Microsampling on the Technicon SMAC System - CiteSeerX

CLIN. CHEM. 29/8, 1531-1534

(1983)

Microsampling on the Technicon

SMAC

System

Trevor A. Walmsley, Richard 1. Fowler, and Maxwell H. Abernethy The sample volume needed for a Technicon SMAC continuous-flow analyzer has been reduced for routine operation. Two options are available: 141 L for a 17-test profile, or 224

jL, which allows the direct-sampling assays for creatinine and iron to be included. The sample decrease is achieved by the sequential dialysis of creatinine and iron, an increased sample dilution from sixfold to ninefold, the strict minimization

of diluted sample stream wastage, and development of more sensitive methods for glucose and alkaline phosphatase to allow greater use of the diluted sample stream. A glycinecontaining diluent increases the sensitivity ofthe iron method by 25% and prevents the protein precipitation that plagues the continuous-flow analysis for iron in plasma. No deterioration in performance of the analyzer has been detected during

nine months of routine operation at the reduced sample size. Added advantages are the decreased consumption of calibration materials and an increased ability to do repeat tests.

unchanged at 235 jiL. O’Leary et al. (3) progressively reduced the predilution sample size from 235 zL to 105 L while maintaining the flow rate of the diluted sample stream. No deterioration in performance was detected. They selected a predilution sample volume of 176 j.L at an overall dilution of 1 in 8.6 for routine operation (personal communication, T. Hurley). Alkaline phosphatase and glucose were fed from the diluted sample stream; iron and creatinine were measured by direct sampling, except for pediatric samples, when these tests were omitted. Our strategy was to use sequential dialysis (4) for measurement of creatinine and iron, reduce the sample and diluent flow rates in the predilution system, and measure alkaline phosphatase and glucose in the diluted stream. By these means we decreased the sample size from 435 tL to 224 L for a 19-test profile or, optionally, 141 p1 for a 17test profile.

Materials and Methods AdditIonal Keyphrases: sequential dialysis microanalysis

.

pediatric chemistry

.

plasma iron continuous-flow analysis .

A new potential of the SMAC system (Technicon Instruments Corp., Tarrytown, NY 10591) can be realized by reducing the sample size to a volume compatible with clinical circumstances where less sample is available than usual. Perhaps the most direct advantage is the ability to produce a multiple test proffle routinely, not only eliminating the necessity for a separate microanalysis facility, but also avoiding the frustration of sample wastage inherent in multiple aliquotting. Other advantages of small sample size are decreased calibration costs and increased ability to schedule repeat tests on any patient’s sample. The addition of a test channel for magnesium (1) and the ability to operate the SMAC out of routine hours make it an extremely valuable analytical tool. Initially, the SMAC installed in this laboratory had its sample stream split five ways: 27 tL for glucose and 27 L for alkaline phosphatase (ALP), 63 jL for iron, 83 iL for creatinine, and 235 L, diluted sixfold, for the assay of sodium, potassium, chloride, carbon dioxide, urea, uric acid, calcium, cholesterol, phosphate, total protein, albumin, total bilirubin, conjugated bilirubin, aspartate aminotransferase (AST), and glutamyltransferase. Magnesium (1) was later added in place of cholesterol. The total sample volume was

435 1L. Previous attempts to decrease the sc sample size have been reported. Stamper and Robertshaw (2) did not include iron or glucose in their profile and developed methods for alkaline phosphatase and creatinine, feeding from the diluted sample stream. Their predilution sample size remained

Department of Clinical Biochemistry, Christchurch Hospital, P.B., Christchurch, New Zealand. Received Jan. 13, 1983; accepted May 3, 1983.

