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DOCTOR'S SECTION » Diagnostic Technical Information

WIDAL SLIDE TEST KITS
For semi-quantitative, In-Vitro determination of specific antibodies present in serum against Salmonella typhi ‘O’ & ‘H’ and S. paratyphi A(H) & B(H) antigens.

Principle:
Killed salmonella suspensions displaying the somatic ‘O’ and flagellar ‘H’ antigens agglutinate with the antibodies present in the serum of patients exposed to these organisms.

Contents, reagents and material provided:

1.	5ml S. Typhi ‘O’ antigen suspension

2.	5ml S. Typhi ‘H’ antigen suspension

3.	5ml S. Paratyphi ‘A’(H) Ag.suspension

4.	5ml S. Paratyphi ‘B’(H) Ag.suspension

5.	1ml Positive Control serum

6.	Glass slide 1 No.

Mix the reagents gently before use.

Stability:
The reagents are stable upto expiry date when stored at 2-8°C.
Do not freeze.

Specimen:
Clear fresh serum sample is required.
However, the specimen may be stored at 2-8°C for upto 48 hrs. and at -20°C for 72 hours.
Do not heat or inactivate serum sample.

Supplementary material required but not provided:
10 x 75 mm test tubes
1ml pipette with 0.05ml graduations
0.1 ml pipette with 0.01 / ml graduations

Pipetting scheme:

A. Qualitative analysis (Slide test):

Bring the reagents and specimen to room temperature.
Mix the antigen suspension gently prior to use.

Drop/pipette on to separate cells of the slide
Serum sample	1 drop
Positive Control serum	1 drop
Antigen suspension O, H, A(H), B(H) to respective cells	1 drop
Antigen suspension on Control cell ‘O’	1 drop
Antigen suspension-any of the 'H', on the Control cell ‘H’	1 drop

Mix the contents of each cell, spreading the reagent mixture over the entire area of the cell.
Tilt the slide back and forth to ensure thorough mixing.
Observe results at the end of 1 minute under high intensity light.

B. Semi-quantitative analysis (Slide test):

To a clear glass slide add as follows:

Cells	Serum Volume	Antigen Volume	Corresponding titre
1.	0.08 ml	1 drop	1:20
2.	0.04 ml	1 drop	1:40
3.	0.02 ml	1 drop	1:80
4.	0.01 ml	1 drop	1:160
5.	0.005 ml	1 drop	1:320

Mix for 1 minute and observe for agglutination.

Interpretation of results:
Read the results under high intensity light. Regardless the degree of reactivity and test result showing slight but definite agglutination is reported as reactive or positive. Complete absence of agglutination and a clear suspension indicates non-reactive or negative result.

Quality control-Good Laboratory Practice:
The positive control may be used for routine performance check.

Diagnostic Value:
Salmonella are motile organisms with ‘O’ (somatic) & ‘H’ (flagellar) antigens. ‘O’ antigens of many species have many antigenic components in common and hence the only one antigen is species specific. S.paratyphi A & B are commonly encountered in our country, hence are employed in the tests. Agglutination appears at the beginning of the 2nd week, reach a maximum during the 3rd week and may persist for weeks or months after convalescence. Cross agglutinations with the ‘O’ suspensions of S.paratyphi ‘A’ & S.paratyphi ‘B’ often take place due to possession of some common epitopes of the somatic antigens.

The titre over 1:120 with either ‘H’ or ‘O’ suspension is practically considered significant.

In case of S.paratyphi A & S.paratyphi B infections, the ‘H’ agglutination are more marked and high titres are usually recorded.

Precautions:

1.	Allow all reagents to attain room temperature before use.

2.	Discard hemolyzed or contaminated sample.

3.	Use clean and dry glassware.

4.	Shake antigen vials well before use, to make a homogenous suspension.

5.	The test card must be rocked gently.

6.	Improper mixing of the sample with the antigen suspension may lead to erroneous results.

7.	All reagents of human source have been tested for HBsAg and anti-HIV antibodies and proved to be negative. However, inspite of negative results, all reagents should be treated as potentially infections.

References:
Cruickshank, R.(1965), Medical Microbiology, 11th Edit., P.907
Felix, A.(1942), brit. Med. J., 11,597
Huddleson, I.F. and Abell, e.(1928),J. Infect. Dis. 42,242
Protell, R.L., et al., (1971), Lancet, 11,330.

VDRL QUIK-RPR TEST KITS
Rapid non-treponemal test for qualitative & semi-quantitative in-vitro determination of Reagin antibodies in serum or plasma for Syphilis.

Pack size:
100 Tests, Complete test kit

Principle:
The antigen used is a modified Cardiolipin antigen in which micro particulate carbon particles are suspended, in addition, it contains a balanced quantity of Cholesterol and Lecithin. In the presence of Reagin antibodies, clumps or aggregates appear which can be visualized as black clumps against white background of the slide, macroscopically.

Contents, reagents and material provided:

1.	Carbon / RPR antigen suspension for 2 x 50 Tests. Mix gently before use. Microparticulate smooth-sieved carbon particles suspended with cardiolipin and with a balanced quantity of cholesterol and lecithin.

2.	0.5 ml, Positive Control Serum. Stabilized Serum Control, reactive with RPR antigen suspension.

3.	0.5 ml, Negative Control Serum. Stabilized Serum Control, non-reactive with RPR antigen suspension.

4.	13, Disposable slides with 8 cells.

5.	100, Disposable applicator sticks.

6.	100, Disposable plastic droppers with 2 rubber teats.

7.	2, Plastic antigen delivery dropper with rubber teat.

Stability:
The reagents are stable upto expiry date printed on the labels when stored at 2-8°C. Do not freeze.

Specimen:
Use fresh Serum or Plasma. The specimen should be non-haemolysed and free from contamination.

The specimen may be stored at 2-8°C for upto 5 days and at –20°C for upto 4 weeks.
(Use EDTA, Heparin or Oxalate as anticoagulant)

Pipetting scheme:

A. Qualitative analysis:

Bring the reagents and specimens to room temperature.
Mix the antigen suspension thoroughly prior to use.

Drop / pipette onto separate cells of the slide:
Specimen	1drop
Positive Control Serum	1drop
Negative Control Serum	1drop
RPR antigen suspension ( with antigen delivery dropper provided on to all cells in use)	1 drop (15-20µl) each

Mix with separate sticks and spread the fluid over the entire area of the particular cell.
Tilt the test card back and forth slowly for 6 minutes or place the card on an automated rotator and rotate at 100 rpm for 6 minutes.

Interpretation of results:
Read the test results under strong source of light. Regardless the degree of reactivity, clumping of Carbon particles is reported as reactive or positive. Complete absence of black aggregates and presence of a uniform greyish suspension indicates non-reactive or negative result.

B. Semiquantitative analysis:

Prepare dilutions of the specimen to be tested with physiological saline (0.9%) as indicated:
1:2, 1:4, 1:18, 1:16, 1:32
Proceed as in qualitative analysis.
The titre is reported as the reciprocal of the highest dilution which shows a positive result.

Quality Control – Good Laboratory Practice:
The positive and negative controls may be used for routine performance check.

Diagnostic values:
Syphilis is a sexually transmitted disease caused usually by direct but sometimes by indirect contact. The causative organism Treponema palladium can gain entrance to the body through minute lesions on the skin or mucous membrane.
Infection is usually rendered evident by the development of a primary lesion or chancre. This appears within a month of infection, and is accompanied by enlargement of the local lymphatic nodes.
From 6 to 12 weeks after the appearance of the primary chancre, the secondary stage of the disease sets in, marked by constitutional symptoms, cutaneous lesions, sometimes infection of the bones, joints, eyes and other organs. Thereafter, the disease becomes latent and can be detected by serological tests.

Certain non-syphilitic conditions which causes tissue damage can give false positive results. Common conditions like malaria, typhoid, leprosy, tuberculosis, certain viral diseases like viral pneumonia, infectious mononucleosis, pregnancy and some autoimmune disorders can cause positive reactions in low titre to appear.
Positive sample should be confirmed with specific tests like TPHA, FTA, etc.

Precautions:

1.	Kit reagents are for in-vitro diagnostic use only.

2.	Strictly follow the instructions mentioned in the Product insert.

3.	Bring specimens and reagents to room temperature before use.

4.	Don’t use lipaemic, haemolysed or contaminated specimens.

5.	Glassware used for specimen collection and test should be detergent free and dry before use.

6.	Antigen suspension should be mixed gently before use.

7.	While dispensing reagents/specimens hold pipette/dropper vertically straight.

8.	Improper mixing of specimen/control with the antigen suspension may lead to erroneous results.

9.	Make sure that the cap of each reagent vials are properly and promptly applied to the same vial. Interchanging of the vial caps and/or droppers will lead to contamination of reagents which might lead to false results.

