INTRODUCTION

Glucose is monosaccharide which is the simplest form of carbohydrates. It is metabolised by

oxidation to carbon dioxide and water through a series of reactions which yield 2840 KJ/mol.
Under anaerobic conditions the energy yield is much less.
Glucose is the primary source of energy for the human body. It is derived from the breakdown of
carbohydrates in the diet (grains, Starchy vegetables, and legumes) and in 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 calorie
intake, endogenous glucose formation occurs from non carbohydrates sources (e.g. Amino acids,
Lactate and glycerol).
The reaction of glucose metabolism are reactions under the control of several hormones viz.
insulin, glucagon, epinephrine, glucocorticoids etc.The glucose level inboxed blood is thus
maintained within fairly narrow range under diverse conditions (feeding, prolonged fasting,
severe exercise) through their effect on these hormones, as well as other regulatory processes

Specimen collection And Processing

Clenicians are dependent upon laboratory investigation to diagnose a disease, and follow its
progress and response to treatment. Many factors, besides disease affect the composition of body
fluids, like (a) pre analytical (b) analytical and (c) biological. These should be properly control
prior to investigation of specimen. Clinical Biochemistry Laboratory receives different types of
the body fluids for different investigations.
These are:
 Blood
 urine
 CSF
 Synovial fluid
 Pleural Fluid
 Pericardial Fluid
 Ascitic Fluid
 Amniotic Fluid etc
 Correct method of collection of each specimen for investigation is equally important as that
of is appropriate and requisite analysis.
 Collection Of Blood
 Blood specimen for analysis may be obtained from veins, arteries, capillaries, depending
upon the investigation concerned. Before collecting a specimen of any type :
 (i) A technical person or clinician must ensure that specific specimen container labelled
with complete patient demographics should be ready
 (ii) A technical person or clinician should put on disposable latex rubber gloves, and apron
to protect against any type of infection
 Collection Of Venous Blood
Method: Venous blood is usually the specimen of choice, for which venipuncture is the
method for obtaining this specimen. Before performing venipuncture the technical person
or the clinician should verify that the patient is instructed properly( e.g. fasting etc) to
obtain medically useful results. Selection of vein for puncture is facilitated by palpation.
The most preferred site for venipuncture in adults is the median cubital vein in the
anticubital fossa, because the vein is large and also close to the surface of the skin. Veins
on the dorsum of the hand or at the ankle may be used. The area around the puncture site
must be cleaned with a methylated spirit ( Methyl alcohol with blue colouring) swab or
gauze, in a circular motion and from the site of puncture and then allowed to dry. Do not
touch the cleaned area without wearing a sterile disposable gloves. After cleaning the site, a
tourniquet is applied four to six inches above the puncture site, veinipunture performed,
and, as the blood starts flowing into the evacuated tube, the tourniquet is realesed. If this is
not done, the composition of blood will be altered. The patient bar code should be stuck on
the tube before sampling.

Collection Of Blood With Evacuated Blood Tubes

Routinely, evacuated blood tubes are used for sampling. Evacuated blood tubes are more
convenient, provide easier sampling and prevent damage to RBC and vessel wall that
occure during force full syringing. The needle is screwed on to the collection tube holder,
venipuncture is performed and than the tubes to be filled are genteelly inserted to this
holder one by one. This will prevent the aspiration of the additive into the patients vein.

Collection of Arterial Blood

Arterial blood is mainly used for blood gas analyses and is collected by arterial puncture
technique. Arterial puncture require considerable skill and are usually performed only by
the clinician or specially trained personnel.

Collection Of Capillary Blood

Capillary blood is frequently collected by skin puncture specially in young children and
small amount of blood and when a small amount of blood is required.

Anticoagulants And Preservatives For Blood

An anticoagulant or preservatives must be added to the blood specimen during collection, if

whole blood or plasma is required for investigation.
 Heparin: For 1 ml of blood about 20 units of heparin is required
 Ethylenediamine Tetracetate (EDTA): EDTA is particularly useful because this is a
chelating agent which preserves the cellular components of blood. For 1ml of blood,
1-2 mg of EDTA is required
 Sodium Fluoride (NaF): For blood glucose, NaF is usually employed as a
preservative, inhibits the glycolytic process and thereby does not allow utilization of
glucose in the standing sample; however it also acts as a weak anticoagulant.
 Citrate: Sodium Citrate in solution at a concentration 3.4 or 3.8 g/dL in a ratio of
1:10 is widely used for coagulation studies.
 Iodoacetate: Sodium Iodoacetate, at a concentration of 2 g/L is an effective
antigycolytic agent and sometimes used as a substitute for NaF.
 For all the analytes estimated in a Clinical Biochemistry Laboratory, we require
serum (blood to be collected without any preservative) except for the following
parameters:
 Glucose (Sodium Fluoride is required to arrest glycolysis and maintain the glucose
level in the sample).
 Lactate (Sodium Fluoride is required to arrest glycolysis and maintain the lactate
level in the sample).
 HbA (EDTA whole blood is required).
 Ammonia (blood is collected in EDTA, centrifuged within half an hour and the
plasma analysed immediately).
 Homocysteine (blood is collected in EDTA, centrifuged in half an hour and the
plasma analysed immediately or stored frozen till analysis).

