Laboratory Hematology 8:85-90 2002 Carden Jennings Publishing Co.. Ltd. Abbott Cell-Dyn Reticulocyte Method Comparison and Reticulocyte Normal Reference Range Evaluation T. SCHISANO, L. VAN HOVE Abbott Diagnostics, Santa Clara, California ABSTRACT Reticulocyte enumeration has been shown to be a reliable indicator of changes in erythropoietic activity in the bone marrow. For many years the clinical laboratory has relied on the manual slide method for enumerating reticulocytes; this method is both time consuming and imprecise. With the advent of automated and semiautomated reticulocyte methods, the clinical laboratory has the ability to provide accurate and precise reticulocyte results in a timely and efficient manner. Abbott has 3 systems that provide an automated reticulocyte assay they are the Cell- Dyn 4000, Cell-Dyn 3700, and the Cell-Dyn 3200. The Abbott Cell-Dyn 4000 is a fully automated random-access hematology analyzer, for use in a high-throughput clinical setting, that provides basic hematologic analysis with a rapid and sensitive reticulocyte method. The Cell-Dyn 4000 system directly quantifies reticulocytes using fluorescence technology with an RNA-bound dye complex. Since the release of the Cell-Dyn 4000, Abbott has added a supravital staining method using New Methylene Blue dye to the Cell-Dyn 3700 and recently to the Cell-Dyn 3200. We compared blood specimens from healthy and anemic individuals for correlations in reticulocyte percentage and absolute count using Abbott s Cell-Dyn 4000, Cell-Dyn 3700, and Cell-Dyn 3200 reticulocyte methods against the manual (microscopic) reticulocyte slide method as well as performing intermethod comparisons of the Cell-Dyn systems. In addition, we evaluated imprecision of each method and developed normal reference ranges for the Cell-Dyn and MAPSS are registered trademarks of Abbott Laboratories. Correspondence and reprint requests: Thomas V. Schisano, Abbott Diagnostics, 5440 Patrick Henry Dr, Santa Clara, CA 95054. Received February 8, 2002; accepted February 22, 2002 reticulocyte parameters based on results from in-house donors at Abbott Diagnostics in Santa Clara. The data revealed that the Abbott Cell-Dyn methods are accurate, showing excellent correlation between the manual (microscopic) method and intermethod assays; the Abbott methods also produced lower levels of imprecision than the manual (microscopic) reticulocyte slide method. The Abbott methods provide the clinician with a more reliable means of evaluating erythropoietic activity both in anemia and in bone marrow transplantation patients than does the tedious and relatively imprecise manual method. Lab Hematol. 2002;8:85-90 KEYWORDS: Reticulocyte Normal reference range INTRODUCTION Reticulocytes are immature erythrocytes in the bone marrow, at the end-stage of hemoglobin synthesis, released into the peripheral blood stream. Reticulocyte enumeration has been shown to be a reliable indicator of changes in erythropoetic activity in the bone marrow [1-8]. Early-stage reticulocytes contain greater amounts of ribosomes, mitochondria, and Golgi bodies compared to late-stage reticulocytes. This material is made easily visible for microscopic enumeration by precipitation with supravital stain techniques such as New Methylene Blue and with fluorescent staining dyes. The degree of precipitation and fluorescent staining is directly proportional to the amount of these cellular components. Until the advent of the automated procedures, the clinical laboratory had to rely on the manual technique, which has shown technical problems with both accuracy and precision [9,10]. The purpose of this study was to compare the clinical performance of the Abbott Cell-Dyn 4000 fluorescence method (Abbott Diagnostics, Santa Clara, CA)-along with the 85