Except as described here, no changes were made to individual SMAC methods. The sensitivity and linearity of all methods were kept within the original SMAC specifications. Sample volumes have been calculated from the nominal flow rate of the sample pump tubes, given that the sample probe aspirates each sample for 22 s. Carryover was determined (5) by using Technicon TCQ Chemistry Controls H and L. Dwell times are given in seconds. The Technicon product numbers of the parts required to carry out modifications are given in parentheses. Predilution manifold. On the regular predilution manifold on SMAC, 642 jiL of sample is mixed per minute with a total of 3210 p1 of water containing, per liter, 1 g of Triton X100 (supplied by five 642 p1/ruin pump tubes). This diluted sample stream is used for most test channels. We reduced the sample pump tube to one with a 385 41 mm rate and replaced the sample-inlet stem (178 G273 01) with transmission tubing (116 0537 07) as recommended by O’Leary et al. (3). The intersample air bubbles were phased to prevent a false increase in the results for the last two specimens (6, 7). The rate for the dilution fluid was reduced to 2914 pJJmin (using one 642 and four 568 pL/min pump tubes) to minimize the volume of diluted sample flowing to waste. The performance of the standard and modified dilution manifolds were assessed for within-day precision by analyzing 45 samples of control material (Interlab QAP Level I; Dade, American Hospital Supply Corp., Miami, FL 33152) over three recalibration cycles. For the correlation study between methods we prepared five samples by mixing “high” (H) and “low” (L) specimens to give 100%H, 75%H:25%L, 50%H:50%L, 25%H:75%L, and 100%L. Each sample was analyzed 18 times over at least three recalibration cycles. For most tests, specimens prepared from Technicon TCQ Chemistry Controls H and L were suitable. Synthetic magnesium and conjugated bilirubin specimens were prepared as follows: Interlab QAP Level II was reconstituted CLINICAL CHEMISTRY, Vol. 29, No. 8, 1983 1531

with 3.0 mmol/L magnesium iodate and 770 pmol/L N-inaphthylethylenediamine (8) for the high specimen. This was blendedwith Level II reconstituted with distilledwater as the low specimen. Aspartate aminotransferase specimens were prepared by mixing a high-activity pool of patients’ sera (estimated at 400 UIL at 37#{176}C) with a low-activity pool

(approximately 10 UIL at 37 #{176}C). The between-day precision was assessed at normal levels, by analyzing Level I during 90-day periods before and after the modifications. Glucose. This method was modified to feed from the diluted sample stream. To compensate for the decrease in sensitivity we increased the sample flow rate from 74 to 118 4/mm, decreased the saline diluent from 568 to 385 ph mm, increased the pathlength of the dialyzer from 109 mm (4.3 inch) to 305 mm (12 inch), and changed the membrane from Type ‘C’ to Type ‘H.’ The dwell time changed from 149 to 205 s and the carryover factor was set to 5. Alkaline phosphatase. In the regular alkaline phosphatase method, 74 ph of undiluted sample is mixed per minute with 385 ph of p-nitrophenol phosphate substrate per minute and incubated at 37#{176}C in a 1.2-mL glass coil. The pnitrophenol is dialyzed and the absorbance measured at 420

am. In the modified method, 226 ph of the diluted sample stream is mixed per minute with 287 p1 of substrate per minute. To compensate for decreased sensitivity we prolonged the incubation by using a 3.3-mL Kel F coil (178

8822 01) at 37 #{176}C. No other changes were made to the manifold. The dwell time was increased from 257 to 487 s and the carryover compensation was set to 5. Iron, creatinine. In the regular manifold, 166 p1 of sample is mixed per minute with 166 ph of ascorbic acid reagent (1 mol/L sodium chloride, 0.2 mol/L hydrochloric acid, and 1 g/L Triton X100; just before use, ascorbic acid is added to give a final concentration of 55 mmol/L) per minute and dialyzed against ferrozine reagent. In the regular creatinine manifold, 226 ph of sample is mixed per minute with 166 p1 of a 155 mmol/L solution of sodium chloride containing, per liter, 1 g of Triton X100 per minute, and dialyzed against water. Sequential dialysis of creatinine and iron was achieved as follows. The 10-turn glass mixing coil on the creatinine manifold was replaced by a five-turn coil adapted from eight turns of a Kel F coil(178 B959 01).To the waste stream from the donor side of the creatinine dialyzer we added, via a reagent/air inlet block (178 B482 02), 74 p1 of glycine/ ascorbic acid reagent (0.6 mol/L glycine adjusted to pH 2.3 with hydrochloric acid and 1 g/L Triton X100; before use,

ascorbic acid is added to give a final concentration of 175 per minute. This stream is mixed in a coil made from the remaining three turns of the Kel F coil, and becomes the sample stream for the iron dialyzer (Figure 1). The shutdown wash solution to the creatinine manifold was changed from hydrochloric acid to 0.1 mol/L sodium hydroxmmol/L)

ide;this was found to be essential in

to

from

Prediluted Manifold SMAC system, the air segments in the stream are removed by the #{176}LOAI” (Liquid Out Air In) pump and four large inter-sample air bubbles are introduced. In this process, 164 ph of the diluted stream is lost per minute. The flow rate of the remaining diluted stream suffices for sampling any combination of the test profiles available on src. However, our SMAC configuration required only 2670 ph of the diluted stream per minute, and the remaining 1018 p1/mm represents a wastage of 26%. This was minimized by decreasing the flow rate of the diluted stream. The effect of this change was studied by investigating dwell times and carryover of the last four test channels of the sequence sampling from the diluted stream (chloride, carbon dioxide, calcium, and total protein). The measured waste flow was reduced from 1068 to 198 pLfmin by progressively changing the five diluent tubes from 642 4/ruin each to 482 ph/mm. The carryover on the channels studied was unaffected by the flow rate of the diluted stream (Table 1). The dwell times on the last test to be resampled (total-protein test channel) increased by a maximum of 165 and remained well within the specifica-

