Table 1. Within-Day Precision and Stability In ... - Clinical Chemistry

0 downloads 0 Views 467KB Size Report
Chase AM. The osmotic resistance (fragil- ity) of human red cells. J Clin Invest 26,. 636-640 (1947). Efamol Res. Inst. P.O. Box 818. Kentville,. Nova Scotia.
acidi4-aminophena.zone chromogenic sysin direct enzymatic assay of uric acid in serum and urine. Clin Chem 26, 227-231

tem

(19801.

2. Price CP, Spencer K. Multiple wavelength spectrophotometry and the centrifugal analyser. In Centrifugal Analysers in Clinical Chemistry, CP Price, K Spencer, Eds., Praeger Publishers, New York, NY, 1980, pp 213-229.

Malcolm Rodger

lyzed. For long-term stability studies, aliquots from each vial were aspirated into glass syringes after the initial reading had been made. These syringes were also sealed with Luer-lock tips, and syringes containing erythrocytes or hemoglobin control material were stored at 4 #{176}C, in ice water. The fluorocarbon-based control, which contained no blood components, was stored at room temperature. All mea-

surements were made with an Instrumentation Laboratory Micro 13 blood-

Dept. Clin. Bmochem. The Hosp. for Sick Children Grand Ormond St. London WC1N 3JH, UX.

gas/pH instrument. Our results exhibited the same pattern of precision and stability for pH,

Pco2, and Po measurements at all control values. 2This pattern is shown in Table 1 for a high

Precision and Long-Term Stability of Newer Controls for Blood pH and Blood Gases To the Editor.’ Recently developed commercial control materials for blood pH and blood

gases better simulate the gas affinity of whole-blood specimens than did previous aqueous buffer controls. One prepa-

ration (“Quantra Plus”; American Dade, Miami, FL 33152), involving fixed human erythrocytes, reportedly is more stable with respect to gas content than are aqueous buffer controls when exposed to room air, but it has technique-dependent oxygen capacity at low tensions (1). The performance of

another preparation (“Prime”; Fisher Scientific, Orangeburg, NY 10962), which contains human hemoglobin, has so far not been evaluated

in the

literature. A fluorocarbon preparation (“abc”; Instrumentation Laboratory, Lexington, MA 02173) has been compared with aqueous buffer controls in proficiency testing (2) and found to be superior to buffer for P02 values, owing to its high oxygen content. Because

these control materials have not been inter-compared in the literature, we evaluated them, along with tonometered whole blood, with regard to within-day precision and stability in sealed glass syringes.

Tonometered whole blood was prepared by equilibration for 20 mm at 37 #{176}C with suitable gas tension in an Instrumentation Laboratory Model 237 tonometer. Blood was withdrawn from the tonometer into 1- and 3-mL gas-equilibrated glass syringes for

and stability

ice water. Commercial controls were prepared according to each manufac-

turer’s directions. For within-day precision studies, 10 ampules of each control concentration and type were ana336

improved

progressively

from whole blood to the media least like blood, the best within-day precision being obtained with the fluorocar-

bon-emulsion control medium, which, as expected, was also the most stable, After 4 h of storage, all control media were stable, as judged by within-

day changes of less than two standard deviations. After 6 h, all but the fluorocarbon control had changed by greater than three within-day standard deviations; however, this instability

at 6 h

was unique to the high value for Po2 In other studies at all values of pH and at Pco2 and P02 within the range 30 to

70 mmHg, all of the control materials were stable for as long as 6 h when stored under the described conditions. No change in the fluorocarbon control occurred until after 12 h at room temperature. Specific data regarding these findings may be obtained from the authors. In conclusion, we found that the fluo-

rocarbon-emulsion

control

material

exhibited the best within-day and between-day precision of the control media studied. If a later second reading on

Jessie L. Hansen Dorilyn J. Forleo Northeastern Univ. College of Pharmacy and Allied Health Professions Boston, MA 02115

Rapid Measurement of Erythrocyte Osmotic Fragility To the Editor:

There are several advanced techniques for determining osmotic fragility curves (1, 2). These techniques are reliable but require expensive equipment and are not optimal for routine use. The conventional multiple-tube procedure (3), on the other hand, is time-consuming and tedious. We have developed a method that is simple, fast, and reliable. The use of a microcentrifuge facilitates the separation of cell debris from hemoglobin released in the hypotonic solution. The reading of the percentage of hemolysis is obtained directly with an automated analyzer

like that used in clinical

laboratories

for enzymatic methods of measurement. Blood from overnight-fasted normal subjects was collected in a tube containing EDTA (1 g/L). A 1-mL aliquot of blood was diluted with 2 mL of 9 g/L

