Validation of Sysmex XT-2000iV generated quantitative bone marrow differential counts in untreated Wistar rats

Authors


Abstract

Background

Preclinical drug trials frequently require assessment of bone marrow toxicity in animals to evaluate hematopoietic safety. Since the gold standard, cytologic evaluation, is time consuming and requires highly trained individuals, automated methods remain intriguing.

Objective

The Sysmex XT-2000iV hematology analyzer allows user-developed customizable gating. This study was conducted to validate the gating of bone marrow cell populations in Sysmex cytograms from untreated rats.

Methods

B- and T-lymphocytes and myeloid cells were experimentally depleted from Charles River Wistar Han IGS (CRL: WI [Han]) rat whole bone marrow suspension using a magnetic cell sorting (MACS) method. The positively and negatively selected populations were used to verify select gates within the Sysmex cytogram. Intra- and inter-animal precision, comparability between right and left femur, as well as agreement with microscopic myelograms based on 500 counted cells, were determined.

Results

Intra-sample precision and right-to-left femur comparability confirmed that gating was reproducible and stable. In 50 tested rats, myeloid to erythroid ratios (M:E) were 1.32 ± 0.33 in males and 1.38 ± 0.29 in females by Sysmex compared to 1.36 ± 0.32 in males and 1.42 ± 0.32 in females by microscopic evaluations. Bland–Altman differences between methods was ≤ ±0.35 units for M:E, ≤ 5.4% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, ≤ 6.0% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, and ≤ 4.1% for lymphocytes.

Conclusions

In untreated control Charles River Wistar Han IGS (CRL: WI [Han]) rats, the Sysmex XT-2000iV produced an automated M:E and 5-part differential count equivalent to microscopic differential counts.

Introduction

Automated hematology analyzers have been used for many years to perform routine peripheral blood cell counts and differentiation of WBC. For approximately the past 10 years, a variety of investigators have attempted cytologic analysis of human bone marrow using automated hematologic analyzers with varying degrees of success.[1-6] During that same period, we have validated and implemented a flow cytometric method for bone marrow analysis in rats.[7, 8] Bone marrow evaluation is an important step in drug development to understand the potential hematotoxic effects of a new drug. Although individual animal data are always important, determination of hematotoxic effects in a new compound is strongly influenced by grouped data, where treatment groups are compared with controls. Under these conditions, it is even more imperative to ensure quantitative reproducibility among substantial numbers of animals. Additionally, as nearly all rodents undergo a terminal necropsy, performing bone marrow evaluation on a suspension of bone marrow cells becomes a legitimate option.

A variety of marketed automated hematology analyzers have been shown to provide accurate and reproducible cell counts and WBC differential counts on peripheral blood. However, when bone marrow suspensions are analyzed on these same analyzers, a total nucleated cell count (TNCC) is often compromised due to contaminating elements such as fat droplets, microfibers, or cell aggregates in the sample. Limitations of analytical performance and insufficient separation of the numerous cell types and precursors have limited replacement of cytologic examination of bone marrow slides with automated bone marrow differential counts.[1, 3, 9, 10] Instead, in a clinical setting, utilization of hematology analyzers for bone marrow analysis serves as a preliminary assessment before microscopic evaluation. It provides a rapid assessment of such characteristics as overall cellularity, excessive proportion of blasts, and myeloid to erythroid ratio (M:E).

Human bone marrow analysis compared on 2 hematology analyzers, using the Cell Dyn 4000 and Sysmex XE-2100, showed a bias when compared with manual evaluations. Both analyzers showed good correlations for TNCC and myeloid cells.[4, 6] However, both methods also showed an underestimation of lymphocytes and M:E, and an overestimation of erythroid precursors compared with microscopic myelograms. This suggests that late erythroid nucleated cells and lymphoid cells cannot be adequately distinguished by forward and side scatter analysis alone. Specifically, bone marrow lymphocytes and erythroblasts of particular maturational stages can be similar in size and morphologic detail. Using cell sorting techniques, we had previously identified that rodent bone marrow lym-phocytes and erythroblasts occupied the same cytogram location when evaluated by flow cytometry.[7, 8] It seemed feasible that a reliable, automated bone marrow differential could be produced using the Sysmex XT-2000iV hematology analyzer if the mixed lymphocyte-erythroblast population could be differentiated by developing and validating gating standards of defined bone marrow populations within the Sysmex cytogram.

