Critical care analytes: pre-analytical factors affecting result

Journal of Automatic Chemistry Vol. 11, No. 5 (September-October 1989), pp. 201-205

Critical care analytes: pre-analytical factorsaffecting result quality combined bloodgas and electrolyte systems

R. F. MoranTechnical Liaison, Ciba-Corning Diagnostics, Medfidd, Massachusetts 02052,USA

and A. FeuilluCentre Hospitalier Rggional Pontchaillon, Laboratoire de Services d’Urgences et deRganimation, Rennes 35033, France

Introduction

’Incorrect test results are much more often due to patientand specimen collection considerations than to labora-tory variability or error’ [1], according to one USA basedprofessional organization. Shapiro [2] noted in a studyinvolving thousands of patients, more than 5% of theindividually collected specimens were questionable basedon pre-analytical error, and, indeed, more than 15% ofthe specimens received from the emergency room wereassociated with significant pre-analytical error! Since’blood gas and pH analysis has more immediacy andpotential impact on patient care than any other labora-tory determination’ [3], it is evident that while themedical laboratory analyst must be primarily aware ofthe impact of analytical error, pre-analytical consider-ations need to be taken into account by others in theassessment of the reliability of results to be reported. Anunderstanding by physicians, nurses and other directpatient care practitioners of the ’Cycle of qualityassurance’ (see figure) for blood gases and other criticalanalytes will minimize the chances that pre-analyticalerror will significantly effect patient care. The laboratoryprofessional must be involved in the process of educationabout these issues.

Pre-analytical considerations

Of major impact on the utility of blood gas, totalhaemoglobin and electrolyte results are (1) the physio-logic stability of the patient; (2) the effects of bloodmetabolism and collection/transport conditions on theanalytes measured; and (3) the timeliness ofthe collectionand reporting of results.

A comprehensive review ofvarious pre-analytical aspectsof blood gas analysis is given in a document prepared bythe USA-based National Committee for ClinicalLaboratory Standards (NCCLS) [3]. This article concen-trates on certain key points of that document, as well asproviding some more recent additional information.

Physiologic stability

The ventilatory process is a key consideration affectingblood gas values. Patient postural changes affect ventila-tory rate and depth, thus causing real changes in bloodgas values, especially in the Pco2(aB), but also in thepo2(aB). Circulatory compromise, such as that seen inarrested patients during resuscitative efforts, can causesamples from peripheral collection sites to be non-representative of whole body conditions. The effects ofthese types ofconditions could either be recognized by theclinician or the changed results assumed to be ’labora-tory’ error, thus masking a need for intervention, orbringing about inappropriate intervention. Patients oncontrolled ventilation, or supplemental oxygen, ideallyshould be assessed only after new settings have been ineffect for at least 20 min. In other cases, especially when’weaning’ a patient from mechanical ventilation (PEEP,CPAP, IMV) or supplemental oxygen, a result may beurgently required between 5 and 10 min after eachchange.

Collection and transport conditions

The collection process itself, being potentially painful,may bring on an apprehensive hyperventilation by thepatient, causing an immediate effect on Pco(aB),lowering the value.

Collection devices are also subject to limitation. The idealsystem incorporates a collection device (syringe) thatminimizes or eliminates exposure to air, includes thecorrect extent ofheparin anticoagulation (20-50 U/ml ofblood) and enables identification of the collection site asbeing arterial (by responsiveness to arterial pressure). Ifsubsequent or simultaneous measurement of sodium,potassium or ionized calcium on the same sample isanticipated, the effects ofthe anticoagulant must be takeninto account. (For example the use of sodium heparinatewill normally elevate the measured sodium by 1-3 mmol/1, or even more ifthe syringe is not completely filled. Also,the binding effects of heparin on ionized calcium canlower the sample’s ionized calcium significantly if theheparin dose is >20 U/ml of blood.)

One of the more significant sources of unrecognized errorin the Pco2 measurement is the use of too great a volume(as opposed to units) of liquid heparin relative to theamount of blood collected (underfilling a syringe), whichleads to a falsely low value. The pH may also be lowered,

0142-0453/89 $3.00 ( 1989 Taylor & Francis Ltd.201

R. F. Moran and A. Feuillu Critical care analytes

but only when the underfilling is severe. Use ofa low-dosedried lithium heparin anticoagulant or a ’calciumbalanced’, or ’titrated’, heparin should eliminate theseproblems.