86 T Schisano and L.Van Hove supravital staining method of the Cell-Dyn 3700 and Cell- Dyn 3200-against the manual (microscopic) New Methylene Blue method. We also compared the Abbott Cell-Dyn methods against each other. To establish the reticulocyte parameter normal reference ranges for each Cell-Dyn system, we obtained 67 whole blood specimens from healthy in-house donors at Abbott Diagnostics in Santa Clara. We obtained the central-core 95% of both the male and female groups by calculating the 2.5. and 97.5-percentile limits. For our abnormal sampling we obtained 41 specimens from our contracted clinical sites in the San Francisco Bay area. All samples were run in duplicate. Linear regression analysis between methods and paireddifference precision analysis were performed. METHODS Sample Selection Sixty-seven in-house donors from Abbott Diagnostics in Santa Clara who were considered in general good health were used for the normal population sampling for our normal-range evaluation using an institutional review board-approved protocol. We selected 33 males and 34 females between the ages of 20 and 60 years. Forty-one abnormal unlinked retention specimens collected from our regional contracted sites were used for the abnormal population sampling in this study. These abnormal specimens contained various anemia cases including iron deficiency, sickle cell anemia, thalassemia, anemias of chronic disease, and normocytic and macrocytic anemias. All samples were run in duplicate and were collected in tubes containing K 3 EDTA anticoagulant. Reticulocyte Counting Methods Abbott Cell-Dyn 4000. All blood samples were run using R8-2E version software. The Cell-Dyn 4000 integrates fully automated random-access reticulocyte analysis with basic hematology on a single analyzer platform. This integration allows the user the option of running a reticulocyte with a complete blood count (CBC) using only 112.5 ul of whole blood. No added steps are required in sample preparation. The absolute and percentage reticulocyte, along with the IRF (immature reticulocyte fraction), are included. The Cell-Dyn 4000 reticulocyte method uses an argon laser and fluorescence flow cytometry by incorporating a DNA- and RNAbinding dye that is excited at 488 nm and emits enhanced fluorescence at 530 nm. The intensity of the fluorescence that is emitted from the RNA-bound dye complex is directly proportional to the RNA concentration of the reticulocyte. Abbott Cell-Dyn 3700. All blood samples were run using 1.1 version software. The Cell-Dyn 3700 provides a semiautomated reticulocyte count based on the National Committee for Laboratory Standards and the International Council for Standardization in Haematology (NCCLS/ICSH) H44-A guideline for supravital staining. The method involves a single dilution of blood using 20 ul of whole blood in the reticulocyte staining reagent thiazine dye New Methylene Blue N. Au incubation time of approximately 30 minutes at room temperature was used. The user then selected a single soft key to initiate the reticulocyte analysis. Both red blood cells and reticulocytes are measured by multiangle polarized scatter separation (MAPSS) technology (Abbott Diagnostics) using an HeNe (helium-neon) laser. Red blood cell information is collected for each cell and the red blood cells and reticulocytes are distinguished from other cells and interferences through the use of algorithm gating and threshold setpoints. Reticulocytes are reported in percentages. The IRF is provided and the instrument will automatically calculate the reticulocyte absolute value if a red blood cell (RBC) count is entered. Abbott Cell-Dyn 3200. All blood samples were run using prerelease software version 1.92, intended for research use only, and were rested in this study prior to the release of software version 2.0, which officially added the reticulocyte assay to the Cell-Dyn 3200. As with the Cell-Dyn 3700, the Cell- Dyn 3200 provides a semiautomated reticulocyte count based on the NCCLS/ICSH H44-A guideline for supravital staining. The method involves a single dilution of blood using 20 ul of whole blood in the reticulocyte staining reagent thiamine dye New Methylene Blue N. After an incubation time of approximately 30 minutes at room temperature, reticulocyte analysis of the sample is initiated when the user selects a single soft key. Both red blood cells and reticulocytes are measured by MAPSS technology using an HeNe laser. RBC information is collected for each cell and the red blood cells and reticulocytes are distinguished from other cells and interferences through the use of algorithm gating and threshold setpoints. Reticulocytes are reported in percent. The instrument will automatically calculate the reticulocyte absolute value if an RBC count is entered. Manual (Microscopic) Method. Manual reticulocyte counts were performed following the NCCLS guidelines. One thousand RBCs were counted. Duplicate slides were made for each sample and both slides were counted. Data analysis results were analyzed using duplicate run samples for correlations, paired difference precision, and percentile ranking using Excel 2000 (Microsoft, Redmond, WA). RESULTS Linear regression analysis using the manual (microscopic) reticulocyte counting method compared to the Abbott Cell- Dyn methods gave an r range between 0.956 and 0.963 for reticulocyte percentage counts. Intermethod correlation of the reticulocyte percentage counts observed when comparing the Abbott Cell-Dyn 4000 against the Cell-Dyn 3700 and Cell-Dyn 3200 gave an r range between 0.950 and 0.959. The Cell.