10.	The slide should be titled back and forth gently to avoid disturbance to the reaction pattern.

11.	Drying of the test mixture at the periphery of the cell may lead to erroneous interpretation of results.

12.	Interpret results exactly at 6 minutes.

13.	The reagents contain sodium azide as preservative. Do not swallow. Avoid contact with skin & mucous membrane.

14.	All reagents of human source have been tested negative for HBsAg & anti-HIV antibodies. However, the same should be treated as potentially infectious.

References:
Portnoy, J., Brewer, J.H., Harris, A.D. (1962). Pub. Health Rep.,77,645-652
Portnoy, J. (1963). Amer. J. Clin Path., 40,473-479.
Falcone, V.H., Stout, G.V.Moore, M.B. (1964). Pub. Health Rep., 79,491-495.
Scotti, A.G., Mackey, D.M., Trautman, J.R. (1970). Arch. Derm.,101,328-330.
Walker, A.N. (1971). Brit. J. Vener. Dis., 47,259-262.
Dorwart, B.B., Myers, A.R. (1974). Brit. J. Ver. Dis., 50,435-436.
Kaufman, R.E., Weiss, S., Moore, J.D., Falcone, V., Wiesner,
P.J. Vener. Dis., 50,530-353.

ANTI-A, ANTI-B AND ANTI-AB BLOOD GROUPING REAGENTS

Slide and Saline tube test

Intended Use:
Monoclonal Anti-A, Monoclonal Anti-B, Monoclonal Anti-AB, are designed for in-vitro diagnostic and professional use only. They are intended for Human Red Blood Cell grouping.

Composition:
Monoclonal Anti-A, Monoclonal Anti-B, Monoclonal Anti-AB, are mouse monoclonal IgM antibodies raised against human blood group antigens A and B.

Source:
Monoclonal Anti-A, Monoclonal Anti-B, Monoclonal Anti-AB, are obtained by immunizing a Balb / C mouse with red cells of blood groups A and B, and fusing the splenocytes of that mouse with myeloma Sp2 / 0 cells.

Storage:
Monoclonal Anti-A, Monoclonal Anti-B, Monoclonal Anti-AB, should be well preserved within utility limit till the expiry date, if stored at 2-8°C. Do not freeze.

Principle:
The procedure used with these reagents are based on the principle of haemagglutination. Red blood cell antigens A, B or AB, when mixed with their respective antibodies, agglutinate. Phenotyping (grouping) of them is done by reacting the blood sample with Monoclonal Anti-A, Monoclonal Anti-B, Monoclonal Anti-AB. Presence of haemagglutination determines the group of the tested blood.

Specimen:
Properly stored anti-coagulated blood or 10% RBC-Saline suspension should be used.

Preparation of 10% RBC-Saline suspension
Steps :

1.	Add approximately 5 volumes of isotonic saline to the whole blood (Washing of RBC’s).

2.	Centrifuge for 2 minutes.

3.	Remove the supernatant and wash the sedimented RBC’s three more times with normal saline as above.

4.	After final wash, take 100µl of sedimented red cells, dilute to 1ml with saline and mix thoroughly before use.

Procedure:
1) Macroscopic slide test:
Bring the reagents and samples to room temperature.
Steps :

1.	Place 1 drop of Monoclonal Anti-A, Monoclonal Anti-B, Monoclonal Anti-AB, in separate areas on glass slide.

2.	Label the respective area as A, B & AB, and also with name or code number of the patient.

3.	Add 1 drop of whole blood sample or RBC-Saline suspension adjacent to each drop of Monoclonal Anti-A, Monoclonal Anti-B, Monoclonal Anti-AB reagents.

4.	Mix the reagent drop and the sample with an applicator stick and spread over an area of about 1 square inch within the circle.

5.	Gently tilt the slide forward and backward at room temperature for a maximum period of 2 minutes.

6.	Read the slides for haemagglutination. Do not interpret fibrin strands as agglutination.

2) Microscopic tube test (for enhanced sensitivity):
Use 8 x 50mm small glass test tubes.
Steps :

1.	For each specimen , take 4 tubes and label them with the name or code number of the patient. Also mark the tubes A, B, AB and saline.

2.	Add one drop of Monoclonal Anti-A, Monoclonal Anti-B, Monoclonal Anti-AB and saline to respective tubes.

3.	Add one drop of 10% RBC-Saline suspension to each tube.

4.	Shake each tube thoroughly and centrifuge for 1 minute at 1000 rpm at room temperature.

5.	Gently dislodge the sedimented cells and read for haemagglutination, either macroscopically or microscopically.

Interpretation:
Agglutination of red blood cells within two minutes indicates the presence of the corresponding antigens in the patient’s red blood cells. Absence of agglutination indicates the absence of such antigens on the red blood cells.

Agglutination results are interpreted as follows for phenotyping :

Red cells sample reacted with

Anti-A	Anti-B	Anti-AB	Saline	Result
+	-	+	-	'A' Group
-	+	+	-	'B' Group
+	+	+	-	'AB' Group
-	-	-	-	'O' Group
-	-	+	-	Weaker variants of ‘A’ / ‘B’ Grp
+	+	+	+	(*)

(*) Suggestive of auto-antibody in the blood giving a non-specific reaction. The entire test is to be repeated using 10% saline suspension of red cells.

Even though Monoclonal Anti-A and Monoclonal Anti-B, antiseras are sufficient for ABO phenotyping of blood, it is advisable to use Monoclonal Anti-AB sera also to rule out any doubt arising due to weaker variants of subgroups of A and B.
Run positive and negative test controls for each batch of blood grouping sera every time before proceeding with the actual test samples.

Precautions:

1.	The blood drop on the slide should not be allowed to dry, partial drying of the blood could be misinterpreted as agglutination.

2.	Centrifugation should be perfect. Over-Centrifugation or under Centrifugation may result in false negative interpretation.

3.	Dislodgement of sedimented red cells in tube test should be done as gently as possible, rough dislodgement may disrupt small or weak agglutinates, hence may lead to false negative interpretation.

4.	The entire procedure should be carried out at room temperature. Warm or cold antibodies in the tested blood can cause agglutination and may lead to wrong interpretation.

5.	Haemolysed blood samples should not be used.

6.	Improper antigen-antibody concentration may cause false or delayed agglutination.

7.	Coomb’s test should be carried out when necessary.

Presentation:
Ready to use vials/bottles containing antibody solution preserved in 0.1% sodium azide.

  REAGENT                                 PACK
Monoclonal Anti-A                      1 x 5 ml
                                              1 x 10 ml

Monoclonal Anti-B                      1 x 5 ml
                                              1 x 10 ml

Monoclonal Anti-AB                    1 x 5 ml
                                              1 x 10 ml

For further readings :

1.	Vox sanguinis, 1989, 56, 122.

2.	Bio test bulletin, 1998, 3, 177.

3.	WHO Expert committee on Biological standardization, Technical reports sera 1977, 610, WHO Geneva.

4.	American Association of blood banking technical Manual, 1990, 345.

ANTI-D BLOOD GROUPING REAGENT

Slide and Saline tube test

Intended Use:
Monoclonal Anti-D IgM is designed for in-vitro diagnostic and professional use only. It is intended for detection of Rhesus D antigen in Human Red Blood Cells.

Composition:
Monoclonal Anti-D IgM antibody.

Source:
Monoclonal Anti-D IgM is obtained by raising lymphoblastoid cell lines in, in-vitro culture from EBV transformed, antibody secreting Human B lymphocytes.

Storage:
Monoclonal Anti-D IgM will be well preserved within utility limit till the expiry date, if stored at 2-8°C. Do not freeze.

Principle:
The procedure used with this reagent is based on the principles of haemagglutination. Red blood cells with Rhesus D antigens agglutinate when mixed with anti-Rhesus (D) antibody. Phenotyping (grouping) of them is done by reacting the blood sample with Monoclonal Anti-D IgM. Presence of haemagglutination determines the positive Rhesus D antigen and the blood is categorized as Rh +ve.

Specimen:
Properly stored anti-coagulated blood or 10% RBC-Saline suspension should be used.

Preparation of 10% RBC-Saline suspension:

Steps :

1.	Add approximately 5 volumes of isotonic saline to the whole blood. (Washing of RBC's)

2.	Centrifuge for 2 minutes.

3.	Remove the supernatant and wash the sedimented RBC's three more times with normal saline as above.

4.	After final wash, take 100µl of sedimented red cells dilute to 1ml with saline and mix thoroughly before use.

Procedure :
1) Macroscopic slide test:
Bring the reagents and samples to room temperature.
Steps :

1.	Label the respective area as ‘D’ and also with name or code number of the patient.

2.	Place 1 drop of Monoclonal Anti-D IgM on a glass slide.

3.	Add 1 drop of whole blood sample or RBC-Saline suspension adjacent to each drop of the Monoclonal Anti-D reagent.

4.	Mix the reagent drop and the sample with an applicator stick and spread over an area of about 1 square inch within the circle.

