Polyethylene Glycol Precipitation for Detection of Eight Macroenzymes
Sara P. Wyness1, Joshua J.H. Hunsaker1, Sonia L. La’ulu1, Lokinendi V. Rao, PhD2,William L. Roberts, MD, PhD1,3
1ARUP
Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 2UMASS Memorial Medical Center, Worcester, MA, 3Department of Pathology, University of Utah, Salt Lake City, UT
American Association for Clinical Chemistry Annual Meeting, Anaheim, California.
ABSTRACT
July 25 – 29, 2010
RESULTS
Table 4. Passing-Bablok Regression Summary (Neat Concentration vs. PEG Supernatant)
RI samples (N=120)
Analytes
Table 2. Summary of Imprecision
Table 1. Non-parametric Reference Interval Summary
Neat
(U/L )
Supernatant
(U/L)
Recovery
(%)
30 – 112
28 – 124
49 – 136
Analytes
ALP
9 – 47
ALT
2 – 14
Analyte
ALP
AMYL
23 – 112
7 – 32
41 – 81
AST
13 – 33
4 – 18
25 - 89
AMYL
AST
30 – 313
CK
18 – 257
35 – 85
CK
8 – 68
GGT
6 – 48
50 - 124
GGT
LDH
110 – 205
26 – 88
18 - 53
LIP
18 - 68
8 – 30
32 - 75
Sample
Mean
Concentration
(U/L)
Within
Run
CV(%)
Between
Day
CV(%)
Neat
278.7
0.4
4.4
4.4
17.9
1.8
7.5
7.7
Neat
180.6
0.8
2.2
2.3
Supernatant
26.7
3.8
4.0
5.5
Neat
275.8
0.5
1.1
1.3
Supernatant
88.4
1.1
3.7
3.8
Neat
229.4
0.6
1.7
1.7
Supernatant
61.7
1.7
3.3
3.7
Neat
586.6
0.6
1.4
1.6
Supernatant
156.0
0.6
3.5
3.6
Neat
123.9
0.6
1.7
1.9
Supernatant
12.6
5.3
6.4
8.3
Neat
LDH
LIP
Mean
Total Recovery
CV(%)
(%)
Supernatant
10 - 57
ALT
ALP
0.5
1.4
1.5
Supernatant
37.0
3.8
6.4
7.4
Neat
142.7
377.4
0.9
1.6
1.8
Supernatant
26.7
1.8
5.8
6.1
12.8
29.5
20.3
19.6
37.4
Samples with total enzyme activities > URL
1
N
Mean % recovery,
(± 2SD)
N with mean %
recovery, < LRL2
N with mean %
recovery, < -2SD
120
80 (49-111)
3
3
29 (4-54)
148
29 (8-50)
4
1
AMYL
57 (39-75)
182
58 (26-90)
11
2
AST
52 (23-81)
240
46 (20-73)
9
4
CK
60 (38-82)
79
62(37-88)
3
3
GGT
86 (49-123)
179
51 (18-84)
90
3
Mean % recovery,
(± 2SD)
ALP
96 (66-125)
ALT
LD
33 (16-50)
244
32 (2-61)
43
1
LIP
49 (28-70)
137
40 (18-63)
35
1
AMYL
50
60
40
20
0
0
0
1500
% Recovery
% Recovery
100
Neat Concentration (U/L)
100
80
80
60
40
20
0
1000
2000
3000
4000
500
1000
1500
0
15000
3.1
112-1158
ALT
0.669
0.29
-0.3
6-71
148
0.988
0.35
-6.6
47-3753
AMYL
0.887
0.53
1.6
18-133
182
0.966
0.66
-19.2
112-1731
AST
0.513
0.50
0.0
12-45
240
0.996
0.47
-2.2
33-10923
CK
0.961
0.56
2.0
21-501
79
0.971
0.63
-6.1
201-19020
GGT
0.951
0.67
3.3
7-79
179
0.866
0.50
-0.2
68-3126
LDH
0.485
0.51
-29.3
86-219
244
0.764
0.32
-1.7
205-5160
LIP
0.803
0.40
2.4
17-80
137
0.987
0.36
6.7
68-9313
6000
4000
2000
y = 0.65x + 31.6
R = 0.997
0
100
50
20000
1000
2000
3000
Neat Concentration (U/L)
10000
4000
10000
15000
LIP
60
40
0
0
2000
4000
6000
8000
Non-macroenzyme CK activity
by electrophoresis
• All analytes demonstrated good correlation between neat and PEG supernatant results for samples >URL with r ≥ 0.95
with the exception of GGT and LD, r = 0.87 and 0.76 respectively. All slopes agreed well with the calculated mean
recoveries (Table 3) with a maximum difference of 8%.
