Science. Reagent Carryover Studies: Preventing Analytical Error with Open Clinical Chemistry Systems

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1 Reagent Carryover Studies: Preventing Analytical Error with Open Clinical Chemistry Systems Joseph Boneno, MS, SC(ASCP), 1 Michelle Fokakis, MT(ASCP), 2 Dave Armbruster, PhD, DABCC, FACB, C(ASCP) 3 ( 1 Our Lady of the Lake Medical Center, Baton Rouge, LA, 2 Abbott Laboratories, Global Service Organization, Dallas, TX, 3 Abbott Laboratories, Scientific Affairs, Chicago, IL) DOI: /MMLEYHNBY4WA12J6 Received Revisions Received Accepted This study examined the validation of performance of open clinical chemistry analyzers. Specifically, user defined applications on open clinical chemistry analyzers and analytical errors due to reagent carryover/cross contamination are examined. Control of laboratory errors associated with the analytical phase of testing was also examined. The modern practice of clinical chemistry relies ever more heavily on automation. Automated analyzers employ robotic pipetting and cuvette washing systems, designed to continuously sample patient specimens, dispense reagents, and clean specimen and reagent probes and cuvettes. Clinical chemistry systems can hold a large number of different assay reagents and perform a wide variety of tests. Analyzers typically use 1 or 2 probes to dispense reagents, and these probes are exposed in rapid succession to a wide spectrum of different reagents. Likewise, cuvettes are exposed to the same variety of different reagents as they are used over and over. A very real concern with analyzers is reagent carryover, that is, reagent from an initial assay clinging to a reagent probe and contaminating the reaction mixture of the next test immediately following the initial assay. Inefficient washing of cuvettes can also result in residual reagent from one test remaining in a cuvette and contaminating the reaction mixture for the next assay whose reagents are pipetted into the same cuvette. In many cases, reagent carryover has no effect on patient results, or it is clinically inconsequential. However, reagent carryover can result in a significant positive or negative bias, shifting patient test results falsely high or low to the extent that there are clinical consequences and potential adverse patient outcomes. Even if reagent carryover is not clinically significant, any amount of false depression or elevation of results due to this phenomenon represents analytical error. No laboratory analysis is error free and, in fact, a certain amount of error due to imprecision and bias is always expected and is acceptable, as long as the total error does not exceed a medically significant limit. Clinical laboratory professionals should understand the sources of analytical error inherent in their testing systems and assure themselves that the observed error is within acceptable limits. Many IVD manufacturers of clinical chemistry analyzers also supply the reagents to be used with the analyzers, and the combination of analyzer and reagents comprises an analytical system. Manufacturers perform verification and validation testing to ensure that systems meet performance claims. Such testing includes examining reagent carryover. Situations in which reagent pairs demonstrate an unacceptable degree of interaction are identified and the carryover is eliminated, or decreased to an acceptable level, by optimizing reagent probe and cuvette washing. Many modern clinical chemistry analyzers are open systems and they can perform user defined assays, that is, tests using reagents other than those supplied by the manufacturer of the analyzer. The versatility of an open system is an advantage because a laboratory can purchase reagents from many different suppliers and adapt them to an analyzer to meet its unique testing requirements. Common reasons for developing user defined applications are to add tests to the menu that are not available from the analyzer manufacturer or to substitute a third party assay that offers better performance than one that is available from the analyzer manufacturer. It is the prerogative of a laboratory to use whatever reagents it deems appropriate and most suitable. However, analyzer manufacturers are not obligated to support assay applications other than those for the reagents that they provide. It is the responsibility of the laboratory to consider the potential for reagent carryover and to ensure that the third party assays that it adapts to the analyzer do not cause analytical errors, resulting in falsely low or high test results. Here we describe the use of a reagent carryover protocol used to evaluate reagent crosscontamination on the Abbott c8000 analyzer between 32 Abbott clinical chemistry reagents and 15 specific protein and other assays from EQual Diagnostics. Materials and Methods Analyzer: Testing was performed on an Abbott Architect c8000 clinical chemistry analyzer. The c8000 is a multipurpose, open system instrument and it may be programmed to use third party reagents, in addition to Abbott reagents. The reagent carryover evaluation provided in the Architect c8000 System Assay Applications Guide was used. Downloaded from labmedicine.com November 2005 Volume 36 Number 11 LABMEDICINE 705

