Development of an enzymatic assay to measure lactate in perchloric acid-precipitated whole blood
Jun Lu1, Brady S. Pulsipher2, David G. Grenache1, 3
1ARUP
Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA
2ARUP
3Department
Laboratories, Salt Lake City, UT, USA
of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA
Introduction
• Specimen collection and handling:
- Whole blood anticoagulated with heparin or
EDTA was collected from healthy donors and
immediately transferred to a 1 mL pre-chilled
tube containing 2 mL 8% (w/v) perchloric acid.
- Samples were mixed well for 30 sec, placed on
ice for 10 min, then centrifuged without delay to
obtain a protein-free supernatant.
• Lactate measurement:
Lactate was oxidized to pyruvate by lactate
oxidase and the resulting H2O2 produced was
oxidized by a chromogen system to a colored
chromogen that was detected at 540 nm on a
Roche cobas c501 chemistry analyzer.
• Analytical validation:
Sample processing effects, accuracy, linearity,
imprecision, analytical sensitivity, and stability
were
studied.
Reference
intervals
were
established from 116 healthy adults.
• The study was approved by the University of Utah
Institutional Review Board.
Acknowledgement
Support for this study was provided by the ARUP
Institute for Clinical and Experimental Pathology.
y = 1.00x + 0.05
R² = 1.00
12
Lactate measured, mM
incubation, supernatant separated
incubation, supernatant not separated
ANOVA P value < 0.0001
1.5
Figure 5. The lactate non-parametric reference interval was 0.312.00 mmol/L using samples obtained from 116 healthy adults.
14
10 min, supernatant separated
2.0
10
8
6
4
2
0
0
1.0
2
4
6
8 10 12
Expected value, mM
14
Table 1. Delayed addition of whole blood to perchloric-acid
significantly increased lactate concentrations.
ANOVA P value = 0.0033
0.5
N
0.0
1
2
3
4
5
6
Subject
7
8
9
10
Figure 2. Lactate measured in an acid-precipitated whole blood
supernatant was comparable to lactate measured in plasma.
Lactate in acid-precipitated WB, mM
Method
Series
Chilled collection tube, kept on ice for
1
Series
Room temperature collection tube, no
2
Series
Room temperature collection tube, no
3
Lactate, mM
• Pyruvate is the end product of glycolysis and the
immediate and only precursor of lactate.
• Blood pyruvate concentrations have clinical utility
only when measured in conjunction with lactate in
the same sample in order to calculate the
lactate:pyruvate (L:P) ratio. But pyruvate is
measured in whole blood added to perchloric-acid
while lactate is measured in whole blood or
plasma.
• The objective of this study was to develop a
clinical assay for the quantitative determination of
lactate in whole blood specimens sent to ARUP
for pyruvate testing, so the L:P ratio can be
established from the results of these two tests in
one specimen.
Results
Figure 4. The analytical measuring range was linear between
0.18 and 13.15 mmol/L.
Figure 1. Alternate methods of sample collection and processing
using perchloric acid collection tubes significantly decreased
lactate concentrations.
11
% increase
5
0
1.29
–
5
30
1.60
24.0
5
60
2.02
56.9
5
120
2.27
76.0
Table 2. Precision was determined by measuring lactate in two
patient pools in three replicates once each day for 10 days.
y = 0.93x - 0.23
R² = 0.95
10
Time (min) Mean lactate (mM)
9
Mean concentration
8
(mM)
(%)
(%)
(%)
7
10.89
0.6
0.9
1.1
1.58
5.7
2.3
6.1
6
5
4
Repeatability Between‐day
Within‐laboratory
Table 3. Recovery was determined by adding lactate at two
different concentrations to 4 blood samples prior to adding the
sample to perchloric acid.
3
2
1
At 11.89 mmol/L , %
At 6.29 mmol/L , %
Sample #1
105.9
111.6
Sample #2
105.9
108.8
Sample #1
106.8
108.2
Sample #2
103.4
112.7
0
0
1
2
3
4
5
6
7
8
9
10 11
Lactate in plasma, mM
EDTA
Analytical sensitivity: The limit of blank was 0.02 mmol/L .
The limit of detection was 0.18 mmol/L.
Heparin
Figure 3. The stability of lactate in a protein-free supernatant was evaluated by four specimens at two concentration levels after storing
the samples at three different temperatures over various times. Lactate is stable for 8 hours at ambient, 21 days at 4-8 °C and 30 days
at -20 °C .
12
12
12
10
10
10
8
8
8
6
6
6
4
4
4
2
2
2
0
0
t0
2 hr
4 hr
Ambient
Figure 6. The distribution of the L:P ratio was 6-39 determined
from lactate and pyruvate measurements obtained from samples
collected from 116 healthy adults.
8 hr
t0
1 day
7 days
14 days 21 days
4-8 °C
0
t0
14 days
21 days
-20 °C
30 days
Conclusions
• Lactate can be quantified in the same proteinfree supernatant used for the measurement of
pyruvate.
• Alternative methods of sample collection and
processing result in falsely decreased
concentrations of lactate.
• Delayed addition of whole blood to perchloric
acid
significantly
increased
lactate
concentrations.
• In a protein-free supernatant, lactate is stable
for 8 hours at ambient, 21 days at 4-8 °C and
30 days at -20 °C.
• The lactate reference interval was determined
to be 0.31-2.00 mmol/L.
• Determining the L:P ratio from a single sample
likely avoids pre-analytical sources of error
that can occur when two separate samples are
collected and processed for lactate and
pyruvate testing.