In the regular

diluted sample

tions set by the manufacturer. O’Leary et al. (3) claimed that it is necessary to maintain the specified flow rate of the diluted stream, but presented no data. Our results demonstrate that the flow rate is not critical. The sample size was progressively decreased from 642 to 287 p1/mm while maintaining minimal flow rates of the diluted stream. We chose a sample flow rate of 385 p1/mm plus 2914 ph/mm diluent, to give the maximum sample dilution compatible with the manufacturer’s sensitivity specifications; it representsa dilution of 1 in 8.6. The waste flow rate was 380 4/mm. We compared results obtained from SMAC before and after the dilution manifold was modified. Linear regression analysis showed that in all cases the correlation coeffIcient exceeded 0.985. The only significant discrepancies detected (p < 0.05) were for albumin, where the inherent nonlinearity of this method leads to the poor linear correlation (nevertheless, the maximum difference in results was < 1 g/L), and aspartate aminotransferase, where substratedepletion occurredat 300 U/L (37 #{176}C) in the standard method as compared with 430 U/L (37 #{176}C) for the modified method. No overall deterioration in precision was observed with use of the decreased sample

volume. Glucose. There was an improvedresolutionof peaks with the modified method, and sensitivity was increased by 22%. Analysis of 25 patients’ samples in duplicate, covering a

Table 1. Effect of Dilution-Stream Flow Rate on

prevent protein buildup

the iron dialyzer. The dwell time of the iron manifold

changed

Results and Discussion

185 to 213 s.

was

Carryover No. of prsdliutlon pump p1

S..pI.

Sir...

Prom

Cr..tlnln.

642

DillyI.,

LJmIn WI.,.

45 tOrI

Dwell

Waste flow

time, total

C.rryo var,

482 &LImIn

rlte, L/mIn

protein test, a

Ci

CO2

Ca

protein

Total

1068

234 238

8 8

7

5 5 5 5

5

0

4

1

877

3 2

2 3

692 532

241

9

244

8

7 7

6 5 5 5

1

4

367

247

9

7

6

5

6

5

8

250 9 7 5A was used throughout. bpyover compensation set to 0 before the expeflment. 0

FIg. 1. Modif led Iron manifold 1532

CLINICAL

CHEMISTRY,

Vol. 29, No. 8, 1983

%5

5

198 sampletube of 642 L/mIn

range from 0 to 20 mmol/L, by both the modified (y) and the standard method (x) gave by linear regression analysis a slope of 0.985 and intercept 0.1 mmol/L (r = 0.999 and S,1,, = 0.17 mmoJJL). The paired standard deviation was 0.18 mmol/L for x and 0.20 mxnollL for y. No nonlinearity or nonspecificity was detected. Alkaline phosphatase. Improved resolution of the peak shapes with the modified method and a sensitivity reduction

of 10% were observed. Analysis of 25 patients’ samples in duplicate, covering a range from 30 to 500 UIL (37 #{176}C), gave a slope of 1.06 and intercept 1 U/L (r = 0.999 and Si,,, = 5.2 UIL). The paired standard deviation was 2.3 and 3.3 UIL at 37#{176}C, respectively, and no nonspecificity or nonlinearity was detected. The slope was significantly greater than 1.00, and we reduced the set point by 6% to compensate. Iron. Protein precipitation in the iron manifold seriously interferes with the analysis of plasma samples (9, 10). We routinely use lithium heparin anticoagulated plasma samples and in the past have experienced serious blockages when using the recommended sodium chloride-hydrochloric acid diluent. We eliminated this problem by using as diluent a glycine buffer at pH 2.3 to stabilize gamma globulins against spontaneous precipitation (11). At the optimum glycine concentration of 0.1 mol/L there is an increase in sensitivity of 25% over the conventional sodium chloridehydrochloric acid diluent. (The use of glycine buffer is generally applicable to continuous-flow iron manifolds; no precipitation problems have occurred in our six years of experience with AutoAnalyzer H manifolds.) Analysis of 20 patients’ serum samples in duplicate, covering a range from 2 to 50 Mmol/L, by the modified method (y) and the regular method (x) gave a slope of 1.04 and intercept 0.4 imoI/L (r =0.998 and S = 0.67 p.mol/L). The paired standard deviations were 0.28 and 0.21 j.anol/L, respectively.

