SUMMARY AND EXPLANATION

Elevated levels of lactate dehydrogenase are associated primarily with myocardial infarctions. A maximum level is reached usually  within 48 hours after onset of pain and then decreases slowly over the next 10 days. The degree of elevation is used in assessing the extent of damage to the heart muscle. Increased LD levels have also been noted in liver disease, pernicious anemia, renal disease and some cases of skeletal muscle trauma.

Wroblewski and LaDue (1) presented the first UV kinetic procedure for the determination of LDH activity. Their reagents utilized the pyruvate to lactate reaction which have become the method of choice because it offers a wider range of linearity and no preincubation  step. The  EAGLE DIAGNOSTICS PROCEDURE is based on this basic reaction and has been optimized for greater sensitivity and linearity.

PRINCIPLE

The following reaction is catalyzed by lactate dehydrogenase:

Lactate + NAD    LD   Pyruvate dehydrogenase

Formation of the reduced nicotinamide adema dinucleotide (NHDH) produces an increase in absorbance at 340 nm. The rate of absorbance change is directly proportional to the activity of LD in the sample.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent set Cat. No. 3270 provides:

LDH (LD) REAGENT - (Cat. No. 3271)

REACTIVE INGREDIENTS:

After reconstitution, the solution will have the following approximate concentration: DL-Lactate-90 mM; NAD-7 nM; Buffer, Stabilizer and Filters have been added. pH=9.0+0.2.

REAGENT PREPARATION :

Reconstitute the reagent with the amount of distilled water stated on the vial label. Reseal the vial, swirl gently to dissolve. Do Not Shake.

PRECAUTIONS:

Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amount of water.

STORAGE AND STABILITY:

Store at 2-8ºC. Stable until expiration date if vial remains sealed and unopened. After reconstitution, the reagent is stable for 7 days stored at 2-8ºC or for 8 hours stored at room temperature.

DETERIORATION:

In a sealed vial, the reagent should be a dry, white powder. After reconstitution the reagent should be a clear colorless solution. Turbidity indicates deterioration and the reagent cannot be used. In addition, if the reconstituted reagent has an absorbance of greater than 0.8 when read against DH20 at 340 nm, it should not be used.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 340 nm and a heating block capable of maintaining 30ºC or 37ºC.

SPECIMEN COLLECTION

Use fresh, unhemolyzed serum. Red cells contain large amounts of LDH.

SAMPLE STORAGE:

LDH in serum is reported to be stable for two days at room temperature. Do not freeze or expose to high temperatures as this will inactivate the LDH isoenzymes.

ADDITIVES:

No special additives or preservatives are required.

INTERFERING SUBSTANCES:

Some drugs and substances will affect LDH activity. Young et al (3) have reviewed drug effects on serum.

PROCEDURE

MATERIALS PROVIDED:

LDH Reagent (Cat. No. 3271)

MATERIALS REQUIRED BUT NOT PROVIDED:

1. Instrument capable of reading at 340nm

2. 0.025 micropipettor

3. 1.0ml pipette or dispenser

4. Incubator capable of maintaining 30ºC or 37ºC.

5. Timer, test tubes and rack

REACTION CONDITIONS:

Wavelength 340nm

Incubation Temperature 30ºC or 37ºC

Prewarm Substrate 5  minutes

Preincubation Time 1 minute

Sample Volume 0.025  mL

Reagent Volume 1.0 mL

Total Volume 1.025 mL

Calibration Factor 6592

Normal 80 - 285 IU/L

PREPARATION OF WORKING REAGENT:

Reconstitute the reagent with the amount of distilled water specified on the vial label. Swirl to dissolve. Do Not Shake.

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE:

For instruments requiring a total volume 1.0 mL  to read, increase Reagent  Volume to 2.0mL and Sample Volume to 0.05mL. Proceed as outlined.

1. Pipette 1.0 mL of reconstituted reagent in tubes labeled Control, Sample 1, etc.

2. Prewarm tubes at 37ºC for five minutes.

3. Adjust instrument to zero absorbance at 340nm using distilled water.

4. Place 0.025 mL specimen into appropriate tube, mix and incubate at 37ºC for one minute.

5. After one minute Preincubation, record the absorbance (A1 reading). Return  the  tube to the 37ºC incubator.

6. After "exactly one minute",  read and record the absorbance (A2 reading).

NOTE:  If  the instrument  used  is equipped with a temperature controlled cuvette, the reaction mixture may be left in the cuvette while the absorbance readings are taken.

STABILITY OF FINAL  REACTION:

The reaction is ongoing, the timing of the readings must  be accurate.

CALCULATION OF RESULTS

1. One  International  Unit  (IU/L) is  defined as the amount of  enzyme  that catalyzes  the transformation of  one micromole of  substrate per minute. Calculation factor = 6592  as derived  from the following equation.
 
 

          (A2 X A1) x 1.025  x 1000

IU/L    --------------------------   = (A2 X A1) x 6592

                 1 x 6.22 x 0.0245

Where:

(A2 x A1)   = Change in absorbance

1.021      = Total reaction volume in mL

1000        = Conversion of IU/mL  to  IU/L

1             = Light path in cm.

6.22         = Millimolar absorptivity of NADH

0.025       = Specimen volume in mL

Example: The initial reading (A1) Abs = 0.495,  the second reading (A2) Abs. = 0.513, the (A1- A1) =0.036 x 6592 (factor) =  237 IUL Conversion to SI Units (nkat/L); IU/L x 16.67 = SI

CALIBRATION:

The reliability of test results should be monitored routinely using suitable quality control materials  (normal and abnormal) analyzed in the same manner used for 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 values are reported.

LIMITATIONS

Specimens with values greater than 800 IU/L should be diluted 1:1 with saline and

reassayed multiplying the result by two.

EXPECTED VALUES

MALES 50  - 166 IU/L (30ºC)

80  -  285 IU/L (37ºC)

FEMALES 60  -  132 IU/L(30ºC)

103 -  227 IU/L(37ºC)

This range represents the 95% confidence interval from a  clinically normal population. Each laboratory should establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

PRECISION:

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

MEAN / STD.DEV. / %CV

Normal 145 / 2.7 / 1.8

Abnormal 431 / 6.1 / 1.4

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

Normal 146 / 2.4 / 1.6

Abnormal 431 / 6.1 / 1.4

SPECIFICITY:

A comparison of this LDH PROCEDURE  with  another widely used commercial method

showed  a 99%  correlation   with 25 serum specimens in the normal and abnormal range.

SENSITIVITY:

This LDH PROCEDURE has sensitivity of 6.5 IU/L per 0.001 absorbance units.

REFERENCES

1. Wroblewski, F.,La Due,  J.S.,Prac. Soc. Exp. Bio. Med., Vol. 90, p.120(1955)

2. Kreutzer, H.H., et al., Clin. Chem. Acta 9, p. 94 (1964).

3. Young, D.S., Pestaner, L.C. and Gibberman, V., Clin. Chem.21, p. 272D (1975).