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Capillary plasma lipid profiles
Clinica Chimica Acta, 204 (1991) 189-198 0 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50
189
CCA05176
Capillary plasma lipid profiles Rebecca Mace, Bernadette May, Carol McCray, Trent Tracy, James Speirs and Charles J. Glueck Cholesterol Center of the Jewish Hospital, Cincinnati, OH (USA)
(Received 15 July 1991; revision received 18 September 1991; accepted 20 September 1991) Key words: Lipid profiles; Low density lipoprotein cholesterol (LDLC); High density lipoprotein cholesterol (HDLC); Coronary heart disease; Lipid Research Clinics (LRC) cholesterol; Standardization program
Introduction
Since the National Cholesterol Education Program (NCEP) [1,2] recommended that all adults have total cholesterol measured (in physicians’ offices), there has been increased emphasis on cholesterol sampling and methodology. Capillary plasma lipid profiles [3-91 provide results within 15 min, and require only 400 ~1 fingerstick blood. Whether they are as accurate and precise as the slower turnaround, venous blood, enzymatic techniques is a central question [9]. In 430 hyperlipidemic outpatients, our specific aim in the current study was to compare accuracy, precision, interchangeability of results, costs, and utility of the capillary plasma fasting lipid profile (AbbottVision) vs our Lipid Research Clinics (LRC) standardized [lo], central laboratory enzymatic method [l l] on the Hitachi 736-plus instrument using venous blood plasma. Materials and methods Study design andpatients
We prospectively studied 430 patients consecutively referred to our Cholesterol Center because of hyperlipidemia, l/91-5/91, without bias in selection of patients, who were sampled in the temporal sequence of their referral. Venous blood was drawn into an EDTA tube after a 12-h fast. Capillary blood was obtained separately Correspondence to: C.J. Glueck, Cholesterol Center, Jewish Hospital, 3200 Bumet Avenue, Cincinnati, OH 45229, USA.
190
by fingerstick. Of the 430 patients, paired comparisons (Hitachi vs Vision) could be made in 410 for total cholesterol, 414 for triglyceride, 417 for HDLC, and 377 for LDLC (Table I). Laboratory methodology for the Hitachi and Vision instruments
Instruments, controls, reagents, and calibrators were obtained from the respective diagnostic companies; the enzymatic cholesterol measurement [l l] on the Hitachi was calibrated through the LRC standardization program [lo]. Triglyceride was quantitated by enzymatic methods [12] as was HDLC, assayed in the supernate plasma after precipitation of LDL and VLDL by phosphotungstate-magnesium [ 131. The Vision lipid profile was carried out as previously described [3-91. For both
TABLE I Intraclass correlations (95% lower and upper confidence limits), relationships, and method differences, Vision and Hitachi
Total cholesterol Triglyceride HDLC LDLC
n
Intraclass correlation r,
Lower limit (95%) intraclass correlation rL
Upper limit (95%) intraclass correlation ru
410 414 417 377
0.96* 0.98* 0.90* 0.96*
0.77 0.98 0.58 0.95
0.98 0.99 0.95 0.97
* P
z (SD), mg/dl
y (SD) A, mg/dl
P (paired
Wilcoxon)
Total cholesterol Triglyceride HDLC LDLC
n
Hitachi
Vision
Hitachi-Vision
410 414 417 377
208+38 146+78 45*14 133k32
217k39 151&78 50+ 14 135+33
-8.3f8.0 -5.3k13.3 -4.5 +4.5 -2.6k8.3
P=O.O012 NS P=O.OOOl
NS
Relationship between lipids by the two methodologies: vision as regressor, Hitachi as response variable
Total cholesterol Triglyceride HDLC LDLC
n
y=a+bx
r-value, P-value
410 414 417 377
y=3.9027+1.0212~ y=8.3549+0.9789~ y = 2.8862 + 1.0359x y= 1.7744+1.0063x
r=0.98, P~O.0001 r=0.99, PCO.0001 r=0.95, P
191
instruments,
LDLC levels were calculated
[14] (when triglycerides
were ~400
mg/dl). Capillary blood was obtained as follows. The finger was cleansed with alcohol, dried (since alcohol can affect the specimen), and then stuck with a lancet. The first drop of blood was wiped away. Blood for HDLC quantitation was collected in an EDTA anticoagulant tube (minimum of 300 ~1). After this tube was filled, it was capped, mixed, and set aside until the rest of the capillary blood specimens were collected. Blood for cholesterol and triglyceride measurements was collected in 2 separate 50-~1 heparinized capillary tubes which were then inserted into the channel in each test pack. Then, 300 ~1 from the EDTA tube was added into the AbbottVision whole blood cholesterol pre-treatment tube with a transfer pipette. This 300 ,ul fills the pre-treatment tube to the required amount. The tube was mixed vigorously for 10 s and allowed to sit for > 5 but < 15 min. We allow this tube to sit for no more than 10 min because the cholesterol and triglyceride can only sit for that long. Using the Abbott-Vision transfer pipette, 2 drops from this pre-treatment tube are added to the test pack. All 3 packs are then run on the Abbott-Vision under the section, lipid profile l-9.2 [3-91. It takes approximately 11 min for the panels to be analyzed. Precision and accuracy
Within-day and between-day precision were assessed as summarized in Table II. The Hitachi Instrument was standardized with the LRC cholesterol standardization program [lo] (Washington University, St. Louis, MN), and met LRC criteria for precision and accuracy. Accuracy for the Vision machine was determined by paired comparison of venous blood plasma samples on the Hitachi vs capillary blood plasma samples on the Vision (Table I). Situations in which capillary plasma lipidpro$les were incomplete
Of the 430 patients tested, in 46 (10.7%), capillary lipid profiles were incomplete. In 10 patients, lipid levels exceeded the reporting range for the Vision method, including a maximum of triglyceride > 500 mg/dl, cholesterol > 500 mg/dl, and HDLC > 100 mg/dl. In 6 patients, values for either cholesterol, triglyceride or HDLC were lower than the reporting range (< 50 mg/dl for cholesterol, < 10 mg/dl for triglyceride, < 10 mg/dl for HDLC). In 14 patients, the specimen was hemolyzed, and in 6 not enough blood was obtained to carry out the full lipid profile. In 2 patients, the machine reported degraded reagent, or interference substance in the blood, and in 1 patient, the reaction would not reach the endpoint. In 7 patients, in the measurement for HDLC, the machine reported ‘volume high’, indicating either anemia or excess specimen added to the machine.