Cross contamination of standards. The 50% reduction in sample volume from 435 p1 to 224 pL enables a total of 10 recalibration cycles to be completed from one ifiling of the first standard vial and 15 from the second. During each recalibration cycle a small amount of wash solution is carried by the sample probe to the standard vials and causes a gradual dilution (12). Figure 2 shows that this effect of

V

No Probi With

WIp.r

Probe

Wlp.r

2.55-

2.50-

2.45-

E 3

Table 2. Results (CV, %) of Between-Day Precision Analysis Before

modification Sodium Potassium Chloride

Carbon dioxide Glucose Urea Creatinine Uric acid Calcium

0.7 1.9 1.3 5.5 3.4 4.5 4.0 3.7

1.8

Magnesium Phosphate

2.4 2.2

Total protein Albumin Total bilirubin ALP AST

1.1 4.1

4.4 3.4 7.8 Glutamyltransferase 7.7 2.2 Iron ‘Of Dade Level I samples. n =90 throughout

After modification 1.0 1.7 1.4 5.4 2.4 4.3 6.4 4.4

1.8 1.6 2.4

1.7 2.3 4.2

4.1 8.8 6.4

2.1

standard dilution on calcium results is apparent after five recalibrations. By 10 recalibration cycles, calcium results are falsely increased by 0.14 mmol/L (6%). The use of a probe-wiper sponge on the sampler wash reservoir as designed for the SMAC H analyzer (107 0708 01 and 107 0462 01), minimizes calibration errors for all tests. Effect of sample volume variability. Problems relating to sample volume variation have been reported (6). We divided a patients’ sample pool into 24 sc “short sample cups” (178 4469 01), using alternate volumes of 5 mL and 250 ph.

The samples were analyzed sequentially on SMAC. The only significant differences found (p < 0.05) in the results from the 5-mL samples and the results from the 250-ph samples were a decrease in the means of the sodium results from 138.8 to 130.0 mmol/L and a decrease in the means of the urea results from 10.4 to 10.3 mmol/L. Otherwise, accuracy and precision remained unchanged. Overall performance. Leng-term performance, assessed by determining precision before and after the modifications, showed no overall deterioration (Table 2). The percentage of specimens that had to be repeated on sIAc owing to malfunction of one or more of the tests was 22.9% before the modification and 23.2% afte; this compares favorably with the re-analysis rate of 23.4% reported by Mazza (13). With use of the modifications recommended here, sample volume has been reduced from 435 ph to 224 ph for our 19test profile. We have used this reduced sample size routinely for over nine months for the analysis of more than 75000 samples with no problems attributable to the change. For analysis of pediatric samples and other small-volume samples we now have the option of using a 224-ph sample or, where specimen volume dictates, the creatinine-iron stream is bypassed, giving a 141-ph sample size for a 17-test proffle. We thank the Medical Research Council of New Zealand for and Mrs. J. McConefor technical assistance.

2.4O

financial support

a U

References

2.35-

I

I

I

I

I

I

I

1

2

3

4

5

6

7

8

No

of

Recalibration

9

10

Cycles

FIg. 2. Changes In results for calcium arising from serial recalibratlons with use of a single fill of calibration standard vials

1. Fowler RT, Abernethy MH, Walnisley TA, Taylor HW. Measurement of magnesium by continuous flow colorimetry. Clin Chem 28, 523-525 (1982). 2. Stamper R, Robertahaw DW. Decreasing sample volume used in the “Sequential Multichannel Analyzer Computerized” (s7sAc).Clin Chem 26, 778-780 (1980).

CLINICALCHEMISTRY,Vol. 29, No. 8, 1983 1533

N, Hurley T, Stapleton M, et al. Simple adaption of the sssc system to operate 18 channels with reduced plasma. Ann Clin Biochem 18, 112-117 (1981). ZK, Turner JC. Reduction in sample size on the Technicon SMA 6/60 continuous-flow analyzer. Clin Chem 22, 1107-1109 (1976). 5. Broughton PMG, Gowenlock AN, McCormack JJ, Neill DW. A revised scheme for the evaluation automatic instruments for use in clinical chemistry. Ann Clin Biochem 11, 207-218 (1974). 6. Walrnsley TA, Abernethy MH, Fowler RT. Compression of sample-line air segments-a source of error in the Technicon SMAC system. Clin Chem 26, 530-531 (1980). Letter. 7. Abernethy MH, Walmsley TA, Fowler RT. Further evidence for the importance of inter-sample air compression as a source of error in a continuous flow (Technicon sMAc) system. Clin Chem 28, 19911992 (1982) Letter. 8. Furda J, Morgenstern S, Snyder LR. Derivatives of 1-napthyl 3. O’Leary Technicon volumes of 4. Shihabi