2 Assays: Thirty-two Abbott routine clinical chemistry assays (eg, electrolytes, enzymes, lipids, metabolites, etc) comprised the original c8000 test menu. To expand the menu, 15 assays (specific protein assays, ammonia, cholinesterase, HDL, and lithium) from EQual Diagnostics, were adapted to the c8000. Abbott c8000 Assays Acid Phosphatase Albumin (Bromcresol Purple) Alkaline Phosphatase ALT Amylase AST Calcium Cholesterol CK CO 2 Cl Creatinine Direct Bilirubin GGT Glucose Hgb A1c EQual Diagnostics Assays Ammonia C3 C4 Cholinesterase CRP Ferritin Haptoglobin Direct HDL Iron K Lactic Acid LD Direct LDL Lipase Na Mg Phosphorus TIBC Total Bilirubin Total Protein Triglycerides Urea Uric Acid Urine Protein IgA IgG IgM Lithium Microalbumin Prealbumin Transferrin Reagent Carryover Protocol: The protocol from the c8000 applications guide allows users to identify reagent carryover and to eliminate or control crosscontamination by programming reagent probe washes. All assays are tested in pairs, one assay designated as the donor and the other as the recipient. A saline assay, a simulated assay used to clean the reagent probes, is also used. The saline assay is configured like a typical test, except that 0.85% or 0.90% saline is used in place of a reagent. To determine if carryover occurs between the third party reagents (donor assays) being adapted to the system and the manufacturer s reagents (recipient assays), the following test order scheme is used: Position Sample Test Replicates Purpose 1 Saline Saline 5 Cleans reagent probes 2 Control Recipient 5 Target value for uncontaminated assay recipient assay 3 Saline Saline 5 Cleans reagent probes 4 Control Donor 5 Potentially contaminates recipient assay assay via carryover 5 Control Recipient 5 Observed value for potentially assay contaminated recipient assay The control material used for donor and recipient assays must be appropriate for each test (eg, microalbumin and urine glucose require urine controls, serum glucose requires a serum control, etc). Controls should contain analytes at normal reference range concentrations. The protocol may be repeated as appropriate for a given assay with different levels of controls to approximate the medical decision levels if appropriate. To determine if the manufacturer s reagents interfere with the third party reagents, the same test order scheme as above is used, but the manufacturers reagents are now the donor assays and the third party reagents are now the recipient assays. In addition, the third party reagents must also be tested in donor/recipient pairs to check for carryover between them. The carryover study results are interpreted as follows: Position Sample Test Calculation 1 Saline Saline None 2 Control Recipient assay Mean of 5 Uncontaminated Replicates = Target Value 3 Saline Saline None 4 Control Donor assay None 5 Control Recipient assay Calculate % difference from target for each potentially contaminated replicate; Replicate 1 Target x 100 = % Difference Target Reagent carryover causing crosscontamination is potentially demonstrated if the first replicate of the recipient assay replicates areas higher or lower than the recipient assay target value, calculated using uncontaminated recipient assay values. The value observed for the first replicate represents the worst case scenario, that is, the first replicate is most likely to demonstrate the maximum amount of carryover. Even if reagent carryover is not a problem, it is unlikely that recipient assay values will be exactly equal to the target values. In fact, they are expected to demonstrate some small difference consistent with the inherent variability of the assay system. Thus, a laboratory must specify an acceptance limit for each assay based on clinical criteria. If the percent difference for the recipient assay is within less than or equal to the predetermined acceptance limit, reagent carryover is judged to be clinically insignificant. However, if the difference exceeds the limit, the reagent carryover is considered to be potentially clinically significant. If reagent carryover is confirmed and is considered to be unacceptable, the c8000 is configured for a SmartWash to eliminate or reduce the cross contamination of the recipient assay by the donor assay. The reagent probe SmartWash configuration allows 4 wash solution options: water, detergent A, 0.5% acid wash, and detergent B. The wash volume is variable from 20 to 345 µl, and 1 to 5 probe washes may be selected. The reagent carryover protocol is then repeated with various SmartWash options configured for the donor/recipient assay pairs that are affected by cross contamination. The SmartWash configuration that eliminates or reduces carryover below the acceptance limit of the affected recipient assay is chosen for routine use. Results There were 32 standard c8000 assays and 15 new EQual original equipment manufacturer (OEM) assays. Each