Certified Reference Material for Measurements of Ionized Calcium and Electrolytes in Human Serum

JCCRM 321-6 (Na,K,Cl,IP,iCa,TCa&TMg)

Certificate of Analysis
The guideline of JCCRM JCCRM321 was established by the Committee on Blood Gases and Electrolytes
(JSCC/CBGE).of the Japan Society of Clinical Chemistry and this JCCRM321-6 was prepared and
certified by the Reference Material Institute for Clinical Chemistry Standards(ReCCS), This CRM meets
requirements for a human serum

reference material

for ISE measurements of ionized calcium as defined in IFCC Recommended Reference Method for the
Determination of the Substance Concentration of Ionized Calcium in Undiluted Serum,
Plasma or Whole Blood issued

by IFCC WG on ISE.

Direction for Use:

(1) For the Measurements of Ca

The serum in ampoule should be analyzed after full gas-liquid equilibrium under a
constant temperature is attained. As the certified value is defined at an incubation
temperature of 30.0, make sure to use the material according to the operating
procedure described on the next page.
(2) For the Measurements of Na, K, Cl, total Ca ,total Mg, and IP
In this case, there is no need to keep the serum in strict gas-liquid equilibrium. Thaw
the serum in ampoule at a room temperature and mix the serum by stirring the ampoule
at horizontal position to the right and left quickly more than 30 times. The serum
should be measured for several hours after thawing. For how to open the ampoule refer
to the operating procedure on the next page.

Notice and Warning to Users:

JCCRM 321-6 is a human source material, and handle as a biohazardous material capable of
transmitting infectious disease. This product was shown to be noncreative for HBs antigen, HCV
and HIV antibodies by our test methods. However, no known test method can give complete
assurance of absence of HIV, HCV antibodies, HBs antigen, and any other infectious agents.
Thus assume that JCCRM 321-6 is infectious, and handle it at the biosafety Level 2. Although
HBs-Ag, HCV and HIV antibodies have not been detected, exercise the same caution for
handling any other clinical specimens with the risk of infectious diseases.

Storage:
This certified reference material is shipped frozen (with dry ice) and, upon receipt,
should be stored in a freezer as soon as possible. A freezer temperature of 20 is
acceptable for storage up to one month. If a longer storage time, up to 9 months, is
anticipated, the material should be stored at or below70 C.
Specifications:
High and medium concentrations: 3 ampoules for each level, a total of 6 ampoules
(1.2 ml/ampoule)

Procedure for Measuring Ca2

Required equipments: Protective gloves, thermo-stated bath (the temperature should

be adjustable within 30 0.5), thermometer (readable in the
unit of 0.1 at around 30), syringe for sampling (two), and
putty (to seal the hematocrit tube)

Carry out the following operations at room

temperature in the range of 18 30.

Incubation
Immediately put the certified reference material
taken out from the refrigerator into a water bath
thermo-stated at a temperature of 30.0 0.5 and
leave it for 10 15 minutes as shown.
Note: Do not leave it in water for more than one
hour.

Gas-Liquid Equilibrium
In order to obtain gas-liquid equilibrium of the
serum strongly shake the ampoule to the right and
the left and up and down in the bath for more than
30 seconds. When too many air bubbles are formed,
they should be decreased by the ampoule being left
in the water for approximately one minute after
equilibrium.

Carry out the following operations as quickly

as possible. Do not over one minute.

Opening
Take out the ampoule from the bath and wipe the
water off with a sheet of paper, and drop the serum
attached onto the inner wall of the ampoule lightly
patting the ampoule by a finger. Break the neck of
the ampoule to the opposite direction of the white
dot. Take sufficient care not to damage the finger
by wearing protective gloves.

Sampling
Take 0.2 ml of the serum with a syringe and discard
the serum all together with air bubbles. Take
approximately 0.8 ml of the serum again within 3
4 seconds without involving air bubbles.

Measurement
Immediately introduce the serum taken in the
syringe into the measurement instrument. The
following introducing method is recommended.
In the case of injection method: Introduce the serum
by pressing the syringe to the sample inlet of the
instrument.

In the case of suction method: Remove the capillary

at the edge of the syringe. Insert the nozzle of the
instrument into the syringe and introduce the serum.
The serum left in the syringe can be separately
measured three times within five minutes if it is kept
in such a way not to contact the outside air. Note
that the serum in the syringe discharges the CO 2
gas if it contacts the outside air and increase its pH
level (the Ca2+ level decreases).

JCCRM 321-6
Certified Values and Expanded Uncertainties:

Medium Level

High Level

JCCRM 321-6M

JCCRM 321-6H

Ionized Calcium

1.220.03

1.520.04

(Ca)

(pH: 7.40)

(pH: 7.40)

Unit

mmol/l

Gas-liquid equilibrium temperature : 30

Note(1) The certified values of Ca2+ can be obtained at a measurement temperature of 37.
Note(2) As Ca2+ depends on the temperature of the gas-liquid equilibrium in the ampoule, make
sure to thaw the serum at 30.0 in a temperature-controlled water bath.
The expanded uncertainty U (95% confidence interval) shown in the above table for each
certified value was calculated using the equation U=ku, where u is the combined standard
uncertainty calculated according to the ISO Guide 35 and k is a coverage factor(k=2).
Traceability to SI units was assured though calibration against NMIJ primary standards and
other pertinent standards traceable to SI units as well as observance of quality assurance
manual in accordance with ISO Guide 35. Standard solutions and sample solutions used in
4

the measurements were prepared by gravimetric method using a calibrated balance.

