LDH SCE mod.
Calculation
Using the absorbance readings calculate the mean absorbance change
liquiUV Test per minute ( A/min).
Lactate Dehydrogenase (EC 1.1.1.27) Calculate the LDH activity in the sample by multiplying A/min using the
following factors:
Package Sizes
[REF] 12214 16 x 5 ml Complete M-Test Kit Procedure 1
12014 10 x 10 ml Complete Test Kit Hg 334 nm 340 nm Hg 365 nm
12024 8 x 50 ml Complete Test Kit U/l (25°C, 30°C) = 10275 10080 18675
[IVD] A/min. x
Method 1 U/l (37°C) = 20390 20000 37060
A/min. x
"Modified method" based on the recommendations of the SCE
(Scandinavian Committee on Enzymes). Procedure 2
Principle Hg 334 nm 340 nm Hg 365 nm
LDH U/l (25°C, 30°C) = 8250 8095 15000
Pyruvate + NADH + H+ Lactate + NAD+ A/min. x
U/l (37°C) = 16345 16030 29705
Contents A/min. x
[REF] 12214 12014 12024
Conversion factor of the traditional units (U/l) in SI-units (kat/l):
[BUF] 16 x 4 ml 10 x 8 ml 8 x 40 ml
[SUB] 1 x 16 ml 2 x 10 ml 8 x 10 ml 1 U/l = 16.67 x 10-3 µkat/l
[BUF] Buffer/Substrate 1 µkat/l = 60 U/l
TRIS buffer (pH 7.35) 62.5 mmol/l Factor to convert results to the new IFCC recommended method:
Pyruvate 1.5 mmol/l
Sodium azide 0.095 % U/l (LDH SCE) x 0.4796 = U/l (LDH IFCC).
[SUB] Substrate Performance Characteristics
NADH 0.75 mmol/l Linearity
Sodium azide 0.095 %
If the absorbance change per minute ( A/min.) exceeds 0.150 at Hg
Reagent Preparation 334 nm, 340 nm or 0.070 at Hg 365 nm dilute 0.1 ml of the sample with
Procedure 1 with reagent start 0.9 ml physiological saline (0.9%) and repeat the assay using this dilution.
Multiply the result by 10.
The reagents are ready for use.
Typical performance data can be found in the Verification Report,
The reagents are stable, even after opening, up to the stated expiry date accessible via:
when stored at 2...8°C. [BUF] must be kept light protected. Contamination
of the reagents must be avoided! www.human.de/data/gb/vr/en-ldhuv.pdf
www.human-de.com/data/gb/vr/en-ldhuv.pdf
Procedure 2 with sample start
[²REF²] 12024: Pour the entire contents of one bottle [SUB] into one bottle Reference Values 2, 3
[BUF], mix thoroughly. Temperature 25°C 30°C 37°C IFCC4
[²REF²] 12214: Pipette 1 ml from bottle [SUB] into one bottle [BUF], mix Adults [U/l] 120-240 160-320 225-450
thoroughly.
Men [U/l] < 243
[²REF²] 12014: Pipette 2 ml from bottle [SUB] into one bottle [BUF], mix
Women [U/l] < 244
thoroughly.
The working reagent is stable for 3 weeks at 2...8°C and 3 days at Children [U/l] (up to up to 500
15...25°C. The working reagent must be kept light protected. 12 months)
Specimen Quality Control
Serum, heparinised or EDTA plasma. All control sera with LDH values determined by this method can be
employed.
Avoid hemolysis!
We recommend to use our animal serum based HumaTrol or our human
Loss of activity within 3 days 8% at + 4°C, 2% at 15...25°C. serum based SERODOS quality control sera.
Assay Automation
Wavelength: Hg 334 nm, 340 nm, Hg 365 nm Proposals to apply the reagents on analysers are available on request.
Optical path: 1 cm Each laboratory has to validate the application in its own responsibility.
Temperature: 25°C, 30°C or 37°C
Measurement: against air (decreasing absorbance) Note
[BUF] and [SUB] contain sodium azide (0.095%). Do not swallow. Avoid
Warm the reagents and the cuvettes to the desired temperature.
contact with skin and mucous membranes.
Temperature must be kept constant ( 0.5°C) for the duration of the test.
References
Procedure 1*
1. Z. Klin. Chem. Klin. Biochem. 8, 658 (1970), 10, 182 (1972)
Pipette into cuvettes 25°C, 30°C 37°C
2. Weißhaar, D. et al., Med. Welt 26, 387 (1975)
Sample 20 µl 10 µl
3. Witt, I., Trendelenburg, C., J. Clin. Chem. Clin. Biochem. 20, 235 - 242
[BUF] 1000 µl 1000 µl (1982)
Mix, incubate for 1 - 5 min. at 25°C, 30°C or 37°C. 4. Schumann G. et al., Clin.Chem.Lab.Med. 40, 643-648 (2002)
[SUB] 250 µl
Mix, read the absorbance after 1 minute and at the same time start EN-LDHUV INF 1221401 GB 02-2011-17 |
the stop watch. Read the absorbance again exactly after 1, 2 and 3
minutes.
Procedure 2*
Pipette into cuvettes 25°C, 30°C 37°C
Sample 20 µl 10 µl
Working reagent 1000 µl 1000 µl
Mix, read the absorbance after 1 minute and at the same time start
the stop watch. Read the absorbance again exactly after 1, 2 and 3
minutes. Human Gesellschaft für Biochemica und Diagnostica mbH
Max-Planck-Ring 21 · 65205 Wiesbaden · Germany
* Semi-micro method; for macro methods double the volumes. Telefon +49 6122-9988-0 · Telefax +49 6122-9988-100 · e-Mail human@human.de