5.	Gently tilt the slide forward and backward at room temperature for a maximum period of 2 minutes.

6.	Read the slides for haemagglutination. Do not interpret fibrin Strands as agglutination.

2) Microscopic tube test (for enhanced sensitivity):
Use 8 x 50mm small glass test tubes.
Steps :

1.	For each specimen, take a tube and label it with the name or code number of the patient. Also mark the tubes for D and saline.

2.	Add one drop of Anti-D IgM and saline to the respective tubes.

3.	Add one drop of 10% RBC-Saline suspension to each tube.

4.	Shake each tube thoroughly and centrifuge for 1 minute at 1000rpm at room temperature.

5.	Gently dislodge the sedimented cells and read for haemagglutination, either macroscopically or microscopically.

Interpretation:
Agglutination of red blood cells are interpreted as follows:
Rh D+ve: Red cells sample positive for haemagglutination with Monoclonal Anti-D IgM.
Rh D-ve : No agglutination of red cells with Monoclonal Anti-D IgM.

Note :

1.	If any doubt arises in the interpretation, the entire test should be repeated after thoroughly washing the red cells in saline and resuspending them before use.

2.	Monoclonal Anti-D IgM agglutinates Rh D+ve cells and most of the weaker sub types of Du antigens. A few of the Du antigens may however be negative for direct haemagglutinating reaction.

3.	To detect such weak variant of Du antigen, use a polyclonal anti-D sera or blend of polyclonal and monoclonal sera with IgG and IgM, by Coomb’s test procedure.

Precautions :

1.	Drop of blood on the slide should not be allowed to dry. Partial drying of the blood could be misinterpreted as agglutination.

2.	Centrifugation should be perfect. Over-Centrifugation or under-Centrifugation may result in false negative interpretation.

3.	Dislodgement of sedimented red cells in tube test should be done as gently as possible, rough dislodgement may disrupt small or weak agglutinates and hence may lead to false negative interpretation.

4.	The entire procedure should be carried out at room temperature. Warm or cold antibodies in the tested blood can cause agglutination and may lead to wrong interpretation.

5.	Haemolysed blood samples should not be used.

6.	Improper antigen-antibody concentration may cause false or delayed agglutination.

7.	Coomb’s test should be carried out whenever necessary.

Presentation :
Ready to use vials / bottles containing antibody solution preserved in 0.1% sodium azide.

Reagent:                              PACK
Monoclonal Anti-D IgM            1 x 5 ml
                                          1 x 10 ml

For further readings :
1. Race RR Sanger R : Blood groups in amn 6th ed. Oxford Blackwell Scientific 1975, 179.
2. Widmann fk ed.Technical manual 9th ed. Arlington VA: American Association of Blood Bank 1985, 130

CHEK-AIDS HIV 1+2 RAPID CARD TESTS

INTRODUCTION
Human Immunodeficiency Virus type-1 (HIV-1) and type-2 (HIV-2) are the etiological agents of Acquired Immunodeficiency Syndrome (AIDS). Current data indicate that the HIV is transmitted through sexual contact, exposure to blood (including sharing of contaminated needles and syringes) or certain blood products or from an infected mother to her child during the perinatal period. People with increased risk of HIV infection includes intravenous drug users, homosexuals and haemophilics. The presence of antibodies to HIV-1 / HIV-2 indicates previous exposures to HIV-1 / HIV-2 virus.

Chek AIDS HIV 1+2 RAPID CARD TEST is an indigenously developed rapid test device used for the detection of HIV-1/2 antibodies in human serum/plasma. This is only a screening test for HIV-1 & 2 antibodies. If the sample gives a positive result, confirmatory tests such as Western Blot, P C R, should be performed.

Principle:
HIV Recombinant Protein antigens gp41, C terminal of gp120 and gp36 (a peptide) representing the immunodominant regions of HIV-1 & HIV-2 envelope genes structure respectively are immobilized on a nylon membrane. As the sample passes through the membrane HIV antibodies if present, bind to the above mentioned immobilized antigens. These bound antibodies are visualized by reacting with Protein A Gold conjugate, which binds to the HIV antibodies, giving a distinct red spot against a white background. Proper test performance is verified by the appearance of a red spot next to ‘C’ produced by binding of Protein A gold to the control antigen immobilized next to ‘C’.

Storage:
The Reagent box has to be stored at 2–8°C. DO NOT FREEZE. Other contents of the kit may be stored at room temperature (20–30°C) or at 2-8°C. Reagents must be brought to room temperature prior to use.

When not in use, return the Reagent Box to 2–8°C.

Contents of the Kit

1.	Test Device 20 Units

2.	Buffer Solution 9 ml (READY TO USE)

3.	Gold Conjugate 2 x 1.5 ml (READY TO USE)

4.	Droppers 20 Nos.

Specimen Processing:
Use only serum or plasma for testing. The specimen should be clean and transparent. Viscous or turbid specimens should be centrifuged at 10,000 rpm for 15 minutes before use. Specimen should be refrigerated if not used on the same day of collection. Specimen should be kept frozen if not used within 3 days after being collected. Do not use repeatedly frozen and thawed samples.

Assay Procedure:
Gold conjugate is stable for 12 months from the date of manufacturing if stored at 2–8°C and should be avoided repeated exposure to room temperature for longer time.
Use fresh gold conjugate vial only after finishing the one used earlier.

1.	Bring all the reagents and specimens to room temperature (25-30°C)

2.	Add 2 drops of buffer solution to the test device.

3.	Add 2 drops of serum / plasma.

4.	Add 2 drops of buffer solution.

5.	Add 2 drops of gold conjugate.

6.	Add 2 drops of buffer solution and read the result.

7.	Read the result immediately. Do not read after 5 minutes

HOLD THE DROPPER VERTICALLY AND ENSURE FREE FALL OF DROPS. AT EACH STEP ALLOW THE SOLUTION TO DRAIN THROUGH THE MEMBRANE BEFORE ADDING THE NEXT SOLUTION.

INTERPRETATION OF RESULTS

1. Negative Result :
If only one red spot (Control spot) appears as shown in Fig 1, the specimen does
Contain antibodies either to HIV-1 or HIV-2.

2. Positive Result :
If two red spots (Control spot and Test spot) appears as shown in Fig 2, the specimen is reactive for antibodies to HIV-1 / HIV-2.

3. Invalid Test :
If no spot appears after the test is complete, as shown in Fig 3, the test has been performed incorrectly.
Repeat the test with a new Device.

Limitations of the test:

1.	Assay procedure and the interpretation must be followed exactly to avoid erratic results.

2.	Because a variety of factors may cause non-specific reactions, samples found to be reactive must be re-tested by using a confirmatory test for HIV, such as Western Blot, P C R etc.

HEPA-Sign One Step HBsAg Rapid Card Test

The most common agent of acute hepatitis is a virus. Of all the viruses that result in hepatitis, Hepatitis B Virus is responsible for the most serious form of the disease. HEPA-Sign HBsAg Test Kit is a rapid, qualitative, one-step immunoassay based on the immunochromatographic principle. This method employs a unique combination of monoclonal-dye conjugate (colloidal gold) and polyclonal solid phase antibodies to selectively identify Hepatitis B surface Antigen with a high degree of sensitivity.

Storage:
Store below 30°C. Do not freeze. The kit is stable till the expiry date mentioned on the label.

Specimen:
Fresh Serum / Plasma.

Precautions:
Handle all specimens carefully as potentially infectious material. On completion of assay, dispose-off specimen after autoclaving at 151b & 121°C for 20 minutes.

Assay Procedure:

1.	Remove Test pack from its foil pouch.

2.	Using dropper add 1-2 drops of specimen into the specimen Window, S.

3.	Visually interpret positive results in 10 minutes and negative results in 30 minutes.

Procedure notes:

1.	Use only serum or plasma specimen. Furthermore, do not use umbilical cord blood, because it prevents colloidal gold from migrating and can interfere with results.

2.	Assay should be conducted at room temperature (15-35°C).

3.	Refrigerated specimen must be brought to the room temperature (15-35°C) before testing.

4.	Specimen should not be repeatedly frozen and thawed.

5.	Specimen with extremely high concentrations of red blood cells, fibrin should be re-centrifuged before using.

INTERPRETATION OF TEST RESULTS

1. Negative: If only one pink colour line appears in the Control Zone, Interpret the result as NEGATIVE. This shows that the concentration of HBsAg in the specimen is under the detection limit. In case the presence of HBsAg is in doubt the result should be confirmed by another method even though the result is negative.