References
80
% Recovery
% Recovery
% Recovery
10000
5000
LD
100
0
5000
0.81
• PEG precipitation appears to recover macroenzymes sufficiently compared to electrophoresis (R = 0.9972) (Figure 2).
Neat Concentration (U/L)
20
Neat Concentration (U/L)
0.949
40
2000
80
0
60
Neat Concentration (U/L)
150
0
120
• Using the LRL of recovery for RI samples as cutoffs proved useful for ALP, ALT, AST and CK. (Table 3 and Figure
1). For AMYL, GGT, LD and LIP setting the cutoffs closer to the -2SD for samples greater than the URL may work
better because the LRL cutoff resulted in to many possible macroenzymes.
0
0
GGT
CK
50
28-126
•Reference intervals after PEG precipitation are slightly lower then RI established for neat samples, but overlap was
observed for most analytes. The range of recoveries observed for RI samples is dependent upon analyte being measured
(Table 1).
20
Neat Concentration (U/L)
150
100
Range
-3.5
• PEG precipitation can be a useful method for identifying macroenzymes. We have established method-specific reference
intervals.
AST
100
% Recovery
80
1000
Intercept
1.00
• Total imprecision was acceptable for all analytes. There was an increased variability seen after PEG precipitation total
CVs ranging from (3.6 – 7.7%). The CVs prior to PEG precipitation ranged from (1.3 – 4.4%) (Table 2).
• Mean % recoveries for all eight enzymes ranged from 29 – 96% for the healthy subjects and 29 – 80% for the samples
with activities above the URL.
ALT
ALP
150
500
Slope
0.906
DISCUSSION and CONCLUSIONS
Figure 1. Recovery Plots
0
R
Figure 2. Comparison of non-macroenyzme CK activity
detected by electrophoresis versus PEG precipitation
53.2
URL = Upper Reference Limit
LRL = Lower Reference Limit established from healthy subjects % recoveries
% Recovery
•PEG precipitation was performed using a PEG 8000 solution (250 g/L in PBS) in a 1:2 dilution
with patient samples. Samples were vortexed for a minimum of 30 seconds, incubated at room
temperature for a minimum of 10 minutes prior to being centrifuged at 10,000x g for 5 minutes.
•Supernatant was removed from specimen and both neat and PEG supernatant were analyzed on
Roche Modular Analytics P (Roche Diagnositic, Indianapolis, IN), for the following analytes:
ALP, ALT, AMYL, AST, CK, GGT, LD, and LIP.
•RI were determined with 120 serum samples from apparently healthy subjects (60 males and 60
females, age 19-60 years).
•Imprecision was assessed using quality control material from Bio-Rad (Bio-Rad Laboratories,
Irvine, CA). Control material was precipitated with PEG solution and the neat and PEG
supernatant were analyzed on all analytes, once a day, for twenty days, in replicates of two, using
a fresh aliquot for each run.
•PEG precipitation was performed on various numbers of specimens (n = 79-244) with total
activities that were greater than the URL from healthy subjects.
•An additional 40 CK-macroenzyme samples, initially tested and confirmed by electrophoresis,
were used for comparison between non-macroenzyme activity detected by electrophoresis versus
non-macroenzyme activity detected after PEG precipitation (Figure 1).
•Along the x-axis is the non-macroenzyme activity detected by electrophoresis. This was
determined by multiplying the calculated non-macroenzyme activity (based on removing the
total % macroenzyme determined by electrophoresis) by the total CK enzyme activity.
•On the y-axis is the non-macroenzyme activity measured after PEG precipitation. The PEG
supernatant concentration was divided by the mean % recovery (0.6) as seen in the healthy
population, to correct for the reduced recovery after PEG precipitation.
•All study specimens were de-identified and stored at -20˚C or colder prior to analysis.