Exposure of the sample to air has the obvious effects onboth Po2 and Pco2 due to potential differences in thetensions of the gases in air versus the sample. Anunrecognized cause of error is the common practice of’flicking’ the syringe to rid it of air bubbles prior to

measurement, since this excessive agitation can causeformation of tiny air bubbles which will equilibrate withthe sample more readily than a large bubble. This willtypically lower Pco2 and elevate Po2 values; however, ifthe patient is breathing high concentrations ofoxygen, aircontamination may cause a lowering ofpo values.

The use of ’arterialized’ capillary blood for the measure-ment ofpo should be recognized as a last resort only.

While acceptable for pH and Pco, the results are areliable estimate ofarterial values only at oxygen tensionsless than 8"0 kPa (60 mmHg). Above that level, theopposing effects of tissue metabolism and air contamina-tion during collection create a large potential uncertaintyin results at oxygen tensions where accuracy andprecision are paramount. (For example at or near 13"0kPa [approximately 100 mmHg], the point at whichoxygen therapy levels are most critical in neonates.)

Vacuum tubes are suitable collection devices only forvenous blood and acid base/electrolyte measurement,since, because of the vacuum, they aspirate blood fromany vessel, and thus reduces the ability to assessplacement of the needle in an artery. The gas bubble inthe tube can change sample results (lower Pco andelevatepo). Ifa vacuum tube is used the report should belabelled as to source, and the appropriate symbol used[for example pH(vB), Pco(vB)].

PRE ANALYTIC POST ANALYTIC

;; - DiagnosisAssessment "’, Action

Action Plan / Interpretation

/ ’’ "J/’ Result// / Patient \ Reporting

7, ( 41 4<,Collection \ II/

i Result\ Physicia.n)/

ii ../i Review

FMeasTransport/Tracking" urement

ANALYTICInstrument Performance

or"Quality Control"

Storage

The cycle ofquality assurance. (The area enclosed by the dashed line represents thatfor which the laboratory typically assumes responsibility.)() 1990 R. F. Moran

202


Due to frequency of multiple blood gas analyses on thesame patient over short time intervals, recording of theexact collection time on each specimen is necessary. Ifmore than one specimen on the same patient is in theblood gas laboratory simultaneously without the collec-tion time or sequence being labelled, the results should beheld from reporting until the issue is resolved.

The immediate impact that blood gas results can have onclinical management and decisions affecting morbidityand mortality emphasizes the need for strict adherence toproper identification of the patient, patient conditionsand the sample while at the same time treating the sampleas quickly as possible.

Metabolic effects

Once collected, the specimen should be analysed in a fewminutes (less than 10) or placed in an ice-water slurry.(Use of crushed ice alone will cause uneven cooling andartificial coolants may cause freezing of portions of theblood sample.) The effect of this slurry is to reducecellular metabolism which in turn minimizes the utiliza-tion of oxygen and production of carbon dioxide by this’living tissue’ sample. A combination of immediate icingand analysis as soon as possible may be required withknown severe leucocytosis (leukaemia) or reticulocytosis,or with severe sepsis, since the increased numbers ofmetabolically active cells can cause rapid changes in thegases, especially oxygen.

If simultaneous or subsequent analysis of potassium onthe same sample is anticipated, the analysis should occurwithin 30 min ofcollection and placement in the ice-waterslurry. Potassium values on samples kept in an ice-waterslurry for longer times may be elevated by 0"2 mmol/1 or

greater [6]. Some data indicates that samples frompatients with cold agglutinins may be substantiallyelevated after only a few (<15) minutes in the ice-waterslurry [7]. Both of these findings were observed when novisible haemolysis was present.

Turnaround time

While post-collection intervals as long as two hours maynot invalidate the measurement of pH, Po2 and Pco2 in aproperly iced sample, most clinical needs are for a muchshorter duration (15 to 30 min turnaround time) [8].