As the Sysmex XT-2100iV hematology analyzer is not capable of lineage-specific fluorescence labeling, identification of myeloid, erythroid, or lymphoid cells by cell sorting is not possible. However, the Sysmex XT-2000iV has the unique option of customizable gating, thereby allowing the operator to select and analyze discrete populations. We subsequently utilized magnetic bead separation of myeloid cells and lymphocytes to accurately identify the position of myeloid, erythroid, and lymphoid cell types in the Sysmex bone marrow cytogram of untreated rats. This manuscript describes the validation process for the XT-2000iV bone marrow differential count in untreated control rats.

Materials and Methods

Animals

Random-bred Charles River Wistar Han IGS (CRL: WI [Han]) rats (Charles River Breeding Laboratories, Raleigh, NC, USA) were used in this evaluation. All animals were housed individually at 75°F ± 5°F and 50 ± 10% relative humidity. The animal facility in which these studies were conducted was AAALAC international-accredited. All animal husbandry and experimental procedures were in compliance with United States Department of Agriculture Animal Welfare Act Regulations, the Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee. The number of animals used in each segment of this study is shown in Table 1.

Table 1. Number of rats used for validation experiments of bone marrow 5-part differential count on a Sysmex XT-2000iV
Study SegmentNumber of Rats/Gender
Validation of Sysmex Gating
Lymphoid Cells10 Males
Myeloid Cells10 Males
Proliferating Myeloid and Erythroid Cells10 Males
Reproducibility
Intra-Sample Precision10 Males
Right-to-Left Femur Comparability10 Males
Blinded Comparison of Sysmex and Microscopic Bone Marrow Differentials in Rats Up to 5 Months of Age

25 Males

25 Females

Bone marrow sample collection, processing, and analysis

Rats were anesthetized with isoflurane gas and then euthanized by exsanguination. One femur was excised from each animal immediately after death and the bone marrow was flushed into a 15-mL conical tube with approximately 5 mL of fetal bovine serum using a 5-mL syringe and a 20-gauge needle. The contents were mixed with a pipette and the tubes were immediately placed on wet ice. The collection tubes were filled with 1x Thermo Scientific Hyclone phosphate-buffered saline (PBS) (SH3002802; Hyclone Laboratories, Logan, UT, USA). Sample contents were mixed by inversion and centrifuged for 5 min at approximately 300g at 2–8°C. The supernatant was decanted without disturbing the bone marrow pellet. Two mL of 1x PBS were added to each collection tube. The pellet was aspirated into a one-mL syringe with a 19-gauge needle and then the suspension was expelled back into the tube. This process was repeated several times to dissociate the bone marrow aggregates. Samples were then diluted to a volume of 10 mL with 1x PBS. Bone marrow samples were stored on wet ice until processing was completed within 8 h of collection. This included cytocentrifuge preparations of the bone marrow cell suspension (Shandon cytocentrifuge; Phicom Applied Technology, Sewickley, PA, USA).

A TNCC was performed on the Sysmex XT-2000iV (Sysmex Corporation, Kobe, Japan), equipped with veterinary software. This instrument performs analysis of WBC with an optical detector block based on the flow cytometry method using a semiconductor laser. The WBC value obtained on the Sysmex XT-2000iV when analyzing bone marrow samples was multiplied by 10 (the dilution factor in the washed bone marrow sample) to represent the TNCC. Over the course of all experiments, there were no changes in the optics of the Sysmex analyzer. Rat-specific profiles were selected on the Sysmex analyzer for determination of TNCC values. Additionally, the bone marrow gating established was also rat-specific.

Validation of lymphoid, myeloid, and erythroid gating

To verify the true position of rat bone marrow lymphoid, myeloid, and erythroid lineages within the Sysmex cytogram, antibody-specific magnetic capture and separation of lymphoid and myeloid lineages was performed. Bone marrow was collected from 10 untreated male Wistar rats as described above to verify lineage position and set the appropriate gating for each of the defined populations. The washed cells were incubated with biotinylated anti-rat CD11b for the sorting of myeloid cells (554981; BD Bioscience, San Jose, CA, USA), or magnetic separation reagents such as OctoMacs magnetic separation unit (130-042-108; Miltenyi Biotec, Auburn, CA, USA), MS columns (130-042-201), anti-rat CD45RA microbeads (130-090-494), or rat anti-T-cell microbeads (130-090-320) and anti-biotin microbeads (130-090-485).