192 TABLE II Within- and between-day coefficients of variation for total cholesterol, triglyceride, HDLC, LDLC (mg/dl) Enzymatic methods, LRC standardized Hitachi, venous blood
Enzymatic methods, Abbott-Vision, capillary blood
Within-day
Within-day
n
x
SD
CV(%)
n
I
SD
CV(%)
Cholesterol
20
199”
2.1
1.07
Triglyceride HDLC
20 20
1438 43’
1.9 0.4
1.32 1.04
10 10 10 10
105s 258b 149s 28b
1.8 3.7 3.4 1.1
1.73 1.42 2.32 3.96
Between-day (30-day period)
Between-day (30-day period)
Cholesterol Triglyceride HDLC
n
Z
SD
CV(%)
n
x
SD
CV(%)
121 123 125 120 137
13@ 215d 93d 192’ 41e
2.5 3.7 2.2 4.1 1.3
1.9 1.7 2.4 2.1 3.1
30 30 30 30 30 30
106b 255b 146b 263b 31s 57s
1.2 5.4 6.5 20.4 0.9 1.1
1.2 2.1 4.5 7.8 2.9 2.0
a Fresh pooled plasma. b Frozen sera pool. ~Precitrol A Lot #XPS-152 (lyophilized serum reconstituted with buffered saline). d Precitrol N Lot #XL!&69 (lyophilized serum reconstituted with buffered saline). eFrozen plasma pool.
Statistical analyses
We used several statistical tests specifically suited for method comparison studies [15-181. To assess for agreement between 2 methods, we used the intraclass correlation (rt) and the 95% lower (Q) and upper (ro) confidence limits (Table I) [17]. Least square parameters were calculated (Table I). Estimates of bias and standard deviation of difference [15-181 were calculated, and the paired plasma samples were compared by paired Wilcoxon tests (Table I). All variables, with the exception of triglyceride (by both methods) and HDLC (by Hitachi) were normally distributed.
193
Precision
Within-day coefficients of variation for total cholesterol were excellent for both instruments (1.07% for the Hitachi, 1.73% and 1.42% for the Vision) (Table II). Within-day coefficients of variation for triglyceride and HDLC on the Hitachi were, respectively, 1.32% and 1.04% and on the Vision, 2.32% and 3.96%. Between-day coefficients of variation for cholesterol on the Hitachi using a low and high control (lyophilized serum reconstituted with buffered saline) were, respectively, 1.9% and 1.7%, for triglyceride 2.4% and 2.1%, and for HDLC (on a frozen plasma pool) 3.08% (Table II). Between-day coefficients of variation for the Abbott-Vision (frozen serum pool) were 1.2% and 2.1% for cholesterol, 4.5% and 7.8% for triglyceride, and 2.9% and 2.0% for HDLC. Thus, between-day coefficients of variation differed predominantly for triglyceride where the coefficients of variation were about twice as high on the Abbott-Vision as the Hitachi (Table II). Part of this difference might possibly arise from differing types of analytes, a reconstituted lyophilized serum pool (Hitachi) vs a frozen serum pool (Vision). Side-by-side comparison in 417 patients’ plasma samples
The 417 patients covered the broad range of total cholesterol, triglyceride, HDLC, and LDLC levels that might be encountered in the clinical laboratory (Table I). Plasma total cholesterol was 4% higher by the Vision (P=O.O012 (Table I), and HDLC was 10% higher by the Vision (P=O.OOOl) (Table I). Triglyceride and LDLC did not differ (P > 0.1). The 2 methods were highly correlated, with correlation coefficients for total cholesterol being 0.98, for triglyceride 0.99, for HDLC 0.95, and for LDLC 0.97 (all Pvalues < 0.0001) (Table I). The intra-class correlations (rr) for total cholesterol were rr=0.96, triglyceride II= 0.98, HDLC rI = 0.90, and LDLC r = 0.96 (P < 0.0001 for all, Table I). Total cholesterol, triglyceride, and LDLC by both the Vision and Hitachi methods were comparable by virtue of the lower limit (rL) of the 95% confidence interval of the intraclass correlation > 0.75 [17] (rL=O.77, 0.98, 0.95, respectively) (Table I). However, rL for HDLC was 0.58, identifying a lack of method interchangeability for HDLC 1171. In the 410 subjects having both measurements, mean total cholesterol by the Hitachi was 8.3 mg/dl(4%) below that of the Vision. The mean (+ 2 SD) of the difference was used to assess the ‘limits of agreement’, i.e., the 95% confidence interval [15,17,18]. Total cholesterols in venous plasma on the Hitachi may be anywhere between 24 mg/dl(12%) below to 8 mg/dl(3.7$) above those quantitated by the Vision, a spread which is close to, but above that (& 5%) set as a currently acceptable
194