Analyser maintenance

For consistently reliable results, it is important that allroutine maintenance be carried out in the manner andfrequency specified by the manufacturer. While changesin maintenance may not affect results immediately,substantial changes in practice will, over time, alterperformance. Changes in performance may also result ifreagents and solutions other than those specified by themanufacturer are used. Again, the changes may be subtleand may take a long time to become obvious. The endresult, however, will be the failure of the analyser toperform as reliably as it is capable of doing.

Analysis

When introducing the sample to the analyser, it isessential to carefully read and follow the instructions forthe particular instrument. Each analyser’s sample-handling protocol may be different, and differences intechnique on the same analyser may affect results.Complete mixing of the sample prior to analysis, by acombination of rolling and inversion of the samplecontainer, is important to ensure satisfactory results inthe measurement ofpH, Pco and Po, and it is critical ifthe measurement of total haemoglobin (tHb) or packedcell volume (pcv or Hct) is to be performed.

Reporting results

After analysis, results for each analyte should be com-pared with the history, patient temperature, past results,ventilatory/supplemental oxygen conditions at the timeof collection, and with prior ’blood gas’ values on thesame patient to see if they are compatible. An excellentcheck for within-sample result consistency is applicationof the modified ’alveolar air equation’:

Po2(A)* FIO2[Patm Primo] l’25[Pco(aB)] (1)* (A) refers to alveolar gas.

By substitution of the fraction of inspired oxygen (FIO2),the atmospheric pressure (Patm), the water vapourpressure PI4O) and the measured Pco2(aB), the alveolarpartial pressure of oxygen can be estimated and com-pared with the arterial value as measured. Given theexpected difference between alveolar and arterial Po,[A-aDO], an assessment of ’mistakes’ in the measure-ment can be made before reporting the results. Theminimum criterion is that the A-aDO be >0, althoughtypically it is 0"7-2 kPa.

The usual precautions hold true in reporting blood gasresults by whatever means (verbal, written or automatic)but again, as with the patient and specimen identifica-tion, the immediate and critical impact of the blood gasresults serves to emphasize that no exceptions to standardprotocol can be tolerated.

Temperature ’correction’ of blood gas values

The values for pH and gas tensions unquestionably varywith respect to temperature. As a result, many labora-tories report blood gas values ’corrected or adjusted’ tothe temperature of the patient.

While some clinicians may have practical experience and’know’ what to expect at temperatures other than 37 C,there are no widely accepted reference values at differenttemperatures. Next, is the issue of what to do with the’adjusted’ values obtained. For example, does one try toregulate the patient’s pH and Pco2 to 7"40 and 5.33 kPa(40 mmHg) respectively? Finally, what algorithms doesone choose to use to make the adjustment from 37 C tothe patient’s temperature?

From the perspective ofthe blood gas laboratory, the firsttwo issues are most reasonably addressed by accepting

203


the recommendations ofAshwood et al. [9]. For the acid-base values (pH and Pco2) and calculated quantities,such as actual bicarbonate, report values at the measur-ing temperature of 37C. For Pco2 and Po, used inassessing gas exchange and possibly compared withexpired gases, report temperature ’adjusted’ values. On apractical basis, this means that the carbon dioxidetension and oxygen tension values should be reported atboth temperatures, while pH should be reported only for37 C. The report itselfshould clearly distinguish betweenthe values at the two temperatures.

The last issue is easiest to address, since, for most bloodgas laboratories, the algorithm used is that chosen by themanufacturer of the analyser. NCCLS document C-12T2[4], a frame of reference accepted by many, states that itsstandard is met if the algorithm used to make the’correction’ gives results that are quantitatively similar tothose specifically documented in the standard.Algorithms chosen by the three major blood gas manufac-turers meet those criteria.

Assessment of measuring system performance:quality control

A properly maintained blood gas analyser, calibratedeither with buffer and gases prepared by the manufac-turer or traceable to them 10], will give years of reliableservice.

As noted earlier, many or even most blood gas result’errors’ occur outside the area of laboratory analysis [1,2]. Additionally, many modern blood gas analysersincorporate automatic devices which signal when theanalyser is not performing adequately.