Myeloid and lymphocyte capture and evaluation

One mL of the processed bone marrow preparation was added to one mL of BD Pharm Lyse Lysis buffer (555899; BD Biosciences) and mixed for 10 min at room temperature. Tubes were centrifuged, the cell pellet washed once, and the final pellet was suspended in 80 μL of BD Pharmingen Stain Buffer (555899; BD Biosciences). For myeloid cell capture, a total of 15 μL of rat-specific anti-CD11b was added to each pellet, mixed, and incubated at 4°C for 10–15 min followed by an additional 10 to 15 min with anti-biotin microbeads. One to 2 mL of stain buffer was added to each tube, which was spun, the cell pellet washed, and the tube re-centrifuged. The cellular pellet was suspended in 500 μL of stain buffer and run over prepared MACSMS separation columns (Miltenyi Biotec Inc.) under a magnetic field. The columns were washed once with stain buffer. The columns were removed from the magnetic field and 1.5 mL of stain buffer was applied to each column. Any remaining sample was flushed out using the provided plunger. Flow through was collected, spun, and suspended in 500 μL PBS. This sample was then analyzed directly on the Sysmex platform. For lymphocyte capture, samples were treated and processed as described for myeloid capture, except that 15 μL of rat-specific anti CD45RA and anti-T CD3 lymphocyte microbeads were utilized. During both the myeloid and lymphoid capture experiments, a cytocentrifuge slide was prepared from each of the 10 flow-through samples to microscopically confirm the absence of myeloid or lymphoid cells in the respective samples.

Sysmex gating for proliferating and maturing myeloid and erythroid populations

Clusters of cell populations in rat bone marrow occupied a consistent position throughout all experiments. Magnetic bead-mediated purified cell populations allowed defined custom gating of distinct bone marrow cell lines. To further evaluate and adjust our gating, microscopic differential counts were performed on 10 whole bone marrow cytocentrifuge slides prepared during the myeloid magnetic bead purification procedure. Sysmex gates were adjusted to achieve results comparable to microscopic differentials for proliferating (myeloblasts, promyelocytes, and myelocytes) vs maturing myeloid cells (metamyelocytes, band and segmented neutrophils). A similar approach was chosen for the 10 whole bone marrow samples in the magnetic bead lymphocyte purification procedure to define the Sysmex gates for proliferating (erythroblasts, prorubricytes, and basophilic rubricytes) and maturing erythroid cells (polychromatophilic rubricytes and metarubricytes), as well as the lymphocyte population. The number of lymphoid cells was determined directly on the antibody-labeled sample, and the maturing erythroid population was derived by calculation (total mixed lymphoid-erythroid population – lymphoid population = maturing erythroid population).

Overall gating

Gating maps for the 5-part differential were constructed by shaping of the cytogram and comparison with microscopic differential counts. Once confirmed, gating proved to be stable and was not altered for any of the remaining experiments. Importantly, this basic gating procedure is a generic process that needs to be re-established in each laboratory and confirmed with each new instrument.

Intra-sample precision

To evaluate between-run reproducibility (precision) within a single sample, 10 rat femoral bone marrow samples were collected and processed by methods previously described.[7, 8] Each of the 10 bone marrow samples was run 10 successive times on the Sysmex analyzer. Proliferating myeloid, maturing myeloid, proliferating erythroid, maturing erythroid, lymphoid (the 5-part differential), and M:E ratio were determined using gating preset from the magnetic bead isolation experiments.

Right-to-left femur comparability

To further evaluate reproducibility of the assay, the right and left femurs of 5 male rats were collected and processed accordingly. Samples were analyzed for the 5-part differential on the Sysmex and the M:E ratio was calculated from the 10 samples.