CLIN. CHEM. 29/8, 1534-1536

ethylenediamine dihydrochioride forstandardization of direct bilirubin assays done on the Technicon SMAC. Clin Chem 22, 10421046 (1976). 9. Whitby LG, Proffitt J, Taylor RH. Seven years’ experience of the sMAcI system.Technicon AutoAnalyzer SingaporePte Ltd., Singa-

pore, 1982. 10. O’Leary N, Duggan PF. Some improvements in operating of the Technicon sattc system. Clin Chem 25, 793 (1980). Letter. 11. Kabat EA, Mayer MM. Properties of gamma globulins. In Experimental Immunochemistiy. 2nd ed., Charles C Thomas, Springfield, IL, 1961, pp 764. 12. Percy-Robb 1W, Whitby LG. Report of the evaluation of the Technicon Sequential Analysis plus Computer (sMAc) system. Scottish Health Service Common Services Agency, Trinity Park House, South Trinity Road, Edinborough, 1977. 13. Mazza L. saic-one year of experience. In SMAC Today, Technicon International Division, Geneva, 1977, pp 19-26.

(1983)

Transcutaneous Carbon Dioxide for Short-Term Monitoring of Neonates Gerald J. Kost, John L Chow, and Margaret A. Kenny We studied transcutaneous pco monitoring in 70 neonates, most of them premature with respiratory distress syndrome. Measurements were at 44#{176}C. Calibration drift was large in some instances. Least squares linear regression analyses of

is wasteful of blood and can lead to vascular and infection complications. Additionally, the trauma of the sampling procedures can alter blood gas values, rendering them unreliable for clinical management (1). We found that tc

transcutaneous Pco (y) vs arterial p (x) in kilopascals showed, for all observations (n = 516), or one observation

Pco2 data aided respiratory

randomly selected from each patient (n = 70), and for the first observation from each patient (n = 70): y = -0.28 + 1.80x, y = 0.01 + 1 .74x, and y = 0.73 + 1 .63x, respectively. Regression lines for individual patients with 14 or more observations each were not coincident (F = 2.80, p < 0.002). Transcutaneous pco2 monitoring was most useful clinically as a means of following short-term trends in arterial continuously during extubation and afterward when avoiding re-intubation. In view of the potential for error associated with drift, we recommend that intervals between calibrations be limited to about 3 h.

Methods and Materials

Additional Keyphrases: blood gases

arterial CO2 tension

We studied noninvasive monitoring of transcutaneous (tc) carbon dioxide tension (pco2) over a period of 18 months in our Neonatal Intensive Care Unit. Bedside measurement of tc pco2 offers a means of following trends in arterial Pco2 (paco) continuously without the need for repetitive sampling. Particularly in premature infants, invasive sampling Department of Laboratory Medicine, SB-b, University Hospital, University of Washington, Seattle, WA 98195. Received Mar. 7, 1983; accepted May 6, 1983. 1534

CLINICALCHEMISTRY, Vol. 29, No. 8, 1983

intubation,

weaning

management, especially during from a respirator, and extubation.

To measure tc Pco2 we used microprocessor-based TCM2O monitors and matching electrodes (Radiometer America, Inc., Cleveland, OH 44145), with Radiometer S44716 electrode filling solution. For two-point calibration, we used the tonometer cell mounted on the TCM2O unit, in combination with a Radiometer A7405 calibration unit. Carbon dioxide contents of the calibrating gases were 50 mLIL (about 38 Torr’ or 5 kPA) and 100 mLfL (about 76 Torr or 10 kPa). The temperature setting of the tc pco2electrode was 44#{176}C during both calibration and clinical monitoring. Electrode heat is used to produce vasodilation of the capillaries in the skin immediately opposite the electrode sensing area surface, thereby facilitating to pO2 responses to systemic changes in paco2. We placed the to Pco2electrode on the chest or abdomen. For in vitro (37 #{176}C) measurements of arterial blood gases from umbilical, radial, or dorsalis pedis sites, we used an 1L813 blood-gas analyzer (Instrumentation Laboratory, Lexington, MA 02173), with accepted quality-control routines (2, 3) for small sample volumes (about 0.3 mL). carbon dioxide

1

1 Torr

133.3 Pa