of the original 32 c8000 assays was tested with each of the 15 EQual assays, each being tested both as the donor and as the recipient assay. Thus, for each c8000 assay there were 64 assay pair combinations (each assay tested both as a donor and as a recipient with each EQual assay), for a total of 960 donor/recipient combinations (64 donor/recipient pairs per each assay x 15 EQual assays). In addition, each of the EQual OEM assays was checked for reagent carryover against each other on the c8000 for a total of 208 donor/recipient pair combinations. Table 1 lists some examples of donor/recipient pairs that did not exhibit reagent carryover exceeding the acceptance limit. For these assay pairs, representing standard c8000 reagents and EQual OEM reagents, no change in assay parameters were 706 LABMEDICINE Volume 36 Number 11 November 2005 labmedicine.com Downloaded from

3 Table 1_Examples Of Some Donor/Recipient Assay Pairs That Did Not Demonstrate Reagent Carryover Exceeding Cross Contamination Limits Donor Assay Recipient Assay Recipient Assay Target Value Recipient Assay Observed Value % Difference (Target Observed) Glucose Ferritin Ferritin Glucose Haptoglobin ALT ALT Haptoglobin Cholinesterase Urea Urea Cholinesterase C4 Creatinine Creatinine C Lithium Cholesterol Cholesterol Lithium Ferritin Haptoglobin Haptoglobin Ferritin Recipient assay target values are the means of 5 replicates. Recipient assay observed values are the means of 5 replicates of samples exposed to potential reagent carryover. necessary. A difference of +/-10% between the recipient assay target value and the observed recipient assay value following the donor assay was chosen as the acceptance limit. This was a practical, first pass decision and it was recognized that, for some analytes, differences less than +/-10% could be clinically significant. In most cases, test values that were increased or decreased by 10% due to reagent carryover were considered to make no difference in patient treatment. All differences were examined and in cases in which the 10% limit was considered to be inappropriate, additional testing with the donor/recipient pair was conducted and SmartWashes were programmed if appropriate. Data was reviewed to determine if a systematic reagent carryover problem truly existed. On a random basis, for 2 donor/recipient assay pairs, 1 replicate of the 5 recipient assay values exhibited a difference exceeding the 10% limit. Repeat testing of these pairs demonstrated that the unacceptably high recipient values were aberrant results and not reproducible. The CRP assay exhibited reagent carryover from 5 donor assays, GGT, LD, total protein, urea, and lactic acid, with target to observed % differences greater than +10%. However, the positive biases demonstrated were not deemed to be clinically significant for this analyte as CRP is a nonspecific marker of inflammation, and therefore it was not necessary to institute additional reagent probe washing. Table 2 lists the donor/recipient pairs that exhibited reagent carryover that exceeded the acceptance limit. If the result for an assay pair exceeds the acceptance limit for the recipient assay and there is a concern that the result may be aberrant, testing can be repeated to confirm that the initial observed degree of reagent carryover is reproducible. For donor/recipient pairs demonstrating unacceptable carryover, SmartWash testing was conducted to decrease the carryover to an acceptable level. Table 3 lists the donor/recipient pairs for which SmartWash programming was required. It lists the original % difference and the % difference after SmartWash and the wash fluid used. Typical normal reference ranges are also listed. In 2 cases, institution of a SmartWash to decrease the effect of reagent carryover was deemed unnecessary. Although carryover of CRP can decrease IgM values by about 15%, the normal reference range of IgM is so wide and the prevalence of CRP testing in this laboratory so infrequent that additional probe washing was not warranted. Cholinesterase carryover can increase total bilirubin by about 15%, which could have a clinical impact. The laboratory performs only about 2 cholinesterase tests per month and cholinesterase assays are segregated and performed separately to avoid the potential for a carryover effect on total bilirubin results. Discussion The practice of clinical chemistry has come a long way since its infancy when specimens were analyzed for individual analytes using manual procedures. The development of the continuous flow autoanalyzer was a significant leap forward as large numbers of specimens could be tested for individual analytes in an automated fashion. By combining autoanalyzer units, each representing a separate analyte channel, many different tests could be performed simultaneously in panels. However, these continuous flow systems were complex and demanded considerable Table 2_Donor/Recipient Assay Pairs That Exhibited Unacceptable Reagent Carryover Donor Assay Recipient Assay Recipient Assay Target Value Recipient Assay Observed Value % Difference (Target Observed) Ferritin Alkaline Phosphatase 354 IU/mL 316 IU/mL Ferritin CK 1007 IU/mL 901 IU/mL Ferritin Fe 234 µg/dl 209 µg/dl CRP IgM 1,300 mg/dl 1,100 mg/dl Cholinesterase Total bilirubin 0.52 mg/dl 0.60 mg/dl Lactic acid Ferritin 72.2 ng/ml 55.7 ng/ml Urea Ferritin 70.4 ng/ml 62.8 ng/ml HDL CO meq/l 15.4 meq/l -7.2 IgA CO meq/l 12.8 meq/l Downloaded labmedicine.com from November 2005 Volume 36 Number 11 LABMEDICINE 707

