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HEMATOPATHOLOGY Original Article Automated Reticulocyte Counting and Measurement of Reticulocyte Cellular Indices Evaluation of the Miles H*3 Blood Analyzer CARLO BRUGNARA, MD, MARY JO HIPP, MT (ASCP), CLS (NCA), PATRICIA J. IRVING, MT (ASCP), HAZEL LATHROP, MT (ASCP), CLS (NCA), PATRICIA A. LEE, MT (ASCP), SH, ELAINE M. MINCHELLO, MT (ASCP), AND JAMES WINKELMAN, MD This study evaluated reticulocyte counting and measurement of reticulocyte cellular indices with the Miles H*3 blood analyzer, a new instrument that combines the Technicon/Miles technology for blood cells counting with a staining technique allowing counting of reticulocytes, quantification of staining intensity and measurement of reticulocyte cellular indices. Reticulocyte counts obtained with the Miles H*3 analyzer were compared with those obtained by manual counting, flow cytometry (thiazole orange method) and by the Sysmex R-3000 (Baxter Diagnostics) reticulocyte analyzer. Reticulocyte counting with the Miles H*3 showed excellent precision, and linearity in the range tested (1.1-49% and 1-72% reticulocytes, respectively, with two different protocols) with no significant carryover. Reticulocyte counts were stable after storing blood samples for 72 hours at 4 °C. Comparison of the four different methods, showed an acceptable intraclass correlation between Miles H*3 and Sysmex R-3000 (intraclass correlation coefficient, [r,] = .952), Miles H*3 and flow cytometry (r, = .922), and Sysmex R-3000 and flow cytometry (r, = .938). There was no satisfactory correlation between any of the three automated methods and the values obtained with manual counting of reticulocytes (r, = .538-.7S5), consistent with the well known imprecision of the manual technique. For a group of normal pediatric subjects, age 1-10, we obtained the following values (±SD) of reticulocyte indices: mean corpuscular volume 97.6 ± 4.7 fL; cell hemoglobin concentration mean 28.2 ±1.4 g/dL; cell hemoglobin content 26.7 ±1.6 pg. We determined the direct cost, including depreciation, of the manual and instrumental methods. Cost/test varied from $1.61 for manual method to $6.03 for the Sysmex R-3000. Cost/test for flow cytometry and Miles H*3 were $3.34 and $3.49, respectively. (Key words: Anemia; Blood cell count; Erythrocyte count; Erythrocyte indices; Erythrocytes; Erythrocyte volume; Hematologic tests; Reticulocyte volume distribution width; Reticulocyte; Reticulocyte counting) Am J Clin Pathol 1994;102:623-632. laboratory to have the option of performing CBC and reticulocyte counting on the same instrument. The new H*3 (Miles, Diagnostics Division, Tarrytown, NY) blood analyzer is a further refinement of the established Technicon H* 1 /H*2 technology9"12 with the addition of reticulocyte counting capability. This is based on a new reticulocyte staining method, which utilizes light absorptive properties of the nucleic acid dye Oxazine 750. This instrument is the first to our knowledge which combines the capabilities of a routine CBC and 5 part differential blood analyzer with those of a reticulocyte analyzer. Reticulocyte cellular indices such as mean cell volume (MCVr), cell hemoglobin concentration mean (CHCMr), cell hemoglobin content (CHr), and their respective distribution widths (RDWr, HDWr, and CHDWr) can also be measured. Reticulocyte indices change considerably in normal subjects receiving recombinant human erythropoietin subcutaneously. The decrease in the CHr induced by subcutaneous recombinant human erythropoietin therapy has provided evireplete normal From the Departments of Laboratory Medicine and Pathology, The dence for iron-deficient erythropoiesis in iron 13 Children's Hospital; the Clinical Laboratories, Brigham and Women's subject, despite oral iron supplementation. Hospital; and Harvard Medical School, Boston, Massachusetts. We tested the Miles H*3 blood analyzer operating capabili- Automated reticulocyte counting has become an essential component of the hematology laboratory. Automated reticulocyte counting can be carried out by either a dedicated instrument, (eg, Sysmex R-3000; Baxter Diagnostics', McGraw Park, IL) or by a flow cytometer (eg, thiazole orange method2"7). In the latter case, reticulocyte counting is performed in a batch mode that is scheduled among other tests such as cell surface markers for lymphocytes or other cells, platelet antibody testing or cell cycle analysis. These automatic techniques have lead to significant savings in labor costs and significant improvement in the accuracy and precision of the reticulocyte enumeration compared with manual counting. 8 A dedicated reticulocyte analyzer or a dedicated flow cytometer adds a significant expense to the laboratory capital budget and also have substantial maintenance costs. Thus, it would be desirable for a hematology ties for reticulocyte counting (carryover, precision, linearity, stability, and time dependence of the staining reaction) and compared the values obtained with this instrument with those obtained by flow cytometry, Sysmex R-3000 automated reticulocyte analyzer and manual counting in a large number of pa- Manuscript received July 20, 1993; revision accepted November 15, 1993. Address reprint requests to Dr. Brugnara: Department of Laboratory Medicine, The Children's Hospital, 300 Longwood Avenue, Bader 760, Boston MA 02115. 