Collection Of Urine
These fluids are collected from different cavities of the body by the clinicians under
specific conditions.
 Random specimen : Random urine is an untimed specimen which is suitable for
biochemical investigations.
 Timed specimen : Timed investigations, collection periods should be long enough to
minimize biological variations for most of the quantitative investigations (e.g.
creatinine clearance), 24 hours specimen are usually collected.

Preservative for Urine

If the urine specimen have to be kept, it may be necessary to suitable preservative to reduce
different type of changes like bacterial or chemical decomposition or even precipitation of
solubilized constituents.
Refrigeration of urine specimen immediately after collection is one of the most satisfactory
forms of preservation. The most common preservatives and the investigations for which
they are required are as follows:
 HCl : 10 ml concentrated HCl for 24 hours ‘urine specimen’. 50 ml of 2NHCl may
also be used for estimation of urea, nitrogen, ammonia, total nitrogen, calcium, and
uric acid.
 Toluene: About 15ml of toluene required for 24 hours’

Collection for estimation of protein, creatinine etc.

 Other Chemical Preservatives, like formalin glacial acetic acid, thymol and light
petroleum can also be used.
In our Clinical Biochemistry Laboratory, the preservatives we used for 24 houres’
urine are :
 Toluene (30ml) for urinary protein, creatinine and creatinine clearance.
 20%HCL : 10 ml of Ca and P , 15ml of VMA

Processing of Specimen for Investigation

 Identification of Specimen: Proper identification of properly collected and preserved
specimen must be maintained for valid results and their correct interpretation. Every
specimen container must be properly labeled even if it is very small, like a capillary
blood tube.
 Transport and Preservation of Specimen: Each specimen must be properly treated
during its transportation to the laboratory, and also the time interval between
collection, separation and analyses must be as per required specifications.
 Separation and Storage of Specimen: Serum or plasma should be separated from the
cells as soon as possible usually within 2 hours by centrifuging in a horizontal head
rotar at 3000 rpm for 10 minutes.
However, premature separation of serum may lead to hemolysis or permit continued
formation of fibrin clot and thence lead to obstruction of sample prob of the testing
analyser. If the separated specimen cannot be analyzed immediately, then these
sera/plasma/fluids should be stored in a capped tube at 2 -8 degree Celsius til
analysis
 Programming on computerized auto analyzer : the bar coded sample tubes are
numbered and then centrifuged. While the sample is being centrifuged to separate
 serum/plasma, a work list of the sample, along with the test requisitioned, is
generated , and subsequently, details are noted in a register in an instrument- friendly
order i.e. these sample are listed under a particular sample/cup number. These listings
are programmed onto the analyzer
 Numbering and Organization of Sample Cups: The plastic sample cups are numbered
(with sample numbers) and placed in the appropriate cup slot of the appropriate sector
to the corresponding to listing. The sample is then pippettd into the correspondingly
numbered cup and once all the cups in a sector (numbered at source) are thus filled,
the sector is loaded onto the analyzer

