UREA

Code No.25927 DAM METHOD

PRINCIPLE:
Urea reacts with hot acidic Diacetylmonoxime in presence of Thiosemicarbazide and a rose purple coloured complex ,which is measured calorimetrically.

ADVANTAGE:
1 Simple, rapid and one step method. 2 No need for deproteinization.

SAMPLE:
Serum or plasma [0.02ml is required] : [Do not anticoagulants containing ammonium salts] Urine: Dilute 1:20

REAGENTS:[Supplied in the kit]

Reagent 1: Urea reagent Reagent 2: Diacetylmonoxime (DAM) Reagent 3: Working urea standard , 30 mg%

PREPARATION OF WORKING SOLUTIONS:

Solution 1: Dilute 1ml of Reagent 1 to 5ml with distilled water. Reagent 2 and Reagent 3 (standard) are ready for use.

STORAGE AND STABILITY:

All reagents are stable at 2-8 C till the expiry date mentioned on the individual label. Solution 1 has to be prepared fresh.

PRECAUTIONS: 1 Use chromic acid and washed dry glassware. 2 Bring all the solutions to room temperature before use. 3 Prepare a standard for each series of determinations. 4 Mark the test tubes properly as Blank(B), Standard(S), and Test (T) before proceeding for the estimation , since markings may come off when the tubes are placed in a boiling water bath. PROCEDURE: A.FOR CALORIMETER: Blank(B) 5.0ml Test(T) 5.0ml 0.02ml Mix well Reagent 2: 0.50ml Diacetylmonoxime(DAM) 0.50ml o.50ml Standard(S) 5.0ml 0.02ml

Solution 1 Sample Reagent 3:working urea standard , 30 mg%

Mix well and keep the tubes in the boiling water exactly for 10mins.,mix by inversion and measures the colour intensity within 10min using a green filter against blank. B. FOR SPECTROPHOTOMETER: All the volumes mentioned under colorimeter procedure can be halved. Rest of the procedure remains unchanged. Measure the O.D. at 525nm. CALCULATIONS: Serum/plasma: Urea in mg/ = O.D. test * 30

O.D. test Urea Nitrogen in mg/100ml = (A) *0.467 Urine: Urea in g/l,(B) = O.D. test/O.D. test * 30*20/100 NORMAL VALUES: Serum : 20-40 mg urea(10-20mg urea nitrogen)/100ml Urine: 20g urea (10g urea nitrogen)/litre

NOTES: 1. In case micropipettes are not available ,use 0.2ml(for spectrophotometer) of sample and standard after diluting 1-10 with normal saline. Rest of the procedure and calculations remain unchanged. 2. If the values are more than 60mg urea/100ml serum or 60g urea /litre urine, repeat the estimation after during the serum 1-2 and urine 1-40 with normal saline and multiply the final result so obtained by 2.0 PRESENTATION: Kit containing sufficient reagents for 20 & 40 estimations: REAGENTS PACK SIZE 20 ESTIMATIONS CODE NO.25927-A 64 ml 32 ml 2 ml 40 ESTIMATIONS CODE NO.25927-B 125 ml 64 ml 4 ml

Reagent 1:Urea Reagent Reagent 2:Diacetylmonoxime Reagent 3: working Urea Standard, 30 mg%

URIC ACID CODE NO.25917 PHOSPHOTUNGSTATE METHOD

PRINCIPLE: Mixed in alkaline medium reduces phosphotungstatic tungsten blue coloured complex ,which is measured calorimetrically. ADVANTAGES: 1. 2. 3. 4. Simple and very popular method. The results are highly reproducible. Standard is specially stabilized. Highly economical.

SAMPLE: Serum (1.0 ml is required) REAGENTS :(Supplied in the kit) Reagent 1 : Sulphuric acid , 2/3N Reagent 2 : Sodium tungstate 10% W/V Reagents 3 : Sodium carbonate, 14% W/V Reagents 4 : Phosphotungstate Reagent 5 : stock uric acid standard ,100 mg% PREAPARATION OF WORKING SOLUTION Working standard : Dilute 0.5ml of stock uric standard to 50ml with distilled water and mix well. All the other reagents are ready for use. STORAGE & STABILITY: Reagent in box no. 1 * (reagents 4&5) are stable at 2-8 degree C till the expiry date mentioned on the individual label. Reagents in box no. 2 are stable indefinitely at room temperature Working standard has to be prepared fresh , everyday.

