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Calcium

The document provides instructions for using the Arsenazo III method to determine calcium levels in serum or plasma samples through a colorimetric assay where calcium combines with Arsenazo III to form a colored complex, with the intensity of the color proportional to the calcium concentration. Normal calcium levels in serum/plasma are listed as 8.7-11.0 mg/dl, with the procedure being linear up to a calcium level of 20 mg/dl, requiring sample dilution for values exceeding that limit.

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Dinesh Sreedharan
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811 views1 page

Calcium

The document provides instructions for using the Arsenazo III method to determine calcium levels in serum or plasma samples through a colorimetric assay where calcium combines with Arsenazo III to form a colored complex, with the intensity of the color proportional to the calcium concentration. Normal calcium levels in serum/plasma are listed as 8.7-11.0 mg/dl, with the procedure being linear up to a calcium level of 20 mg/dl, requiring sample dilution for values exceeding that limit.

Uploaded by

Dinesh Sreedharan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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CALCIUM ASSAY PROCEDURE:

(Arsenazo III method) Wavelength/ Filter : 630nm


For the determination of Calcium in serum or plasma Temperature : R.T
(For In vitro Diagnostic Use Only)
Light Path : 1 cm

SUMMARY: Pipette into clean dry test tubes labelled as Blank (B), Standard (S), and Test (T):
Calcium, in the body, is found mainly in the bones (approximately 99%). In serum calcium Addition Sequence B(µl) S(µl) T(µl)
exists equally in a free ionized form and in a bound form (with albumin). Hence a decrease
in Albumin causes lower Calcium levels and vice versa. The levels of Calcium in serum Calcium Reagent(A1) 1000 1000 1000
depends on the parathyroid hormone. Increased Calcium levels are found in bone Standard(S) - 10 -
tumours, hyperparathyroidism. Decreased levels are found in hypoparathyroidism, renal Sample - - 10
failure, rickets, Vitamin D deficiency and pancreatitis.
Mix well and incubate at R.T. for 3 min. Measure the absorbance of the Standard (Abs.
S), and Test Sample (Abs. T) against Blank.
PRINCIPLE:
Calcium combines specifically with Arsenazo III at neutral pH to form a blue purple
CALCULATIONS:
colored complex. Intensity of the color formed is directly proportional to the amount of
Abs.T
calcium present in the sample. Calcium in mg / dl = -------------------------- x 10
Calcium + Arsenazo III Blue Purple coloured Complex Abs.S

CONTENTS: NORMAL REFERENCE VALUES:


Serum/Plasma : 8.7 - 11.0 mg/dl
PACK SIZE CALCIUM REAGENT(A1) STANDARD (S)
Urine : 100 - 400 mg/24 hours
1x50ml 1x50ml 5ml
LINEARITY:
STORAGE & STABILITY: This procedure is linear up to 20 mg/dl. If values exceed this limit dilute the sample
Reagents are stable at RT till the expiry mentioned on the labels. with distilled water and repeat the assay. Calculate the value using the proper dilution
factor.
REAGENT PREPARATION: NOTE:
Reagents are ready to use. Protect from bright light. As calcium is a very widely distributed ion, care should be taken to avoid any
contamination. All glassware being used for the test should first be rinsed with 1% or
SAMPLE MATERIAL: 0.1 N HCL and then with good quality deionised water before use. It is suggested that
Serum/ Heparinized plasma. Calcium is reported to be stable in serum for 7 days at 2- after the rinsing of the tubes with HCL the reagent be pipetted in their respective tubes
8°C. and the tubes be rinsed with the reagent. The reagent then should be pooled together
in the 'blank' tube and repipetted out into the 'standard' and 'test' test tubes. This will
GENERAL SYSTEM PARAMETER: ensure that any remaining contamination will be carried over equally in all the tubes.
Reaction Mode End Point Sample Volume 10 µl For flow cell cuvettes it is suggested that some reagent be aspirated before the blank
to take away any contamination in the flow through tubing or cuvette which may cause
Wavelength 630 nm Reagent volume 1000 µl a higher than the actual blank of the reagent. Chelating agents such as EDTA, present
Blank Reagent Blank Standard 10mg/dl even in traces, prevent the formation of the colour complex, hence necessary care
should be taken during the assay.
Incubation 3 min at R.T Reaction Slope Increasing
Delay Time 5 Sec Linearity 20 mg/dl REFERENCE :
1. Smith, H.G. Jr.et al, (1979) Biochem 18:5067.
Read Time 5Sec Units mg/dl

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