4 Table 3_Donor/Recipient Assay Pairs That Exhibited Significant Carryover Donor Assay Recipient Assay Initial % Carryover % Carryover Between Donor Description of Typical Normal Between Donor and and Recipient Assays after SmartWash Program Reference Range Recipient Assays Programming SmartWash Ferritin Alkaline Phosphatase Water wash, 345 µl IU/mL Ferritin CK Water wash, 345 µl IU/mL Ferritin Fe Water wash, 345 µl µg/dl CRP IgM N/A * mg/l Cholinesterase Total Bilirubin N/A * mg/dl Lactic Acid Ferritin Water wash, 345 µl ng/ml Urea Ferritin % acid wash, 345 µl ng/ml HDL CO Detergent A wash, 345 µl meq/l IgA CO Water wash, 345 µl meq/l * The observed decrease or increase in recipient assay values due to reagent carryover was not deemed to be clinically significant based on the normal reference range for the recipient assay and/or medical decision levels. The table lists the percent difference before and after application of sample probe washes to minimize the observed carryover. laboratory technologist time and effort to maintain them and keep them synchronized. The next generations of analyzers moved away from the continuous flow model to discrete analysis. Discrete analysis, sometimes colorfully known as sip and spit, allows individual specimens to be tested for a single analyte, a few analytes, or a whole panel of analytes. Today, discrete analyzers represent the bulk of systems in use by clinical laboratories. They do not use analyte specific channels but integrate the operations of various subsystems (sample and reagent pipettes, cuvettes or reaction vessels, spectrophotometers, wash assemblies, etc) to process many different specimens (and types of specimens) and reagents very rapidly, one after another, reusing all of the subsystems time and again. A disadvantage of the old continuous flow methodology was that it required duplication of the basic subsystems, or channels. For example, multiple reagent probes, cuvettes (actually, flow cells), spectrophotometric or other signal generating systems were required. Discrete analysis, by the continuous reuse of the subsystems, eliminates the need for duplication and allows for more compact analyzers. Ironically, the duplicative nature of continuous flow systems allowed laboratory personnel to focus on individual channels when performing maintenance, repair, and troubleshooting instead of balancing the operation of the system in toto. A problem with a single channel would impact the results for a single analyte but would not affect all of the others. The advantages of discrete analysis come at the price of increased complexity and require many subsystems to work in harmony. A problem with one subsystem (eg, a partially clogged sample pipettor, a scratched or clouded cuvette, or a malfunctioning photodiode for a given wavelength) has the potential to impact many test results for all assays and may require the entire system to be shutdown for repair. Discrete analyzers tend to be classified as closed or open. Closed systems are restricted to the use of reagents from the manufacturer of the analyzer, as the reagents and the analyzer constitute an integrated system by design. Only tests for which the manufacturer offers reagents may be performed with a closed system. Open systems are more flexible and reagents from a variety of manufacturers may be adapted to them. Manufacturers that provide both analyzers and reagents perform reagent carryover studies to ensure that test results are not affected. Reagent carryover studies are repeated by manufacturers any time a new assay is added to the test menu. Laboratories consider open systems to offer a distinct advantage because of the versatility that they offer. Indeed, being able to add any assay to an analyzer for which suitable reagents are available is an attractive feature. What may go unrecognized is the potential for reagent carryover to cause an unacceptable increase or decrease in test results for some analytes any time a new assay is added. Manufacturers cannot anticipate all possible assay reagent pair combinations or offer any guarantee that unacceptable reagent carryover will not occur. It is the responsibility of every laboratory that modifies an analytical system by adding third party reagents to them to ensure that unacceptable reagent carryover is not a problem. A literature search found relatively few papers discussing specimen or reagent carryover prior to Krouwer, Stewart, and Schlain proposed a multi-factor testing scheme to evaluate random access analyzers for imprecision, agreement with a reference method (slope of regression line), nonlinearity, linear drift, and reagent carryover. 