Sampling Guidelines
 No sample should be drawn from a site through which intravenous administration of
fluids/medication is being done.
 All samples for ammonia and homocysteine must be collected in EDTA vacutainers
and immediately transported to the biochemistry laboratory ( within 10 mins).
 All samples for homocysteine , vitamin B12 , Folic acid , and lipid profile must be
drawn after an overnight fast (12 to 14 hours).
 Appropriate vacutainers must be used for sampling and samling must be performed
by vacutainers needles on their appropriate holders. Where paedriatric samples are to
be collected, a vein flow may be used. Also when samples have to be collected from
patient with collapsed/thrombosed veins, a vein flow is still the method of choice.
 Use of Vacutainers also obviates the need to first calculate the total amount of sample
required and then the capacity of syringe required for that.
 If a syringe must be used, then a 21 gauze needle should be used, a smaller gauze
needles gauze hemolysis.
 Samples that cannot be transported to the laboratory for sometime need to be allowed
to stand for approximately 30 minutes at room temperature, and then refrigerated. For
assays to be performed on serum or plasma the sample should be centrifuged and the
serum/plasma separated and stored at 2-8 degree Celsius.
 For samples drawn in anti coagulant, the sample must be inverted and straightened 8-
10 times to ensure proper mixing. For plain samples, too, invert the vacutainer once
 very gently and then let it stand erect. The inner wall of the vacutainer is lined with
clot promoter and hence this maneuver facilitates clotting of the sample.
 Before drawing samples for folic acid and vitamin B12, please ascertain that the
patient has not had any vitamin supplement/vitamin injection for the proceeding 48
hours.
 When samples are sent for estimation of a drug, please note the time of the last dose
and the time of sampling.
 For all timed urine collections, ensure proper collection by the patient and ascertain
that the appropriate preservative has been added.
 While drawing samples for GTD, the patient must consume the whole amount of
glucose given within ten minutes and time of further sampling must be counted from
that point onwards. Also, the patient must not eat or drink anything (but water) for the
duration of the test. If the patient vomits, the test must be discontinued.
 When stones are sent for analysis, the source must be specified, i.e. whether it is from
the gall bladder, urinary bladder, ureter or kidney.

Policy For Receiving Samples

 Check label on specimen and requisition form.
 Enter date and time for receiving specimen.
 Check test requisition and whether sample is in appropriate container.
 For timed urine specimen, check the time period and preservatives used.
 For ammonia and homocysteine requisition check the time of sampling.
 If urgent reporting is requested send the sample for processing urgently in a separate
lot.
 All sample from ICU, IMCU, Pediatric ICU, Transplant unit are to be processed as
urgent, whether or not specified on the requisition.
 Sample for ammonia are to be analyzed within 30 mins. Of sampling.
 Samples for ammonia and homocysteine are to be centrifuged immediately.
 Policy For Sample Processing
 Samples for ammonia are to be analyzed within 30 minutes of sampling
 Samples for homocystine are to be centrifuged immediately, and the plasma separated
from the cells and stored appropriately depending upon when they are to be analyzed.
 All plain samples must be allowed to stand for 30 minutes before centrifuging to
allow proper and complete clotting.
 All samples received in the laboratory must be centrifuged if received in a separated
state.
 After centrifugation, where hemolysis icterus or lipemia is present, the status of the
sample must be clearly mentioned on the requisition form and the consultants
informed it should also be entered in the analyzer data.
 Blood Glucose Test

METHODS
 The following procedures are commonly employed for the measurement of glucose:
 Alkaline Copper Reduction method:
Which is the oldest, but also measures other reducing g substances in the sample, as well,
e.g Uric Acid, Ascorbic acid, creatinine etc. Examples are - Folin Wu, Somogyi-Nelson,
neosporine, ferricynaide.
 Orthotluidine Method:
A derivative of ortho-tolidine is used in the "Sangur" test sticks manufactured by
Boehringer Mannheim. These are intended for the detection of blood in urine in clinical
situations but are equally useful as a screening test for dried bloodstains.This procedure is
not specific for glucose, And other carbohydrates produce variable amounts of colour in the
reaction ( fructose has little effect, but Galactose produces a strong reaction).
 Enzymatic Procedures are the method of choice.
They employ a variety of enzymes e.g. glucose dehydrogenase, glucose oxidase and
peroxidase, hexokinase, etc.Glucose oxidase is an enzyme extracted from the growth
medium of Aspergillus niger. Glucose oxidase catalyse the oxidation of Beta D- glucose
present in the plasma to D glucono -1 ,5 - lactone with the formation of hydrogen peroxide;
the lactone is then slowly hydrolyzed to D-gluconic acid. The hydrogen peroxide produced
is then broken down to oxygen and water by a peroxidase enzyme. Oxygen then react with
an oxygen acceptor such as ortho- toluidine which itself converted to a cultured compound,
the amount of which can be measured calorimetrically.

Reference Method:

 The reference method for glucose estimation is the Enzymatic Method that uses hexokinase
who’s affinity for glucose is more than foe any other hexose and their for this method is the
most specific.
 The increase of absorbance at 340nm( due to increase in concentration of NAD(P)H is a
direct measure of glucose concentration in the sample
 Glucose oxidase (GOD) Peroxidase(POD): Method has a specificity only marginally less
than that of the hexokinase method and is more cost effective than that method.

 Glucose is determined after enzymatic oxidation in the presence of glucose oxidase. The
hydrogen per oxide formed reacts, under catalysis of per oxidase, with phenol and 4-
aminophenazone to form a red-violet quinonemine dye, which is measured at 510nm.