2. Positive: If two lines appear in the Result Area, interpret the result as POSITIVE.

3. Retest: If no lines appear in the Result Area, it is likely that not enough specimen was added or there was some other procedural mistake. Please check procedure and retest with a new HEPA-Sign HBsAg Test Kit.

Performance Characteristics:
HEPA-Sign one step rapid card test are found to detect HbsAg levels is serum as low as 1ng / ml in 10 minutes.

hCG PREGNANCY DETECTION RAPID CARD TEST KITS:

Intended use:
hCG Pregnancy Detection Rapid Card Test Kit is one Step, Fast, Accurate, Easy-to-use, Simple Immunoassay for the Qualitative Detection of hCG (human Chorionic Gonadotropin) in Urine for the early detection of Pregnancy.

Summary and Principle:
hCG is a glycoprotein hormone produced by the placental trophoblastic cells shortly after the fertilized ovum is implanted in the uterine wall. The primary function of the hCG is to maintain the corpus luteum during early Pregnancy. The appearance of hCG in both urine and serum soon after conception and its rapid rise in concentration make it an excellent marker for detection and confirmation of Pregnancy. The hormone may become detectable in both Urine and Serum as early as 5 to 10 days after conception.

The test is a solid-phase, two-site immuno-assay in which combination of Monoclonal and Polyclonal antibodies are used to detect the levels of hCG in urine. In the test procedure, sample is added to the sample well and the sample is allowed to soak in. If hCG is present in the specimen, it will react with the conjugate dye, which binds to the antibody on the membrane to generate coloured dye.

Storage and Stability:
hCG Pregnancy detection rapid card test kit should be stored at 2 to 30°C in the original sealed pouch. The test kits are stable till the expiry date.

Specimen Collection:
hCG Pregnancy detection rapid card test kit can be performed at any time of the day. Collect random urine sample in a clean container, first morning specimen is recommended for best results. If the testing is not performed immediately, the specimens should be stored at 2 to 8°C. Do not freeze. If urine has been refrigerated, let it warm to room temperature before using.

Procedure:

1.	Remove the test cassette from its foil pouch and place it on a flat, dry surface. Test device must be allowed to stand at Room Temparature for atleast 30 minutes prior to testing.

2.	Label the device with the Patient Name or Control Number.

3.	Place the end of the dropper in the urine sample. Draw urine into the dropper.

4.	Hold the urine dropper atleast one inch above the cassette, add two drops of urine into the Sample Well. Do not lift or shake the device.

5.	Read the result in the Result window after 3 minutes, but within 10 minutes.

READING THE RESULTS

1. Negative Result: Only One Pinkish-purple Coloured Straight line in the Control Window (C) means the patient is not Pregnant. The result has to be interpreted after 3 minutes, but within 10 minutes.

2. Positive Result: Two Pinkish-purple Coloured Straight lines, one each in Test (T) and Control (C) Window, means the patient is Pregnant. The result has to be interpreted after 3 minutes, but within 10 minutes.

While you are waiting for results, one line may become visible quickly. However, you need to wait the full time to see if the second line also appears.

3. Inconclusive or Invalid Result: If there is no distinct Pinkish-purple Straight line in the Control Window (C), the test is Inconclusive or Invalid. A Control Line (C) should always appear, the absence of Pinkish-purple line in the Control window (C) means the test is invalid. It is recommended that, in this case the test be repeated or a fresh specimen is obtained and re-tested. The result has to be interpreted after 3 minutes, but within 10 minutes.

Important:
Sometimes one line is darker and the other is lighter and vice versa. Even if line(s) is dark or light, the results has to be interpreted accordingly. Intensity of the colour lines have no clinical significance.

Expected values:
hCG Pregnancy detection rapid card test kit is capable of detecting hCG level of 25 mIU / ml of Urine.

What the result mean
A Positive result indicates that urine contains hCG and the patient is Pregnant. Patient should consult doctor who is able to further advise on proper parental care. A Negative result means that no hCG has been detected and the patient is not Pregnant. If a week passes and the patient still have not started menstruating, do another hCG Pregnancy Detection Rapid Card Test. If the result is still Negative, there could be health-related reasons for not menstruating, consult doctor without delay.

If menstrual cycle is irregular, it may be difficult to know the day of first missed period. If the test is indicated that the patient is not pregnant and the period does not begin within few days, you may have miscalculated your cycle and should undergo a second test.

In laboratory studies, hCG Pregnancy detection rapid card tests was shown to be greater than 99% overall accurate and also shown to be 98% accurate when used by consumers.

Limits of the test
hCG Pregnancy detection rapid card test kit is not re-usable. The test works only if instructions are followed precisely. Although, hCG Pregnancy detection rapid card test kit is highly accurate in detecting pregnancy, a low incidence of false results (positive when no pregnancy exists or negative when pregnancy exists) can occur. Check with doctor, if the Test results are unexpected or inconsistent. Certain health conditions can cause a false or irregular result with the test.

Do not use after expiration date shown on package.

For further queries contact your doctor.

COMBIKIT
ALBUMIN / TOTAL PROTEINS
B.C.G / BIURET METHOD

Order Information:
Kit Size          Cat. No.
2 x 100 ml      IKCBC001

Method:
Colorimetric End Point Chemistry

Principle:
ALBUMIN:
The measurement of serum albumin is based on its quantitative binding to the indicator BromoCresol Green (BCG). Cupric ions, in an alkaline medium, interact with peptide bonds resulting in formation of a coloured complex. The intensity of colour is proportional to the amount of Albumin in the sample.

TOTAL PROTEINS:
Protein in serum forms a blue colored complex when reacted with cupric ions in an alkaline solution. The intensity of the violet colour is proprotional to the amount of Protein in the sample.

Reagents:
Reagent I                      : BCG Reagent
Reagent II                     : Biuret Reagent
Albumin standard           : 4 g / dl
Total Protein standard     : 6 g / dl

Sample:
Serum or Plasma.

Reagent Preparation:
The reagents are ready to use.

Stability:
The reagents are stable upto the expiry date when stored at 2-8°C.

AUTOMATED PARAMETERS

	ALBUMIN	TOTAL PROTEINS
Wavelength	620 nm	540 nm
Cuvette light path	1 cm	1 cm
Reaction Temperature	R.T.	R.T.
Measurement	Against Reagent Blank	Against Reagent Blank
Reaction	End Point	End Point
Sample / Reagent Ratio	1:200	1:50
Incubation	5 minutes	10 minutes
Low Normal	1.8 gr/dl	6.6 gr/dl
High Normal	5.1 gr/dl	8.3 gr/dl
Linearity	8.0 gr/dl	10 gr/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBE
ALBUMIN

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	5µl
STANDARD	-	5µl	-
REAGENT	1000µl	1000µl	1000µl

TOTAL PROTEINS

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	20µl
STANDARD	-	20µl	-
REAGENT	1000µl	1000µl	1000µl

Mix well and wait for 5 minutes for Albumin and 10 minutes for Total Proteins at R.T.

Measure the absorbance of the Sample (Ac) and Standard (As) against reagent blank at the respective wavelengths indicated.

CALCULATION AND LINEARITY
Ac/As x Conc. Std. = gr/dl Albumin/Total Proteins

This method is linear upto a concentration of 8.0 gr/dl for Albumin and 10 gr/dl for Total Proteins.

If the concentration exceeds this value, the sample should be diluted in the ration of 1:1 with 0.9% saline solution and re-assayed. Multiply the result by 2.

Reference values:
Albumin           : 1.8 – 5.1 gr/dl
Total Proteins   : 6.6 – 8.3 gr/dl

Clinical Significance:
Total proteins constitutes one among the major nutrients of an living organisms.
The values increases in Liver cirrhosis, Heaptic Jaundice
The values decrease in anaemia, dehydration etc.,

References
E.M. Gindler and J.O. Westgard Clin. Chem., (1973), 6,4.
J.O. Westgard, M.A. Poquette, Clin. Chem., (1973) 19, 647.

CALCIUM
O-Cresolphthalein (OCPC) method

Order Information
Kit Size         Cat. No.
2 x 50 ml      IKCAL250

Method:
Colorimetric End Point Chemistry

Principle:
Calcium ions form a purple color complex with O-Cresolphthalein complexone in an alkaline medium.
                                                                    Alkaline Medium
Calcium + o-Cresolphthalein Complexone ------------------------> Calcium-Cresophthalein Complexone Complex (purple color)

Reagents
Reagent I                : AMP Buffer reagent
Reagent II               : OCPC Color reagent
Calcium standard     : 10 mg/dl

Sample:
1) Serum, heparinised plasma,
2) 24 urine diluted in the ratio of 1:3.