1 These authors recognized that random access instruments differed from batch analyzers in that they are susceptible to reagent cross contamination in addition to sample carryover. They found that an AST donor assay could carryover into a recipient LD assay and falsely elevate the patient LD value because the AST reagent contained LD in high concentration on the Ciba Corning 550 Express analyzer. The analyzer was modified to decrease the reagent carryover to an acceptable level. The authors concluded that a 12 sample multi-factor protocol was a useful screening method but that more involved protocols would allow a better estimate of system performance. They specifically noted that a complete estimate of reagent carryover would require testing all possible reagent pairs available on an analyzer. Haeckel defined carryover as a process by which materials are carried into a reaction mixture to which they do not belong. These materials can be either parts of a specimen, or reagents including the diluent or wash solution. 2 Carryover can be classified by material (eg, specimen, diluent, reagent, reaction mixture, wash solution) or by site of carryover (eg, specimen cup, sample probe, reagent probe, wash station). The following types of carryover have been observed in practice: (1) specimen carryover in a sample probe, (2) carryover from diluent to specimen in a specimen cup, (3) carryover from reaction mixture to reaction mixture, and (4) carryover over from reagent to reaction mixture from a reagent probe. Haeckel proposed various formulae for quantitating carryover and describes various specific scenarios to that should be considered. He concludes by noting that new analytical systems should be designed to avoid any type of carry-over as far as possible. Downloaded 708 from LABMEDICINE Volume Number November 2005 labmedicine.com

5 Dixon classified carryover in selective access analyzers as physical (specimen) carryover or chemical (reagent) carryover and proposed formulae for quantitating both types. 3 Brenna and Prencipe described studies to characterize specimen carryover, reagent cross contamination, and cuvette-related carryover on the Bayer Axon for 12 analytes. 4 Specimen carryover was found to range between 1.50% to +0.66%; reagent cross contamination was found to be statistically significant only for CK and cholesterol (negative carryover of 2.5% and 1.9%, respectively); no cuvette-related carryover was noted. Lovric and colleagues examined specimen carryover and reagent cross contamination on the Olympus AU 800 analyzer using 2 approaches: (1) a screening method, and (2) a multifactorial method, but only for ALT and LD. 5 Carryover was not found to be statistically or clinically significant. Ferguson, Mee, and Wong reported the carryover of PSA on the Abbott IMx and AxSYM analyzers was identical at 0.02 ng/ml, or 0.003%. 6 Schlain and colleagues evaluated interassay carryover (reagent crosscontamination) biases on the Abbott AxSYM. 7 They used a 2-stage approach to examine a total of 231 possible assay pair configurations. In the first stage, those assay pairs that did not demonstrate carryover biases were eliminated from further consideration and in the second stage, those assay pairs could potentially result in large carryover biases were studied further. Their statistical model involved constructing 95% confidence intervals for test results from assay pair interactions. If the observed carryover bias was contained within the defined confidence interval, it was considered not to be either statistically or clinically significant. Increasing pipettor probe wash volumes was found to be an effective means of controlling carryover. Sadler, Murray, and Turner searched the literature for carryover papers in 1996 for the period of 1991 through June of 1995 and found only 12 in which the word carryover appeared either in the title or the abstract. 8 Four of the papers concerned testing for drugs of abuse and reported significant carryover problems, a issue that is not unusual in that type of analysis. The other 8 papers report no significant carryover, but few details were given. For example, specimens were described simply as high or low with no quantitative values provided. In other cases, the difference in concentration between high and low specimens was not considered to be great enough to provide a realistic carryover challenge. These authors reported on their own work involving thyroid stimulating hormone (TSH) samples with concentrations of about 30, 12, 3, 0.4, and 0.1 mu/l that were pipetted in different patterns using a fixed, glass capillary pipette for a 2nd generation TSH immunoradiometric assay (IRMA). They estimated sample carryover to be 0.086% for the 0.1 mu/l sample. Several papers discussing sample or reagent carryover appeared in the literature from 2000 to present. Skinner and Waterson reported increased imprecision and a negative bias for urine oxalate on the Roche MIRA S analyzer due to reagent carryover, as demonstrated by external quality assessment scheme performance. 