Analytic Range: 2-512mg/dL

Analytical sensitivity: 2mg/dL

SAMPLE
 Plasma from blood collected in vials containing sodium fluoride which inhibit the
glycolytic process and thus preserves the plasma level of glucose.
 The fasting glucose estimation, 2ml of blood inn fluoride after 12-14 hours fast
 For post-prandial glucose estimation, 2ml of blood in fluoride. 2 hours after 75 grams of
glucose , or alternately, 2 hours after high glucose meal.
 For Oral Glucose Tolerance Test
 A fasting sample of 2 ml blood in a fluoride vial
 75 grams of glucose (as per WHO specification) to be ingested with 2 glasses of water and
time noted (of ingestion).
 4 serial samples of 2 ml blood in a fluoride vial to be taken every half hour, i.e, half hour,
one hour, one and half hours and two hours after ingestion of glucose load.
 Simultaneously, half hourly samples of urine are to be collected and sent for glucose
estimation

Precaution
 During this two hour period, patient is not allowed to eat or drink anything except 1-2
glasses of water. 24 hours’ urine must be collected with glacial acetic acid as preservative
 and the containers must be amber tinted and kept at 2 degree celsius to 8 degree celsius
during collection. Final pH of 4-5 needed

EQUIPMENT: LX-20 Pro or CX-5 Pro

Reagents : IL glucose (oxidase)-0018250840

 Ready to use kit containing
 Glucose oxidase
 Per oxidase
 4-amino antipyrine
 Phenol
 Buffer
 Sodium Azide(<0.1%)
 Storage of Reagents
 The reagent is stored at 2-8 degree celsius, at which temperature it is stable till the date of
expiry. On board (opened) reagent is stable for 35 days.
 Safety Precautions
 The reagent should be clear and reddish.
 Though sodium azide concentration is <0.1%, it may form explosive azides with metal
plumbing. Therefore, it must be diluted with water before disposal
 The waste stream contains about 0.14 mg/L of phenol per test, it must not be emptied
directly into the drains.
 Avoid eye contact with skin and eyes
Calibration
Multical from Beckman is used to calibrate the reagent. Calibration is valid for 14 days but
due to high turnover, fresh reagent is used everyday and calibration also performed
everyday.
Quality Control
 Control material of different levels obtained from Beckman, Bio-Rad and Roche are
estimated everyday at different times of the day. Pooled serum is also estimated. In case of
 any discrepancy, reports withheld, corrective measures are performed, samples are rerun
and the fresh reports dispatched.

Technique: Fully automated immonuturbidimitry.

 After centrifuging the fluoridated sample, the supernatant plasma is pipetted out into a
plastic cup which fits into a slot on a sector of the instrument. This is programmed on the
computer of the instrument under the sector number and the cup-place number. The sector
is then loaded onto the machine which is automatically pipettes the sample and
corresponding reagent, incubates them, reads the absorbance at the appropriate time,
calculates the result of the sample and prints it out.
 Interference
 Urine and CSF measurements are subject to interference by uric acid and ascorbic acid.
 No interference has been observed with the following:
 lipemia upto 1100 mg/dL, triglycerides,
 hemolysis upto 250 mg/dL, haemoglobin
 Icterus upto 5 mg/dL bilirubin

Clinical Significance
 Blood glucose estimation is primarily required for the diagnosis and management of
diabetes mellitus. Diabetes mellitus is a group of disorders of carbohydrate metabolism in
which glucose is under-utilised producing hyperglycaemia and preventing the direct
production of energy from glucose. This one disease alone is responsible for a majority of
the morbidity in today’s world. Its incidence in india is approaching 30% and it has been
predicted to reach colossal proportion in the next decade.
 There are two major classes of DM-type 1 and type 2(IDDM) present with sudden onset of
symptoms(polyurea, polydypsia and loss of weight) usually in early childhood. Type 2 or
non insulin dependent DM(NIDDM) usually occurs later in age(middle age or older) and is
characterised by an insidious onset of symptoms. The maximum morbidity of DM lies in its
complications; renal failure, neuropathy, stroke, gangerine or coronary disease.
 Hyperglycemia is diagnostic of this condition, i.e., DM. Depending on the time of blood
glucose estimation with respect to intake of food or glucose, various criteria have been set
down for diagnosis of DM by the American Diabetes Association. The three most
important factors to be noted are:
 glycoseurea
 maximum blood values of glucose.
 rate at which glucose returns to fasting level
 In the GTT, most population have plasma glucose value that exhibit a unimodal
distributions,i.e., distribution curve that is skewed to the high end but becomes bell shaped
on a logarithmic axis.

Abnormalities of the GTT.