accuracy goal by the Laboratory Standardization Panel of the NCEP [19]. Mean triglyceride was 5.3 mg/dl(5%) lower on the Hitachi, and may be 32 mg/dl(22@ below to 21 mg/dl (14%) above those by the Vision, outside of the f5% NCEP goal for accuracy [19]. Mean HDLC in venous plasma on the Hitachi was 4.5 mg/dl (10%) below that of capillary plasma (Vision). HDLC on the Hitachi may be anywhere from 14 mg/dl below (30%) to 4.5 mg/dl (10%) above the Vision, a spread outside of the f 5% NCEP goal for accuracy [19]. Mean LDLC was 2.6 mg/dl lower on the Hitachi as compared to the Vision, with LDLC levels ranging from 19 mg/dl below (14%) to 14 mg/dl above (11%) those on the Vision, again outside of the + 5% NCEP goal for accuracy [ 191. Unit costs andpatient throughput The per-unit cost for the full lipid profile was $ 15 by the Vision and $ 6.40 by the Hitachi. On the Vision, 8 lipid profiles per hour could be done by one technician who also collected the capillary blood. The rate-limiting step for the capillary lipid profile was the HDLC determination. It takes approximately 11 min for the 3 panels (cholesterol, triglyceride, HDLC) to be analyzed and with some overlap, a single technician can, working very efficiently, do 8 lipid profiles per hour. Discussion For total cholesterol, our data are very comparable to those of Burke and Fisher [3] who compared 84 venous plasma specimens (using the Abbott-Vision) against results obtained in a LRC standardized laboratory. Burke and Fisher [3] reported that the two methods correlated closely for total cholesterol (r=0.98), identical to that (r=0.98) for our current study. Burke and Fisher [3] also reported a slope of 1.06 compared to ours of 1.02, and a y intercept of 1.0 mg/dl compared to ours of 3.9. The mean total bias for cholesterol reported by Burke and Fisher [3] was + 3.9% and was + 4% in our study on the Vision. Our data for total cholesterol are also generally comparable to those of Hicks and Iosefsohn [5] and Nanji et al. [8]. For accuracy in measurement of total cholesterol, the Abbott-Vision was close to the currently acceptable bias criteria of the NCEP of rfr5% or less [19]. Moreover, the cholesterol determinations in venous blood by the Hitachi and capillary blood by the Vision were comparable using the criteria of Lee et al. [ 171 and Bland and Altman [18]. Although there was a 4% positive bias on the Vision methodology in capillary plasma, the intraclass correlation of the 2 methods was highly significant (rr=0.96, P~0.0001), and the lower limit of the 95% confidence interval of the intraclass correlation was > 0.75, suggesting method interchangeability [ 17,181. Triglyceride and LDL cholesterol on the Hitachi and the Vision were also comparable [17,18]. For HDLC, however, both methods were not interchangeable. There was a significant positive bias of 4.5 mg/dl (10%) on the Vision, and the lower limit
195 TABLE III Capillary blood lipid profile values (Vision, mg/dl) calculated from venous blood, LRC standardized levels on the Hitachi Instrument
Hitachi Calculated Vision
HDLC
LDLC
Total cholesterol
Triglyceride
200
240
250
500
35
40
130
160
208
249
253
498
39
44
133
163
of the 95% confidence interval of the intraclass correlation was ~0.75. Moreover, HDLC by the 2 methods could range as much as 14 mg/dl(30%) lower to 4.5 mg/dl (10%) higher comparing the Hitachi to the Vision, a range well beyond the + 5% accuracy targets of the NCEP [19]. Our HDLC results compare favorably to those of Ng and Altaffer [7]. Cholesterol, triglyceride, HDLC, and LDLC levels on the Vision instrument that correspond to the LRC standardized values on the Hitachi instrument can be calculated by multiplying Hitachi venous plasma levels by the slope of the regression line and adding the intercept value, an exercise carried out by Burke and Fisher [3]. The actual Hitachi values and the calculated Vision values for cholesterol, triglyceride, and LDLC agreed closely (Table III). However, the calculated Vision HDLC was 10-l 1% higher than the Hitachi values, which has diagnostic import [2]. Precision of total cholesterol and HDLC measurements by both instruments was well within the NCEP guidelines of a 5% coefficient of variation with an ultimate goal of 3% or less [19]. Naito [20] and Cooper et al. [21] have reported that HDLC has a mean within-person total coefficient of variation of 4.5% daily, 7.7% monthly, and 8.4% yearly. Between-day coefficients of variation for triglyceride were excellent on the Hitachi at 2.1% and 2.4% for pools of 192 and 93 mg/dl, but were higher on the Abbott-Vision, 4.5% for 146 mg/dl and 7.8% for 263 mg/dl. Our between-day coefficients of variation for triglyceride (Vision) were higher than those reported by Hicks and Iosefsohn (l.l%, 1.3%) [5] or by Nanji et al. (1.8+3.2%) [8]. Bachorik et al. [9] in comparing the Vision against a central LRC standardized laboratory, has reported that ‘. . . screening HDL-cholesterol values were too variable to establish the HDL-cholesterol level reliably’, a conclusion similar to ours. Moreover, the overall positive bias of 4% for cholesterol measurements in capillary samples vs venous measures reported by Bachorik et al. [9] was identical to our 4%, reflecting in part the EDTA-mediated slight dilution of lipoproteins in our venous blood samples vs the lack of osmotic effect of the heparinized capillary tubes [9].
