Karunapala
Ms. E.A.N.S. Peiris
Mr. B. D. Lankananda
Ms. S.K. Nanayakkara
Ms. N. D. Wijekoon
Support staff
Mr. N. A. H. H. Nissanka
Mr. J. M. Wijesinghe
Mr. D. L. Upasena
Mr. T. V. Anton
Ms. W. Pushpika Perera

Manual On Standard Operation Procedures, Sample Collection and Reference Ranges For Clinical Chemistry

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Manual On Standard Operation Procedures, Sample Collection and Reference Ranges For Clinical Chemistry

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MANUAL ON

STANDARD OPERATION PROCEDURES, SAMPLE COLLECTION AND

WORLD HEALTH ORGANISATION,

THE DEPARTMENT OF BIOCHEMISTRY, MEDICAL RESEARCH INSTITUTE

Dr. Meliyanthi M. Gunatillaka

Ms. D. K. Daya Silva

Mr. M. Muhammed Hunais

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

We thank the Director General of Health Services Dr. Athula Kahandaliyanage,

We appreciate the assistance of administrative staff of World Health Organisation

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

Clinical laboratory services have become an important component of modern

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

Specimen which do not conform to the requirements or are of inadequate quality

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

GLASSWARE AND EQUIPMENT

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

2.2 CLINICAL SIGNIFICANCE

2.3 PRINCIPLE OF THE METHOD

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

2.4 SPECIMEN TYPE, COLLECTION AND STORAGE

APPARATUS AND CHEMICALS

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

PREPARATION OF CALIBRATION GRAPH

Working standard No (01) (02) (03) (04)

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

Where T=the absorbance of the test

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

3.2 CLINICAL SIGNIFICANCE

3.3 PRINCIPLE OF THE METHOD

3.4 SPECIMEN TYPE, COLLECTION AND STORAGE

3.5 APPARATUS AND CHEMICALS

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

NOTE: Avoid contamination of the pipette with saliva

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 6% should be

ROUTINE CONDITIONS VARIANCE: This value should not exceed 12 %

REFERENCE VALUES: Approximate reference values: 70 – 340 U/L

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

4.2 CLINICAL SIGNIFICANCE

4.3 PRINCIPLE OF THE METHOD

4.4 SPECIMEN TYPE, COLLECTION AND STORAGE

4.5 APPARATUS AND CHEMICALS

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

GLASSWARE: Automatic pipette (50µl and 100µl)

NOTE: The quality of the disodium 4 – Nitrophenyl phosphate should be checked to

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

PREPARATION OF CALIBRATION GRAPH

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 10% should be

APPROXIMATE REFERENCE VALUES

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

5. ASPARTATE AMINO TRANSFERASE

5.2 CLINICAL SIGNIFICANCE

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

5.3 PRINCIPLE OF THE METHOD

5.4 SPECIMEN TYPE, COLLECTION AND STORAGE

5.5 APPARATUS AND CHEMICALS

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

Amount of pyruvate formed = (A Test-A Sample Blank) x 4x1x1000

With the reagent blank set to zero the spectrophotometer at 505 nm

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005