 Impaired Glucose Tolerance is diagnosed in individuals who have fasting blood glucose
levels than those required for a diagnosis of DM but have a plasma glucose response during
the oral glucose tolerance test between normal and diabetic.
 Diminished Glucose Tolerance is where risen glucose level is normal but return fasting
level is delayed. This is seen in:
 diabetes.
 hyperactivity of thyroid, pituitary or adrenals.
 glycogen storage disease of the liver(type 1,3,4)
 Increased Glucose Tolerance
 is seen in
 steatorrhoea
 coeliac disease, partial gastrectomy
 Lowered Renal Threshold (Renal Glycosuria)
 is the term used when glycoseurea is observed after meal whereas the GTT curve is
normal. This occurs due to a defect at the level of tubular reabsorption.
 In later stages of pregnancy, the patient may present with gestational diabetes mellitus
which is carbohydrate intolerance of variable severity with onset or first recognition during
pregnancy.
 Raised Renal Threshold.
 If GFR is reduced, a higher concentration of glucose in plasma maybe reached before the
absorptive capacities of the tubules is saturated. As a result, a blood glucose of over 200
mg/dL may not produce glycoseurea. This occurs:
 with increasing age.
 diabetic nephropathy: In this, the return to normal occurs within expected time, but the
peak is higher than the threshold causing transient glycoseurea. This is due to a higher rate
of glucose absorption from the intestine following rapid emptying of the stomach
 Potential abnormality of glucose tolerance : This is statistical risk class including person at
increased risk for the development of DM- e.g. an identical twin or first degree relative of a
patient with NIDMM. The purpose of identifying this category is for preventive measures
to be taken by these i ndividuals.

Factors Affecting GTT

 Starvation/ ingestion of high fat diet

 Illness
 Drugs
 Pregnancy
 Endocrine disorders
 Non Endocrine Disorders (Leukemia)
 Obesity

Biological Reference Intervals

 Plasma Glucose (fasting) = 70-100 mg/dl
 Plasma Glucose (2hr.PP) =90-140 mg/dl
 Plasma Glucose (Random) =70-160mg/dl
 Plasma Glucose (Spot Test)=70-140mg/dl
 KIDNEY FUNCTION TEST

Blood urea nitrogen (BUN)

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.
Its is synthesis from amino nitrogen derived ammonia which is carried out exclusively by
hepatic enzymes of the urea cycle. Therefore its production is de3pendent on diet as well as these
hepatic enzymes.
Over 90% urea is excreted through the Kidneys, and most of the remaining is lost through the
gastrointestinal tract and skin. This is because urea is neither actively reabsorbed nor secreted by
tubules but is filtered by the Glomeruli. The back diffusion of urea is dependent on urine flow
rate, so that in states like Chronic Renal Failure where there is osmotic dieresis in the functional
nephrons, the urinary urea clearance is high

Methods
The various methods of estimating urea/urea nitrogen are:
 Urease nesselerisation method of Slyke: The blood is deproteinised with isotonic sodium
sulphur and sodium tungstate. The supernatant is treated with urease, and the ammonium
carbonate released is reacted by nesseler’s reagent with resultant formation of a coloured
product. The disadvantage is that the production of color may obeys Bear’s law
 Berthelot reaction: Ammonia (released from hydrolysis of urea by urease) is reacted with
phenol and sodium hypochlorite to form a blue indophenol dye
 Diacetal Monoxime (DAM): Oxidated blood is deproteines by sodium tungstate and
sulphuric acid. The supernatant is treated with diacetal Monoxime and sulphuric
phosphoric acid. “The Fearan Reaction produces a yellow color which is not stable for
prolonged periods
 Urease Hydrolysis: Where the need for deproteinization has been obviated by selection of
small samples volumes and optimization of the reagent system.
 Principle
Urea is hydrolyzed in the presence of water and urease to produce ammonia and carbon
dioxide. The ammonia produces in the first reaction combines with alpha-oxoglutrate
andNADH in the presence of glutamate dehydrogenase (GLDH) to yield glutamate and
NAD. The rate of oxidation of NADH which is measured as the rate of decrease in
absorbance at 340nm, is proportional to the BUN concentration.

Analytical Range: 0-300 mg/dL.

Analytical Sensitivity: 0.02 mg/dL.
Sample: 2ml blood in plain vial.

Equipment: LX-20 Pro

Reagent:
 IL Urea Nitrogen
 Ready to use kit containing
1. Alpha-oxoglutarate
2. ADP
3. Urease
4. Glutamate dehydrogenase
5. BUN-NADH
6. NADH
7. Tris buffer
8. Preservatives.

Safety Precautions:

 Working reagent should be clear and colorless.