PRECAUTIONS: 1. 2. 3. 4. 5. Serum should be free from any haemolysis Use clean and dry glassware Bring all the solutions to room temperature before use Prepare a blank and a standard for each series of determinations Mark the test tubes properly as Blank(B),Standard(S), and Test(T) before proceeding for the estimation.

PROCEDURE: A. FOR COLORIMETER Step A. Deproteinization of the sample : Serum 1.0ml

Distilled water Reagent 1 : sulphuric acid,2/3N

8.0ml 0.5ml

Reagent 2 : sodium tungstate,10% W/V

0.5ml

Mix well and stand for 10mins centrifuge or filter through whatman no.1 filter paper.

Step B. Color development:

Mix well and stand in dark for 15mins and measure the O.D. of Blank(B),Standard(S), and Test(T) against distilled water on the colorimeter using red filter. B.For spectrophotometer: All the volumes mentioned under colorimeter procedure can behalved. Rest of the procedure remains unchanged measure the O.D. at 710nm. CALCULATIONS: Serum Uric acid in mg /100ml = O.D. test-O.D. test/O.D. test-O.D. blank *10 NORMAL VALUES: Men : 2.5-7.0mg/100ml

Women : 1.5-6.0mg/100ml NOTES: 1. Uric acid is stable for served days, if the serum is stored at 2-8 degree C , addition of thymol increases the stability. 2. If the O.D. of the test exceeds 0.8 repeat the estimation after diluting the serum properly with normal saline. Multiply the final result so obtained with the appropriate dilution factor.

PRESENTATION:

REAGENTS Reagent 1: Sulphuric acid ,2/3N Reagent 2 : Sodium tungstate,10% W/V Reagent 3 : Sodium Carbonate ,14% W/V Reagent 4 : Phosphotungstate Reagent 5 : Stock Uric Acid standard , 100mg%

PACKING 25ml 25ml 125ml 125ml 20ml

AMYLASE Code. No. 25908 Street and close method CLINICAL SIGNIFICANCE: Increased activity of serum Amylase is found in various forms of pancreatitis and carcinoma of pancreas. A variation in Urinary Amylase reflects alterations in serum Amylase so long as renal function is normal. In renal failure, the clearance of amylase is reduced and serum amylase activity is increased while urinary amylase excretion is normal. PRINCIPLE: The enzyme -Amylase degrades starch into reducing dextrin and smaller saccharine. The -Amylase is allowed to react under defined conditions. Iodine solution is added and the resulting blue colour of the test is compared with a control. The reactive decreases in blue colour of test are the measure of -Amylase activity. ADVANTAGES: 1. Simple, convenient and rapid method. 2. Amy lose, a more uniform compound is used as a substrate instead of starch. SAMPLE: Serum and /urine In case of serum, dilute it 1 to 10 with normal saline. In case of urine , a 24hr urine specimen is preferred. Collect 24hr urine specimen and measures its volume. Dilute 1 to 100 with normal saline. REAGENTS :( Supplied in the kit) Reagent 1: Buffered substrate Reagent 2: Stock Iodine solution Auxiliary: Normal saline PREPARATION OF WORKING SOLUTION: Working Iodine Solution: Just before use, dilute 0.2ml of reagent 2 to 2ml with distilled water. Reagent 1 is ready for use.

STORAGE & STABILITY: Both reagents (1&2) are stable at R.T. till expiry date mentioned on the individual label. Once opened, transfer reagent 2 to the empty brown glass vial supplied in the kit and store at 2-8 degree C. PRECAUTIONS: Since saliva contains very large amount of amylase, pipetting by mouth should be avoided. Use rubber bulb for pipetting. PROCEDURE: A. For colorimeter: .. Control(C) Reagent 1 : Buffered Substrate 1.0ml Test(T) 1.0ml

Incubate at 37 degree C for 3 min Serum /Urine (Diluted) 0.1ml Mix well and incubate at 37 degree for 15min

Distilled water Working Iodine Solution

8.6ml 0.4ml

8.5ml 0.4ml

Mix well and measure the optical density of control (C) and Test (T) using a red filter against distilled water. B.For Spectrophotometer: All the volumes mentioned under the colorimeter procedure can be halved. Rest of the procedure remains unchanged. Measure the O.D. at 620nm.

