CHEMISTRY REAGENT KITS
                    ------->CHLORIDE(Thiocyanate) End-Point
                                             
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CLINICAL SIGNIFICANCE
Low Chloride levels In blood are associated with gastrointestinal or renal salt losing conditions or metabolic acidosis and Addison's disease. Increased levels are observed in cases of dehydration, renal obstruction, nephritis, congestive heart failure and Cushing's syndrome.

PRINCIPLE
2CI  +  Hg+(SCN)-2---®    HgCI2+(SCN)

(SCN) +  2 Fe++   ----®   2Fe++(SCN)

The formation of REDDISH BROWN COLOURED ferric thiocyanate can be measured at 460nM and is directly proportional to Chloride concentration.

COMPOSITION
1 CHLORIDE REAGENT      

4 x 50 mL

Ferric Nitrate

100mMol/L

Mercuric Thiocyanate 10 mMol/L
Methanol

3 mol/L

Contains preservatives and stabilizers.

2 CHLORIDE STANDARD 3 mL
Sodium Chloride 100 mMol/L
Contains surfactants and stabilisers.
REAGENTS SUPPLIED
Cat. No. LS 325

4x50mL

Reagent 1 4x50mL
Standard (100 mMol/L)

3 mL

Preparation Of Working Reagent
All reagents are ready to use.

STABILITY
All reagents are stable untill expiry date marked on labels at 2-8°C away from bright light

SAMPLE
Specimen can be unhaemolysed serum, heparinised plasma, CSF, bodyfluidsorurine. Urine sample to be diluted 1 to 2 with glass distilled water and result to be multiplied by 2 (dilution factor).

SYSTEM PARAMETERS

Reaction

End-point

Temperature 30-35°C
Wavelength 570 nM (530-590 nM)
Absorbance Range 10 mGs/dL
Cuvette Path Length

0-1°A

Reaction Time 1 cM
Dynamic Range 70-140 mMol/L
Max. limit of Blank Reagent

0.20

Final Colour Stability 30 Mins.
Reagent Volume 1000 µL
Sample Volume 10 µL

METHOD FOR AUTOANALYSERS

Chloride Colour Reagen............................................IIOOOpL
Sample or Standard....................................................10 ^L

Mix well. Incubate at R.T. for 5 mins. Read OD at 460 nM (440-500 nM) against Blank. The final colour is stable for 30 minutes away from bright light.
Note: Programme the analyser using system parameters. A specific data sheet may be provided for each analyser on request.

MANUAL METHOD
1. Wash all glasswares, pipette tips and pipettes thoroughly with glass distilled water. DRY.

2.
Pipette into 3 Test tubes Blank mL Standard mL Test mL
Chloride Colour Reagent 3.00 3.00 3.00
Distilled Water 0.02 - -
Standard - 0.02 -
Sample - - 0.02

Mix well. Incubate for 5 minutes at R.T. Read Optical Density (OD) at 460 nM (440-500nM) or BLUE filter against Blank. Final colour is stable for 30 minutes away from bright light.
Note : Reagent and sample volume can be altered proportionately.

RESULTS

                                        O.D. Test
Chloride in mMol/L = -----------------------   x  100
                                        O.D. STD

                         O.D.TEST - O.D.Blank
in mEq/L  =  ----------------------------------    x    100
                         O.D.STD  -  O.D. Blank

mGs/dL = mEq/L x 3.545

EXPECTED.VALUES

Serum : 96

-106 mEq/L

  340  - 375.8 mGs of Chlorine/dL
  561 -619.4 mGs of NaCI/dL
CSF  : 123 "135 mEq/L
  436 " 478.6 mGs of Chlorine/dL
  728.8 " 788.9 mGs of NaCI/dL
Urine  : 170 250 mEq/24 hours

602.7 - 886.3 mGs of Chlorine/24hrs 993.5- 1461mGsofNaCI/24hrs Values vary subject to the infusion of sodium chloride. As with all diagnostic methods, the final diagnosis should not be made on the result of a single test as well as laboratory diagnosis must be confirmed with clinical manifestations.

LIMITATIONS
This reagent system is highly sensitive to chloride radicals commonly found as contaminants in tap water and detergents etc. Test tubes and glasswares washed in tap-water may lead to false results.
This methodology is not useful to measure outside dynamic range of 70-140 mMol/L.
For levels out side the dynamic range of methodology increase or decrease sample volume and multiply the result appropriately. For more accuracy each kit should be tested against a set of standards with different concentrations and a graph be plotted.

QUALITY CONTROL
To ensure adequate quality control, each kit should be tested against a standard control sera. It should be realised that the use of quality control material checks both instrument and reagent function together. Factors which might affect the performance of this test include proper instrument function, temperature control, cleanliness of glasswares and accuracy of pippetting.

It is appropriate to establish each laboratory's accuracy constant and interpret values accordingly. Similarly, laboratory findings should be established by clinical manifestations.

WARNING
This reagent system is for in vitro Diagnostic use only. This reagent system is containing preservatives and components that have not established for safety if contacted on broken skin or eye or taken orally. In case of such incidents wash off with plenty of water, or consult a physician.

BIBLIOGRAPHY

1.  

    SHOENFELD, R.G. Clin. Chem. 10,533.

    2.  ZALL, D.M. et. al. Anal. chem. 28,1665,1956