 Avoid eye contact with skin and eyes.
Calibration:

Multical from Beckman is used to calibrate the reagent. Calibration is valid for 14 days but due
to high turnover, fresh reagent is used everyday and calibration also performed everyday.

Quality control:
Pooled sera as well as control sera of different levels are estimated everyday and quality of
results assessed.
 Creatinine Test
A creatinine blood test measures the level of creatinine in the blood. Creatinine is a waste
product that forms when creatine breaks down. Creatine is found in your muscle. Creatinine
levels in the blood can provide your doctor with information about how well your kidneys are
working.

The kidneys are a pair of fist-sized organs located at the bottom of the rib cage. One kidney is on
each side of the spine. Each kidney has millions of small blood-filtering units called nephrons.
The nephrons constantly filter blood through a very tiny cluster of blood vessels known as
glomeruli. These structures filter waste products, excess water, and other impurities out of the
blood. The toxins are stored in the bladder and then removed during urination.

Creatinine is one of the substances that your kidneys normally eliminate from the body. Doctors
measure the level of creatinine in the blood to check kidney function. High levels of creatinine
may indicate that your kidney is damaged and not working properly.

Creatinine blood tests are usually performed along with several other laboratory tests, including
a blood urea nitrogen (BUN) test and a basic metabolic panel (BMP) or comprehensive
metabolic panel (CMP). These tests are done during routine physical exams to help diagnose
certain diseases and to check for any problems with your kidney function.

Principle:

The method is based on the Jaffe reaction. Creatinine reacts with picrate ion formed in alkaline
medium to develop a red-orange colour. The colour produced from the sample is then compared
in a colorimeter at wavelength of 505 nm with that produced by a known amount of creatinine
under the same condition.

The Jaffe reaction is known to be nonspecific when applied to the measurement of creatinine in
plasma. Many compounds, including protein, glucose,ascorbic acid, acetone, and keto acids can
also form the Jaffe reaction and cause overestimation of the creatinine in plasma.
Deproteinisation is an important step in creatinine determination of plasma.
Analytical Range: 0-25 mg/dL.

Analytical Sensitivity: 0.1 mg/dL.

Sample: 3ml of blood in a plain vial of the close sample. Urine sample should be collected in
toluene ; sample in 20% HCL is also accepted.

For CCR= 12 or 24 hour urine sample is obtained along with a blood sample collected in a plain
vial sometime within the period of urine collection.

Calculations:

CCR = UV/P, where

U is the urinary creatinine concentration

V is the volume of urine excreted per minute

P is the plasma/serum creatinine

 CX-5 pro

Reagent: Beckman CR-T , Picric Acid, Buffered to pH>13.3, Antifoam.

SAFETY PRECAUTIONS :
Consider all plasma or serum specimens as potentially positive for infectious agents including
HIV and the hepatitis B virus. We recommend the hepatitis B vaccination series for all analysts
working with whole blood and/or plasma. Observe universal precautions; wear protective gloves,
laboratory coats. Place disposable plastic, glass, and paper (pipette tips, gloves, etc.) that contact
plasma and any residual sample material in a biohazard bag and keep these bags in appropriate
containers until disposal by maceration chlorination. Wipe down all work surfaces with Sani-
Cloth HB, Germicidal Disposable Wipe when work is finished. Handle acids and bases with
extreme care; they are caustic and toxic. Handle organic solvents only in a well-ventilated area
or, as required, under a chemical fume hood.

Calibration:
Multical from Beckman is used to calibrate the reagent. Calibration is valid for 14 days but due
to high turnover, fresh reagent is used everyday and calibration also performed everyday.
 Quality control:
Pooled sera as well as control sera of different levels are estimated everyday and quality of
results assessed.

Technique:
 One drop of antifoam is added to reagent compartment A of the creatinine reagent
cartridge. It is mixed by inverting gently few times.
 The reagent is then calibrated using multical as the calibrator.
 The serum is prepared and the instrument programmed.
 The system automatically proportions the sample and reagent volumes into cuvette. The
system monitors the change in absorbance at 520nm. This change is directly proportional
to the concentration of cratinine in the sample.
 The system perform all calculations internally to produce the final result, which is
expressed as mg of creatinine/dL.
 For diluted samples, the dilution factor has to be fed along the programming to obtain the
final result.

Clinical Significance:

Its major clinical utility lies in the diagnosis of renal disease and, in conjugation with BUN, it
differentiate between pre- renal and post renal azotemia. In PRE-RENAL azotemia, BUN
increases without a concomitant increase in serum creatinine, in RENAL azotemia, increase in
BUN and increase in creatinine go hand in hand i.e. it is proportionate; in POST RENAL
azotemia there is an increase in BUN exceeds the increase in creatinine.

