1.0 INTENDED USE
This reagent is intended for the quantitative
determination of chloride in serum.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Chloride has been determined by a range of analytical
methods. Schales and Schales (10.1) described a titration method in which
chloride is reacted with mercury. The
endpoint of the titration is recognized by the formation of a blue color when
excess mercury ions react with diphenyl carbazone. The method was modified by Schoenfeld and
Lewellen (10.2) who used mercuric thiocyanate to produce mercuric chloride and
free thiocyanate ions. The thiocyanate
ions react with ferric ions to produce a red‑brown color which is
proportional to the chloride concentration. This procedure for chloride is a
modification of the Schoenfeld and Lewellen technique.
2.2 TEST PRINCIPLE
2 Cl‑ + Hg(SCN)2 ‑‑‑‑‑‑>
HgCl2 + 2 SCN‑
3 SCN‑
+ Fe+++ ‑‑‑‑‑‑> Fe(SCN)3 (Red Brown)
The absorbance of the ferric thiocyanate at 500 nm is
proportional to the concentration of chloride in the sample.
2.3 CLINICAL SIGNIFICANCE (10.5)
Chloride and bicarbonate are the principal anions in
blood; sodium and potassium the principal cations. The balance between these electrolytes is
frequently affected in disease states, thus meaningful interpretation of
chloride levels requires knowledge of other electrolyte concentrations.
Increased chloride levels can occur in nephritis,
prostatic obstruction, eclampsia and dehydration. Decreased levels are often associated with
impaired gastro-intestinal or renal function.
3.0 SPECIMEN COLLECTION AND HANDLING
3.1 PATIENT PREPARATION
No special
patient preparation is required.
3.2 SPECIMEN COLLECTION.
Fresh,
clear, unhemolyzed serum is the preferred specimen.
Use a
standard venipuncture tube to draw patient sample.
The amount of sample required will depend on the analyzer
used. The amount of serum required is in
the range of 5-25 µl. Call Biotron’s
technical service department at 1-800-595 8766 for the recommended sample
volume for your analyzer.
Record the patient's name, date and time of sample
collection and preparation.
3.3 SPECIMEN STORAGE
Serum should be separated from the cells as soon as
possible and may be stored at 2‑8°C (refrigerated) for up to one week.
It is recommended that testing be done as soon as
possible after sample collection and preparation. If testing cannot occur immediately, store
the sample properly using the guidelines above.
4.0 MATERIALS (2 X 125 ml)
(1 X 500 ml)
Reagents necessary for the determination of chloride are
included in the kit.
4.1 REAGENT
Chloride
reagent contains:
mercuric thiocyanate 0.031%
ferric nitrate 1.52%
nitric acid 0.24%
methanol 7.5%
4.2 WARNINGS AND PRECAUTIONS
For In Vitro Diagnostic Use. Not for Internal use in Humans or
Animals. In Vitro Diagnostics reagents
may be hazardous. Avoid ingestion and
skin or eye contact.
The reagent contains mercury and hence is toxic. Avoid contact with skin and eyes. Flush contacted area with water. Contact physician immediately if reagent is
taken internally.
4.3 REAGENT PREPARATION
The
reagent is ready to use as is.
4.4 REAGENT STORAGE AND STABILITY
All reagents included in the kit are stable until the
expiration date stated on the label when stored at 18‑26° C (room
temperature) and away from light.
4.5 ADDITIONAL MATERIALS REQUIRED
4.5.1 Spectrophotometer
or colorimeter capable of reading absorbance accurately at 500 nm.
4.5.2 1 cm cuvettes
or a flow cell capable of transmitting light at 500 nm.
4.5.3 Test tubes
capable of holding 3 ml.
4.5.4 Pipettes
capable of delivering the required volumes.
4.5.5 Distilled or
deionized water for preparing the reagent blank.
4.5.6 A timer for a
five minute incubation.
4.5.7 Normal and
abnormal control for quality control.
5.0 TEST PROCEDURE
The
following is a general procedure for use on a manual instrument.
5.1 PROCEDURE CONDITIONS
Wavelength 500
nm
Temperature 18‑26°
C
Mode Endpoint
Reaction time 5
minutes
Sample volume 10
µl
Reagent volume 2.0
ml
Total volume 2.01
ml
Sample to reagent
ratio 1:200
5.2 INSTRUMENT
Any instrument capable of reading absorbance accurately
with a sensitivity of 0.001 absorbance at 500 nm may be used. The band width should be 10 nm or less, stray
light 0.5% or less, and the wavelength accuracy within 2 nm.
5.3 CALIBRATION
The chloride assay is calibrated by referencing the
absorbance of the unknown sample to the absorbance of the calibrator.
5.4 PROCEDURE
5.4.1 Into separate
test tubes pipette 10 µl of distilled water, calibrator, or serum to be
assayed.
5.4.2 Add 2.0 ml of
chloride reagent and mix well.
5.4.3 Incubate at
18‑26°C (room temperature) for 5 minutes.