•The Institutional Review Board of University of Utah, Salt Lake City, UT, approved all studies
using human samples.
2
% Recovery
MATERIALS AND METHODS
N
0
Reference Interval Samples
1
Range
53.7
Table 3. Mean % Recovery
Analytes
Intercept
60.1
INTRODUCTION
Macroenzymes are enzymes in serum that have formed high-molecular-mass complexes,
either by self-polymerization or by association with other serum component. There are a number of
enzymes typically measured in a clinical laboratory that have been found to form macroenzyme
complexes. The formation of these complexes results in an unexpected increase in serum enzyme
activity. Detection of these complexes is important because of the diagnostic confusion and
therapeutic errors which can be caused by macroenzyme interference. Laboratory methods for
detecting macroenzymes are often complicated and required specialized equipment. Polyethylene
glycol (PEG) precipitation methodology is both simple and cost effective; however reference interval
data is limited. The aim of this study is to establish reference intervals for eight macroenzymes using
PEG precipitation as well as evaluate the overall performance of the methodology.
Slope
Non-macroenzyme CK activity
by PEG precipitation
Serum macroenzymes may cause elevations in total enzyme activity leading to diagnostic
confusion. Polyethylene glycol (PEG) precipitation is a useful technique which can help detect
macroenzymes in serum. However, reference intervals (RI) need to be established for serum
samples after PEG precipitation. We analyzed alkaline phosphatase (ALP), alanine
aminotransferase (ALT), amylase (AMYL), aspartate aminotransferase (AST), creatine kinase
(CK), γ-glutamyltranspeptidase (GGT), lactate dehydrogenase (LD) and lipase (LIP) before and
after PEG precipitation on the Roche Modular Analytics P. We evaluated 120 samples from
healthy subjects to establish RI. For comparison, an additional 79-244 samples with enzyme
activities greater than the upper reference limit (URL) were tested for each analyte. RI and mean %
recovery were determined for all analytes. Using the URL’s established from the total enzyme
activity of the healthy subjects, the mean % recoveries were determined for all samples with
activities > URL. A URL of 200 U/L was used for CK. Mean % recoveries for all eight enzymes
ranged from 29-96% for healthy subjects and 29-80% for samples above the URL. Using a -2SD
cutoff from the mean % recovery for CK samples above the URL, we were able to identify 3
possible macro-CK samples of which 2 were confirmed to contain macro-CK by electrophoresis.
An additional 25 confirmed macro-CK identified by electrophoresis were analyzed for macro-CK
by PEG. Of the 25 samples, 17 had recoveries below the -2SD cutoff. In the current study, ALP,
ALT, AMYL, AST and CK had mean % recoveries for samples with activities above the URL that
compared well to those established using samples from healthy subjects. However, GGT, LDH and
LIP had recoveries after PEG precipitation that differed between the healthy subject and greater
than URL sample sets. Therefore, further analysis including definitive macroenzyme detection
needs to be performed on these three analytes before PEG precipitation can be implemented for
clinical use.
Samples > URL
R
1.
60
40
2.
20
3.
0
0
2000
4000
Neat Concentration (U/L)
6000
0
2000
4000
6000
8000
Davidson DF, Watson DJ. Macroenzyme dectection by polyethylene glycol precipitation. Ann Clin Biochem
2003; 40(Pt 5):514-520.
Remaley AT, Wilding P. Macroenzymes: biochemical characterization, clinical significance, and laboratory
detection. Clin Chem 1989; 35:2261-2270.
Turecky L. Macroenzymes and their clinical significance. Bratisl Lek Listy 2004; 105:260-263.
10000
Neat Concentration (U/L)
Figure 1. Recovery plots represent samples >URL of the healthy subjects, identified by . The mean of the sample set is represented by the solid black line, ±2SD from the
mean is the dashed black lines. The LRL established from the % recoveries of the healthy subjects is the solid blue line. The CK plot has four samples that were confirmed as
CK-macroenzymes by electrophoresis after PEG precipitation and testing, identified by the ▲. An additional subset of samples that were initially tested and confirmed as CKmacroenzymes by electrophoresis prior to PEG precipitation and testing are identified by ■.
Acknowledgements
The ARUP Institute for Clinical and Experimental Pathology for financial support.