196
Conclusion Capillary plasma lipid profiles by the Abbott-Vision method are quick (approximately 11 min), require only 400 ~1 blood, avoid the need for venesection, and provide reasonable comparability for total cholesterol, triglyceride, and LDLC to those by the LRC standardized [IO] venous blood plasma methodology. On the other hand, differences in accuracy between the methods, particularly for HDLC [9], are enough so that longitudinal follow-up or intervention should probably rely on the more accurate and equally precise venous plasma methodology. Acknowledgement Supported by Jewish Hospital Medical Research Council Grant No. 739. References 1 NIH Consensus Development Conference. Lowering blood cholesterol to prevent heart disease. J Am Med Ass 1985; 253: 2080-2086. 2 Report of the National Cholesterol Education Program expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. Arch Intern Med 1988; 148: 3669. 3 Burke JJ, Fisher PM. A clinician’s guide to the office management of cholesterol. J Am Med Ass 1988; 259: 3444-3448. 4 Schultz SG, Holen JT, Donohue JP, et al. Two-dimensional centrifugation for desk-top clinical chemistry. Clin Chem 1985; 31: 1457-1463. 5 Hicks JM, Iosefsohn M. Another physician’s office analyzer: the Abbott ‘Vision’ evaluated Clin Chem 1987; 33(6): 817-819. 6 Maturen A, Francoeur TA, Wynn AL, Reid DL, Anderson CP. Clinical performance of the Abott ‘Vision System’: reference intervals and specimen type comparisons. Clin Chem 1987; 33(6): 10781079. 7 Ng RH, Altaffer M. HDL-cholesterol assay with the Abbott ‘Vision System’ evaluated. Clin Chem 1987; 33(10): 1943. 8 Nanji AA, Poon R, Hinberg I. A physician’s office analyzer. Arch Path01 Lab Med 1988; 112: 242-244. 9 Bachorik PS, Cloey TA, Finney CA, et al. Lipoprotein-cholesterol analysis during screening: accuracy and reliability. Ann Int Med 1991; 114: 741-747. 10 Myers GL, Cooper GR, Winn CL, Smith SJ. The centers for disease control - National Heart, Lung and Blood Institute Lipid Standardization Program. An approach to accurate and precise lipid measurements. Clin Lab Med 1989; 9: 105-135. 11 Allain C, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974; 20: 470475. 12 McGowan MW, Artiss JD, Strandbergh DR, Zak B. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin Chem 1983; 29: 538-543. 13 Assman G, Schriewer H, Schmitz G, Hagele EO. Quantification of high density lipoprotein cholesterol by precipitation with phosphotungstic acid/Mg& Clin Chem 1983; 29: 20262030. 14 Friedwald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without use of the preparative vitra centrifuge. Clin Chem 1972; 18: 499-502. 15 Glueck CJ, McCray C, Speirs J. Measurement of serum Apo Al and Apo B: comparison of immunoturbidimetric and rate nephelometric techniques. Clin Chim Acta 1991; 197: 123-132.
197 16 Westgard JO, Hunt MR. Use and interpretation of common statistical tests in method-comparison studies. Clin Chem 1973; 49-57. 17 Lee J, Koh D, Ong CN. Statistical evaluation of agreement between two methods for measuring a quantitative variable. Comput Biol Med 1989; 19: 61-70. 18 Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; i: 307-310. 19 National Cholesterol Education Program. Current status of blood cholesterol measurements in clinical laboratories in the United States. Clin Chem 1988; 34: 193-201. 20 Naito HK. Reliability of lipid, lipoprotein, and apolipoprotein measurements. Clin Chem 1988; 34: B84-B94. 21 Cooper GR, Myers GI, Smith SJ, Sampson EJ. Standardization of lipid, lipoprotein, and apolipoprotein measurements. Clin Chem 1988; 34: B95-B105.