CALCULATIONS: Amylase activity Serum : O.D control O.D. test/O.D. control * 100 Urine : Street Close Units /100ml Street-Close Units/24hrs 1 street & close unit= 3.3 Simonyi units = 0.61 International Units NORMAL VALUES: Serum: 9-50 street-Close units /100ml Urine: 23-380 Street-close units /100ml or 370-3000 Street-close units /24hrs. NOTES: 1. In order to achieve the stated linearity, note that the absorbance of control(C) at 620nm. Will read 0.8+/-0.05. 2. If the values are more than 80 street-close units/100ml.repeat the estimation using more diluted sample, or by shortening the incubation period to 5 min. In either case, the result so obtained should be multiplied by an appropriate factor. PRESENTATION: Kits containing sufficient reagents for 10 estimations: = O.D control O.D. test /O.D. control*10*24hrs urine volume in ml = O.D control-O.D. test/O.D. control *1000

Reagents 0

CHLORIDE METHOD: Thiocyanate. Endpoint Colorimetric. Single Reagent Chemistry. PRINCIPLE: Chloride ions reacts with a solution containing ferric ,mercuric , nitrate and thiocyanate ions in equilibrium to form yellow-brown ferric thiocyanate. Absorbance measured at 505nm is proportional to the concentration of chloride in the specimen. 6 cl2 + 2 Fe2 + 3 Hg(SCN)3 REAGENTS: Reagent 1 Chloride Reagent B 05111 1*100ml Mercuric nitrate, Nitric acid, Ferric Nitrate, Thiocyanate. 3 HgCl2 + 2 Fe(SCN)

Reagent 2 Chloride Standard 100 mEq/L B 05112 1*3.0ml Hydrochloric acid, Diluents.

PREPARATION OF WORKING REAGENTS: Both Reagent 1 ,and Reagent 2 are ready for use as supplied. REAGENT STABILITY: Reagent 1 is stable at room temperature (15-30 degree C) until the expiry date printed on the container label. Reagent 2, Chloride Standard is stable at 2-8 degree C until the expiry date printed on the container label. It is suggested that, upon opening the kit ,the Chloride Standard should be refrigerated immediately. SPECIMEN: Serum or plasma collected in heparin and free from haemolysis, or CSF. Specimen is stable for 7days at 2-8 degree C. Separate serum or plasma with minimum delay. Bring specimen to room temperature (15-25 degree C ) before as-saying. Anticoagulants such as sodium fluoride and potassium EDTA cause negative errors in the assay.

Bromide, haemoglobin and iodine in concentrations exceeding 9mEq/L, 250 mg/dL, and 1.5 mEq/L respectively cause positive errors in the assay. For assay on urine, collect a 24hr specimen. Dilute the specimen two times with deionised water. Multiply results by 2. EQUIPMENT: Auto span reagents for chloride estimation are suitable for use on all automated and semi automated analyzers and on colorimeters having a 490-530 nm filter. PROGRAMME: Method sheets for specific analyzers are available on request. The basic assay parameters are : Mode Wavelength Temperature Optical path length Blanking Sample volume Reagent volume Concentration of standard `Incubation Maximum absorbance limit Linearity Stability of colour Units End point 505nm (490-530n,) Room temperature 1 cm Reagent Blank 10l or 5l 1000l or 500l 100 mEq/L 3min 2.0 70-140 mEq/L 30 min mEq/L

NOTE: 1. The sample volume and the reagent volume may be halved in case of analyzers having a cuvette or flow-cell capacity of 500 l. 2. For samples containing less than 70mEq/L of chloride repeat the test using double the recommended volume of sample. Divide results by 2. 3. For samples containing more than 140 mEq/L Chloride, dilute the sample 1:2 with distilled or deionised water. Multiply results of the diluted sample by 2. 4. Do not use normal saline for diluting the sample.

PROCEDURE: MICRO METHOD Pipette into tubes marked Blank Standard Serum,plasma,urine Chloride standard 5l Chloride reagent 0.5ml o.5ml mix well.incubate at room temperature or at 37 degree C for 3 minutes. MACRO METHOD Pipette into tubes marked Blank Standard Serum,plasma,urine Chloride standard 10l Chloride reagent 1.0ml 1.0ml Mix well.incubate at room temperature or at 37 degree C for 3 minutes. 2.Programme the analyzer using parameters given above. 3.Blank the analyzer with the reagent blank. 4.Aspireate standard. 5.Aspirate tests. METHOD FOR 3 ml CUVETTE Pipette into tubes marked Blank Standard Test Serum,plasma,urine 30l Chloride standard 30l Chloride reagent 3.0ml 3.0ml 3.0ml Mix well.incubate at room temperature or at 37 degree C for 3 minutes.read absorbance at 505 nm against blank. CALCULATION Serum chloride {mEq/L} =absorbance of test/abrobance of standard *100 REFERENCE RANGE Serum CSF Urine : : : 97 - 107mEq/L 115 - 132mEq/L 170 - 250 mEq/24hours Test 10l 1.0ml Test 5l 0.5ml