NaCl solution. Test solutions (not buffered), in NaC1 concentrations of 6 g/L or less, were prepared by sequentially

diluting 500 mL of a 6 g/L NaCl solu-

a control specimen is necessary, the fluorocarbon emulsion is the most suitable, being the most stable. Added ad-

tion with a liquid dispenser, successively replacing 10-mL aliquots of solution with 10-mL portions of distilled

vantages are that the fluorocarbon ma-

water

and thoroughly

terial does not require ice-water storage and does not subject personnel to

steps.

This

the potential

infectious

hazard

of the

blood-based controls.

process

C,,

1. Leary ET, Graham G, Kenny MA. Commercially available blood-gas quality con-

mixing between was repeated

20

times or more. The NaCl concentration in each aliquot removed (C,,) can be calculated as follows:

References

studies of within-day precision and long-term stability, respectively. The syringe tips were sealed with Luer-lock caps (Becton Dickinson Co., Ruther-

ford, NJ 07070) and stored at 4 #{176}C, in

value. Precision

P0,

trols compared with tonometered blood. Clin Chem 26, 1309-1316 (1980). 2. Hansen JE, Clausen JL, Mohler JG, et al. Blood gas proficiency-testing materials: A multilaboratory comparison of an aqueous solution and a fluorocarbon-containing emulsion. Clin Chem 28, 1818-1820 (1982).

=

Co e

where C0 is the initial

solution concen-

Table 1. Within-Day Precision and Stability In Controlsa Within-day precision

Change (In mmHg) alter

Control

SD, mmHg

CV, %

4h

Whole blood

3.39

2.19

+2.5

+

Erythrocytes Hemoglobin

2.52

Emulsion

1.71

1.61 1.23 1.13

+5.2 +1.0 -0.9

+21.3 +20.4 +1.8

CLINICAL CHEMISTRY, Vol. 30, No. 2, 1984

2.11

150-1 65 mmHg (about 20-22 kPa).

6h

12 h

19.8 +1.8

tration, 6 g/L; f is the dilution factor, 0.02; and n is the number of dilutions. To measure hemolysis, we pipetted 0.5 mL of test solution and 10 tL of the threefold-diluted blood into an 0.8-mL disposable

polypropylene

sample

Chase AM. The osmotic resistance (fragility) of human red cells. J Clin Invest 26, 636-640 (1947).

lot

Y. S. Huang

75

K. Jenkins

cup

M. S. Manku

supplied for the Cobas Bio micro-centrifugal analyzer (Hoffmann-La Roche Ltd., Vaudreuil, Quebec). For the complete-hemolysis standard we added 10 L of diluted blood to 0.5 mL of dis-

J. Davignon 50

Efamol Res. Inst. P.O. Box 818 Kentville, Nova Scotia Canada B4N 4H8 and Clin. Res. Inst. of Montreal

0

a

tilled water. Each cup was capped, and the contents

were mixed

gently

and

allowed to stand at room temperature for 30 mm. The cup was then centrifuged in a micro-scale centrifuge at 8000 x g for 2 mm, to pack unhemolyzed cells and membrane debris into the tip of each cup. The cups were then placed, without disturbance and in order of increasing hypotonicity, into the sample disc of a Cobas Bio analyzer. Supernate from the completely hemolyzed erythrocytes (100% hemolysis), in triplicate,

was

pipetted

into

25

I 50

analyzer

were

as follows:

tained with this automated method

Table 1. Reproducibility of the OsmoticFragilityCurve for Blood

0 5; start

1 (water blank); printout mode, 1. The samples and the water blank are automatically pipetted into the disposable cuvette rotor supplied for the Cobas Bio and mixed by centrifugal force, whereupon the measurement is then made and the reading printed out

automatically and directly as the percentage of hemolysis in each sample. The osmotic fragility curve is then constructedby plottingthe percentage of hemolysis vs the concentrations of the test solutions. A typical curve thus obtainedisshown in Figure 1.