189
CCA05176
Capillary plasma lipid profiles Rebecca Mace, Bernadette May, Carol McCray, Trent Tracy, James Speirs and Charles J. Glueck Cholesterol Center of the Jewish Hospital, Cincinnati, OH (USA)
(Received 15 July 1991; revision received 18 September 1991; accepted 20 September 1991) Key words: Lipid profiles; Low density lipoprotein cholesterol (LDLC); High density lipoprotein cholesterol (HDLC); Coronary heart disease; Lipid Research Clinics (LRC) cholesterol; Standardization program
Introduction
Since the National Cholesterol Education Program (NCEP) [1,2] recommended that all adults have total cholesterol measured (in physicians’ offices), there has been increased emphasis on cholesterol sampling and methodology. Capillary plasma lipid profiles [3-91 provide results within 15 min, and require only 400 ~1 fingerstick blood. Whether they are as accurate and precise as the slower turnaround, venous blood, enzymatic techniques is a central question [9]. In 430 hyperlipidemic outpatients, our specific aim in the current study was to compare accuracy, precision, interchangeability of results, costs, and utility of the capillary plasma fasting lipid profile (AbbottVision) vs our Lipid Research Clinics (LRC) standardized [lo], central laboratory enzymatic method [l l] on the Hitachi 736-plus instrument using venous blood plasma. Materials and methods Study design andpatients
We prospectively studied 430 patients consecutively referred to our Cholesterol Center because of hyperlipidemia, l/91-5/91, without bias in selection of patients, who were sampled in the temporal sequence of their referral. Venous blood was drawn into an EDTA tube after a 12-h fast. Capillary blood was obtained separately Correspondence to: C.J. Glueck, Cholesterol Center, Jewish Hospital, 3200 Bumet Avenue, Cincinnati, OH 45229, USA.
190
by fingerstick. Of the 430 patients, paired comparisons (Hitachi vs Vision) could be made in 410 for total cholesterol, 414 for triglyceride, 417 for HDLC, and 377 for LDLC (Table I). Laboratory methodology for the Hitachi and Vision instruments
Instruments, controls, reagents, and calibrators were obtained from the respective diagnostic companies; the enzymatic cholesterol measurement [l l] on the Hitachi was calibrated through the LRC standardization program [lo]. Triglyceride was quantitated by enzymatic methods [12] as was HDLC, assayed in the supernate plasma after precipitation of LDL and VLDL by phosphotungstate-magnesium [ 131. The Vision lipid profile was carried out as previously described [3-91. For both
TABLE I Intraclass correlations (95% lower and upper confidence limits), relationships, and method differences, Vision and Hitachi
Total cholesterol Triglyceride HDLC LDLC
n
Intraclass correlation r,
Lower limit (95%) intraclass correlation rL
Upper limit (95%) intraclass correlation ru
410 414 417 377
0.96* 0.98* 0.90* 0.96*
0.77 0.98 0.58 0.95
0.98 0.99 0.95 0.97
* P
z (SD), mg/dl
y (SD) A, mg/dl
P (paired
Wilcoxon)
Total cholesterol Triglyceride HDLC LDLC
n
Hitachi
Vision
Hitachi-Vision
410 414 417 377
208+38 146+78 45*14 133k32
217k39 151&78 50+ 14 135+33
-8.3f8.0 -5.3k13.3 -4.5 +4.5 -2.6k8.3
P=O.O012 NS P=O.OOOl
NS
Relationship between lipids by the two methodologies: vision as regressor, Hitachi as response variable
Total cholesterol Triglyceride HDLC LDLC
n
y=a+bx
r-value, P-value
410 414 417 377
y=3.9027+1.0212~ y=8.3549+0.9789~ y = 2.8862 + 1.0359x y= 1.7744+1.0063x
r=0.98, P~O.0001 r=0.99, PCO.0001 r=0.95, P
191
instruments,
LDLC levels were calculated
[14] (when triglycerides
were ~400
mg/dl). Capillary blood was obtained as follows. The finger was cleansed with alcohol, dried (since alcohol can affect the specimen), and then stuck with a lancet. The first drop of blood was wiped away. Blood for HDLC quantitation was collected in an EDTA anticoagulant tube (minimum of 300 ~1). After this tube was filled, it was capped, mixed, and set aside until the rest of the capillary blood specimens were collected. Blood for cholesterol and triglyceride measurements was collected in 2 separate 50-~1 heparinized capillary tubes which were then inserted into the channel in each test pack. Then, 300 ~1 from the EDTA tube was added into the AbbottVision whole blood cholesterol pre-treatment tube with a transfer pipette. This 300 ,ul fills the pre-treatment tube to the required amount. The tube was mixed vigorously for 10 s and allowed to sit for > 5 but < 15 min. We allow this tube to sit for no more than 10 min because the cholesterol and triglyceride can only sit for that long. Using the Abbott-Vision transfer pipette, 2 drops from this pre-treatment tube are added to the test pack. All 3 packs are then run on the Abbott-Vision under the section, lipid profile l-9.2 [3-91. It takes approximately 11 min for the panels to be analyzed. Precision and accuracy
Within-day and between-day precision were assessed as summarized in Table II. The Hitachi Instrument was standardized with the LRC cholesterol standardization program [lo] (Washington University, St. Louis, MN), and met LRC criteria for precision and accuracy. Accuracy for the Vision machine was determined by paired comparison of venous blood plasma samples on the Hitachi vs capillary blood plasma samples on the Vision (Table I). Situations in which capillary plasma lipidpro$les were incomplete
Of the 430 patients tested, in 46 (10.7%), capillary lipid profiles were incomplete. In 10 patients, lipid levels exceeded the reporting range for the Vision method, including a maximum of triglyceride > 500 mg/dl, cholesterol > 500 mg/dl, and HDLC > 100 mg/dl. In 6 patients, values for either cholesterol, triglyceride or HDLC were lower than the reporting range (< 50 mg/dl for cholesterol, < 10 mg/dl for triglyceride, < 10 mg/dl for HDLC). In 14 patients, the specimen was hemolyzed, and in 6 not enough blood was obtained to carry out the full lipid profile. In 2 patients, the machine reported degraded reagent, or interference substance in the blood, and in 1 patient, the reaction would not reach the endpoint. In 7 patients, in the measurement for HDLC, the machine reported ‘volume high’, indicating either anemia or excess specimen added to the machine.
