SUMMARY AND EXPLANATION

Chloride represents the major extracellular anion and helps to balance the electrical neutrality of body fluids. It plays an important role in maintaining proper hydration and osmotic pressure. Low chloride values occur with extensive burns, excessive vomiting, internal obstruction, nephritis, metabolic acidosis, addisonian crisis and diarrhea. High chloride can be observed in dehydration, hyperventilation, congestive heart failure, urinary obstruction and excessive treatment with chloride (1)(5).

In 1956, Zail et al (2) introduced a micro-method for chloride determination based on the use of mercuric thiocyanate and ferric compounds. Subsequently, Skeggs and Hochstrasser (3) modified the procedure for serum chloride assay. the EAGLE CHLORIDE PROCEDURE is based on the Skeggs modification and does not require protein precipitation.

PRINCIPLE

Chloride displaces thiocyanate for mercury forming non-ionized mercuric chloride. Thiocyanate reacts with ferric ions to form a red-colored complex which can be measured at 480 nm. The color intensity is proportional to the chloride concentration.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2450 provides:

CHLORIDE REAGENT - (Cat. No. 2451)

REACTIVE INGREDIENTS:

1.0 mM mercuric thiocyanate and 38 mM ferric nitrate in dilute acid solution. Methanol added.

PRECAUTIONS:

CONTAINS MERCURY. May be harmful if swallowed. Do not pipette by mouth. If ingested, perform gastric lavage and call a physician. Also contains methanol. Do not inhale.

STORAGE AND STABILITY:

Store at 15-30ºC. stable until expiration date. PROTECT FROM LIGHT.

DETERIORATION:

The reagent should be a clear, pale yellow solution. Turbidity or a red-brown color would indicate deterioration and the regent should not be used.

CHLORIDE BLANK- (CAT. NO. 2452)

REACTIVE INGREDIENTS:

24 mEq/L sodium chloride.

PRECAUTIONS:

Do not ingest. Toxicity has not been established.

STORAGE AND STABILITY:

Store at 15-30ºC. Stable until expiration date.

DETERIORATION:

The blank should be a clear, colorless solution. Turbidity would indicate deterioration and the blank should not be used.

CHLORIDE CALIBRATOR- (CAT. NO. 2453)

REACTIVE INGREDIENTS:

100 mEq/L sodium chloride.

PRECAUTIONS:

Do not ingest. Toxicity has not been established.

STORAGE AND STABILITY:

Store at 15-30ºC. Stable until expiration date.

DETERIORATION:

The calibrator should be a clear, colorless solution. Turbidity would indicate deterioration and the calibrator should not be used.

INSTRUMENTS

Use suitable spectrophotometer or colorimeter calibrated at 480 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Unhemolyzed serum separated form the blood clot as soon as possible. Plasma may be used.

2. Grossly hemolyzed or lipemic samples should not be used.

3. Do not take blood from a limb receiving an infusion.

4. Cerebrospinal fluid may be used.

SAMPLE STORAGE:

Serum chloride appears stable for 7 days at 2-8ºC.and three month frozen. Store in tightly sealed container.

ADDITIVES:

No special additives or preservatives are required.

INTERFERING SUBSTANCES:

Patients receiving bromide may show falsely elevated chloride levels. Young et al (4) have reviewed drug effects on serum chloride.

PROCEDURE

MATERIALS PROVIDES:

CHLORIDE REAGENT (Cat. No. 2451), CHLORIDE BLANK (Cat. No. 2452), and CHLORIDE CALIBRATOR (Cat. No. 2453).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.02 mL micropipettor

2. 4.00 mL pipetor or dispenser

3. Timer

4. Test tubes and rack

REACTION CONDITIONS:

Wavelength 480nm

Filter Selection 470 - 500 nm

Reaction Type Endpoint

Incubation Temperature 15-30ºC

Incubation Time 5 minutes

Sample Volume 0.005 ml.

Reagent Volume 1.0 ml.