623 HEMATOPATHOLOGY 624 Original Article TABLE 1. WITHIN RUN PRECISION FOR RETICULOCYTE COUNTING, STAINING INTENSITY, AND RETICULOCYTE INDICES £ 50 -i 4-) Normal Control Reticulocylosis Mean ± SD CV (%) Mean ± SD CV (%) % Reticulocytes Staining intensity (%) Low Medium High Reticulocyte indices MCVr RDWr CHCMr HDWr CHr CHDWr 1.55 ± 0.15 9.7 8.8 ± 0.59 6.7 86.2 ± 1.8 11.8 ± 1.5 1.94 ±0.69 2.0 12.7 35.7 55.2 ± 1.71 24.9 ± 0.78 19.9 ± 1.7 3.1 3.2 8.6 C 3 O 40 O n oo>*30 O 20 4-> 91.8 18.0 29.5 3.5 26.2 4.1 ± 1.5 ± 1.7 ±0.35 ±0.27 ±0.26 ±0.36 1.6 9.2 1.2 7.6 1 8.6 126.9 ± 1.23 18.4 ±0.5 25.1 +0.37 4.8 ±0.19 30.5 ± 0.25 4.2 ±0.10 0.9 2.8 1.5 3.9 0.8 2.5 0) Tjl" ™ 0 ^5 0 10 20 Expected Values are lhe mean ± SD of 21 determinations each for one normal control and one patient with reliculocytosis. Similar results were obtained in another normal subject and in one additional patient with reticulocytosis. CV = coefficient of variation; MCVr = reticulocyte mean cell volume: RDWr = reticulocyte distribution widths; CHCMr = reticulocyte cell hemoglobin concentration mean; HDWr = reticulocyte hemoglobin distribution widths; CHr = reticulocyte cell hemoglobin content; CHDWr = reticulocyte cell hemoglobin content distribution widths. 30 40 50 reticulocyte count (%) tients. Since the inclusion of reticulocyte testing by instrumental methods has potentially significant operational consequences, we determined and compared the true direct costs, including depreciation, of the several methods evaluated in this study. MATERIALS A N D METHODS Reticulocyte Analyzer analysis with the Miles H*3 Hematology Reticulocyte indices were measured on a Miles H*3 RTX Hematology Analyzer. Whole blood, collected in potassium ethylenediaminetetraacetic acid (EDTA), was diluted 1:1000 with Reticulocyte Reagent T03-3392-50 (Miles Diagnostics). The reagent spheres the red cells using the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1 -propansulfonate and selectively stains the reticulocytes using the dye Oxazine 750. The stained reticulocytes were discriminated from mature red blood cells by their increased absorption of light. The prepared samples were analyzed on the Miles H*3 and the reticulocyte indices were measured as described previously for red blood cells.9"12 The software used was version V1.01 for the .o o T 20 40 60 80 Expected reticulocyte count (%) 5n £ 4- V) 3- +-> >> o o FIG. 1. Linearity of reticulocyte counting with the Miles H*3 blood analyzer. Top, a reticulocyte-rich fraction was added in different ratios to a reticulocyte-poor fraction of blood from the same patient. Middle, blood from a patient with pyruvate kinase deficiency (95.3% reticulocytes by manual counting) was added in various ratios to an ABO-compatible blood sample (.85% reticulocytes) diluted with saline to a similar number of red cells per microliter. Bottom, the constancy of percentage of reticulocytes at varying hematocrits was examined in blood from a normal subject. A.J.C.P. • November 1994 2- —i r 1y - 1.34 + 0.003x 20 40 r - 0.890 60 Hct (%) 80 BRUGNARA ET AL. 625 Miles H*3 and Reticulocytes TABLE 2. CARRYOVER STUDIES FOR RETICULOCYTE COUNTING ON THE MILES H»3 BLOOD ANALYZER % Reticulocytes Method A (1) Method B (0 (2) % Carryover n Range Mean Range Mean tSD 12 1.3-1.8 1.49 0.09-0.37 0.218 ± 0.083 10 10 12.1-16.1 95 0-0.068 0.061-0.2 0.019 ± 0.025 0.126 ± 0.014 14.3 — Method A = blood from norma] control was prepared with 77.1% hematocrit. Cell-free sample was prepared from spun plasma from the same patient. Method B (1) = blood from a patient with autoimmune hemolytic anemia (hematocrit: 26%, 14.3% reticulocytes). Method B(2) = blood from a patient with pyruvate kinase deficiency (hematocrit: 21%. 95% reticulocytes). In Method A. one aspiration of sample was followed by three aspirations of cell-free plasma for a total of 12 cycles. In Method B, each sample was run in triplicate followed by three cycles of diluent, for a total often cycles. seven tubes was then run three times with the Miles H*3 analyzer in the reticulocyte mode. Mean values and coefficients of variation (CV) were calculated based on 21 determinations for each subject. Imprecision due to sample preparation. Studies were carried out in one normal subjects and in one patient with reticulocytosis. Five separate vials were prepared for each subject, each containing 3 /xL of blood added to 3 mL of reticulocyte reagent. After incubation at room temperature (15-90 minutes), each of the 5 vials was run in triplicate for reticulocyte counting. The data were analyzed with nested analysis of variance14 using the SAS (SAS Institute Inc., Cary, NC) software package. The total standard deviation for each run was broken down into its within preparation and between preparation error components. A F test for a significant preparation effect was performed and the P value calculated. P values greater than .05 5- Miles H*3 RTX system. The Miles H*3 includesflowcytometric analysis of cells, with laser light scattering to quantify cell volume, hemoglobin concentration, and the light absorbance of cells stained with Oxazine 750 to detect reticulocytes and distinguish them from mature cells. A total of 20,000 cells are counted for each sample. Light absorbance is measured over 100 discrete channels. The gate for reticulocytes is established based on the distribution of the negative cells. To establish the three levels of staining intensity, the