ICSH guidelines for the verification and performance of automated cell counters for body fluids

Transcription

1 International Journal of Laboratory Hematology ORIGINAL ARTICLE The Official journal of the International Society for Laboratory Hematology INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY ICSH guidelines for the verification and performance of automated cell counters for body fluids G. BOURNER*, B. DE LA SALLE,T.GEORGE,Y.TABE,,H.BAUM**,N.CULP,T.B.KENG, ON BEHALF OF THE INTERNATIONAL COMMITTEE FOR STANDARDIZATION IN HEMATOLOGY (ICSH) *Gamma Dynacare Medical Laboratories, Brampton, ON, Canada UK National External Quality Assessment Scheme for General Haematology, Watford, UK Department of Pathology, University of New Mexico, Albuquerque, NM, USA Department of Clinical Laboratory Medicine, Juntendo University School of Medicine, Toyko, Japan Japanese Society for Laboratory Hematology, Standardization committee, Tokyo, Japan **Institut f ur Laboratoriumsmedizin, Regionale Kliniken Holding RKH GmbH, Ludwigsburg, Germany Trillium Diagnostics, Bangor, ME, USA Sullivan Nicolaides Pathology, Brisbane, QLD, Australia Correspondence: Gini Bourner, Gamma Dynacare Medical Laboratories, 115 Midair Ct., Brampton, ON, Canada L6T 5M3. Tel.: , ext. 5220; Fax: ; gbourner@rogers.com SUMMARY One of the many challenges facing laboratories is the verification of their automated Complete Blood Count cell counters for the enumeration of body fluids. These analyzers offer improved accuracy, precision, and efficiency in performing the enumeration of cells compared with manual methods. A patterns of practice survey was distributed to laboratories that participate in proficiency testing in Ontario, Canada, the United States, the United Kingdom, and Japan to determine the number of laboratories that are testing body fluids on automated analyzers and the performance specifications that were performed. Based on the results of this questionnaire, an International Working Group for the Verification and Performance of Automated Cell Counters for Body Fluids was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines to help laboratories plan and execute the verification of their automated cell counters to provide accurate and reliable results for automated body fluid counts. These guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus. doi: /ijlh Received 1 December 2013; accepted for publication 2 January 2014 Keywords Automated cell counts, body fluids, cerebrospinal fluid, verification 598

2 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS 599 INTRODUCTION The intended purpose of this guideline is to provide recommendations for the verification of automated cell counters for the accurate enumeration of cells in body fluids. Multiple analyzers are available on the markets today that offer automated solutions to performing enumerations of cells in body fluids. Manual counting is labor intensive, lacks reproducibility, and requires a high level of expertise. Many laboratories already perform automated counts or have started to verify their systems. Numerous articles have been published [1 7], but in many cases, the verification is incomplete. In a few cases, the articles include studies on analyzers that the manufacturer provided no performance specifications [2]; therefore, it becomes the responsibility of the laboratory to perform a complete validation as opposed to verification. A working group was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines to help laboratories plan and execute the verification of their automated cell counters to provide accurate and reliable results for automated body fluid counts. Current laboratory practices A patterns of practice questionnaire was developed by the Quality Management Proficiency Laboratory Services () of Ontario, Canada. This was distributed to laboratories in Ontario that participate in the external proficiency program for body fluid analysis, to laboratories in the United States that participate in the College of American Pathologists (), to the laboratories in the United Kingdom that participate in the National External Quality Assessment Service for General Hematology [ (H)], and to the laboratories that participate in the Japanese Society of Laboratory Hematology () proficiency testing program. The purpose of the questionnaire was to determine whether laboratories were using automated methods for cerebrospinal fluid (CSF) cell counts and other body fluid cell counts and how the performance specifications were determined for these analyzers. The details of the patterns of practice can be found in Appendix 1 of this document. Laboratories were asked to indicate the performance qualifications that were carried out to determine whether the analyzer was acceptable to meet quality requirements for the enumeration of body fluids. Figure 1 shows the percentage of laboratories that performed accuracy and precision. A wide range of laboratories performed precision (19 83%), accuracy (26 86%), sensitivity (11 64%), specificity (5 33%), and reportable range (2 71%). Fewer laboratories performed studies to evaluate sensitivity, specificity, and reportable range compared with precision and accuracy. North American laboratories performed these performance qualifications more frequently than laboratories in the United Kingdom and Japan. The laboratories were also asked questions as to what protocols they had established to determine the Figure 1. Testing performed for validation studies. Percentage of laboratories Precision Accuracy Sensitivity Specificity Report range