192 TABLE II Within- and between-day coefficients of variation for total cholesterol, triglyceride, HDLC, LDLC (mg/dl) Enzymatic methods, LRC standardized Hitachi, venous blood
Enzymatic methods, Abbott-Vision, capillary blood
Within-day
Within-day
n
x
SD
CV(%)
n
I
SD
CV(%)
Cholesterol
20
199”
2.1
1.07
Triglyceride HDLC
20 20
1438 43’
1.9 0.4
1.32 1.04
10 10 10 10
105s 258b 149s 28b
1.8 3.7 3.4 1.1
1.73 1.42 2.32 3.96
Between-day (30-day period)
Between-day (30-day period)
Cholesterol Triglyceride HDLC
n
Z
SD
CV(%)
n
x
SD
CV(%)
121 123 125 120 137
13@ 215d 93d 192’ 41e
2.5 3.7 2.2 4.1 1.3
1.9 1.7 2.4 2.1 3.1
30 30 30 30 30 30
106b 255b 146b 263b 31s 57s
1.2 5.4 6.5 20.4 0.9 1.1
1.2 2.1 4.5 7.8 2.9 2.0
a Fresh pooled plasma. b Frozen sera pool. ~Precitrol A Lot #XPS-152 (lyophilized serum reconstituted with buffered saline). d Precitrol N Lot #XL!&69 (lyophilized serum reconstituted with buffered saline). eFrozen plasma pool.
Statistical analyses
We used several statistical tests specifically suited for method comparison studies [15-181. To assess for agreement between 2 methods, we used the intraclass correlation (rt) and the 95% lower (Q) and upper (ro) confidence limits (Table I) [17]. Least square parameters were calculated (Table I). Estimates of bias and standard deviation of difference [15-181 were calculated, and the paired plasma samples were compared by paired Wilcoxon tests (Table I). All variables, with the exception of triglyceride (by both methods) and HDLC (by Hitachi) were normally distributed.
193
Precision
Within-day coefficients of variation for total cholesterol were excellent for both instruments (1.07% for the Hitachi, 1.73% and 1.42% for the Vision) (Table II). Within-day coefficients of variation for triglyceride and HDLC on the Hitachi were, respectively, 1.32% and 1.04% and on the Vision, 2.32% and 3.96%. Between-day coefficients of variation for cholesterol on the Hitachi using a low and high control (lyophilized serum reconstituted with buffered saline) were, respectively, 1.9% and 1.7%, for triglyceride 2.4% and 2.1%, and for HDLC (on a frozen plasma pool) 3.08% (Table II). Between-day coefficients of variation for the Abbott-Vision (frozen serum pool) were 1.2% and 2.1% for cholesterol, 4.5% and 7.8% for triglyceride, and 2.9% and 2.0% for HDLC. Thus, between-day coefficients of variation differed predominantly for triglyceride where the coefficients of variation were about twice as high on the Abbott-Vision as the Hitachi (Table II). Part of this difference might possibly arise from differing types of analytes, a reconstituted lyophilized serum pool (Hitachi) vs a frozen serum pool (Vision). Side-by-side comparison in 417 patients’ plasma samples
The 417 patients covered the broad range of total cholesterol, triglyceride, HDLC, and LDLC levels that might be encountered in the clinical laboratory (Table I). Plasma total cholesterol was 4% higher by the Vision (P=O.O012 (Table I), and HDLC was 10% higher by the Vision (P=O.OOOl) (Table I). Triglyceride and LDLC did not differ (P > 0.1). The 2 methods were highly correlated, with correlation coefficients for total cholesterol being 0.98, for triglyceride 0.99, for HDLC 0.95, and for LDLC 0.97 (all Pvalues < 0.0001) (Table I). The intra-class correlations (rr) for total cholesterol were rr=0.96, triglyceride II= 0.98, HDLC rI = 0.90, and LDLC r = 0.96 (P < 0.0001 for all, Table I). Total cholesterol, triglyceride, and LDLC by both the Vision and Hitachi methods were comparable by virtue of the lower limit (rL) of the 95% confidence interval of the intraclass correlation > 0.75 [17] (rL=O.77, 0.98, 0.95, respectively) (Table I). However, rL for HDLC was 0.58, identifying a lack of method interchangeability for HDLC 1171. In the 410 subjects having both measurements, mean total cholesterol by the Hitachi was 8.3 mg/dl(4%) below that of the Vision. The mean (+ 2 SD) of the difference was used to assess the ‘limits of agreement’, i.e., the 95% confidence interval [15,17,18]. Total cholesterols in venous plasma on the Hitachi may be anywhere between 24 mg/dl(12%) below to 8 mg/dl(3.7$) above those quantitated by the Vision, a spread which is close to, but above that (& 5%) set as a currently acceptable
194