number of channels between the threshold for reticulocytes and channel 80 (d) is divided by 3. Low-staining- and medium-staining-intensity reticulocytes comprise all the positive cells between threshold, threshold + l/id, and threshold + 2/id, respectively. High-staining-intensity reticulocytes comprise all the positive cells between threshold +2/id and channel 100. The amount of light absorbed by the reticulocyte is directly proportional to its RNA content. For every red cell assessed, the cell volume, hemoglobin concentration, and presence or absence of residual mRNA can be determined. After the volume and hemoglobin concentration of individual mature red blood cells and reticulocytes is measured, the hemoglobin content of individual cells is calculated as volume X hemoglobin concentration. Histograms are generated for each of the measured indices and distribution widths are calculated for each one of the three histograms of reticulocyte indices: RDWr (for MCVr), HDWR (for CHCMr), and CHDWr for CHr. Two cytograms are generated, one plotting cell hemoglobin concentration (scatter high) versus staining intensity (absorbance), the other cell volume (scatter low) versus cell hemoglobin concentration. Mature red cells are displayed in red and reticulocytes in blue. 4- I 3- * * * T 2" ,, " ^ — < l * II T * * I ,1 II ,, I - 0 i 1 > i 1 1 • 1 • 1 • 1 • r 30 Time (min) Precision Within-run precision. Studies were carried out in 2 normal subjects and in two patients with reticulocytosis. For each subject, 5 separate vials were prepared, each containing 3 mL of reticulocyte reagent and 3 /*L aliquot of blood collected in potassium EDTA. After incubation at room temperature (15-90 minutes), the five vials were pooled in a 25 mL Erlenmeyer flask, and aliquoted into seven glass tubes, each containing approximately 2 mL of the reaction medium. Each of these Time (min) FIG. 2. Stability over time of the percentage of reticulocytes and staining intensity after addition of blood to the reticulocyte reagent. Top, mean ± SD for reticulocyte counts in 15 subjects is plotted as a function of incubation time in the reticulocyte reagent. Bottom, the percentage of reticulocytes with low, medium, and high staining intensity is plotted as a function of incubation time in the reticulocyte reagent. •Statistically significant (P < .05) difference (paired /-test) compared with the 15-minute values. Vol. 102 • No. 5 626 HEMATOPATHOLOGY Original Article f^+H+1 15 30 45 60 75 Time 15 90 105 120 30 45 60 75 90 105 120 Time (min) (min) 3 a x 2 :-T-H+++i plasma. The hematocrit of this pool was 5%. The cells collected from the bottom of the conical tube were resuspended in the cell free plasma at the same hematocrit of the reticulocyte pool. The reticulocyte pool was then serially diluted with the bottom pool at 20% intervals. Method B: Blood from a splenectomized patient with red cell pyruvate kinase deficiency (95.3% reticulocytes, determined with manual counting) was combined in different ratios with ABO compatible blood from a normal control previously diluted with physiologic saline to a similar number of cells/VL. Absolute reticulocytes. Blood (150 mL in EDTA) was collected from a normal donor, and spun down for 25 minutes at 4 °C at 750 g. Plasma was removed and spun again for 10 minutes at 750g. This supernatant was used as 0% pool. Buffy coat was removed and the packed cells were pooled (100% pool, hematocrit 77.1%). Mixtures of the 0% and 100% pools were prepared to obtain intermediate values (80, 60, 40, and 20%). Carryover 15 15 30 45 30 60 75 90 105 120 Time (min) 45 75 60 90 105 120 Time (min) 15 30 45 60 75 90 105 120 Time (min) 30 45 60 75 105 120 90 Time (min) FIG. 3. Time course of changes in reticulocyte cellular indices and their relative distribution widths after addition of blood to the reticulocyte reagent. Top, mean reticulocyte cell volume and volume distribution widths are plotted versus incubation time. Middle, reticulocyte cell hemoglobin concentration and distribution widths are plotted versus incubation time. Bottom, reticulocyte hemoglobin content and distribution widths are plotted versus incubation time. *Statistically significant (P < .05) difference (paired Mest) compared with the 15-minute values. Values are the mean ± SD for 15 subjects. indicate that the preparation error is not statistically significant. Method A. The 100% pool used for determination of linearity for absolute reticulocyte measurements (see above, hematocrit 77.1 %) was also used for carryover studies. After the appropriate dilution with the reticulocyte reagent, the specimen was run and the plasma used for the 0% pool was measured consecutively in triplicate. This was repeated 12 times. Carryover was determined as (D1-D3/S-D3) X 100, where S is the number of cells counted in the 100% pool and D are then numbers of cells counted in the respective runs of 0% pool.1 Method B. Bloods from one patient with a moderately increase reticulocyte count (14.3%) and one patient with pyruvate kinase deficiency (95.3% reticulocytes) were used for this study. For each patient, 10 vials were prepared, each containing 3 ML of blood and 3 mL of reticulocyte reagent. Each vial was run in triplicate followed by three runs of diluent, and the carryover calculated as detailed in ref. 1, based on a total of 10 cycles. Reticulocyte Percentage of reticulocytes. Method A: Thirty-five 