3 600 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS quality of their results. These protocols included whether the laboratories ran body fluid control samples, performed background counts prior to running a sample, and had protocols to handle spurious results. The results are shown in Figure 2. One notable observation is that few laboratories use a separate body fluid control. Most of the new analyzers on the market have a body fluid mode, but it is important that the manufacturer s performance claim is verified for each type of body fluid that is to be run including CSF, serous fluids (pericardial, pleural, ascitic), and synovial fluids. It is particularly important that it is shown that the analyzer can provide accurate counts at the low levels which are more often found in the different body fluids, particularly CSF [8]. VALIDATION/VERIFICATION OF AUTOMATED SYSTEMS It is important for laboratories to understand the difference between validation and verification. Validation is usually carried out by the manufacturer and provides the performance characteristics of a method. If validation has not been carried out by the manufacturer, then it must be carried out by the laboratory. Validations performed by the laboratory should follow any local guidelines and recommendations for establishing a laboratory-developed test (LDT) [8]. Verification is a confirmation of the validation performed by the manufacturer that provides evidence that the analyzer can meet specific requirements within a given test site. This verification must be carried out by the each individual laboratory before the analyzer is used for testing. Verification or a transference verification (described below) must be performed on each individual analyzer that will be used for testing clinical samples. The recommendations in this guideline are for the verification of a manufacturer s claim. Manufacturer s intended use Manufacturers are required to have a statement of intended use, which indicates the type of body fluids that have been validated on the analyzer. The analytical measurement range (AMR) for each type of body fluid is provided by the vendor in the specifications. It is the laboratory s responsibility to be aware of this statement. If the laboratory s intent is to run other fluids that are not included in the manufacturer s claim, then the performance of the analyzer for counting these fluids must be validated as this is considered a LDT. The hematology analyzers most commonly used to perform body fluid enumeration are listed in Table 1 with the fluids and parameters that have been validated by the manufacturers. Specimen handling pre-analytic variables Percent of laboratories Controls Background each test Spurious results Figure 2. Use of body fluid controls, background count, and spurious results in laboratories surveyed Laboratories should be aware of the pre-analytic variables during specimen collection, including the type of container, transportation, and storage that can have an effect on the reportable result [8]. Each laboratory must have a standardized operating procedure that follows recommended guidelines for their locale. This point is beyond the scope of this document. Sample stability It is well documented that cellular deterioration, cell lysis, and bacteria growth can occur within hours of specimen collection, depending on the time elapsed, storage conditions, and type of sample [8]. Sample testing should occur within the time the sample remains stable, and the correlation between methods should be within 2 h of each other [9].