Reagent Preparation:
Mix equal volumes of Reagents I and II to give enough solution for the number of samples being run.

Stability:
The reagents are stable for 6 hours at R.T.

AUTOMATED PARAMETERS

Wavelength	570 nm
Cuvette	1cm
Reaction Temperature	R.T.
Measurement	Against reagent blank
Reaction Type	End point
Sample / Reagent Ratio	1:40
Incubation	5 minutes
Reagent Blank Abs limit	<=0.5
Low Normal	9 mg/dl
High Normal	11 mg/dl
Linearity	15 mg/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	25µl
STANDARD	-	25µl	-
REAGENT	1000µl	1000µl	1000µl

Mix well and incubate at R.T. for 5 minnutes. Measure the absorbance of Sample (Ac) and standard (As) against reagent blank.

CALCULATION AND LINEARITY
Ac/As x C         = mg/dl Calcium in SERUM
Ac/As x C x 3   = mg/dl Calcium in URINE

This method is linear upto a concentration of 15 mg/dl

Reference values
Serum 9-11 mg/dl
24 hours Urine 50-400 mg/24h

Clinical Significance
Increased serum calcium levels are observed in primary hyperparathyroidism, hpovitaminosis D and multiple myeloma. In addition some neoblastic disorders of bone may be accompanied with increased calcium levels. Decreased serum calcium levels are observed in hypoparathyroidism, tetany steattorea. (due to decreased absorption) in nephritis (due to loss of protein and decreased reabsorption).

References
Faulker W.R, and Meites.S., Caly J.P. et.al Giteman,Richterich.R,59,836.

TOTAL / DIRECT BILIRUBIN
COLORIMETRIC ENDPOINT
BASED ON JENDRASIK AND GROF METHOD

Order Information
Kit Size       Cat. No.
2 x 50 ml    IKBIL250

Method:
Colorimetric End Point Chemistry

Principle:
Total bilirubin in the sample reacts with diazotised sulphalinic acid in the presence of Caffiene.
Direct bilirubin (conjugated bilirubin) reacts in an acid environment with diazotised sulphalinic acid.The coloured formed azobilirubin is measured photometrically at 546 nm.

Reagents
Reagent I      : Total Bilirubin Reagent
Reagent II     : Total Nitrite Reagent
Reagent III    : Direct Bilirubin Reagent
Reagent IV    : Direct Nitrite Reagent

Sample:
Serum or EDTA Plasma.

Reagent preparation:
The Reagents are ready to use.

Stability:
The Reagents are stable till Expiry period.

AUTOMATED PARAMETERS

	TOTAL	DIRECT
Wavelength	546 nm	546 nm
Cuvette Light Path	1cm	1cm
Reaction Type	End Point	End Point
Reaction Temperature	37°C	37°C
Measurement	Against Sample Blank	Against Sample Blank
Sample / Reagent Ratio	1:20	1:20
Incubation	5 minutes	5 minutes
Factor	20.2	20.2
Low Normal	0 mg/dl	0 mg/dl
High Normal	1.2 mg/dl	0.25 mg/dl
Linearity	20 mg/dl	20 mg/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

TOTAL BILIRUBIN

	SAMPLE	BLANK
REAGENT I	1000µl	1000µl
REAGENT II	20µl	-
SAMPLE	50µl	50µl

DIRECT BILIRUBIN

	SAMPLE	BLANK
REAGENT III	1000µl	1000µl
REAGENT IV	20µl	-
SAMPLE	50µl	50µl

Mix well and incubate at 37°C for 5 mins.
Measure absorbance of the Sample (Ac) against respective Blank (Ab) before 8 minutes at 546 nm.

CALCULATION AND LINEARITY
(Ac-Ab) x 20.2 = mg/dl Total/Direct Bilirubin.

The method is linear upto a Concentration of 20 mg/dl.

Reference values:
Total Bilirubin: Upto: 1.2 mg/dl
Direct Bilirubin: Upto: 0.25 mg/dl

The reference values are to be considered as indicative only. Every laboratory should establish its own normal ranges.

Note:
Haemolysis may cause false low values in Bilirubin assays.

Clinical Significance:
Biliribin is formed from Hemoglobin in the RE system. It is circulated attached to plasma albumin in low concentration. Increase in Jaundice, Hepatitis etc.,

References:
Tietz, N.W., text book of clinical Chemistry. Gambino, S R.
Michaelson, M Gambino S R et al, Jana, W B Saunders Co. (1983).

HEMOGLOBIN
(Cyanomethemoglobin)

Order Information:

Kit Size    1 x 1000 ml
Cat.No.    IKHGC019

Method:
Colorimetric End Point Chemistry

Principle:
In cyanomethemoglobin method, erythrocytes are lysed by a stromatolytic reagent in the presence of a surfactant and release their hemoglobin into the solution. Hemoglobin is oxidised to methemoglobin by ferricyanide and the methemoglobin is converted into the stable cyanomethemoglobin by addition of Pottasium Cyanide. The absorbance of cyanomethemoglobin is measured at 540 nm and the color intensity is directly proportional to hemoglobin concentration.

Reagents:
Reagent I                       - Cyanomethemoglobin reagent
Hemoglobin Standard      - 60 mg/dl (Store at 2-8°C)

Sample:
Collect whole blood with EDTA using aseptic techniques.
Whole blood collected with EDTA is stable for one week at 2 - 8°C.

Reagent Preparation:
The reagents are ready to use.

Stability:
The reagents are stable till the expiry date. (Store the Reagents away from sunlight).

AUTOMATED PARAMETERS

Wavelength	540 nm
Reaction Type	End Point
Cuvette	1 cm light path
Reaction Temperature	R.T.
Measurement	Against reagent blank
Sample / Reagent Ratio	1:250
Incubation	5 minutes
Low Normal	10.0 gr/dl
High Normal	18.0 gr/dl
Linearity	20.0 gr/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	20µl
STANDARD	-	5000µl	-
REAGENT	5000µl	-	5000µl

Mix and Incubate at R.T. for 5 minutes. Measure the absorbance of the Sample (Ac) and Standard (As) against the Reagent Blank at 540nm (520–550 nm).

CALCULATION AND LINEARITY
Ac/As x Conc.of Std. = gr/dl Hemoglobin

The hemoglobin assay shows a linearity upto 20 gr/dl. If the hemoglobin values are more than 20 gr/dl, dilute the sample in the ratio of 1:1 with deionised water. Multiply the result by 2.

Reference values:
Adult Males           13.0-18.0 gr/dl
Adult Females       11.0-16.0 gr/dl
Children               10.0-14.0 gr/dl
Newborns             14.0-23.0 gr/dl

Clinical Significance:
It is the main constitutent of the RBC’s and carries out the importance function of transportation of oxygen from lungs to various parts of the body.
Increases in polycythemia, malignant tumors, etc.,
Decreases in anamai, multiple myeloma

References:
Eilers R.J., Am. J. Clin. Path., 47:212 (1967).
Tietz N.W., Fundamentals of Clinical Chemistry.
2nd Ed. W.B. saunders Co., Philadelphia p 411 (1976).

GLUCOSE
Enzymatic (GOD-POD) Colorimetric Test

Order Information:
Cat. No. Kit Size
IKGLE001 5 x 100 ml

Method:
Colorimetric End Point Chemistry

Principle:
Glucose is determined after enzymatic oxidation in the presence of glucoseoxidase. The hydrogen peroxide thus formed reacts, under the catalysis of peroxidase, with 4 hydroxy benzoic acid and 4-aminoantipyrine to form a red quinoneimine dye as indicator.

                                    Glucose Oxidase
D-Glucose + H₂O + O₂ ----------------------> H₂O₂ + D-Gluconate

                                                                       POD
H₂O₂ + 4-Aminoantipyrine + Hydroxybenzoate --------> Quinoneimine dye + H₂O

Reagents:
Reagent I                - Enzyme reagent
Glucose standard     - 100 mg/dl

Sample:
Serum, Plasma,
Serum and Plasma stability: Upto 7 days at 2 - 8°C

Reagent preparation:
Dissolve the enzyme reagent with volume of distilled water as specified on the enzyme vial.

Stability
Working Reagent is stable for 60 days at 2 - 8°C. (Store protected form light)

AUTOMATED PARAMETERS

Wavelength	505 nm (490 - 550 nm)
Reaction Type	End Point
Cuvette	1 cm light path
Reaction Temperature	37°C
Measurement	Against Reagent Blank
Sample / Reagent Ratio	1:100
Incubation	15 minutes
Low Normal	60 mg/dl
High Normal	110 mg/dl
Linearity	500 mg/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	10µl
STANDARD	-	10µl	-
Reagent	1000µl	1000µl	1000µl

Mix and Incubate for 15 minutes at 37°C or 30 minutes at RT. Measure absorbance of Sample (AT) and Standard (AS) against Reagent Blank. The colour is stable for 30 minutes at R.T.