7-mL potassium EDTA tubes were collected from a single patient undergoing therapeutic phlebotomy for idiopathic hemochromatosis. Blood was pooled into a 500 mL bag and filtered with a Fenwall filter to remove leukocytes. Blood was then centrifuged at 2,000 g for one hour at 4 °C using 15 mL conical tubes. The cell free plasma was collected and reserved for later use. The upper 2-3 mm layer of red cells was collected from each tube, as well as one fraction of cells from the bottom part of one conical tube. The collected upper layer was recentrifuged using Wintrobe tubes for 30 minutes at 2,000g at 4 °C. The uppermost layer of pale red cells was removed from each Wintrobe tube and diluted to a final volume of .5 mL with the cell free Stability Blood from 30 donors was drawn into EDTA tubes and analyzed in duplicate for percentage of reticulocytes on the day of collection and after 72 hours storage at 4 °C. This time was chosen to replicate working conditions in the Clinical Laboratories, with specimens being drawn on Friday and run the following Monday. Reticulocyte Linearity Sample Prepared Sample Stability The protocol was designed to evaluate the stability of the prepared sample over a period of 15-90 minutes from the time of preparation. Blood from 15 different donors was added to the reticulocyte reagent and assayed at 15-minute intervals over a period of two hours. Agreement Between Methods Values for reticulocyte counts obtained with the Miles H*3 system were compared with those obtained with the following methods: Manual counting of reticulocytes. The NCCLS new methylene blue method was used and reticulocyte were counted with a Miller disk by two different technologists.15 Average values of A.J.C.P. • November 1994 BRUGNARA ET AL. Miles H*3 and Reticulocytes 627 TABLE 3. COMPARISON OF FOUR METHODS FOR RETICULOCYTE COUNTING: INTRACLASS CORRELATION (r,) COEFFICIENTS, PEARSON PRODUCT-MOMENT (r) VALUES, REGRESSION INTERCEPTS, AND SLOPES Intraclass Correlation 95% ConfidenceLimits H*3/SYS H*3/FLOW MAN/SYS MAN/FLOW MAN/H*3 SYS/FLOW Pearson s Product-Moment Ct jrrelation n rt Lower Upper r Intercept Slope 175 158 184 176 158 175 0.952 0.922 0.538 0.755 0.610 0.938 0.938 0.883 0.447 0.487 0.533 0.922 0.973 0.957 0.620 0.861 0.677 0.950 0.974 0.938 0.980 0.940 0.940 0.954 -0.326 1.082 0.211 1.440 0.665 1.195 1.014 1.170 1.050 1.307 0.977 1.242 H*3 = Miles H*3; SYS => Sysmex R-3000; FLOW == flow cytometry MAN •= manual counting. these two counts were used for comparison with the other methods. Flow cytometry with ihiazole orange. The method used was based on that described by Lee and Chiu.2 A FACS/SCAN (Becton Dickinson) flow cytometer was used. Sysmex R-3000. Reticulocyte were counted on blood samples collected in EDTA with a Sysmex R-3000' in the manual mode, following manufacturer's specifications. Studies in Pediatric Normal Controls and Hematologic Patients Reticulocyte count and red cell and reticulocyte indices were measured in 110 hematologic normal controls (51 males and 59 females, age 1-10 y), using "leftover" samples from the blood collected for routine CBC measurement. All the subjects studied were outpatients with normal hematological parameters, according to our established, age-adjusted, normal range. Various pediatric and adult patients with hematologic and non-hematologic disorders were also studied. Data Analysis Group data are expressed as mean ± standard deviation (SD). Paired Student's Mests were used to compare data within groups and unpaired Student's Mests to compare data between groups. Correlation studies used standard linear regression analysis with the Pearson's product-moment correlation coefficient. Comparison of reticulocyte counts obtained with different methods was carried out with the intraclass correlation coefficient using SAS.16"18 To determine if two methods can be used interchangeably, the Pearson's product-moment correlation (r) is customarily used. However, r is just an estimation of trend rather than concordance.17 The intraclass correlation (r,) quantitates the extent of agreement between methods. Meaningful agreement is achieved if the lower limit of the 95% confidence interval of the intraclass correlation is at least .75.17 The pattern of agreement between any methods can also be visually analyzed by plotting the mean reading of the two methods for each subject against the difference in reading between the two methods.16 RESULTS Within Run Precision Table 1 presents data on the intraassay precision for one subject with normal reticulocyte counts and one with an increased reticulocyte counts. Precision was estimated from the CV of 21 separate measurements performed on the same blood as detailed in Methods. The CV was 9.7% for the normal control and 6.7% for the sample with high reticulocyte counts. We also examined the precision of the values obtained for staining intensity of reticulocytes. As expected, in normal blood, there is a relatively larger variability in estimating the percentage of high intensity staining reticulocytes, due to the small number of cells present in normal conditions. When the reticulocyte count increases, the CV for the medium and high intensity staining reticulocytes decreases significantly. The CVs for the three classes of staining intensity in samples with increased reticulocyte content ranged from 3.1% to 8.6% (Table 1). Excellent precision was found in