Despite these facts, there is an increasing level of activityin the use ofinternal and external quality control schemesto check on analyser performance. Quality controlschemes, of course, only check on performance of theanalyser on the test material actually chosen, and at thetime the control material is actually measured. As such,any such quality control scheme needs to be considered asonly a part of a complete programme that addresses thevarious issues discussed in this report. The requirementsand complications ofquality control protocols for a bloodgas analyser are substantially different from otheranalyses performed in the clinical laboratory environ-ment. This is due substantially to the combination ofturnaround time requirements for the patient sample, thefact that the patient sample is fresh whole blood--thatis--living tissue, and of course, the various pre-analyticalitems discussed earlier in this report.

The optimum technique for establishing the inaccuracyand imprecision ofthe gas channels ofan individual bloodgas analyser is the use ofwhole blood tonometry [11, 12],using fresh anticoagulated whole blood, carefully equili-brated with a known standard mixture ofoxygen, carbondioxide and nitrogen at 37C. Properly done, thistechnique enables determination of the performance ofthe analyser when measuring blood at the partial

204

pressures of the tonometry gas mixtures. In essence,tonometry more closely resembles a primary standard ina true sample matrix, not merely a control material. Thetechnical and economic advantages of whole bloodtonometry must be balanced by hazard potential and thelabour-intensive nature of the process.

The alternative to whole blood tonometry is the use ofcommercially available pre-packaged materials, eachtype having its own ’mix’ of advantages. There arecurrently three major types of commercially availablecontrols used for blood gas quality control each of whichis gas-equilibrated; aqueous buffer solutions, blood-based(haemoglobin-containing) materials, and perfluoro-carbon/oil emulsions. Their effectiveness varies and isdependent on the particular analyte and the physical andchemical characteristics of the material itself. While, forblood gases alone, one might consider the purportedmarginal advantages ofnon-aqueous materials, the fact isthat aqueous buffers are economical and acceptable forroutine performance assessment of pH and blood gas.However, if electrolytes are also to be measured on thesame analyser as the blood gas, it is probably mostfeasible to use the aqueous-based materials, since ac-ceptable values for the electrolytes are also available.While not as good as whole blood (patient) duplicatesrun on separate analysers offer the best combination ofeconomy, feasibility and analytical quality.

The fundamental issue with respect to any non-bloodcontrol is that their physical and chemical properties donot, in many respects, match those of whole blood. As aresult, they may not detect certain problems or they maysignal problems that are not there. The former situationcan result in clinical problems; the latter creates financialissues due to the cost of repeat controls, labour, andpossibly, unnecessary service calls.

Selection of a particular type of control must be based onevaluation of its technical and other merits in the contextof a complete blood gas quality control programme. Inthat context, the pre-packaged controls can serve aprimary role in assessing blood gas analyser performance,while remembering that when a true performance issuearises (with respect to the reliability of patient results),only reference methods/materials may be able to settle itsatisfactorily 12].

Duplicate analysis of the same blood sample can be auseful tool, but should not be used as the sole method forassessing instrument performance. Duplicates measuredon two different analysers are most useful in detectingindividual sample errors due to the improbability ofsimilar errors occurring simultaneously on two differentinstruments. Certain types of pH system malfunction(reference circuit) can only be reliably detected usingwhole blood. In an emergency situation, where analyticalreliability and turnaround time are equally important,duplicate analysis ofthis type may be a requirement. Bearin mind, however, that duplicate analysis on the sameinstrument has a marginal usefulness at best, and atworst, provides a false sense of security.


Descriptions of available blood gas control materials 12,13] and detailed protocols for their use [3, 13] may assistin choosing the quality control programme that meets therequirement of the particular institution.

For those just beginning to consider a formal qualitycontrol programme for blood gas or any other analyte, itis best to assess the clinical requirements for resultquality, the analyser’s age and performance history, aswell as the control scheme and materials available. Whilethere is no substitute for quality, the extent needed for aparticular analyte group, such as blood gas, is not thesame in all institutions.