4 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS 601 Table 1. List of hematology analyzers most commonly used to perform automated body fluid counts Analyzers Fluids Parameters reported Beckman LH 750/780 Serous, synovial, cerebrospinal fluid (CSF) WBC, RBC {WBC = TNC} Beckman DxH 800 Serous, synovial, CSF TNC, RBC Sysmex XE 2100, XT 1800i/2000i, Serous, synovial, CSF WBC, RBC Sysmex XT-4000 and XE-5000 Serous, synovial, CSF BF Mode:WBC-BF, TC-BF, RBC-BF, 2 part diff (mononuclear/ polymorphonuclear) Advia 2120, 2120i Peritoneal, pleural, peritoneal dialysate TNC, RBC Iris iq200 and iricell systems CSF CSF, pleural, peritoneal, peritoneal lavage, peritoneal dialysate, pericardial, synovial, general serous TNC, RBC, 5 part diff and PMN/MN% Nucleated count, RBC Performance specifications It is good laboratory practice to provide evidence that the analyzer is capable of reporting reliable results; this is a regulatory requirement in some countries [8, 10]. Body fluids have a different matrix than whole blood and may contain different cell types compared with peripheral blood. Therefore, it is critical to ensure that the analyzer produces true and reliable results for each body fluid type that the laboratory is intending to analyze [8]. Peripheral blood samples should not be used for validation or verification. Many analyzers have a dedicated body fluid mode; therefore, the verification of the complete blood count (CBC) does not satisfy regulatory requirements for body fluid reporting. Each laboratory needs to determine its own goals of acceptability, with a performance specification study. A full verification may be performed on one analyzer at one site for laboratories with multiple analyzers or laboratories that are part of a large group such as an integrated health network. A transference verification can be performed on the other analyzers, providing the instruments are from the same manufacturer and are the same model [11]. This means that a full study does not need to be performed, but the data from the full verification need to be available at the other sites for laboratory accreditation purposes. Table 2 shows what studies should be performed for a full and a transference verification. For example, the patient correlation full validation may be carried out with 40 samples, but the transference can be carried out with 20 samples. Samples may be shared among Table 2. Full verification vs. transference Studies sites, as long as the integrity of the sample can be maintained during transportation of the samples. Accuracy (Trueness) Full verification Transference Precision (repeatability) Yes Yes Accuracy Yes Yes Analytical sensitivity Yes Yes Analytical specificity Yes Yes in include interfering substances Reference range Full study Smaller study Patient correlation Full study Smaller study studies Linearity Yes Yes Limits of detection Yes Yes Carryover Yes Yes Analytical measuring range Yes Yes Accuracy or trueness can be verified using two different approaches [12]. Comparability can be performed using split sample testing, usually with a minimum of 40 patient samples distributed evenly over the reporting range. The results are compared against the laboratory s defined limits to determine whether significant differences occur. This can be challenging where manual counting is the comparative method.

5 602 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS Recovery of expected values from assayed reference materials such as commercial controls may also be used. The laboratory should set its own limits for the acceptable range. Precision (Repeatability) The precision of the analyzer must be assessed in a way that takes into account all of the variables that may influence the analyzer [13]. It is important that the test samples have the same characteristics as clinical samples. It is recommended to test two or more concentrations, usually a high and low concentration, including a sample at any medical decision point(s). It is recommended that the samples should be run a minimum of 10 times to determine within-run precision [13]. Depending on the volume of available sample, it may be difficult to run 10 times, but at least five runs should be performed for valid statistics. Reproducibility should also be determined over a period of time. Due to the instability of the samples, it is not possible to run the same sample on different days, but the precision study may be performed with an automated body fluid control. Patient correlation Samples should be handled and testing performed according to accepted laboratory protocol and the manufacturer s recommendations. Studies may be performed either using the laboratory s current method, the method used by the manufacturer in their claim or a reference method [10]. It should be noted that the predicate used for validation by vendors in most cases is the manual count. It is recommended that a minimum of at least 40 samples should be tested and samples should be included that cover the analytical range, particularly at medical decision levels [9]. Correlation should be performed with each fluid type. Body fluid counts with low and high values are necessary to determine the bias at the limits of the analytical measurement range. This may be difficult for smaller laboratories, but the more samples, the better the correlation data and the greater the opportunity to challenge the system samples with interfering substances. If the reference method is the manual cell counting method, it is recommended that the manual counting be carried out in duplicate to improve the precision of manual counting [8]. The stability of the sample should be taken into account. Samples should be stored under the same conditions and tested within 2 h of each other to avoid sample storage as a variable. Carryover It is important to ensure that a high sample does not cause a positive bias on the following sample and cause a false high result [10]. This is particularly true for CSF samples. As it is recommended that a blank is run prior to running a body fluid sample, it is equally important to ensure that a nonblood aspiration prior to the analysis of the body fluid sample should not cause a dilution and a false low result [10]. Testing should be carried out using a fluid with an elevated count followed by a sample with a low count. The high sample should be run three times, A1, A2, and A3, followed by the a low sample run three times B1, B2, and B3. The carryover is calculated using the following equation: B1 B3 % Carryover ¼ A3 B3 100 As some laboratories run a blank sample prior to analyzing a body fluid, it is also important to ensure that this does not cause a false low result due to a predilution [10]. If the carryover is unacceptable based on your laboratories acceptability, maintenance or service may be required and the carryover study should then be repeated. Lower limits of detection This is probably one of the most critical verifications steps, particularly in the analysis of CSF cell counts. The lower limits must be defined for both total nucleated cells and erythrocytes and should not exceed the limits suggested by manufacturers [8, 14]. The verification should be carried out with all fluid types that the laboratory intends to analyze, to account for the matrix effect of the samples [8]. For verification, it is not necessary to use different reagent lot numbers.