the measurements of the reticulocyte cellular indices in both the normal control and the patient with reticulocytosis (Table 1). The CVs for measurements of MCVr, CHCMr, and CHr were .8% to 1.6%; CVs for the distribution widths of these indices also were excellent (range, 2.5% to 9.2%). Imprecision Due to Sample Preparation Interassay precision was evaluated by comparing reticulocyte counts from the same specimen run asfivetriplicate specimens. Nested analysis of variance was used to identify the two components of the total standard deviation for each run, namely the within preparation and between preparation standard deviations. An F test for a significant preparation effect was performed. P values greater than .05 indicate a statistically not significant preparation error. For a normal control blood (% reticulocytes mean: 1.70, n = 15) the total SD was .18, the within preparation SD. 16 and the between preparation SD. 14, with P = .3261 (difference not significant). For a patient with reticulocytosis (% reticulocytes mean: 8.6, n = 15) the total SD was .71, the within preparation SD and the between preparation SD were .71 and .00, respectively with P = .8408 (difference not significant). Thus, no statistically significant preparation error could be identified. Vol. 102 • N o . 5 628 HEMATOPATHOLOGY Original Article Linearity Linearity was established by obtaining a reticulocyte-rich fraction from a subject with moderately increased reticulocyte counts and diluting it with a fraction with low reticulocyte content from the same subject. As shown in Figure 1, excellent linearity was obtained in the range tested (1-49%). Linearity was also determined by mixing blood of a patient with pyruvate kinase deficiency (reticulocytes = 95.3%) with an ABO compatible normal control, diluted to a similar number of cells/^L. As shown in Figure 1B, linearity was established in the range of 1% to 73% reticulocytes. Undiluted blood of this patient could not be counted with any of the automatic instruments. The effect of varying hematocrit on the percentage of reticulocytes was studied by counting reticulocytes in blood from a normal subject for hematocrit values of 14.9% to 77.1%. As shown in Figure 1C, there was no significant effect of changing TABLE 4. RETICULOCYTE AND RED CELL INDICES OBTAINED IN A PEDIATRIC POPULATION WITH THE MILES H«3 HEMATOLOGY ANALYZER Age (years) % Reticulocytes Red cell indices MCV (fL) CHCM (g/dL) CH (pg) RDW (%) HDW (g/dL) Reticulocyte MCVr (fL) CHCMr (g/dL) CHr(pg) RDWr (%) HDWr (g/dL) CHDWr (pg) Mean ± SD Range 4.5 ± 2.6 1.5 ± 0 . 6 1-10 0.4-4.0 79.3 34.9 27.7 14.0 2.6 ± 3.7 ± 1.0 ± 1.4 ±0.9 ± 0.2 72.0-91.2 31.9-36.8 24.4-31.6 12.4-17.8 2.1-3.1 97.6 28.2 26.7 14.3 3.3 3.3 ± 4.7 ± 1.4 ± 1.6 ±2.0 ±0.7 ± 0.4 87.7-116.5 23.3-31.4 21.4-30.5 10.9-22.0 2.5-7.5 2.6-4.8 Data were collected from 110 normal controls (51 males. 59 females, age 1 -10 years), with normal CBC values as defined by age-adjusted normal range values. MCV = mean cell volume: RDW = red cell distribution widths; CHCM = cell hemoglobin concentration mean; HDW = hemoglobin distribution widths; CH = cell hemoglobin content; CHDW = cell hemoglobin content distribution widths. H*3 - SYSMEX this variable on the values obtained for the percentage of reticulocytes. 10 20 30 10 Mean value (%) H*3 (% 20 30 reticulocytes) Carryover FLOW 0 10 0 20 10 20 30 H*3 (% reticulocytes) Mean value (%) Results of carryover studies are presented in Table 2. Two different methods were used to evaluate carryover. In the first one, a suspension of cells with 77.1% hematocrit was used to evaluate carryover into a cell free plasma from the same subject. This process was repeated 12 times. In the second method, blood from a patient with moderately increased (14.3%) and markedly increased (95.3%) reticulocyte counts were run in triplicate followed by three runs of diluent (as described in ref. 1) for a total often cycles each. In all three cases, carryover was minimal, with average values of .218, .019 and .126%, respectively. MAN - H*3 o o go Reticulocyte ° Sample Stability a 0 5 %3°o o ° 0 o 0 10 0 o 20 Mean value (%) 30 0 10 Manual (% 20 30 reticulocytes) FIG. 4. Left column, relation between mean values and difference in values of reticulocyte counting with the Miles H*3 and the other three methods. Right column, scatter plots and regression equation of reticulocyte counting with the Miles H*3 and the other three methods. Top row, comparison between Miles H*3 and Sysmex R-3000. Middle row, comparison between Miles H*3 and flow cytometry. Bottom row, comparison between Miles H*3 and manual reticulocyte counting. H*3 = Miles H*3; SYSMEX = Sysmex R-3000; FLOW = flow cytometry; MAN = manual counting. To evaluate stability of the reticulocyte measurements over time, 30 blood samples were measured at day 0 and again after 72 hours of storage at 4 °C. Data were then compared with the paired Mest. There were no statistically significant differences after 72 hours of cold storage for percentage of reticulocytes, and MCVr. The CHCMr index was significantly decreased after storage (average decrease of 1.2 ± 1.2 g/dL; P < .01) and this change was accompanied by a significant increase in RDWr (average increase 2.0 ± 4.6%; P < .05) and in HDWr (average increase .5 ± . 5 g/dL; P<. 