accuracy goal by the Laboratory Standardization Panel of the NCEP [19]. Mean triglyceride was 5.3 mg/dl(5%) lower on the Hitachi, and may be 32 mg/dl(22@ below to 21 mg/dl (14%) above those by the Vision, outside of the f5% NCEP goal for accuracy [19]. Mean HDLC in venous plasma on the Hitachi was 4.5 mg/dl (10%) below that of capillary plasma (Vision). HDLC on the Hitachi may be anywhere from 14 mg/dl below (30%) to 4.5 mg/dl (10%) above the Vision, a spread outside of the f 5% NCEP goal for accuracy [19]. Mean LDLC was 2.6 mg/dl lower on the Hitachi as compared to the Vision, with LDLC levels ranging from 19 mg/dl below (14%) to 14 mg/dl above (11%) those on the Vision, again outside of the + 5% NCEP goal for accuracy [ 191. Unit costs andpatient throughput The per-unit cost for the full lipid profile was $ 15 by the Vision and $ 6.40 by the Hitachi. On the Vision, 8 lipid profiles per hour could be done by one technician who also collected the capillary blood. The rate-limiting step for the capillary lipid profile was the HDLC determination. It takes approximately 11 min for the 3 panels (cholesterol, triglyceride, HDLC) to be analyzed and with some overlap, a single technician can, working very efficiently, do 8 lipid profiles per hour. Discussion For total cholesterol, our data are very comparable to those of Burke and Fisher [3] who compared 84 venous plasma specimens (using the Abbott-Vision) against results obtained in a LRC standardized laboratory. Burke and Fisher [3] reported that the two methods correlated closely for total cholesterol (r=0.98), identical to that (r=0.98) for our current study. Burke and Fisher [3] also reported a slope of 1.06 compared to ours of 1.02, and a y intercept of 1.0 mg/dl compared to ours of 3.9. The mean total bias for cholesterol reported by Burke and Fisher [3] was + 3.9% and was + 4% in our study on the Vision. Our data for total cholesterol are also generally comparable to those of Hicks and Iosefsohn [5] and Nanji et al. [8]. For accuracy in measurement of total cholesterol, the Abbott-Vision was close to the currently acceptable bias criteria of the NCEP of rfr5% or less [19]. Moreover, the cholesterol determinations in venous blood by the Hitachi and capillary blood by the Vision were comparable using the criteria of Lee et al. [ 171 and Bland and Altman [18]. Although there was a 4% positive bias on the Vision methodology in capillary plasma, the intraclass correlation of the 2 methods was highly significant (rr=0.96, P~0.0001), and the lower limit of the 95% confidence interval of the intraclass correlation was > 0.75, suggesting method interchangeability [ 17,181. Triglyceride and LDL cholesterol on the Hitachi and the Vision were also comparable [17,18]. For HDLC, however, both methods were not interchangeable. There was a significant positive bias of 4.5 mg/dl (10%) on the Vision, and the lower limit
195 TABLE III Capillary blood lipid profile values (Vision, mg/dl) calculated from venous blood, LRC standardized levels on the Hitachi Instrument
Hitachi Calculated Vision
HDLC
LDLC
Total cholesterol
Triglyceride
200
240
250
500
35
40
130
160
208
249
253
498
39
44
133
163
of the 95% confidence interval of the intraclass correlation was ~0.75. Moreover, HDLC by the 2 methods could range as much as 14 mg/dl(30%) lower to 4.5 mg/dl (10%) higher comparing the Hitachi to the Vision, a range well beyond the + 5% accuracy targets of the NCEP [19]. Our HDLC results compare favorably to those of Ng and Altaffer [7]. Cholesterol, triglyceride, HDLC, and LDLC levels on the Vision instrument that correspond to the LRC standardized values on the Hitachi instrument can be calculated by multiplying Hitachi venous plasma levels by the slope of the regression line and adding the intercept value, an exercise carried out by Burke and Fisher [3]. The actual Hitachi values and the calculated Vision values for cholesterol, triglyceride, and LDLC agreed closely (Table III). However, the calculated Vision HDLC was 10-l 1% higher than the Hitachi values, which has diagnostic import [2]. Precision of total cholesterol and HDLC measurements by both instruments was well within the NCEP guidelines of a 5% coefficient of variation with an ultimate goal of 3% or less [19]. Naito [20] and Cooper et al. [21] have reported that HDLC has a mean within-person total coefficient of variation of 4.5% daily, 7.7% monthly, and 8.4% yearly. Between-day coefficients of variation for triglyceride were excellent on the Hitachi at 2.1% and 2.4% for pools of 192 and 93 mg/dl, but were higher on the Abbott-Vision, 4.5% for 146 mg/dl and 7.8% for 263 mg/dl. Our between-day coefficients of variation for triglyceride (Vision) were higher than those reported by Hicks and Iosefsohn (l.l%, 1.3%) [5] or by Nanji et al. (1.8+3.2%) [8]. Bachorik et al. [9] in comparing the Vision against a central LRC standardized laboratory, has reported that ‘. . . screening HDL-cholesterol values were too variable to establish the HDL-cholesterol level reliably’, a conclusion similar to ours. Moreover, the overall positive bias of 4% for cholesterol measurements in capillary samples vs venous measures reported by Bachorik et al. [9] was identical to our 4%, reflecting in part the EDTA-mediated slight dilution of lipoproteins in our venous blood samples vs the lack of osmotic effect of the heparinized capillary tubes [9].
