Reference. 1. Letellier G, Desjarlais F. A. A simple procedure ... Letellier and Desjarlais. (1). The do- .... References. 1. Daoud EWR, McClellan AC, Scott MG.
the serum
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creating 10 fractions. Therefore, results agree with those obtained Letellier and Desjarlais (1). The gree of concentration of serum did depend on the freezing temperature.
not
and Desjarlais did not examine the effect of temperature on the thawing as we did. We consider that the discrepancy between our results and Hollenders results concerning the thermal effects may be caused by the Letellier
differences in the methods and sampling.
of thawing
Reference 1. Letellier G, Desjarlais F. A. A simple procedure for the preparation of concentrated sera. Clin Biochem 1985;18: 267-7 1. T. Hirano Central Clin. Labs. Tokyo Metropolitan Police Hoop. Fujimi, Chiyoda-ku Tokyo 102, Japan
round
To the Editor: Recently we presented a method for apolipoprotein E (apoE) phenotyping by isoelectric focusing the apoE in agarose with subsequent immunofixation (1). We have now improved this method in three ways: (a) serum is used directly instead of apoE-enriched lipoproteins, which had to be isolated by precipitation; (b) the sample number per plate is increased from 20 to 45; and (c) the requirement of antiserum per immunofixation slot is reduced from 10 to 5 pL per sample. In total, these changes save >50% of both time and material involved. Now, if two focusing chambers are available, a technician can run four plates with 180 samples per day. Instead of isolation of apoE-containing lipoproteins by precipitation, 50 tL of serum is mixed directly with 12 ML of a delipidation solution. This solution is freshly prepared by dissolving 500 mg of urea in an aqueous buffer containing 150 mg of dithiothreitol (Sigma), 120 mg of Tris (Merck), and 23 mL of Tween 20 (Sigma) in 100 mL After the mixture has stood at room temperature for 30 mm, 2 yL of sample is applied per hole of the sample application sheet. To increase the sample throughput, we changed the number of holes in the sample 168
application sheet. Now, 45
munofixation
3.0 the cathodsheet
Abteilung f#{252}r KIm. Chem. Kim. der Albert-Ludwigs-Univ. Hugstetter Str. 55 7800 Freiburg, FR.G.
New Enzymatic CO2 Slide on the Ektachem 700 XR Analyzer Eiimlnates interference from Nitrate/Nitrite ions To the Editor: An abnormal total CO2 as part of a serum electrolyte panel can be the first indication of an acid-base disorder. The need to accurately and rapidly measure serum total CO2 is obvious, and the Kodak Ektachem potentiometric method serves this need well. However, we identified a very significant positive interference with
Reference 1. Luley C, Baumstark MW, Wieland H.
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FIg. 1. An entire isoelectnc focusingplate afterimmunofixation and staining The cathodeisat the right Stainedareas are (from right) sample application spots, nonspecifically precipitated matenal, and immunofixatedapoE bands in the order E4, E3, and E2 with the wellknownadditional mono- and dislalo apoE bands. The apoE phenotypeofeachsampleisgivenat the left. A small artifactualspot mimicks an apoE4 band In lane 9; the strong E3 and E2 bands, however,disguisethe sampleas an E2/3 phenotype
CLINICALCHEMISTRY,Vol.38, No. 1, 1992
top-
ical silver or cerous nitrate therapy for burns or skin exfoliation (1). We demonstrated a direct correlation between the extent of the interference and nitrate or nitrite concentration. Indeed, the difference between the Ektachem potentiometric method and the standard enzymatic total CO2 method (2) could be used to estimate the serum nitrate/nitrite concentration in these patients. In some patients this difference was >25 mmol of CO2 per liter. This and other factors prompted the manufacturer to develop an alternative slide for total CO2 for the Ektachem analyzer, based on the standard enzymatic method with phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37) and measuring decreased absorption at 340 nm to monitor NADH-i.NAD (3). The preproduction evaluation of the new enzymatic slide showed an analytical range of 5-41 mmolIL, as determined by examining the deviation from linearity of results for samples diluted in parallel with a 70 g/L solution of human serum albumin. The new slide demonstrated excellent precision, as follows: interassay precision (CV) was 1.7% and 3.7% for qualitycontrol material having CO2 content of 13 and 23 mmol/L, respectively (n = 27); intraassay precision was 2.2% and 1.7% with the same materials (n = 27); and interas8ay precision was 5.0%
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J Lipid Res 1991;
Claus H. Luley Brigitte Haas Bernhard B#{252}hrer Heinrich Wieland
uted apoE phenotypes.
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in agarose.
32:880-3.
ical long side. We also changed the slots in the immunofixation sheet to use less antiserum for immunofixation. Now, the width and length of the slot are 1.5 and 40 mm, respectively, and only 20 1zL of diluted antiserum is used per slot [antiserum against apoE (Daiichi, Tokyo, Japan) diluted fourfold in saline]. Thus, the antiserum requirement is 5.0 .tL per sample. All other steps of the procedure-such as preparation of the agarose plate, focusing conditions, and immunofixation-are unchanged. Figure 1 shows a plate with 45 immunofixated samples from German blood donors with randomly distrib-
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Improvement of ApoilpoproteIn E Phenotyping by Isoeiectric Focuslng/Immunoflxatlon
Rapid apolipoprotein E phenotyping by Ira-
holes (1.5 mm diameter) are
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and 2.0% for patients’ samples having mean total CO2 of 7 and 35 mmol/L, respectively (n = 27). Least-squares linear-regression analysis of the total CO2 values obtained from the potentiometric (x) and new enzymatic slides (y) for 113 clinical samples (range 15-41 mmol/L) showed the following relationship: y = 0.92 x + 0.35 (r = 0.976, P