CALCULATION AND LINEARITY
Total Glucose (mg/dl) = AT/AS x Conc. of Standard.

This method is linear upto a concentration of 500 mg/dl. If the values are above this concentration then dilute in the ratio of 1:1 and multiply the result by 2.

Reference values:
Serum/Plasma      : 60-110 mg/dl (Random)

Clinical significance:
Glucose determination is mainly useful in the diagnosis of Diabetes of Mellitus in which the bloods glucose levels are elevated. Other diseases like Hyperthyroidism and Hyperpituitatism also lead to Hyperglycemia. Hypoglycemia occurs frequently as a result of overdosage of Insulin or anti-diabetes treatment. Hypoglycemia may also be noticed in conditions like Hyperinsulinemia, Hypothyroidism, Hypopituitarism and Hypoadrenocorticism.

References:
Teitz, N.W., Fundamentals of Clin. Chem., Philadelphia. W.B.
Saunders (1970) Trinder.P., “Determination of Blood Glucose Using 4 Aminophenazone”.

UREA REAGENT
(BERTHELOT METHOD)

Order Information:
Kit Size         Cat. No.
2 x 100 ml     IKURE210

Method:
Colorimetric End Point Chemistry

Principle:
The colorimetric urea procedure is modification of the Berthelot reaction. Urea is converted to ammonium by the use of urease enzyme. Ammonium ions then reacts with a mixture of salicylate, sodium nitroprusside and hypochlorite to yield a blue-green chromophore. The intensity of the color formed is proportional to the urea concentration in the sample.

Reagents:
Reagent I            : Urea Enzyme reagent
Reagent II           : Urea Color developer
Urea standard      : 50 mg/dl

Sample:
Serum or Plasma.

Reagent preparation:
Reagent I    : Dissolve contents of Reagent I with volume of distilled water as
                    specified on the vial.
Reagent II : Ready to use.

Stability: :
Reagent I      : 45 days at 2 - 8°C
Reagent II     : 90 days at 2 - 8°C

AUTOMATED PARAMETERS

Wavelength	630 nm
Cuvette	1 cm light path
Reaction Temperature	37°C
Measurement	Against Reagent Blank
Reaction Type	End Point
Sample / Reagent Ratio	1:200
Incubation	5 + 5 minutes
Low Normal	15 mg/dl
High Normal	50 mg/dl
Linearity	200 mg/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

	BLANK	STANDARD	SAMPLE
REAGENT I	1000µl	1000µl	1000µl
STANDARD	-	10µl	-
SAMPLE	-	-	10µl

Mix well and incubate for 5 minutes at 37°C or 10 minutes at R.T.

REAGENT II

1000µl

Mix well and incubate for 5 minutes at 37°C or 10 minutes at R.T.

Measure absorbance of Test (AT) and Standard (AS) against Reagent Blank at 630 nm.

CALCULATION AND LINEARITY :
Urea (mg/dl) = AT / AS x Conc. of Std

The procedure is linear upto 200 mg/dl Urea.

Samples with values above 200 mg/dl should be diluted in the ratio of 1:1 with 0.9% saline, re-assayed and the results to be multiplied by 2.

Reference values:
15 – 50 mg/dl
The reference values are to be considered as indicative only. Every Laboratory should establish its own normal range.

Clinical Significance:
Azotemia is a biochemical designation referring to any significant increase in the Plasma/Serum concentratin of non-protein nitrogenous compounds principally urea.
Azotemia is frequently categorised as prerenal, renal and post renal.

References:
Teitz, N.W; Fundamentals of clinical chemistry, Philadelphia,
W.B. Saunders & Co., - Philadelphia, PA, p991(1976)., Talke H, Schubert GE, KlinWchers., (1965), 43, 174.

URIC ACID
Enzymatic Colorimetric method

Order Infomation:
Kit Size       Cat.No.
5 x 10 ml    IKUCE003

Method:
Colorimetric End Point Chemistry

Principle:
Uric acid is converted by uricase to Allantoin and Hydrogen Peroxide, which under the catalytic influence of peroxidase, oxidises 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to form a Chromogen compound.

                                   Uricase
Uric acid + O₂ + 2H₂O -----------> Allantoin + CO₂ + H₂O₂
                                                        HPD
2H₂O₂ + 4-Aminoantipyrine + DHBS --------> Chromogen + 4H₂O

Reagents:
Reagent I                  : Buffer reagent
Reagent II                 : Enzyme reagent
Uric Acid Standard      : 5 mg/dl

Sample:
Serum, Heparinised or EDTA Plasma, Urine.
Dilute Urine in the ration of 1:10 with distilled water. Multiply the result by 10.

Reagent preparation:
Dissolve contents of one vial of enzyme reagent with the volume of buffer reagent as specified on the enzyme vial.

Stability:
The re-constituted reagent is stable for 60 days at 2-8°C/ 15 days at 15-25°C (Store protected from light)

AUTOMATED PARAMETERS

Wavelength	520 nm
Cuvette	1 cm light path
Reaction Temperature	37°C
Measurement	Against Reagent Blank
Reaction Type	End Point
Sample / Reagent Ratio	1:40
Incubation	10 minutes
Maximum Blank Absorbance	0.50
Low Normal	2.4 mg/dl
High Normal	7.0 mg/dl
Linearity	20 mg/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	25µl
STANDARD	-	25µl	-
REAGENT	1000µl	1000µl	1000µl

Mix well and incubate for 10 minutes at 37°C or 15 mins at 20-25°C.
Measure absorbance of Sample (Ac) and Standard (As) against Reagent Blank. The color is stable for 60 mins at 20-25°C.

CALCULATION AND LINEARITY
Ac/As x C           = mg/dl Uric Acid (Serum/Plasma)
Ac/As x C x 10    = mg/dl Uric Acid (Urine)
C = Concentration of standard

This method is linear upto a concentration of 20 mg/dl. Dilute samples above this concentration in the ratio of 1:1 with 0.9% saline and re-assay. Multiply the result by 2.

Reference values:
Serum
Men         : 3.4-7.0 mg/dl
Women    : 2.4-5.7 mg/dl

Urine       : 250-750 mg/24hours

Clinical Significance:
Increased levels are observed in Gout. Other conditions in which Uric Acid level increases are Leukaemia and Polycythemia.

Low leves may be found in Wilson's disease and in Fanconi's Syndrome.

References:
Caraway, W.T., Clin Cem.4, 239 (1963),
Morin L.G.,Clin Chem, 20, 51 (1974)
Trivedi R.C., Rebar L., Berka E., Strong L., Clin.
Chem., (1978), 24, 1908.

ALKALINE PHOSPHATASE
Kinetic test optimized according to
IFCC recommendations

Order information:
Kit Size         Cat.No.
5 x 10 ml      IKALP510

Method:
Colorimetric Kinetic Point Chemistry

Principle:
p-Nitrophenyl phosphate is converted to p-nitrophenol and phosphate by alkaline phosphatase. The increase of absorption at 405nm is proportional to the alkaline phosphatase concentration in the sample.

                      Alk. Phos.
p-Npp + H₂O --------------> p-Nitrophenol + H₃PO₄

Reagent:
Reagent I    : Buffer reagent
Reagent II   : Substrate reagent

Sample:
Serum or Plasma.

Reagent preparation
Dissolve the contents of one vial of substrate reagent with the volume of buffer reagent as specified on the substrate vial.

Stability:
The re-constituted reagent is stable for 21 days at 2-8°C.

AUTOMATED PARAMETERS

Wavelength	405 nm
Cuvette	1 cm light path
Reaction Temperature	37°C
Measurement	Against Distilled water
Reaction Type	Kinetic test
Reaction Direction	Increasing
Sample / Reagent Ratio	1:50
Delay / Lag time	60 secs
Interval time	30 secs
No. of Readings	04
Blank Absorbance limit	<=0.85
Factor	2720
Low Normal at 37°C	25 U/L
High Normal at 37°C	147 U/L
Linearity at 37°C	800 U/L

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

SAMPLE	20µl
REAGENT	1000µl

Mix well and Incubate at 37°C for 60 seconds. Measure Absorbance increase every 30 secs for 2 minutes and determine the ΔA/min.

CALCULATION AND LINEARITY :
ΔA/minute x 2720 = U/L Alkaline Phosphatase

This method is linear upto a concentration of 800 U/L.

Reference values:
ADULTS
At 37°C 25 – 147 U/L

The reference values are to be considered as indicative only.Every Laboratory should establish its own normal ranges.