Total Volume 1.005 ml.

Low Normal 96 mEq/L

High Normal 106 mEq/L

Calibrator Value 100 mEq/L

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTES:

1) For instruments requiring a total volume of 1.0mL., increase Chloride Reagent volume to 2.0 mL and sample volume to 0.01 mL. Proceed as outlined.

2) It is important to use the CHLORIDE BLANK SOLUTION as sample for the Reagent Blank in order to correct for the limited linearity when using single point calibration.

1. Dispense 1.00 mL of reagent into tubes labeled Chloride Blank, Calibrator, Control, Sample 1, etc.

2. Place 0.005 mL Specimen into appropriately labeled tube.

3. Permit the test samples to stand at room temperature (15-30ºC) for 5 minutes.

4. Adjust instrument to zero absorbance at 480 nm using Chloride Blank.

5. Read and record absorbance values for Calibrator, Controls and Unknowns.

NOTE:  For a direct read-out instrument, zero with Chloride Blank and set read-out to concentration value of the Calibrator. Read the Unknown concentrations directly.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read with 60 minutes after color development.

CALIBRATION:

It is not necessary to determine a Calibration curve with this procedure since the reaction is linear in a range of 70-140 mEq/L However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. CHLORIDE CALIBRATOR (CAT. NO.2453) and CHLORIDE BLANK (CAT. NO.2452) are supplied for this purpose.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials ( normal and abnormal) analyzed in the same manner employed for the Unknowns. EAGLE DIAGNOSTICS makes available CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient results are reported.

CALCULATION OF RESULTS

The following equation is used to determine Unknown concentrations

Unknown (mEq/L) =

Abs. Unk.

--------   X   Cal. Conc. (mEq/L

Abs. Cal.

EXAMPLE:

A 100 mEq/L Calibrator had an Abs. = 0.820 while the Unknown Abs. = 0.780. The chloride concentration of the Unknown is:

0.780

-------   X   100 mEq/L  =   95 mEq/L

0.820

LIMITATIONS

1. Exercise care not to touch pipette tips with the fingers since chloride contamination may result.

2. Exposure of samples or reagent to hydrochloric acid fumes may cause high results.
 
 

EXPECTED VALUES  (5)

SERUM  96 - 106 m Eq/L

CSF (ADULT)      118 - 132 mEq/L

CSF (INFANT)     110 - 130 mEq/L

This range represents the 95% confidence interval obtained from a clinically normal population. It is recommended that each laboratory establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linearly in the range of 70 - 140 mEq/L

PRECISION:

Within Run - Normal and abnormal control sera were assayed 20 times each for within run precision.

MEAN / STD. DEV. / %CV

Normal 103.6 / 1.47 / 1.42

Abnormal 113.5 / 1.20 / 1.05

Run to Run - Normal and abnormal control sera were  assayed for 10 working days to establish run to run precision.

Normal 103.7 / 1.42 / 1.37

SPECIFICITY:

A comparison of this CHLORIDE PROCEDURE with another widely used commercial method showed a 97% correlation in the normal and abnormal range.

SENSITIVITY:

The CHLORIDE PROCEDURE HAS A SENSITIVITY OF 0.12 mEq/L per 0.001 absorbance unit.

REFERENCES

1. Tietz, N.W. Fundamentals of Clinical Chemistry, 2nd ed., W.B. Saunders Co., Philadelphia 1966,p. 1183.

2. Zall, D.M., Fisher, D., and Garner, M.Q., Anal. Chem. 10, 918 (1964).

3. Skeggs, L.T., and Hochstrasser, H.C., Clin. Chem. 10, 918 (1964).

4. Young, D.S., Pestaner, L.C. and Gibberman, V., Clin.Chem., Vol 21, p. 277D (1975).

5. Tietz, N.W., Clinical Guide to Laboratory Tests, 2nd ed., W.B. Saunders Co., Philadelphia, p.118 (1990).