Ca was determined by a IFCC reference cell, Umemoto-type ,reference cell with the
capillary tube type liquid junction which minimizes the residual liquid junction potential
difference.
Analyses for the certification and characterization of JCCRM 321-6 were
performed by the Reference Material Institute for Clinical Chemistry Standards.

Katsuhiko Kuwa, Chairman of

Committee on Blood
Gases/Electrolytes,
Japan Society of Clinical
Chemistry

Masao Umemoto, Head of

Reference Material Institute for
Clinical Chemistry Standards

Certified Values and Expanded Uncertainties:

To use the certified values of TCa, TMg, Na, K, Cl ,and IP, shake an ampoule vigorously
before use for getting homogeneous.,
The values below are at 25

Medium Level

High Level

JCCRM 321-6M JCCRM 321-6H

Unit

Total-Ca

9.38 0.08

11.82 0.10

mg/dL

Total-Mg

1.94 0.03

3.40 0.05

mg/dL

3.2 0.1

mg/dL

Na

142.1 0.5

155.7 0.5

mmol/l

4.14 0.02

5.63 0.03

mmol/l

IP (Inorganic Phosphorus)

Cl

104.5 0.4

121.5 0.6

mmol/l

The expanded uncertainty U(95% confidence interval) was obtained by U=ku, where u
was a standard uncertainty and k was a coverage factor (k=2.0 for Ca and Mg;k=2.0 for
IP:k=2.3 for Na:k=2.4 for K:k=2.0 for cl)
Assay Methods

Assay Methods
Total-Ca

atomic absorption spectrometry calibrated with SRM 909b.

Total-Mg atomic absorption spectrometry calibrated with SRM 909b.

IP

Ion chromatography

flame photometry calibrated with JCCRM 111, of which Na concentration

Na

was determined by the ion-exchange separation gravimetry (NIST definitive

method).

flame photometry calibrated with JCCRM 111, of which K concentration

was determined by isotope dilution mass spectrometry.

coulometric titration calibrated with JCCRM 111, of which Cl

Cl

concentration was determined by ion chromatography and coulometric

titration (its accuracy was certified by ID/MS).

Characteristics of the Serum:

This certified reference material for the measurement of ionized calcium in serum uses
human serum as raw material. Its physiochemical property and coexistent components are
as follows.

This certified reference material is shipped frozen (with dry ice) and, upon receipt, should
be stored in a freezer as soon as possible. A freezer temperature of 20 is acceptable for
storage up to one month. If a longer storage time, up to 9 months, is anticipated, the material
should be stored at or below70 .
6

Density

1.024 (25)

g/cm3

Viscosity

1.6 (20)

Pas

*Water conc.

0.930 (25)

kg/l

pH Medium Level

7.40 (37)

High Level

7.40 (37)

HCO

26 (37)

mmol/l

Br

<0.1

mmol/l

NO

<0.1

mmol/l

SO

0.4

mmol/l

NH

0.6

mmol/l

Citric acid

0.1

mmol/l

Lactic acid

mmol/l

Total protein

7.3

g/dL

Albumin

4.4

g/dL

Triglycerides

79

mg/dL

Total-cholesterol

158

mg/dL

Phospholipid

195

mg/dL

Water conc.: water mass concentration.

** Neither germicide nor stabilizer is added.

*** The above values are the ones of the medium concentration level of the serum, and
were measured by the routine methods and should not be used for assessment of
accuracy.

References:

1) Umemoto M, Tani W, Kuwa K, Ujihira Y : Measuring calcium in plasma, Anal.

Chem. 66 : 352A-356A (1994)
2) IFCC Recommended Reference Method for the Determination of Substance
Concentration Ionized Calcium in Undiluted Serum, Plasma or Whole Blood, Clin.
Chem. Lab. Med. 38: 1301-1314 (2000).
3) Committee for Blood Gases and Electrolytes/Japan Society of Clinical Chemistry:
Recommendation for the Concentration Measurement of Ionized Calcium in Blood
using ISEthe Measurement System and Calibration Method with Serum Reference
Material., Japanese Journal of Clinical Chemistry, 22 : 291-299 (1993) in Japanese
4) A reference method for the determination of calcium in serum, NBS Special
Publication 260-36 (1972)
5) Committee for Blood Gases and Electrolytes/Japan Society of Clinical Chemistry:
Recommendation for the Concentration Measurements of Sodium, Potassium and
Chloride in Blood using ISECalibration Method of the Accuracy with Serum
Reference Material. Japanese Journal of Clinical Chemistry, 22 : 279-290 (1993) in
Japanese
Contact Information
Reference Material Institute for Clinical Chemistry StandardsLaboratory of ReCCS
KSP A205, 3-2-1 Sakato
Takatsu-ku, Kawasaki City, Kanagawa 213-0012 Japan
TEL: 81-44-813-0055 FAX: 81-44-813-0224
E-mail: tani-wataru@reccs.net