01). There was also a significant decrease in CHr (average decrease 1.8 ± 4.5 pg; P < .05) with no significant changes in CHDWr. Stability of reticulocyte indices at room temperature or at different times of storage was non evaluated. A.J.C.P. • November 1994 BRUGNARA ET AL. 629 Miles H*3 and Reticulocytes A. NORMAL CONTROL RETICULOCYTE INDICES MCVr CHCMr RDWr HDWr CHr CHDWr 112.2 27.8 16.9 3.68 30.2 3.6 fL g/dL % g/dL pg pg B. IRON-DEFICIENT ANEMIA FIG. 5. Reticulocyte analysis with the Miles H*3 analyzer. Left column, plot of staining intensity (x axis) versus red scatter high (5°-15°; y axis), which is proportional to cell hemoglobin concentration. Right column, plots of hemoglobin concentration (x axis) versus cell volume (y axis). Red dots = erythrocytes; blue dots = reticulocytes; white dots outside of the red cell area = platelets, mononuclear cells, or scattered-light coincident events. A, Normal control, 1.5%. B, Iron deficiency, reticulocytes 1.6%. C, Sickle cell anemia, reticulocytes 18.1%. D, Hereditary spherocytosis, reticulocytes 11.7%. RETICULOCYTE INDICES MCVr CHCMr RDWr HDWr CHr CHDWr 84.7 20.8 21.3 3.70 16.9 3.6 fL g/dL % g/dL pg pg C. SICKLE CELL ANEMIA RETICULOCYTE INDICES MCVr CHCMr RDWr HDWr CHr CHDWr 106.2 29.0 23.3 6.06 29.2 4.5 fL g/dL % g/dL pg pg D. HEREDITARY SPHEROCYTOSIS RETICULOCYTE INDICES MCVr CHCMr RDWr HDWr CHr CHDWr Reticulocyte Prepared Sample 100.6 27.7 20.0 4.74 26.7 4.4 fL g/dL % g/dL pg pg Stability Reticulocyte counts and indices were measured in 15 subjects at 15-minute intervals for 120 minutes after the addition of blood to the reticulocyte reagent. Changes in the variables measured were plotted against time and compared with the values obtained at 15 minutes. As shown in Figure 2, values for percentage of reticulocytes were essentially stable over two hours. With paired /-test analysis, in comparison with the value obtained at 15 minutes, a statistically significant decrease in the percentage of reticulocytes was observed after 45 minutes. Staining intensity showed a small but significant increase in the percentage of reticulocyte in the low intensity fraction starting at 90 minutes. This was associated with a significant decrease in the percentage of reticulocytes in the medium and high intensity staining fractions at the same time points. Study of the reticulocyte cell indices revealed important changes during the 2 hours of incubation with the reticulocyte reagent. Paired /-test analysis showed a significant increase of MCVr beginning at 60 minutes, which was associated with a significant decrease in the CHCMr values (Fig. 3). This indicates that the reticulocytes swell over time in the reticulocyte reagent. A significant increase was also observed in the volume of red cells between 15 and 120 minutes of incubation (P < Vol. 102 • No. 5 630 HEMATOPATHOLOGY Original Article Studies in Pediatric Normal Controls and Hematologic Patients Sysmex Manual FACS/SCAN R-3000 MilesH*3 Values for red cell and reticulocyte indices obtained with the Miles H*3 in a normal pediatric populations are presented in Direct labor Table 4. Included in these group were 110 subjects, age 1-10, Specimen preparation 0.18 0.13 — 0.13 with normal hematologic parameters (hematocrit, hemogloTesting procedure 1.23 0.36 0.39 0.22 bin, red cell indices) based on age-adjusted normal range valConsumables ues. We found no significant differences in the measured paReagents 0.02 1.20 0.89 2.00 rameters between males and female. The data indicate that, Other 0.18 0.05 — 0.05 compared with mature red cells, reticulocytes have a larger Calibrators, quality control — 0.75 0.47 0.49 Proficiency testing — 0.02 0.02 0.02 volume, a reduced cell hemoglobin concentration and a similar Depreciation — 0.53 2.88 0.58 hemoglobin content. Service contracts — 0.30 1.38 — Figure 5 presents data on reticulocyte analysis of a normal control and 3 patients with iron deficiency, sickle cell anemia Total cost/test 1.61 3.34 6.03 3.49 and hereditary spherocytosis, respectively. As shown in Figure Labor cost is based on time studies of a batch of 10 samples for reticulocyte analysis (3 5, staining intensity of reticulocytes can be plotted versus cell minutes 45 seconds for preparation and 6 minutes 10 seconds run time for Miles H*3, 11 hemoglobin concentration and this variable can also be plotted minutes 7 seconds for Sysmex R-3000) and a batch of 12 samples for FACS/SCAN (4 minutes against cell volume. These plots show the presence of hy35 seconds for preparation and 12 minutes 10 seconds run lime) at the hourly rate of $21 pochromic reticulocytes in iron deficient anemia, and a inclusive of fringe benefits. Manual reticulocyte counting: 30 seconds for preparation and 3.5 minutes for reading (Miller's disk technique). If CAP workload units (9 minutes) were used, marked heterogeneity of reticulocytes volume and hemoglobin $3.15 can be attributed as labor cost for manual counting. List prices were used for reagent concentration in sickle cell anemia and hereditary spherocytocosts. Data for Sysmex R-3000 are based on yearly usage in our laboratory (manufacturer's sis, with the presence of dehydrated reticulocytes. total is $0.55/test). Quality control expenses: FACS/SCAN. yearly usage of 12 kit of ReticCheck (Streck Lab. Inc; $85/kit) and 30 control runs/week; Sysmex R-3000, 9 control kits/ We found no interferences with the Miles H*3 method for year (Baxter B3I60/90/AP. $268.80/kit) and 24 control runs/week; Miles H*3, 12 kit/year reticulocyte counting