To assess our method, we repeatedly measured

the values of Hso (the concentration of test solution at 50% hemolysis) and H2575 (the difference be-

tween the concentration of the test solution at 25 and 75% hemolysis) in 14 preparations of normal blood. The results (Table 1) indicate excellent reproducibility. The CVs ranged from 0.22% to 0.75% for H and from 2.84%

to 8.82% for H25_75.The mean of H50 (4.460 g/L) for 14 subjects studied agrees well with the mean reported by Maeda et al. (2), 4.408 (SD 0.091) g/L, who used the multiple-tube method (3). With our procedure, the percentages of hemolysis

of all samples

tested

are

Rawal et al. (Clin Chem 29: 586, 1983) recently concluded that the fluorescence polarizationimmunoassay (FPIA) of serum digoxin (from Abbott

H 4.423 4.637 4.513 4.370 4.335 4.493 4.587

H25..75

± 0.018 ± 0.016 ±

0.022

0.023 0.010 0.018 0.023 4.343 0.008 4.571 ± 0.013 4.455 ± 0.010 4.457 ± 0.012 4.355 ± 0.021 4.492 ± 0.023 4.413 ± 0.033 ± ± ± ± ±

0.228 ± 0.010 0.238 ± 0.021 0.272 ± 0.010 0.267 ± 0.008 0.302 ± 0.012 0.297 ± 0.010 0.377 ± 0.012 0.268 ± 0.017 0.352 ± 0.010 0.225 ± 0.013

0.253

0.022

±

0.342 ± 0.008 0.375 ± 0.012 0.395 ± 0.015

4.460 ± 0.095 0.299 ± 0.059

measured directly and simultaneously vs both the completely hemolyzed sample (100%) and water (0% hemolysis); hence, no further calculation is required. In each run, a sample in 24 different hypotonic test solutions can be measured simultaneously within a few minutes. Several sets of assays can

be preparedat the same time, and the entire procedure, including blood preparation,

incubation,

h or longer for the multiple-tube od.

meth-

References

1. Kitazima K, Shibata S. Coil planet trifugationand itsapplicationto the

cen-

obser-

altered membrane properties of erythrocytes in hepatobiliary disorders. J Lab Clin Med 85, 855-864 (1975). 2. Maeda N, Aono K, Sekiya M, et a!. A computerized method for the determination of the osmotic fragility curve of erythrocytes.AnalBiochem 83, 149-161 (1977). 3. Parpart AK, Lorenz PB, Parpart ER, of

Laboratories, Diagnostics Division, North Chicago, IL 60064) offers many advantages, mainly reagent stability and assay speed, as compared with

radioimmunoassay (RIA). They compared the FPIA and the “Amerlex” (Amersham Corp., Arlington Heights, IL 60005) RIA for determining digoxin in 223 patients’ samples, and the correlation was satisfactory. We assayed samples for digoxin with

the kit we use routinely, the Phadebas digoxin RIA (Pharmacia Diagnostics AB, Uppsala 1, Sweden) and the Abbott FPIA, and were surprised to find a poor correlation for 28 patients’ samples (Figure 1). Although the correlation coefficient (r) was 0.975, there was

a consistent and appreciable bias between the two techniques,with values being lower for the FPLA than for the

RIA.

Common

quality

criteria

(repro-

4

and assay, takes

less than an hour, as compared with 2

vation

FluorescencePolarization Immunoassay To the Editor:

6 6 6 6 6 6 6 6 8 4 6 6 6 6

reagent volume, 0 pL; time of first reading, 0.5 5; time interval, 30 s; number of readings, 2; blanking mode,

Effect of Deprotelnization on Determination of Serum Dlgoxin by

Measured by the Described Procedure Soin concn, mean ± SD, 9/1

tests

540 nm; sample volume,50 tL; diluent volume (water), 25 .tL; reagent voltime,

112W 1R7

from 14 NormalPersons,as

No. of

100; standard 3 concentration, 100; limit, 100; temperature 25 #{176}C; type of analysis, 1 (fixed point); wavelength,

ume, 0 zL; incubation

Canada

4,0

Fig. 1. A typical osmotic fragility curve ob-

the

reaction direction, +; units, own; calculation factor, 0; standard 1 concentration, 100; standard 2 concentration,

4.5 NoCI,

standard cups 1, 2, and 3 in the disposable plastic reagent tray. The settings for the Cobas Bio microcentrifuge

ilOPine Ave. W. Montreal, Qu#{233}bec

U-

z 0

C, 0

DIGOXIN

RIA

Fig.

1. Correlation between results by the FPIA and RIA for serum digoxin (nmol/L):y = 0.856 x -0.36 (r = 0.975, n = 28)

CLINICAL CHEMISTRY, Vol. 30, No. 2, 1984

337