6 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS 603 It is important to understand the different limits of lower levels and their relationship [14]. Limit of blank (LOB) is the highest measurement that should be obtained with a blank sample. The LOB is determined running repeated measurements on multiple blank samples Limit of detection (LOD) or analytical sensitivity is the lowest measurement of an analyte that can be detected in a sample. This is carried out by repeat testing on at least four to six samples with low concentration cell counts that are usually within the range of the LOB to four times the LOB [14]. Limit of quantitation (LOQ) is the lowest measurement of an analyte in a sample that can be detected with acceptable precision and trueness. This must meet your laboratory s requirement for accuracy or total allowable error. The relationship between the limits is LOB < LOD < LOQ. The limits of detection and limits of quantitation are quite often the same value, but must be higher than the limits of blank [14]. How to verify a claimed LOB and LOD The LOB is determined using replicate analysis of more than one different type of fluid without any cells if possible. It is recommended to use body fluids to avoid matrix effects, but if it is not possible to use body fluid samples, diluent may be used. Each sample should be run a minimum of 10 times. The study should be performed on at least two or three different days to take into account different laboratory conditions. Depending on the availability of the samples, it is not necessary that this be performed on consecutive days. If a minimum of three of the replicate analysis are equal to or less than the manufacturer s LOB, it is acceptable to use the manufacturer s claimed LOB, if the manufacturer has specified the LOB. The LOD is determined using the same process with samples with a low concentration equivalent to the manufacturer s claimed LOD, if the manufacturer has specified the LOD. If the results are within 95% agreement, then the manufacturer s claimed LOD may be used [14]. Otherwise, the laboratory must establish their own LOD [14]. The LOQ needs to be performed whether the observed precision of the LOD does not meet the laboratory s acceptable set goals for precision. The same process would be used to determine the LOQ [14]. The LOD or LOQ is the lowest cell count that is reliably detected and meets the laboratory s goal of uncertainty, that is, the bias and imprecision. Analytical specificity to include interferences Manufacturers should identify any interfering substances that may lead to erroneous results. Interfering substances in body fluids can be anything from small clots to crystals, and the laboratory should access what effect these interfering substances have on the results. The study should be defined in advance to take into account the laboratory s specific patient population. It should include all types of body fluids, including those with interfering substances and from those patients with various disease states [8]. This study is can be included with the patient correlation. Analytical measurement range, linearity The analytical measurement range is the range of the cell counts that the analyzer can accurately measure without any pretreatment such as a dilution. The study should be carried out with samples of similar matrix compared with body fluids [10, 15]; therefore, the recommendation is to perform the linearity for each type of body fluid that the laboratory intends to run on the analyzer [8]. The manufacturer must define the AMR; it is the responsibility of the laboratory to verify this claim. This is demonstrated by testing 5 7 concentrations throughout the stated linear range. Three replicates should be tested at 4 6 levels of concentrations [15]. The diluent to be used should be recommended by the manufacturer. It is important to use concentrations of cell counts at medical decision points, at the maximum concentration, and at the lower limit of detection, particularly for CSF samples. It may be difficult to find a sample with high values during the verification period. The linearity may be repeated at a later time if a higher sample is received and laboratory protocol updated to reflect the higher upper limit.