5.4.4 Read and
record the absorbance of each tube using the distilled water sample as the
reagent blank.
5.5 PROCEDURE NOTE
The final
reaction mixture is stable for 30 minutes.
Chloride is a common laboratory contaminant and care
should be taken to make sure that all glassware or plasticware used in the
analysis is free from contamination.
5.6 CALCULATION AND RESULTS
A
Chloride
(mEq/L) = ‑‑---‑‑- X concentration of calibrator
As
A =
absorbance of sample, As = absorbance of calibrator
Example:
0.450
Chloride
concentration = ‑‑‑‑-‑‑‑- X 100 mEq/L
= 82 mEq/L
0.550
with A =
0.450 and As = 0.550, concentration of calibrator = 100 mEq/L
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.4)
The range
of expected values is: 98 ‑ 103 mEq/L
These values are suggested guidelines. It is recommended that each laboratory
establish the normal range for the area in which it is located.
6.2 MEDICAL ALERT VALUES (10.6)
Each laboratory should establish low and high values
beyond which the patient would require immediate attention by a physician. If a "medical alert value" is
reached, always repeat the test to confirm the result and notify a physician if
the result is confirmed.
6.3 LIMITATIONS OF PROCEDURE
6.3.1 Severely
icteric, hemolytic, or lipemic samples require the use of a sample blank which
may be prepared by using 10 µl of sample and 2.0 ml of distilled or deionized
water.
6.3.2 This
procedure measures total halide concentration including chloride, bromide, and
iodide. The latter two do not interfere to a significant degree as their
concentrations in serum are normally less than 1 mEq/L.
6.3.3 A summary of
the influence of drugs on clinical laboratory tests may be found by consulting
Young, D.S. (3) Some examples of drugs
that affect the serum chloride test are: Acetazolamide, Bromides, M.,
Intra-Amniotic Saline, Phenylbutazone.
6.3.4 This
procedure is not recommended for use in determining sweat chloride for cystic
fibrosis.
7.0 QUALITY CONTROL
Standard practice for quality control should be applied
to this system. Commercially available
lyophilized controls can be used to monitor the daily acceptable
variations. Normal and abnormal controls
should be assayed at the beginning of each run of patient samples, whenever a
new reagent or a different lot number is being used, and following any system
maintenance.
A satisfactory level of performance is achieved when the
analyte values obtained are within the "acceptable range" established
by the laboratory.
8.0 CALIBRATION PROCEDURES
The chloride assay is calibrated by referencing the
absorbance of the unknown sample to the absorbance of the calibrator. Refer to your instrument manual for more
details.
Calibration is required with the use of a new lot of
reagent, any system maintenance or whenever indicated by quality control data.
9.0 PERFORMANCE CHARACTERISTICS
9.1 PRECISION
The estimates of precision shown below were obtained from assays of human control serum.
Within-Run
In this
study, replicates of 2 control sera were run.
Number
of
Samples Mean (mEq/L ) SD (mEq/L) CV
(%)
89 107 0.89 0.8
20 90 0.86 0.9
Between-Run
5 assays
were run each day for 10 days on a pool serum specimen.
Number
of
Samples Mean (mEq/L) SD (mEq/L) CV (%)
50 105 0.88 0.8
9.2 CORRELATION
A correlation study was done comparing this method (y)
and a similar comparative chloride procedure (x). The samples range between 61 to 140 mEq/L.
Number Regression Equation Correlation
of
Samples y=Biotron,
x=Comparative Coefficient
45 y = .988 x + 1.20 0.987
9.3 RECOVERY STUDY
Chloride of concentrations 25, 37.5 and 50 mEq/L was
added to a pool of human serum. The
recovery of added chloride averaged 102%.
9.4 LINEARITY
This procedure is linear from 70 to 140 mEq/L. Sample with chloride values above 140 mEq/L
should be diluted 1 to 1 with distilled water.
Re-run sample and multiply the final answer by 2.
9.5 SENSITIVITY
Absorbance values obtained by the above described
procedure show that an absorbance value of 0.005 optical density corresponds to
chloride concentrations of 1 mEq/L. This
may vary slightly from lot to lot.
10.0 REFERENCES
10.1 Schales, O.,
Schales, S.S., J. Biol. Chem. 140, 879 (1941).
10.2 Schoenfeld,
R.G., Lewellen, C.J., Clin. Chem. 10, 533 (1964).
10.3 Young, D.S.,
Effects of Drugs on Clinical Laboratory Tests, 3rd ed., Washington DC, AACC
Press (1990).
10.4 Henry, R.J.,
(ed.), Clinical Chemistry Principles and Technics, Harper and Row, New York,
(1974), p. 720.
10.5 Clinical
Diagnosis, 2nd ed., R.J. Henry, D.C. Cannon and J.W. Wilkinson, Eds., Harper
& Row, New York, NY 1974.
10.6 G.J. Kost,
"Critical Limits for Urgent Clinician Notification at U.S. Medical
Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704
Rev
10//99