-Dyn 3700 versus Cell-Dyn 3200 reticulocyte percentage counts gave an r of 0.989. Intermethod correlation
Abbott Cell-Dyn Reticulocyte Method and Normal Reference Range Evaluation FIGURE I. Manual versus Cell-Dyn reticulocyte percentage. Regression analyses for the manual reticulocyte percentage (Retic %) method compared to the Cell-Dyn Systems: A, Cell-Dyn 4000; B, Cell-Dyn 3700; C, Cell-Dyn 3200. of the reticulocyte absolute counts observed comparing the Abbott Cell-Dyn 4000 versus the Cell-Dyn 3700 and Cell- Dyn 3200 gave an r range between 0.926 and 0.938. The Cell-Dyn 3700 versus Cell-Dyn 3200 reticulocyte absolute counts gave an r of 0.987. Linear regression analysis of the microscopic manual reticulocyte percentage counting method against all the Abbott Cell-Dyn reticulocyte percentage (retic %) methods and the intermethod reticulocyte percentage correlations arc shown in Figures 1 and 2 and summarized in Tables 1 and 2. Correlations of the Abbott Cell-Dyn 4000 reticulocyte absolute count against the Cell-Dyn 3700, and Cell-Dyn 3200 and FIGURE 2. Cell-Dyn versus Cell-Dyn reticulocyte percentage Regression analyses for the reticulocyte percentage (Retic %) intermethod comparison of the Cell-Dyn systems: A, Cell- Dyn 4000 versus Cell-Dyn 3700; B, Cell-Dyn 4000 versus Cell-Dyn 3200; C, Cell-Dyn 3700 versus Cell-Dyn 3200. TABLE 1. Regression Summary: Manual versus Cell-Dyn Reticulocyte Percentage (Retic %) (N = 216)
88 T. Schisano and L.Van Hove TABLE 2. Regression Summary: Cell-Dyn versus Cell-Dyn Reticulocyte Percentage (Retic 96) (N = 216) TABLE 3. Regression Summary: Cell-Dyn versus Cell-Dyn Reticulocyte Absolute (N = 216) Cell-Dyn 3700 reticulocyte absolute counts against the Cell- Dyn 3200 are shown in Figure 3 and summarized in Table 3. The normal reference range evaluation showed excellent tracking between methods and established the normal reference range for reticulocyte parameters on the Abbott Cell- Dyn platforms. Our sample population of 67 in-house donors from Abbott Diagnostics, which contained 33 male and 34 female donors, represents a typical population sampling found in the San Francisco Bay Area of California. Table 4 contains data from the total population of 67 donors, run in duplicate for a total of 134 data points. Tables 5 and 6 summarize the data from 33 male and 34 female donors taken from the total group; they summarize the 95% central core of the normal population as shown by the 2.5- and 97.5-percentile limits. These results, shown in Tables 4 through 6, verified across all platforms what is reported in the literature [11]. The 33 male versus 34 female results in Tables 5 and 6 show slightly higher reticulocyte percentage ranges for each system in the female group, as would be expected [11]. Paired-difference precision showed a coefficient of variation (CV) at 95% between 6.06 and 7.66 for both reticulocyte percentage and absolute counts across all methods excluding the manual method, which showed a CV at 95% of 25.01 for percentage reticulocytes. The paired-difference precision showed that each Cell-Dyn method has a low level of imprecision, and results are well within the manufacturer s specification whereas-as expected-the manual count has a higher level of imprecision, more than double that of the Cell-Dyn methods. The imprecision of the manual method has long been established [9-10]. Paired-difference precision is summarized in Table 7. TABLE 4. Total Population Normal Reference Range: 95% Central Core (2.5-97.5 Percentile Limits) FlGURE 3. Cell-Dyn versus Cell-Dyn reticulocyte absolute count. Regression analyses for the reticulocyte absolute count (Retic Abs) intermethod comparison of the Cell-Dyn systems: A. Cell-Dyn 4000 versus Cell-Dyn 3700; B, Cell-Dyn 4000 versus Cell-Dyn 3200; C, Cell-Dyn 3700) versus Cell-Dyn 3200.