Unfortunately, it is nota very early indicator of renal disease as a 50% loss of renal required to
increase the serum creatinine from 0.1 mg/dL to 2.0 mg/dL.

Other conditions causing an increase in serum craetinine are:

 Diet(increased meat intake)

  Hormonal imbalance as in : ( Gigantism, Acromegaly).
Urinary Creatinine Increases in :

1. Physiologic States:
1.1. Childhood growth
1.2. Pregnancy
1.3. Starvation
1.4. Raw meat diet
2. Increased Formation
2.1. Myopathy, as in (Muscular dystrophy, Crush injury etc)
3. Endocrine Diseases, e.g.
3.1. Diabetes mellitus
3.2. Addison’s disease
4. Increased breakdown as in
4.1. Infections
4.2. Burns
4.3. Leukemia
Biological References Interval:

 Serum creatinine = 0.6-1.3mg/dL

 Urine Creatinine (random)= 6-424mg/dL
 Urine Creatinine (24hr) =600-2000mg/dL
 Amniotic fluid creatinine =2.1-2.5mg/dL
 CCR Male =70-110ml/mt.
Female =80-120ml/mt.
 Uric Acid Test

Higher than normal uric acid levels in the blood is called hyperuricemia and can be caused by the
over-production of uric acid in the body or the inability of the kidneys to adequately remove
enough uric acid from the body. Further investigation is needed to determine the cause of the
overproduction or decreased excretion of uric acid.

There are several genetic inborn errors that effect purine metabolism. Metastatic cancer, multiple
myeloma, leukemias, and cancer chemotherapy can cause increased production of uric
acid. Chronic renal disease, acidosis, toxemia of pregnancy, and alcoholism can cause decreased
excretion.
Increased concentrations of uric acid can cause crystals to form in the joints, which can lead to
the joint inflammation and pain characteristic of gout. Uric acid can also form crystals or kidney
stones that can damage the kidneys.

The American College of Rheumatology published guidelines on the management of gout in

2012 that recommend that target serum urate (uric acid) levels should be below 6 mg/dL for
people diagnosed with the condition.

Low levels of uric acid in the blood are seen much less commonly than high levels and are
seldom considered cause for concern. Although low values can be associated with some kinds
of liver or kidney diseases, Fanconi syndrome, exposure to toxic compounds, and rarely as the
result of an inherited metabolic defect (Wilson disease), these conditions are typically identified
by other tests and symptoms and not by an isolated low uric acid result.
Urine
High uric acid levels in the urine are seen with gout, multiple myeloma, metastatic cancer,
leukemia, and a diet high in purines. Those at risk of kidney stones who have high uric acid
levels in their urine may be given medication to prevent stone formation.
Low urine uric acid levels may be seen with kidney disease, chronic alcohol use, and lead
poisoning.
 Method

Currently, two methods are widely utilized to quantify uric acid. A colorimetric method depends
on the reduction of a chromogen such as sodium tungstate by uric acid to produce a measurable
color change. This technique has been commonly employed in automated hospital screening
(SMA systems). The method measures materials other than urate, such as ascorbic acid.
Colorimetric determinations are generally considered an overestimation of true uric acid levels,
and the normal range is usually 1 mg/dl higher than the more specific enzymatic techniques.

Enzymatic determination of uric acid results from the specific oxidation of uric acid by uricase,
which converts its substrate to allantoin. The differential absorbance of these substances at 293
nm allows quantification.

Clinical Significance

Hyperuricemia

Hyperuricemia may be conveniently divided into two major categories. Symptomatic

hyperuricemia is manifested by gout, nephrolithiasis, and uric acid nephropathy. A larger group
of patients have asymptomatic hyperuricemia. Some of these patients will eventually become
symptomatic.

The risk of acute gouty arthritis increases with the level of serum uric acid and the duration of
hyperuricemia. Acute fluctuations in serum uric acid may be associated with the precipitation of
acute gouty arthritis. Sudden reductions in serum uric acid may accompany the introduction of
antihyperuricemic therapy; hence, these patients often simultaneously begin prophylactic doses
of colchicine.

Nephrolithiasis may accompany gouty arthropathy or occur as an independent problem. Uric

acid forms radiolucent stones or may contribute to the formation of calcium stones. In patients
with gout, the risk of stone formation rises with the level of serum uric acid. A better correlation
exists between stone formation and urinary uric acid excretion. One study found the prevalence
of stones to be 50% in gouty patients excreting greater than 1100 mg of uric acid per 24 hours.