in patients with malaria infection (2 pa($85/kit) and 24 control runs/week; CAP survey, $ 123/year. Instrument list price: FACS/ tients tested, on with P. Falciparum and the other with P. FalciSCAN, $99,500; Sysmex R-3000, $109,000; Miles H*3, $212,000; Miles H , 2, $189,000; Cost parum plus P. Malariae infection), high WBC counts, high of interface with LIS ($6,000) was added to the capital cost of each instrument. Depreciation is calculated as a straight line in 5 years. Service contracts are for 7 days/week coverage: platelets counts, alpha or beta thalassemia, sickle cell anemia, FACS/SCAN $ 12,000; Sysmex R-3000 $ 11,000; Miles H*3 or H*2. $ 13,990. Incremental cost renal failure, bone marrow transplant and neonatal blood. The attributable to reticulocyte counting for service contract and depreciation was based for H*3 Miles could not gate on the negative, mature cells in two FACS/SCAN on the current utilization of 5 of 40 working hours/week (1/8 of total cost) and on an annual test volume of 5,000. For Miles H*3, the incremental cost was based on the price transfused patients, one with Hb H/CS (Constance Spring) and difference compared with Miles H*2 ($23,000). An alternative analysis for Miles H*3 based one with severe Hb H disease. Presence of nucleated red cells on the total instrument cost and annual billable volume of 90,000 CBCs and 8,000 reticulo(one patient, WBC 232,850/>L, corrected WBC 18,310/>L) cyte counts, yielded a cost/reticulocyte test of $0.44 for depreciation and $0.14 for service was associated with overestimation of reticulocyte counts contracts. (2.5% manual, 19.6% Miles H*3, 12.7% Sysmex R-3000). TABLE 5. COST ANALYSIS OF RETICULOCYTE COUNTING .001; paired Mest). A comparison of MCVr/red cell volume ratios at 15 and 120 minutes still showed a statistically significant increase (P < .001, paired Mest), suggesting that the swelling was proportionally greater in reticulocytes than mature cells. These changes did not produce any significant variation in CHr. Thus, CHr values can be compared independently of the reaction time used. The CHr index has been shown to be useful in detecting iron-deficient erythropoiesis.13 Agreement Between Methods Comparison of reticulocyte counts obtained with the Miles H*3, Sysmex R-3000, flow cytometry and manual counting is presented in Table 3 and Figure 4. Pearson's product moment (r) values greater than .93 were obtained for all comparisons, indicating that the different methods had similar trends (Table 3). However, intraclass correlation studies yielded unsatisfactory results when manual counting was compared with the three automated methods, since in all three cases the lower 95% confidence limit for r{ was lower than .75 (Table 3). Good intraclass correlation coefficients (r; for the lower 95% confidence limit > .75) were obtained for the comparisons among the three automated methods, indicating that there is satisfactory concordance among the three instruments. DISCUSSION Our studies of the operating characteristics of the Miles H*3 analyzer indicate that reticulocyte counting with this instrument has good precision, excellent linearity in the range tested (1-72%), and insignificant carryover. Reticulocyte counts obtained with the Miles H*3 instrument are stable when blood is stored for 72 hours at 4 °C. Comparison of four different methods for reticulocyte counting was carried out with two different methods of statistical analysis. With the classic Pearson's product-moment correlation r, there was relatively good agreement between any of the two methods (Table 3 and Fig. 4). However, this kind of analysis only indicates a linear trend for both variables to change in the same direction," and does not permit determination of agreement between any of the two methods. With the intraclass correlation coefficient, the level of agreement can be estimated; a satisfactory level is achieved when the lower limit of the 95% confidence interval is at least .75." Using these criteria, manual counting of reticulocytes can not be considered interchangeable with the three automatic methods. This reflects the significant imprecision of manual reticulocyte counting, mostly based on the interobserver variation in defining a reticulocyte and on the small number of cells counted.8 Excellent level of agreement was achieved with any combination of the three automated methods for reticulocyte counting. As shown in Figure 4, there was a tendency for the flow cytometry method to overestimate counts compared with the H*3, but A.J.CP.-November 1994 BRUGNARA ET AL. Miles H*3 a, the overall agreement was well above the acceptable values. Some of the differences might be due to the fact that the FACS/ SCAN method includes "shift" reticulocytes which are not captured by either the Sysmex R-3000 or the Miles H*3. Autofluorescence and interference by WBC and platelets are a significant interference for the flow cytometry method, but not for the Miles H*3 method. We found no significant interfering factors in the reticulocyte counting with the Miles H*3 analyzer, with the exception of nucleated red cells. In particular, no significant interference was observed in the presence of high WBC counts or platelets counts. Use of red cell indices has become an essential part of the differential diagnosis of anemias.' 