Abbott Cell-Dyn Reticulocyte Method and Normal Reference Range Evaluation 89 TABLE 5. Male Normal Reference Range: 95% Central Core (2.5-97.5 Percentile Limits) TABLE 7. Paired Difference Precision: % Coefficient of Variation = 0.950 DISCUSSION Our data showed good correlation between the Cell-Dyn systems and the manual (microscopic) method for reticulocyte percent. The intermethod correlations between analyzers were also good and showed a better correlation of the Cell- Dyn 3700 against the Cell-Dyn 3200. This correlation exists because both systems use the same technology and the same reticulocyte-staining reagent thiamine dye New Methylene Blue N. Even against the fluorescence flow cytometric method of the Cell-Dyn 4000, both the Cell-Dyn 3700 and Cell-Dyn 3200 correlated well. Reticulocyte enumeration has been well documented in the literature for its use in indicating changes in erythropoietic activity in the bone marrow, diagnosing anemia, and monitoring bone marrow transplantation patients. In the past, the clinical laboratory has relied upon the manual (slide) method to generate a reticulocyte count for the clinician. The manual method is time consuming and thus decreases the throughput in the clinical laboratory. It also has been shown, as in this evaluation, to be more imprecise than automated methods [9,10]. Attempts have been made by other hematology instrument manufacturers to automate the reticulocyte parameter and thus allow the clinical laboratory to maintain a relatively high throughput and improve precision. In the past, standalone reticulocyte analyzers were developed using flow cytometry to measure the staining effect on the reticulocyte with the fluorochrome dye Auramine-0. Although automated, these systems are not designed to allow for generation of multiple hematology parameters and require the addition of a red cell count from an outside source to generate the absolute reticulocyte count. Semiautomated supravital staining methods have been developed by various manufacturers that are run on multiparameter hematology analyzers (such as the Cell-Dyn 3700 and Cell-Dyn 3200) and allow a full CBC report with the whole blood sample. The Cell-Dyn 4000 was the first system to integrate fully automated random-access reticulocyte analysis with basic hematology on a single analyzer platform. We evaluated this technology with the semiautomated supravital staining method of the Cell-Dyn 3700 and the prereleased semiautomated supravital staining method of the Cell-Dyn 3200. Both the Cell-Dyn 3700 and Cell-Dyn 3200 Systems use MAPSS technology and easy access into the reticulocyte mode on a multi-hematology parameter platform. The results showed good correlation between systems, with good correlation to the manual reticulocyte method. Intermethod paired-difference precision was excellent and well within manufacturer s specification whereas-as expectedthe manual slide method showed a greater level of imprecision. The data confirm the sensitivity of the fluorescence method [8]. The goals of this cross-platform reticulocyte evaluation were to compare the clinical performance of each of the 3 Cell-Dyn Systems and to establish normal reference ranges for the reticulocyte parameters. This study accomplished these goals and showed that each system maintains a high level of accuracy with a low level of imprecision. Any clinical hematology laboratory can rely on each of these platforms to enhance the throughput of their reticulocyte assay and can use each system interchangeably in a multianalyzer environment provided that the Cell-Dyn system-specific normal ranges are used. The addition of the reticulocyte assay to the Cell-Dyn 3200 will enhance the capabilities of this platform and further improve on a well-established technology. REFERENCES TABLE 6. Female Normal Reference Range: 95% Central Core (2.5-97.5 Percentile Limits)
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