Acute uric acid nephropathy results from the precipitation of uric acid crystals within the
collecting tubules and ureters. It is a severe form of acute renal failure and is classically
associated with the chemotherapy of leukemias and lymphomas. It may also occur following
strenuous exercise and epileptic seizures. Hyperuricosuria, aciduria, and urine concentration
seem to act in concert to produce this syndrome. The diagnosis can be made by the
demonstration of a uric acid to creatinine ratio greater than 1 in the setting of acute renal failure.

Routine screening of hospitalized patients will identify a substantial number with elevated serum
uric acid and no related symptoms. Most of these patients will remain asymptomatic throughout
their lives. A complete discussion of the management of asymptomatic hyperuricemia is beyond
the scope of this chapter. Suffice it to say that the weight of current evidence speaks against the
normalization of uric acid in asymptomatic patients. Regardless of the level of uric acid, little
seems to be lost by awaiting the onset of the first bout of arthritis or kidney stone.

Hypouricemia

Hypouricemia is commonly defined as a serum urate concentration of 2 mg/dl or less. A low

serum urate concentration may result from decreased production or increased excretion.
Quantification of urinary uric acid can facilitate a distinction between these two mechanisms.
Patients with hypouricemia secondary to impaired production will have little or no urinary uric
acid.

Hypouricemia can be found in about 1% of hospitalized patients. In most cases the cause is
related to drugs, including salicylates, allopurinol, x-ray contrast agents, and glyceryl
guaiacholate. Forced diuresis, used mainly in the treatment of suicide-attempt patients and renal
colic, may result in hypouricemia. Total parenteral nutrition can cause profound hypouricemia in
some patients.

Several malignant diseases have been associated with hypouricemia, including Hodgkin's
disease, sarcoma, glioblastoma, and a variety of carcinomas. In multiple myeloma, light chains
most likely cause tubular epithelial damage and Fanconi syndrome. Other malignancies have
also been associated with tubular dysfunction and increased renal clearance of urate. The
inappropriate secretion of antidiuretic hormone that accompanies some malignancies may lower
serum uric acid. Decreased xanthine oxidase activity, acquired after the development of a
malignancy, appears to be an uncommon mechanism.

Hypouricemia produces no symptoms or known morbidity. Its fortuitous discovery on automated

chemistry screening requires no therapy but should alert the physician to search for an
underlying cause.

Range

The reference ranges for uric acid are as follows:

 Men: 2.5-8 mg/dL
 Women: 1.9–7.5 mg/dL
 Table of content

1. Introduction
2. Specimen Collection and Processing
(a) Collection of Blood with Evacuated Tubes
(b) Collection of Arterial Blood
(c) Collection of Capillary Blood
(d) Anticoagulant and preservatives for Blood
(e) Collection of Urine
(f) Preservatives of urine
(g) Processing of Specimen for Investigation
(h) Sampling Guidlines
(i) Policy for Sample Processing
3. Glucose Test
a. Methods
b. Reference Methods
c. Sample
d. Precautions
e. Calibration
f. Quality Control
g. Clinical Significance
h. Abnormalities of the GTT
i. Factors effecting GTT
4. Blood Urea Nitrogen Test (BUN)
a. Methods
b. Principle
c. Calibration
d. Quality Test
5. Creatinine Test
 a. Principle
b. Calculation
c. Safety Precautions
d. Technique
e. Clinical Significance
6. Uric Acid Test
a. Method
b. Clinical Significance
c. Range
7. References
8. WPR-1
9. WPR-2
10. WPR-3
11. WPR-4
 Abstract

The glucose tolerance test is a medical test in which glucose is given and blood samples taken
afterward to determine how quickly it is cleared from the blood. The test is usually used to test
for diabetes, insulin resistance, impaired beta cell function, and sometimes reactive
hypoglycemia and acromegaly, or rarer disorders of carbohydrate metabolism. In the most
commonly performed version of the test, an oral glucose tolerance test (OGTT), a standard dose
of glucose is ingested by mouth and blood levels are checked two hours later. Many variations of
the GTT have been devised over the years for various purposes, with different standard doses of
glucose, different routes of administration, different intervals and durations of sampling, and
various substances measured in addition to blood glucose.

Renal function, in nephrology, is an indication of the state of the kidney and its role in renal
physiology. Glomerular filtration rate (GFR) describes the flow rate of filtered fluid through the
kidney. Creatinine clearance rate (CCr or CrCl) is the volume of blood plasma that is cleared
of creatinine per unit time and is a useful measure for approximating the GFR. Creatinine
clearance exceeds GFR due to creatinine secretion, which can be blocked by cimetidine. In
alternative fashion, overestimation by older serum creatinine methods resulted in an
underestimation of creatinine clearance, which provided a less biased estimate of GFR