CLINICAL SIGNIFICANCE Serum or plasma:elevated levels of chloride are associated with dehydration and congestive cardiac failure. Conditions such as salt-losing nephritis,diabetic acidosis and renal failure on the other hand,are associated with iow chloride levels. CSF:CSF chloride concentrations decrease in tuberculous meningitis,but remain normal in viral meningitis,and in almost all other neurological states. An increase may be found in hyper chloremic acidosis of chornic renal failure. NOTES 1.rinse all equipment with deionsed water to remove all traces of chloride.avoid use of tap water for cleaning equipment,as tap water may contain chloride in substantial quantities. 2.the reagent contains a potentially harmful mercuric compound.avoid ingestion.avoid contact with eyes,skin and clothing.wash thoroughly after handling. 3.it is recommended that each laboratory establish its own references range among its patient population.

ALKALINE PHOSPHATASE METHOD: PNPP Kinetic , spectrophotometry. Single Reagent Chemistry. PRINCIPLE: At pH 10.30 alkaline phosphatise catalyses the hydrolysis of p-Nitrophenol and phosphate. Change in absorbance measured at 405nm is directly proportional to enzyme activity. REAGENTS: Reagent 1 AMP Buffer B 18111 B 18121 Reagent 2 1*24ml 1*50ml AMP Buffer,pH 10.30 , Magnesium Acetate, Zinc sulphate Chelator PNPP , Stabilizer

PNPP Substrate B 18112 B 18122 20*1.2ml 10*5ml

WORKING REAGENT PREPARATION: Reconstitute 1 vial of reagent 2, PNPP Substrate ,with the volumes of reagent 1 , AMP Buffer as indicated below : For 10*5ml Pack : Use 5ml of reagent 1 For 20*1.2ml Pack : Use 1.2ml of Reagent 1 REAGENT STORAGE AND STABILITY: Unopened reagents are stable at 2-8 degree C until the expiry date printed on the container labels. The working ALP Reagent is stable for 7days at 2-8 degree C and for 2days at room temperature , when protected from light. SPECIMEN: Serum , unhemolysed ,ALP in serum is stable for 24hrs at 2-8 degree C and for several months when stored frozen at -20degree C. Less than 10% activity is lost in 2-3 days at 2-8 degree C.

EQUIPMENT: AUTOSPAN Reagents for ALP estimation are suitable for use on all automated analyzers. PROGRAMME: Method sheets for specific analyzers are available on request. The basic assay parameters are: Method Wavelength Temperature Optical path length Blanking Sample volume Working reagent volume Delay Interval Number of readings Maximum absorbance limit Kinetic factor Linearity Units Kinetic 405nm 25,30/37 degree C 1cm Water 20l 1000l 30 sec 30 sec 4 2.0 2712 Upto 600IU/L IU/L

NOTE: 1. The sample volume and the working ALP reagent volume may be halved in case of analyzers having a cuvette or flow-cell capacity of 500l. 2. For best results , bring samples and reagents to the selected assay temperature before use. Maintain constant temperature throughout the assay. 3. In case ALP activity in the sample exceeds 600 IU/L , dilute the sample suitably with normal saline. Multiply results obtained by the appropriate dilution factor. 4. In case the Multitest packs are being used,protect the working ALP Reagent form strong light and refrigerate unused reagent with minimum delay. PROCEDURE: Pipette into tubes marked Blank Deionized water 1.0ml Working ALP Reagent Serum Mix well and aspirate Blank followed by tests. Test 1.0ml 20l

CALCULATIONS: ALP activity = Absorbance /minute *2712 IU/L Where Absorbance/minute represents the change in absorbance per minute and 2712 is the kinetic factor. NOTE: The kinetic Factor K will change if the sample volume ,working reagent volume or the optical path length are altered. The following formula is useful in calculating the kinetic factor K . K= TV/SV * 1/P * 1/M * 10