1,19'2° The Miles H*3 hematology analyzer has the capability of measuring the values of MCVr, CHCMr, and CHr, and the distribution widths RDWr, HDWr, and CHDWr. One interesting potential clinical application of the reticulocyte indices is detection of iron-deficient erythropoiesis induced by recombinant human erythropoietin13,21,22). Reticulocyte indices and CHr in particular could allow a "real time" evaluation of the appropriateness of the recombinant human erythropoietin-induced erythropoiesis and provide the bases for a more rationale and cost-effective usage of this expensive recombinant red cell growth factor. In our evaluation, we observed that the reticulocyte indices have excellent precision and reproducibility (Table 1). Whereas MCVr and CHCMr have a tendency to change over time when the cells are incubated in the reticulocyte reagent (Fig. 3), CHr remains stable for 2 hours in the reagent buffer. Thus, time of incubation in the reticulocyte reagent is not a critical variable for this reticulocyte parameter. The availability of reticulocyte cell indices with the Miles H*3 is an important advance compared with the currently available methods, which quantify reticulocyte staining intensity, without providing any information on volume, hemoglobin concentration and content of the cells examined. The staining intensity of reticulocytes has been used to obtain insight into erythropoiesis in a variety of clinical conditions. 4,7 It will be important to test whether similarly useful information can be gathered from the study of the reticulocyte indices and CHr in particular, in settings such as bone marrow transplantation, differential diagnosis of anemias and sickle cell anemia. It certainly represents a powerful and unique feature of this instrument as compared with all the other reticulocyte analyzers. The cost-effectiveness for the clinical laboratories of combining blood cell counting and reticulocyte counting in one instrument versus two separate instruments deserves careful consideration. An analysis based exclusively on capital cost indicates that the purchase of the Miles H*3 versus the purchase of an H*2 plus a Sysmex R-3000 is financially very attractive. Capital cost would be $218,000 versus $310,000 (including $6,000 of cost for each interface with LIS), with an annual depreciation expense (5 year, straight line) of $43,600 versus $62,000. Other purchasing options such as leasing or reagent rentals were not considered. Expenses for service contracts are also significant lower in the Miles H*3 ($13,990) compared with the H*2 plus Sysmex R-3000 ($24,990). We analyzed the cost of reticulocyte counting with a manual technique and with three automated reticulocyte analyzers. This analysis is based on our experience at Children's Hospital and Brigham and Women's Hospital (Table 5). In this analysis, we did not consider discounts that might be obtained in purchasing instruments, service contracts, or supplies, and we itemized only the direct components of cost.22 An open tube testing Vol. I' 631 Reticulocytes mode for all the instruments was assumed. Time spent by technologists in starting up and shutting down instruments was not included. Depreciation and service contract costs were estimated for the FACS/SCAN based on the relative utilization of the instrument for reticulocyte counting (one eighth of total time). For the Miles H*3, two different approaches were used, with similar outcomes: the first one, based on the additional cost of an H*3 compared with H*2 system ($23,000 for purchase price, and $0 for service contract) yielded a cost per reticulocyte count of $.58 for depreciation and service contracts. The second one used the purchase price and service contract cost for an H*3 instrument, and calculated the cost per test based on a volume of 90,000 billable CBCs and 8,000 billable reticulocyte counts per year. This second approach yielded a cost per reticulocyte test of $.44 for depreciation and $.14 for service contracts. Our analysis indicates that the cost for reticulocyte counting with a dedicated analyzer (Sysmex R-3000) is significantly higher compared to instruments such as a flow cytometer or the Miles H*3 analyzer that perform other, additional, tests. Changes in annual volume of reticulocyte and CBC billable tests may greatly affect this comparison, which is based on the actual volumes at our two institutions. The manual counting technique is significantly cheaper that the automated methods, due to the absence of depreciation and maintenance expenses and to the low cost of consumables. However, the labor component of this method is significantly higher (3-4 fold) than the other three methods. A 6-8 fold higher value than the automated methods can be obtained for the labor cost of the manual method, if CAP workload units (9 minutes) are used rather than the value obtained in our time studies (4 minutes). For laboratorians facing the constant challenge of containing expenses, the availability of a combined blood and reticulocyte analyzer adds an interesting option regarding the choice of the appropriate hematology analyzer. The availability of reticulocyte cellular indices with the Miles H*3 and the possibility that they may become useful indicators of the appropriateness of erythropoiesis in a variety of clinical conditions also represents a significant advantage compared with other automated reticulocyte analyzers. Acknowledgments. 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