9 The sample probe is used both to pipette specimen and reagent on this analyzer. Improving probe washing was not an option and the problem was resolved by delaying addition of the assay color reagent. Muser and colleagues evaluated the Cobas Integra 400 and examined both sample and reagent carryover. 10 Sample carryover was tested using only albumin, with 3 high concentration samples (50 g/l) followed by 5 low concentration samples (20 g/l), with the low sample values compared to a low reference value. Sample carryover did not exceed the 5% limit specified. Reagent carryover was assessed using 11 potentially interfering assays and 10 potentially sensitive assays in providing and receiving pairs. Carryover was determined using patient samples near the medical decision levels. Replicates (5) of the potentially contaminated assays were compared to the mean control value (n = 10) of the assays and a limit of +/-10% was applied. Reagent carryover occurred for 2 assay pairs: digoxin/antithrombin III and total protein/urine uric acid. An additional automated wash cycle for these assay pairs resolved the carryover problem. Redondo and colleagues performed a similar evaluation of the Cobas Integra Sample carryover was assessed with the same protocol used by Muser and colleagues for the Integra For CK and albumin, carryover was not observed (<5% difference). For reagent carryover, the median control value for recipient assays was determined and donor/recipient pairs were tested in replicate (n = 10). The differences between the median values for the recipient assay control value and the potentially impacted recipient assay were calculated for the triglyceride/lipase assay pair. Reagent carryover was not observed (< 5% difference). Wan and colleagues assessed sample carryover for the Roche E170 immunoanalyzer. 12 High concentration samples for several analytes (ferritin, 67.1 µg/l; AFP, 3,051,200 µg/l; TSH miu/l; free T4, 67.1 nmol/l; and PTH, 2,949 ng/l) were followed by low concentration samples (ferritin, 7.7 µg/l; AFP, 1.6 µg/l; TSH miu/l; free T4, 7.7 nmol/l; and PTH, 6.1 ng/l). The percent carryover was calculated as [(L1 L2)/(H1- L2)] x 100. Carryover was found to be minimal (< 0.2%). In an evaluation of the Architect c8000, Pauli, Seyfarth, and Dibbelt studied specimen carryover by testing 3 replicates of high concentration samples followed by 9 replicates of low concentration samples. 13 No significant carryover was demonstrated for 3 analytes (glucose at 54.4 mmol/l, ALT at 790 U/L, and CK at 40,966 U/L). There are many factors that can affect the accuracy of test results in the clinical laboratory, some more significant than others. The total error associated with a result is usually described as consisting of random error (imprecision) and systematic error (bias). Reagent carryover is an example of an analytical systematic error that may tend to be overlooked. Manufacturers are expected to comprehend reagent carryover and to ensure that it does not exceed reasonable acceptance limits for closed systems and for open systems using the manufacturer s own reagents that have been optimized for a the system. Reagent carryover is an ongoing challenge for manufacturers as carryover studies must be conducted any time that a new reagent is added to a system s test menu, or an existing assay undergoes a significant reformulation. Tempting as it is to argue that some changes to reagent formulations are minor and unlikely to impact results through reagent carryover, this can only be proven by collecting and reviewing data. Likewise, changes to a system s hardware or software, made to improve its performance, could inadvertently affect and change the reagent carryover profile. Again, only by performing reagent carryover studies can a manufacturer know to what extent this phenomenon impacts test results. Understanding reagent carryover is every manufacturer s obligation for its own analyzers and its own reagents. However, for very practical reasons, manufacturers cannot be expected to guarantee the performance of open systems if third party reagents are introduced on them. The onus falls upon each individual laboratory to investigate reagent carryover once it adapts a new third party reagent to a system. The more assays added, the greater the challenge as Downloaded labmedicine.com from November 2005 Volume 36 Number 11 LABMEDICINE 709