196
Conclusion Capillary plasma lipid profiles by the Abbott-Vision method are quick (approximately 11 min), require only 400 ~1 blood, avoid the need for venesection, and provide reasonable comparability for total cholesterol, triglyceride, and LDLC to those by the LRC standardized [IO] venous blood plasma methodology. On the other hand, differences in accuracy between the methods, particularly for HDLC [9], are enough so that longitudinal follow-up or intervention should probably rely on the more accurate and equally precise venous plasma methodology. Acknowledgement Supported by Jewish Hospital Medical Research Council Grant No. 739. References 1 NIH Consensus Development Conference. Lowering blood cholesterol to prevent heart disease. J Am Med Ass 1985; 253: 2080-2086. 2 Report of the National Cholesterol Education Program expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. Arch Intern Med 1988; 148: 3669. 3 Burke JJ, Fisher PM. A clinician’s guide to the office management of cholesterol. J Am Med Ass 1988; 259: 3444-3448. 4 Schultz SG, Holen JT, Donohue JP, et al. Two-dimensional centrifugation for desk-top clinical chemistry. Clin Chem 1985; 31: 1457-1463. 5 Hicks JM, Iosefsohn M. Another physician’s office analyzer: the Abbott ‘Vision’ evaluated Clin Chem 1987; 33(6): 817-819. 6 Maturen A, Francoeur TA, Wynn AL, Reid DL, Anderson CP. Clinical performance of the Abott ‘Vision System’: reference intervals and specimen type comparisons. Clin Chem 1987; 33(6): 10781079. 7 Ng RH, Altaffer M. HDL-cholesterol assay with the Abbott ‘Vision System’ evaluated. Clin Chem 1987; 33(10): 1943. 8 Nanji AA, Poon R, Hinberg I. A physician’s office analyzer. Arch Path01 Lab Med 1988; 112: 242-244. 9 Bachorik PS, Cloey TA, Finney CA, et al. Lipoprotein-cholesterol analysis during screening: accuracy and reliability. Ann Int Med 1991; 114: 741-747. 10 Myers GL, Cooper GR, Winn CL, Smith SJ. The centers for disease control - National Heart, Lung and Blood Institute Lipid Standardization Program. An approach to accurate and precise lipid measurements. Clin Lab Med 1989; 9: 105-135. 11 Allain C, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974; 20: 470475. 12 McGowan MW, Artiss JD, Strandbergh DR, Zak B. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin Chem 1983; 29: 538-543. 13 Assman G, Schriewer H, Schmitz G, Hagele EO. Quantification of high density lipoprotein cholesterol by precipitation with phosphotungstic acid/Mg& Clin Chem 1983; 29: 20262030. 14 Friedwald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without use of the preparative vitra centrifuge. Clin Chem 1972; 18: 499-502. 15 Glueck CJ, McCray C, Speirs J. Measurement of serum Apo Al and Apo B: comparison of immunoturbidimetric and rate nephelometric techniques. Clin Chim Acta 1991; 197: 123-132.
197 16 Westgard JO, Hunt MR. Use and interpretation of common statistical tests in method-comparison studies. Clin Chem 1973; 49-57. 17 Lee J, Koh D, Ong CN. Statistical evaluation of agreement between two methods for measuring a quantitative variable. Comput Biol Med 1989; 19: 61-70. 18 Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; i: 307-310. 19 National Cholesterol Education Program. Current status of blood cholesterol measurements in clinical laboratories in the United States. Clin Chem 1988; 34: 193-201. 20 Naito HK. Reliability of lipid, lipoprotein, and apolipoprotein measurements. Clin Chem 1988; 34: B84-B94. 21 Cooper GR, Myers GI, Smith SJ, Sampson EJ. Standardization of lipid, lipoprotein, and apolipoprotein measurements. Clin Chem 1988; 34: B95-B105.