It is most important to start by reviewing all of the pre-analytical issues discussed above with the medical staffofthe institution, at the same time determining their clinicalrequirements. Once a quality assurance programme toaddress the pre-analytical concerns has been established,then a quality control program for the analyser, sufficientto meet the clinical requirements, can be started.

Typically, this might begin by combining the use of (1)daily duplicate analysis of several blood specimens(preferably on two analysers), plus statistical comparisonof results; and (2) weekly or daily analysis of buffers of adifferent lot number than that used to calibrate theanalyser. In either case, if results of analysis differ to anunacceptable extent, corrective action must be taken andthe results recorded. The next step, after some experienceis gained with the above approach, might be to use asingle level (abnormal--either high or low) of commer-cial pre-packaged control. This is used to test perfor-mance after each change in reagents or electrodes. Aneven better approach would be to use the control daily sothat normal performance can be compared with perfor-mance after electrode or reagent changes.

In all cases, in developing a quality control programme,keep records go that a real value to the quality controlscheme is clear. By observing values obtained, calculat-ing mean values and standard deviation [13] andcomparing results with specific performance problems,an overall scheme can be developed. If done in thisplanned, stepwise manner, it will best meet the particularinstitution’s requirements by balancing costs, time, andquality.

Summary

As indicated at the outset ofthis article, the critical natureof the clinical requirements when blood gas testing isindicated, coupled with the labile nature of the sampleand rapidly changing patient conditions, makes it

imperative that the analyst be aware ofmore thanjust theanalysis itself. Knowledge by the analyst of the effects ofthe changing patient environment, and the manner inwhich the sample is treated in the few short minutes fromcollection to reporting are equally important if one is toassure timely and clinically meaningful results.

References and Bibliography

1. JONES, R.J. and PAULONIS, R. M. (Eds), Laboratory Tests inMedical Practice (AMA Council on Scientific Affairs, 1980),11.

2. SHAPIRO, B. A., HARRISON, R. A., CANE, R. D. andTEXPLIN, R., Clinical Application of Blood Gases, 4th edn.(Year Book Medical Publishers, Chicago, 1988).

3. EICHHORN, J. H., MORAN, R. F. and CogMw.R, A. D., BloodGas Pre-Analytical Considerations: Specimen Collection,Calibration and Controls (NCCLS Publication, C-27T,Villanova, 1989).

4. BURNETT, R. W. et al., Definitions of Quantities and ConventionsRelated to Blood pH and Gas Values (NCCLS publicationC-12T, Villanova, 1982).

5. SIGGAARD ANDERSON, O., The Acid Base Status of the Blood(Munksgaard, Copenhagen, 1978).

6. MORAN, R. F. and GRENIER, R. E., The effects of’standard’blood gas transport and storage conditions on electrolyteand haemoglobin results. Presented at European Workinggroup on Ion Selective Electrodes, 9th Meeting, Stresa,Italy (1988).

7. MxsxANo, D. (Massachusetts [USA] General Hospital),Personal communication (November 1988).

8. VANDERLINDE, R. E., GOODWIN, J., KOCH, D., SCHEER, D.,STEINDEL, S. and CEMBROWSKI, G., Guidelines for ProvidingQuality Stat Laboratory Services (AACC Press, 1987).

9. ASHWOOD, E. R., KOST, G. and KENNY, M., ClinicalChemistry 29, (1985), 1877.

10. TERRA, P., MALEY, T. C. and MORAN, R. F., ASEANJournalof Clinical Science, Supplement (1984), 23-29.

11. FALLON, K., BtRNETT, R. W., CHRXSTIANSEN, T. F.,CLAtJSEN, J. L., DtRST, R. A., EnRME’ER, S., EmHnORN,J. H., LADENSON, J. L., LEGG, K. D., MORAN, R. F.,VANKESSEL, A. L. and WEISBERG, H. F. Devices MeasuringP02 and PCO2 in Blood Samples (NCCLS Press, C-21T,p. 130.

12. HANSF.N, J. E., STON, M. E. ONY, S. T. and VANK.ssL,A. L., American Review of Respiratory Disorders, 125 (1982),480.

13. MORAN, R. F. and GRENIER, R. E., Canadian Journal ofMedical Technology, 50 (1988), 95.

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