Clinical significance:
Increased levels of serum ALP are observed in Hepatic conditions and the elevation tends to be more in post hepatic conditions. Increased serum ALP levels are seen in bone diseases like Paget's disease, Ricketes. High levels of serum ALP are also observed in Bone cancer, moderate elevation seen in Hyperparathyroidism.

References:
Fundamental of Clinical Chemistry, Young D.S, Tietz, N.
Fundamentals of Clinical Chemistry 602/609, Kaplan,
M.M. New England.

CHOLESTEROL
Enzymatic Colorimetric Test

Order information:
Kit Size       Cat.No.
5 x 20 ml     IKCHE005

Method:
Colorimetric Enzymatic End Point Chemistry

Principle:
Cholesterol esters are hydrolyzed to produce Cholesterol. Hydrogen Peroxide is then produced from oxidation of cholesterol by cholesterol oxidase. The indicator Quinoneimine is formed from hydrogen peroxide and 4-aminoantipyrine in the presence of POD. The absorption of the red quinoneimine dye is proportional to the concentration of cholesterol in the sample.

                             C. Esterase
Cholesterol Esters ------------------> Cholesterol + Fatty Acids
                            C. Oxidase
Cholesterol + O₂ ----------------> Cholesterol-3-one + H₂0₂
                                          Peroxidase
2H₂0₂ + 4-Aminoantipyrine ----------------> Quinoneimine (red dye) + 2H₂0₂

Reagents:
Reagent I                     : Buffer reagent
Reagent II                    : Enzyme reagent
Cholesterol standard     : 200 mg/dl

Sample:
Serum, heparinised or EDTA Plasma.

Stability:
6 days at 4 - 25°C / 4 months at -20°C

Reagent preparation:
Dissolve one vial of enzyme reagent with amount of buffer reagent as specified on the enzyme vial.

Stability:
The re-constituted reagent is stable 50 days at 2-8°C. Store protected from light.

AUTOMATED PARAMETERS

Wavelength	520 nm
Reaction Type	End Point
Cuvette	1 cm light path
Reaction Temperature	37°C
Measurement	Against Reagent Blank
Sample / Reagent Ratio	1:100
Incubation	5 minutes
Maximum Blank Absorbance	0.50
Low Normal	<200 mg/dl
High Normal	>240 mg/dl
Linearity	500 mg/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	10µl
STANDARD	-	10µl	-
REAGENT	1000µl	1000µl	1000µl

Mix and Incubate for 5 minutes at 37°C or 10 mins at 20 - 25°C.
Measure the Absorbance of Sample (AT) and Standard (AS) against Reagent Blank.

The colour is stable for 90 minutes at 20 - 25°C.

CALCULATION AND LINEARITY
AT/AS x Conc. of Standard = mg/dl of Total Cholesterol

This method is linear upto a concentration of 500 mg/dl.

Dilute samples above this concentration in the rate of 1 : 1 with 0.9% saline solution and repeat the assay. Multiple the result by 2.

Reference values:
<       200 mg/dl      Normal
220 – 239 mg/dl     Borderline – High
>       240 mg/dl      High

Clinical Significance:
Increases in DM,Cardiac failure, nephrotic syndrome etc.,
Decrease in anaemia

References:
Tietz N.W. Fundamentals of clin. Chem,
Young D.S, Naito, HK.et.al. (1973), 10.79.

HDL CHOLESTEROL
PTA METHOD

Order information:
Kit Size       Cat.No.
5 x 20 ml     IKHDE006

Method:
Colorimetric End Point Chemistry

Principle:
Low density and Very Low Density Lipoproteins (LDL and VLDL) and chylomicron fractions are precipitated by the precipitating reagent. After centrifugation, the HDL-Cholesterol fraction remains in the supernatant and is determined by an enzymatic method.

Reagents:
Reagent I                   : Precipitating Reagent
Reagent II                   : Cholesterol Buffer Reagent
Reagent III                  : Enzyme Reagent
Cholesterol Standard    : 50 mg/dl

Sample:
Serum or EDTA Plasma.

Reagent preparation:
Ready to use

Stability:
The contents are ready to use and are stable upto the expiry date when stored at 2-8°C.

Precipitation step:
Pipette into centrifuge tubes

SAMPLE	REAGENT
500µl	500µl

Mix and allow to stand for 5 minutes at R.T. Centrifuge for 10 minutes at 3000 rpm so as to get clear supernatant and determine the cholesterol content by the CHOD-PAP method. Only clear supernatant should be used.

Reagent preparation:
Dissolve one vial of enzyme reagent with amount of buffer reagent as specified on the enzyme vial.

Stability:
The reconstituted reagent is stable for 50 days at 2-8°C. Store protected from light.

AUTOMATED PARAMETERS

Wavelength	520 nm
Reaction Type	End Point
Cuvette	1 cm light path
Reaction Temperature	37°C
Measurement	Against Reagent Blank
Sample / Reagent Ratio	1:20
Incubation	5 minutes
Maximum Reagent Blank Abs	0.50
Low Normal at 37°C	41 mg/dl
High Normal at 37°C	75 mg/dl
Linearity at 37°C	500 mg/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	50µl
STANDARD	-	50µl	-
REAGENT	1000µl	1000µl	1000µl

Mix and Incubate at 37°C for 5 minutes or 10 minutes at 20 - 25°C.
Measure Absorbance of Sample (Ac) and Standard (As) against Reagent Blank. The colour is stable for 90 minutes at 20-25°C.

CALCULATION AND LINEARITY
Ac/As x Conc. of Standard x 2 = mg% HDL Cholesterol in Serum/Plasma

2 is the serum dilution factor.
The method is linear to a concentration of 500 mg/dl.

Reference values:
MEN          41.0 – 59.0 mg/dl
WOMEN     49.0 – 75.0 mg/dl

Clinical significance:
HDL-Cholesterol has been shown to have an inverse correlation with the incidence of cardiovascular disease. Therefore the determination of HDL is useful in assessing the risk of patients to Coronary Heart Diaease. High HDL levels are associated with lower risk and low HDL levels are associated with increased risks.

References:
Denahcherp N.M., Hijans A.G.M., Vos-janssen H.E, Vant Laar
A.P., Clin. Chem., 26 1775 (1980)
Castelli., ; Current Prescribing, 3, 39 (1977).

TRIGLYCERIDES
Enzymatic Colorimetric Test – GPO-PAP

ORDER INFORMATION
Kit Size       Cat. No.
5 x 10 ml     IKTRG510

Method:
Colorimetric Enzymatic End Point Chemistry

Principle:
Triglycerides in the sample are hydrolyzed by Lipase to Glycerol and Fatty acids. the glycerol is then phosphorylated by ATP to glycerol-3-phosphate (G3P) and adenosine-5-diphosphate in a reaction catalyzed by glycerol kinase (GK). G3P is then converted to dihydroxyacetone phosphate (DAP) and hydrogen peroxide by glycerophosphate oxidase (GPO). The hydrogen peroxide then reacts with 4-aminoantipyrine (4-AAP) and 3-hydroxy-2,4,6-tribromobenzoic acid (TBHB) in a reaction catalysed by peroxidase to yield a red colored Quinoneimine dye. The intensity of the color produced is directly proportional to the concentration of Triglycerides in the samples when measured at 540nm.

                      Lipase
Triglycerides -------------> Glycerol + Fatty Acids
                         GK
Glycerol + ATP --------> G₃P + ADP
                  GPO
G₃P + O₂ --------> DAP + H₂O₂
                       Peroxidase
H₂O₂ + TBHB -----------------> Quinoneimine Dye + 2H₂O

Sample:
Serum, heparinised or EDTA Plasma.
Stability: 3 days at 2 – 8°C, 3 months at -20°C.

Reagent preparation:
Dissolve the contents of one vial of enzyme reagent with the volume of buffer reagent as specified on the enzyme vial.

Stability:
The reconstituted reagent is stable for 40 days at 2 - 8°C. Store protected from light.

AUTOMATED PROCEDURES

Wavelength	520 nm
Reaction Type	End Point
Cuvette	1 cm light path
Reaction Temperature	37°C
Measurement	Against Reagent Blank
Sample / Reagent Ratio	1:100
Incubation	5 minutes
Maximum Blank Absorbance	0.50
Low Normal	40 mg/dl
High NOrmal	165 mg/dl
Linearity	1000 mg/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	10µl
STANDARD	-	10µl	-
REAGENT	1000µl	1000µl	1000µl

Mix well and incubate for 5 minutes at 37°C or 15 mins at 20 - 25°C.
Measure Absorbance of Sample (AT) and Standard (AS) against Reagent Blank.

The colour is stable for 60 minutes at 20 - 25°C.