7 604 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS Limited number of samples The biggest hurdle probably facing most laboratories is the availability of samples to perform the verification or validation studies. Once the decision is made to run automated body fluid counts, data collection can be integrated into the daily routine. Each time a sample is received, it should be tested with both methods, the current method and the method under verification, and the data saved for later statistical comparison. The accumulated data should be reviewed periodically to ensure that all fluid types have been verified and to identify where the correlation is poor, looking for common causes between the samples. The number of samples required to perform the studies will be difficult for smaller laboratories. It is important to have enough samples to obtain valid statistics and to ensure that the analyzer is providing true results, especially at medical decision points. This question was asked in the patterns of practice questionnaire and laboratories used anywhere from 1 to 10 samples, even greater than 80 samples. For valid statistics, it is recommended to perform studies using a minimum of 40 samples per sample type [9]. If the samples are run through an open mode, it is good laboratory practice to clean the outside of the probe as well. The background count must equal to or be less than the lower limit of blank. If not, it should be repeated. If the count is still high after the repeat, then the instrument should be put through a cleaning cycle or daily maintenance. Procedures for handling spurious results Most systems have flags that indicate when the result should not be accepted without further review. Laboratory procedures should have instructions on how to investigate these flags and the actions required when they occur with body fluid samples [8]. These procedures should include how to detect debris or cell clumps that may cause spurious results and indicate if it is necessary to use an alternative counting method. The investigation may be a macroscopic examination or microscopic examination by a wet preparation. If the policy of the laboratory is to perform testing in the samples that are considered irretrievable, then the laboratory report should include a description of these findings and the impact on the accuracy of the results. ANALYSIS OF AUTOMATED BODY FLUIDS The automated counting of body fluid samples requires different handling procedures from peripheral blood samples. Standard operating procedures should include all steps required for handling the sample, changing modes on the analyzer, and running the sample. Treatment of sample prior to running The manufacturer s statement of intended use must state if any special treatment for a sample is necessary before analysis. One example would be the pretreatment of synovial fluid samples with hyaluronidase to reduce viscosity of the samples. Background check Before running any samples, particularly CSF samples, it is important to make sure that the aspiration pathway is clean to avoid any contamination of the fluid. Procedure if results outside reportable range The laboratory must define the upper and lower reporting limits for each fluid when the results are outside the analytical measuring range verified by the laboratory (Section Analytical measurement range, linearity). There should be a written protocol for handling samples that exceed both the upper and lower limits of the reporting range including a procedure for performing a dilution if the result exceeds the AMR [8]. Units of measure Laboratories were asked to indicate the units of measure used to report body fluid cell counts, and the survey results showed that different units of measure are used which can be found in Appendix 1. This can be confusing to the physician. It is recommended that for automated body fluid counts, the same units of measure should be used as for the CBC. This also eliminates the need for a calculation which may result