6 each new assay must be checked both as the donor and recipient assay with each existing assay and with each new assay as well. With luck, relatively few assay pairs will demonstrate clinically significant reagent carryover and require modification of system parameters to decrease the carryover effect to an acceptable level. That was the case in this study. However, performing carryover studies is a time consuming process. The data must be carefully examined and questionable, possibly aberrant results must be confirmed by retesting. Confirmed, unacceptable reagent carryover situations must then be addressed by appropriate modification of the system s operation, for example, use of additional reagent probe washes with an appropriate wash solution, as reported here. This requires even more testing and data scrutiny. Laboratories must decide on reasonable acceptance limits to judge the significance of changes in result values caused by reagent carryover. In some cases, a change of up to +/-10% may be analytically insignificant, but clinically significant, depending on the specific analyte, the normal reference range, the medical decision level, and other considerations. Conversely, some change may seem to be analytically significant (eg, < +/-10%), but ultimately may be clinically insignificant. When making a judgment call about the importance to recipient assays of the demonstrated differences between target and observed values, laboratories should remember that the differences are not totally due to the reagent carryover phenomenon but are dampened or exacerbated by the inherent random variability (imprecision) of the test. The use of 5 replicates of donor and recipient assays is a practical attempt to capture the mean influence of reagent carryover. However, for any given analytical event, the actual impact of reagent carryover may be either greater or less than that predicted by a reagent carryover study. Laboratories cannot realistically expect to eliminate reagent carryover (ie, percent difference between target and observed values equals zero). They can only try to objectively measure reagent carryover and control it. The judicious application of laboratory experience and clinical insight by the laboratory director is called for when making decisions about the amount of reagent carryover that can be tolerated. Manufacturers are faced with a balancing act when designing automated systems. While it may be possible to engineer the probe sampling and washing systems to virtually eliminate the possibility of carryover, such efforts invariably affect other aspects of sample handling, for example, the unwelcome consequence of decreasing specimen throughput. Juggling the practical needs for efficient, high volume sample throughput with minimization of specimen and reagent carryover, means that the potential for carryover is unavoidable. Manufacturers must test the performance envelope of their systems and understand their capabilities and limitations. Users of these systems must take into account the performance information provided by manufacturers when judging the acceptability of patient results and conducting investigations of suspect results. Users of open systems who adapt reagents from third party vendors to them should consider the effect of reagent carryover on results and perform testing to measure it. The c8000 is an example of a general purpose open system clinical chemistry analyzer that allows the user to optimize probe washing to eliminate or control reagent carryover, reducing laboratory analytical phase error and improving patient safety. LM Acknowledgements: Michelle Fokakis of Abbott Laboratories provided technical assistance to Our Lady of the Lake Medical Center to perform reagent carryover studies for EQual specific protein assays (third party reagents) that were adapted to Abbott c8000 clinical chemistry analyzer. 1. Krouwer JS, Stewart WN, Schlain B. A multi-factor experimental design for evaluating random-access analyzers. Clin Chem. 1988;34: Haeckel R. Proposals for the description and measurement of carry-over effects in clinical chemistry. Pure Appl Chem. 1991;63: Dixon K. A theoretical study of carryover in selective access analyzers. Ann Clin Biochem. 1990;27: Brenna S, Prencipe L. Axon clinical chemistry analyzer evaluated according to ECCLS protocol. Clin Chem. 1992;38: Lovric M, Cvoriscec D, Petrovecki M, et al. Evaluation of the Olympus AU 800 clinical chemistry analyzer. Clin Lab. 1995;41: Ferguson RA, Mee VA, Wong PY. Comparative evaluation of serum prostate specific antigen analysis by the Abbott AxSYM and IMx analyzers. J Clin Lig Assay. 1995;18: Schlain B, Frush H, Pennington C, et al. Two-stage procedure for evaluating interassay carryover on random-access instruments. Clin Chem. 1996;42: Sadler WA, Murray LM, Turner JG. Influence of specimen carryover on sensitive thyrotropin (TSH) assays: is there a problem? Clin Chem. 1996;42: Skinner E, Waterson M. Improvement of a standard MIRA method for urinary oxalate by elimination of reagent carry-over. Ann Clin Biochem. 2001;38: Muser J, Bienvenu J, Blanckaert N, et al. Inter-Laboratory evaluation of the COBAS INTEGRA 400 analytical system. Clin Chem Lab Med. 2001;39: Redondo FL, Bermudez P, Cocco C, et al. Evaluation of Cobas Integra 800 under simulated routine conditions in six laboratories. Clin Chem Lab Med. 2003;41: Wan B, Augustin R, Chan MK, et al. Analytical performance and workflow evaluation of the Roche E170 modular immunoassay analyzer in a pediatric setting. Clin Biochem. 2005;38: Pauli D, Seyfarth M, Dibbelt L. The Abbott Architect c8000: analytical performance and productivity characteristics of a new analyzer applied to general chemistry testing. Clin Lab. 2005;51: LABMEDICINE Volume 36 Number 11 November 2005 labmedicine.com Downloaded from