CALCULATION AND LINEARITY
AT/AS x Conc. of Std. = mg/dl Triglycerides

The method is linear upto a concentration of 1000 mg/dl.
Dilute samples above this concentration in the ratio of 1 : 1 with 0.9% saline and re-assay. Multiply the result by 2.

Reference values:
Men        : 60 – 165 mg/dl
Women   : 40 – 140 mg/dl

Clinical significance:
Phospholipids, Cholesterol and Triglycerdes are the three large lipid fractions in the Blood. Determination of Triglycerdies has specific significance in conditions like cardiovascular diseases, diabetes mellitus, billary obstructions, nephrosis and various endocrine related metabolic disturbances.

References:
Buccolo G., David M., Clin. Chem, 19, (1973), 476

CREATININE
Without deproteinisation method

Order information:
Kit Size       Cat.No.
2 x 50 ml     IKCRE250

Method:
Colorimetric Fixed Time Kinetic Chemistry

Principle:
Creatinine in alkaline solution reacts with picrate to form a coloured complex which absorbs at 500 - 520nm. The amount of complex formed is directly proportional to the creatinine concentration.

Creatinine + Sodium picrate --------> Creatinine-picrate complex

Reagents:
Reagent I                  : Buffer reagent
Reagent II                 : Picrate reagent
Creatinine Standard : 2 mg/dl

Sample:
Serum, Urine 24hours diluted 1:20 with distilled water.

Reagent preparation:
Mix equal volumes of Reagent I and II and let it stand for 30 minutes.

Stability:
30 days at 2 - 8°C / 7 days at RT (when stored in a dark bottle).

AUTOMATED PARAMETERS:

Wavelength	500 nm
Measurement	Against Reagent Blank
Cuvette	1 cm light path
Reaction Temperature	Room Temperature
Reaction Type	Fixed Time
Reaction Direction	Increasing
Sample / Reagent Ratio	1:10
Delay / Lag time	30 secs
Interval time	120 secs
No. of Readings	01
Low Normal	0.7 mg/dl
High Normal	1.2 mg/dl
Linearity	25 mg/dl

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

	BLANK	STANDARD	SAMPLE
SAMPLE	-	-	100µl
STANDARD	-	100µl	-
REAGENT	1000µl	1000µl	1000µl

Mix well and after 30 seconds at R.T., read initial Absorbance and start the timer simultaneously. Read again after 2 minutes. Determine ΔA of Standard (ΔAs) and Sample (ΔAc) against Reagent Blank.

CALCULATION AND LINEARITY:
ΔAc / ΔAs x C = mg Creatinine/dl Serum
ΔAc / ΔAs X C X 20 = mg Creatinine/dl Urine
C = Concentration Standard.

The method is linear to a concentration of 25 mg/dl

Reference values:

	MEN	WOMEN
SERUM	0.8 – 1.4	0.7 – 1.2 mg/dl
24 HOUR URINE	1.0 – 2.0	0.8 – 1.8 gr/24h

The reference values are to be considered as indicative only.Every laboratory should establish its own normal range.

Clinical Significance:
It is necessary in the process of muscle contraction. Creatinine is derived from creatine and is a waste product.
Increase in renal failure,hyperthyroidism, etc.,

References:
Henry, J.B, Young D.S.teitz N.W, Vasilades, J,Can, Chem (1972), 18.

SGOT – AST
Kinetic test UV – optimised IFCC method

Order information:
Kit Size Cat.No.
5 x 10 ml IKOTE 009

Method:
Colorimetric Kinetic Chemistry

Principle:
Aspartate transaminase (GOT – AST) catalyses the reaction between alpha-ketoglutaric acid and L-aspartate giving glutamate and oxaloacetate. Oxaloacetate, in the presence of malate dehydrogenase (MDH) reacts with NADH giving malate and NAD, the rate of NADH decrease is determined photometrically and is directly proportional to the GOT acitivity in the sample.

AST
L-Aspartate + α-Ketoglutanate ----------> Oxalacetate + L-Glutamate
MDH
Oxalacetate + NADH + H⁺ ----------> L-Malate + NAD⁺ + H₂O

Reagents:
Reagent I : Buffer reagent
Reagent II : Enzyme reagent

Sample:
Serum or Plasma.

Reagent preparation:
Dissolve one vial of enzyme reagent with amount of buffer reagent as specified on the enzyme vial.

Stability:
21 days at 2-8°C.

AUTOMATED PARAMETERS:

Wavelength	340 nm
Cuvette	1 cm light path
Reaction Temperature	37°C
Measurement	Against Distilled water
Reaction Type	Kinetic test
Reaction Direction	Decreasing
Sample / Reagent Ratio	1:10
Delay / Lag time	60 seconds
Interval time	30 seconds
No. of Readings	04
Blank Absorbance limit	<=0.8
Factor	1746
Low Normal at 37°C	0 U/L
High Normal at 37°C	35 U/L
Linearity at 37°C	350 U/L

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

SAMPLE	100µl
REAGENT	1000µl

Mix well and let it stand for 1 minute at 37°C. Measure Absorbance decrease per minute during 3 minutes (ΔA/min).

CALCULATION AND LINEARITY
ΔA/min x 1746 = U/L AST

This method is linear upto 350 U/L

Reference values:
0 to 35 U/L at 37°C

Clinical significance:
AST is widely distributed in tissues with the highest concentrations found in the liver, heart, kidneys and skeletal muscles.

Diseases involving any of these tissues can lead to elevated levels of AST in serum. Following Myocardial Infraction, AST levels are elevated and reach a peack after 48 to 60 hours.

Hepatobiliary diseases such as cirrhosis, metastatic carcinoma and viral hepatitis can show increased levels of AST.

Elevated levels of AST are also seen in muscular dystrophy, dermatomyositis, acute pancreatitis and infectious monocucleosis.

Notes:
Very low initial absorbance suggest very high activity of GOT, in such cases dilute the samples appropriately and re-assay. Multiply results with dilution factor. Procedure & calculation will be the same.

References:
Expert Panel on enzyme of the IFCC, Clin. Chem. Acta, 70, PM, (1976), Teitz., N.W.

SGPT – ALT
Kinetic test UV - optimised IFCC method

Order information:
Kit Size       Cat. No.
5 x 10 ml     IKPTE010

Method:
Colorimetric Kinetic Chemistry

Principle:
Glutamic-pyruvic Transaminase (GPT – ALT) catalyses the reaction between alpha-Ketoglutaric acid and Alanine giving L-glutamic acid and pyruvic acid. Pyruvic acid, in the presence of lactate dehydrogenase (LDH) reacts with NADH giving lactic acid and NAD. The rate of NADH consumption is determined photometrically and is directly proportional to the GPT activity in the sample.
                                           ALT
L-Alanine + α-Ketoglutarate ---------> Pyruvate + L-Glutamate

                                     LDH
Pyruvate + NADH + H⁺ ----------> L-Lactate + NAD⁺ + H₂O

Reagents:
Reagent I      : Buffer reagent
Reagetn II     : Enzyme reagent

Sample:
Serum or Plasma.

Reagent preparation:
Dissolve one vial of enzyme reagent with amount of buffer reagent as specified on the enzyme vial.

Stability:
21 days at 2-8°C.

AUTOMATED PARAMETERS

Wavelength	340 nm
Cuvette	1 cm light path
Reaction Temperature	37°C
Measurement	Against Distilled water
Reaction Type	Kinetic test
Reaction Direction	Decreasing
Sample / Reagent Ratio	1:10
Delay / Lag time	60 seconds
Interval time	30 seconds
No. of Readings	04
Blank Absorbance limit	>=0.8
Factor	1746
Low Normal at 37°C	0 U/L
High Normal at 37°C	40 U/L
Linearity at 37°C	350 U/L

MANUAL PROCEDURE

PIPETTE INTO TEST TUBES

SAMPLE	100µl
REAGENT	1000µl

Mix well and let it stand for 1 minute at 37°C. Measure absorbance decrease per minute during 3 minutes (ΔA/min)

CALCULATION AND LINEARITY
ΔA/min. x 1746 = U/L ALT

This method is linear upto 350 U/L

Reference values:
0 to 40 U/L

The reference values are only indicative in nature. Every laboratory should establish its own normal ranges.

Clinical significance:
Increased levels of serum ALT are generally associated with damage to the liver and Kidney's, such as hepatitis, cirrohis and hepaticnecrosis.

A moderate increase is also found in obsturctive jaundice, MI and hepatic congestion.
Notes:
Very low initial extinction suggest very high activity of GPT, in such cases dilute the samples appropriately and re-assay. Multiply results with dilution factor. Procedure and calculation will be the same.

References:
Expert Panel on enzyme of the IFCC,Clin. Chem. Acta, 70, PM, (1976), Teitz., N.W.