8 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS 605 in an erroneous result. The units of measure used should be clearly stated on the patient s report. QUALITY CONTROL Internal quality control The analytical system must be controlled for the quantitation of body fluid samples. Commercial body fluid controls are available, but there has been some confusion whether it is necessary to run a separate control or whether the daily commercial CBC control is acceptable. Newer analyzers have specific body fluid modes, and it is important to understand whether this mode is a different channel, aspiration pathway, sample dilution, reporting mode, counting volume, or cell analysis compared with the CBC mode. If the body fluid samples are tested differently, a separate control is necessary. Commercial products are also available that test the analyzer in the lower end of the AMR of body fluids. Other considerations should be if the differential is being reported from the analyzer. Some analyzers report a two-part differential, which is included as part of the body fluid commercial controls. Proficiency testing Proficiency testing is a requirement for laboratory accreditation. If testing samples are not available to your laboratory from an external proficiency program, then proficiency must be demonstrated by other means such as blind testing or interlaboratory sample exchange and comparison to meet regulatory compliance. SUMMARY Performing a validation/verification of an automated analyzer for counting body fluids should not be considered different from performing verification for the automated counting of peripheral blood samples, except that it is be performed with the different body fluids that will be analyzed by the instrument. The most important factors to be determined are the accuracy and precision at the low counts found in body fluids. The purpose of verification/validation is to determine that the analyzer is suitable for its intended use and to identify any potential error that could provide erroneous results that may affect patient care. ACKNOWLEDGEMENTS This guideline would not have been possible without the permission of and the support of, (H), and in the distribution of the patterns of practice. The authors would like to acknowledge this support. We would also like to thank the reviewers. REFERENCES 1. Williams JE, Walters J, Kabb K. Gaining efficiency in the laboratory automated body fluid cell counts: evaluation of the body fluid application on the Sysmex XE-5000 hematology analyzer. Lab Med 2011;42: Keuren JF, Hoffmann JJ, Leers MP. Analysis of serous body fluids using the CELL- DYN Sapphire hematology analyzer. Clin Chem Lab Med 2013 Jun 1;51: Barnes PW, Eby CS, Shimer G. An evaluation of the utility of performing body fluid counts on the coulter LH 750. Lab Hematol 2004;10: Aulesa C, Mainar I, Prieto M, Cobos N, Galimany R. Use of the Advia 120 hematology analyzer in the differential cytologic analysis of biological fluids (cerebrospinal, peritoneal, pleural, pericardial, synovial, and others). Lab Hematol 2003;9: Riedl JA, Dinkelaar RB, van Gelder W. Automated morphological analysis of cells in body fluids by the digital microscopy system DM96. J Clin Pathol 2010;63: Goubard A, Marzouk M, Canoui-Poitrine F, Launay S, Le Thuaut A, Legrand P. Performance of the Iris iq(r)200 Elite analyser in the cell counting of serous effusion fluids and cerebrospinal drainage fluids. J Clin Pathol 2011;64: Danise P, Maconi M, Rovetti A, Avino D, Di Palma A, Gerardo Pirofalo M, Esposito C. Cell counting of body fluids: comparison between three automated haematology analysers and the manual microscope method. Int J Lab Hematol 2013;35: Clinical Laboratory Standards Institute (CLSI). Body Fluid Analysis for Cellular Composition. Approved Guideline: CLSI Document H56-A, (ISBN X), CLSI, 940 West Valley Road, Suite 1400, Wayne, PA, USA, June Clinical Laboratory Standards Institute (CLSI). Method comparison and bias Estimation Using Patient Samples: Approved Guideline, 2nd edn. CLSI document EP9- A2 (ISBN ). CLSI 940 West Valley Road, Suite 1400, Wayne, PA, USA, Clinical Laboratory Standards Institute (CLSI). Validation, Verification, and Qual-

9 606 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS ity Assurance of Automated Hematology Analyzers; Approved Guideline, 2nd edn. CLSI document H26-A2 (ISBN ). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA, USA, Clinical Laboratory Standards Institute (CLSI). Defining, Establishing, and Verifying Reference ranges in the Clinical Laboratory Approved Guideline, 3rd edn. CLSI document C28-A3 (ISBN ). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA, USA, Clinical Laboratory Standards Institute (CLSI). User Verification of Performance for Precision and Trueness Approved Guideline, 2nd edn. CLSI document EP15-A2 (ISBN ). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA, USA, Clinical Laboratory Standards Institute (CLSI). Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline, 2nd edn. CLSI document EP5-A2 (ISBN ). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA, USA, Clinical Laboratory Standards Institute (CLSI). Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline, 2nd edn. CLSI document EP17-A2 (ISBN [print]; ISBN [electronic]). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA, USA, Clinical Laboratory Standards Institute (CLSI). Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline. CLSI document EP6-A (ISBN ). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA, USA, APPENDIX Patterns of practice with summary of responses The patterns of practice survey were distributed to laboratories in Ontario that participate in the external proficiency program for body fluid analysis, to laboratories in the United States that participate in the College of American Pathologists (), to the laboratories in the United Kingdom that participate in the National External Quality Assessment Service for General Hematology [ (H)], and to the laboratories that participate in the Japanese Society of Laboratory Hematology () proficiency testing program. The questions and answers are summarized below. 1. Does your laboratory perform automated body fluid counts (e.g., cerebrospinal fluid (CSF) or other body fluids including synovial, pleural, peritoneal, or any other body fluid) on an electronic hematology analyzer? (H) Laboratories that received survey Laboratories that responded 129 (99%) 396 (38%) 234 (38%) 122 (45%) Laboratories performing automated body fluid counts 58 (45%) 291 (73%) 49 (21%) 60 (49%) Laboratories performing automated CSF counts 5 (4%) 241 (61%) 17 (7%) 25 (20%) 2. How many automated CSF cell counts does your laboratory perform per month? Number of CSF samples per month n = 5 n = 241 n = 16 n = > Only if grossly bloody per year 1 1

10 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS How many automated other body fluids cell counts does your laboratory perform per month? Number of other body fluid samples per month n = 57 n = 291 n = 46 n = > <10 per year 3 4. What analyzer(s) does your laboratory use to perform automated body fluid cell counts? Group of analyzers n = 61 n = 357 n = 53 n = 67 Abbott Cell-Dyn 3200, Ruby 9 1 Abbott Cell-Dyn 3500, Abbott Cell-Dyn 4000/Sapphire Abbott Cell-Dyn Beckman Diff2 1 1 Beckman LH 700 Series/DxH Beckman 500 Series 4 1 Beckman STKS, MaxM, HmX 2 Beckman GenS 2 1 Sysmex XE Sysmex XE Sysmex XT-2000i/XS-1000i Sysmex XT Siemens ADVIA 120/ Iris iq Pentra ABX 1 Automated image analysis system 1 Other Is/are the analyzer(s) used to perform automated body fluid cell counts approved by the appropriate regulatory body, that is, FDA, Health Canada, CE, Japan Ministry of Health? (%) (%) N\A* (%) n = 49 (%) n = 61 Yes No Don t know * Question not included in survey to participants.

11 608 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS 6. Does the manufacturer have a statement of Intended Use for the analyzer(s) that clearly defines which body fluids have been approved by a regulatory agency for automated body fluid cell count testing? (%) (%) N/A* (%) n = 47 (%) n = 57 Yes No Do not know * Question not included in survey to participants. 7. If your laboratory uses an approved analyzer for body fluid cell counts, which of the following test system performance specifications did your laboratory verify/validate/establish prior to testing? (%)* n = 32 (%)* n = 291 (%)* n = 39 (%)* n = 66 Did not perform verification study Accuracy Precision Analytical sensitivity Analytical specificity to include interfering substances Reportable range Reference range study Other 7 Correlation studies 13 Yes Carryover 6 Yes Linearity 9 Yes Limits of detection 3 Not applicable 41 *Multiple responses received. 8. If your laboratory modified an approved test system or uses an analyzer in which performance specifications are not provided by the manufacturer, which of the following test system performance specifications did your laboratory establish/validate prior to testing? (%)* n = 26 (%)* n = 13 (%)* n = 28 (%) N/A Did not perform verification study Accuracy Precision Analytical sensitivity Analytical specificity to include interfering substances Reportable range Reference range study 31 5 Other 15 N/A Correlation studies 15 Yes Carryover Yes Linearity 4 Limits of detection 4 *Multiple responses received. Question not included in survey to participants.

12 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS How many CSF samples were used for your laboratory s performance specification study? Number of CSF samples n = 5 n = 160 n = 13 n = 21 Perform automated counts, but did not perform study > How many other body fluid samples were used for your laboratory s performance specification study? Number of other body fluid samples n = 56 n = 214 (%) n = 41 n = 52 Perform automated counts, but did not perform study >100 samples Commercial BF controls What is your laboratory s reportable range (lower and upper limits) for automated CSF cell counts for the total nucleated count (TNC)? Reportable range n = 4 n = 152* n = 9 n = 13 Established lower/upper limits No lower Limit (<) No upper limit 30 7 No upper limit, perform dilution 7 Laboratories with same limit 2 14 Lower limit = Indicated not applicable 5 2 Not defined 2 2 Note: Laboratories using the same limit indicated that manufacturer s limits were used. Some laboratories counted in two categories.

13 610 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS 12. What is your laboratory s reportable range (lower and upper limits) for automated other body fluid cell counts for total nucleated counts (TNC)? Reportable range n = 52 n = 186 n = 20 n = 32 Established upper/lower limits No lower Limit(<) No upper limit No upper limit stated, indicate would perform dilution 7 36 Laboratories with same limit Lower limit = Indicated upper limit not applicable 5 1 Not defined 1 8 Note: Laboratories using the same limit indicated that manufacturer s limits were used. Some laboratories counted in 2 categories. 13. What unit value does your laboratory use to report automated CSF cell counts? Unit value n = 5* N/A n = 16 n = 22 TNC 10 6 /L /L /ll 17 Other 1 RBC 10 6 /L /L /L 1 8 /ll 7 11 Did not respond 7 Question not included in survey to participants. 14. What unit value does your laboratory use to report automated other body fluid cell counts? Unit value n = 59 N/A* n = 39 n = 57 TNC 10 6 /L /L /ll /L 2 Other 1 RBC 10 6 /L /L /L /ll 23 Did not response * Question not included in survey to participants.

14 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS Do you run quality control samples for automated body fluid cell counts? (%) (%) n = 256 (%) n = 49 (%) n = 62 Yes No Use CBC control If yes to Question 15, how often? n = 11 n = 222 n = 4 n = 4 Each test Daily Each shift 77 Weekly 1 Other 52 1 No response 17. If yes to Question 15, what type of QC is used? Type of control (%) n = 11 (%) n = 246 (%) n = 5 (%) n = 6 Commercial In-house Do you perform background counts prior to automated body fluid cell count analysis? (%) (%) n = 259 (%) n = 49 (%) n = 62 Yes No If yes to Question 18, how often do you perform background counts? n = 100 n = 24 N/A Each test Daily Each shift 2 1 Weekly 1 Other 3 1 Did not provide response 4 Question not included in survey to participants.

15 612 G. BOURNER ET AL. VERIFICATION OF CBC ANALYZERS FOR BODY FLUIDS 20. If yes to Question 17, what is an acceptable background count for total nucleated cells? n = 108 n = 18 n = 14 Accept background of /lL 4 Indicated N/A Do you have a procedure for detecting clumps of cells or debris that could lead to spurious results? (%) (%) n = 256 (%) n = 48 (%) n = 56 Yes No Unsure Are you enrolled in a formal EQA program for automated body fluid cell counts? (%) (%) n = 291 (%) n = 48 (%) n = 60 Yes No What are the EQA programs or in-house program do you participate in: a. b. American Proficiency Institute c. d. No program available in Japan e. Split sample testing (manual vs. automated) f. Split sample testing with another laboratory g. Competency of staff h. Commercial QC material i. Other