Comparison of Sysmex XT-2000iV and microscopic 5-part bone marrow differentials in untreated rats up to 5 months old

To evaluate comparability and agreement between the Sysmex XT-2000iV-generated 5-part and the microscopic differential counts, bone marrow collected from 50 untreated Wistar rats (25 males and 25 females) was analyzed with both methods. Comparisons were conducted in a blinded fashion, and the microscopist had no access to Sysmex data. Rats ranged in age from approximately 2–5 months. For each microscopic bone marrow differential, a minimum of 500 cells were classified by an experienced hematologist. Flushed femur bone marrow samples were processed as previously described and then analyzed directly on the Sysmex platform with our confirmed gating from initial magnetic bead separation experiments.

Statistical evaluation

All statistics were generated using SAS R version 3.0.1. For the intra-sample precision experiment, individual data points, mean ± standard deviation (SD), and intra-animal coefficient of variation (CV) were calculated for the first rat analyzed as representative of the group. Inter-animal percent variance within the group of 10 rats and the maximum difference in 5-part differential and M:E ratio were also calculated. Box and whisker plots were prepared to show intra-sample minimum, median, and 25% and 75% quartiles for each of the 10-replicated analyses on each of the 10 individual rats.

The mean and SD for M:E and the 5-part differential were also determined for the 5 right and 5 left femur samples, and a correlation coefficient was calculated and used to compare left and right femur groups in male rats. The maximum difference between any corresponding right/left femur pair for M:E and 5-part differential was recorded.

Mean ± SD and maximum difference between corresponding variables were also calculated for Sysmex and microscopic differential counts in both sexes. Bland–Altman plots were generated using publicly available plotting code and constructed to assess systematic level of agreement between the Sysmex and microscopic differential count results. The mean difference between the 5-part differential variables assessed by the 2 methods was the estimated bias, and the SD of the differences provided a measure of fluctuation around the mean. The 95% limits of agreement (average difference ± 1.96 standard deviation of the difference) were calculated.

Results

Qualification of rat bone marrow cytogram regions

Although bone marrow is a more complex mixture of cell types due mainly to the many maturational stages, peripheral blood cytograms provide a reasonable estimate of where various bone marrow populations should locate. As can be observed in Figure 1, the x-axis represents the side scatter of light assessed through a principle of flow cytometry, and the y-axis represents RNA- and DNA-fluorescence in an example of human blood. As hematopoietic cells mature, they also become smaller in size. Smaller, more mature cells are located near the base of the Sysmex cytogram, whereas, larger, more immature cells (including blasts) reside higher up on the y-axis of the Sysmex scattergram. Peripheral blood neutrophils (N) reside to the right of the lymphocyte (L) population, and eosinophils (E) are positioned to the most extreme right of the Sysmex cytogram. Peripheral blood monocytes (M) reside between the lymphocyte and immature granulocyte (IG) populations. Nucleated red blood cells (NRBC) are often late-stage (poly- or orthochromatophilic) normoblasts when found in peripheral blood. These small NRBC are typically found beneath the peripheral blood lymphocyte population and to the left of mature neutrophils. Ghost cells, platelet clumps, and other peripheral blood debris are typically isolated at the base of the cytogram (Figure 1C and D).

Figure 1.

Examples of cell gating on a Sysmex XT-2000iV. (A) Peripheral whole human blood cytogram on the Sysmex analyzer (printed with permission of Sysmex, Inc.). The x-axis represents side scatter of light, whereas the y-axis represents RNA- and DNA-fluorescence. Smaller and more mature cells are located near the bottom of the Sysmex cytogram, whereas larger and more immature cells, including blasts, reside higher on the y-axis. Human cell types within cytogram are peripheral blood neutrophils (N), lymphocytes (L), eosinophils (E), monocytes (M), and immature granulocytes (IG) (B) Whole washed Wistar Han rat bone marrow cells separated into 4 regions (Region A, B, C, and D) on the Sysmex cytogram. (C) Position of purified Wistar Han rat bone marrow cells after incubation with biotinylated anti-myeloid (CD11b) followed by magnetic field separation showing position of maturing myeloid cells in region A and proliferating myeloid cells in Region B. Eosinophils = (yellow circle) and macrophage/monocyte region = M (white circle) are also demonstrable. (D) The flow-through portion of Wistar Han rat bone marrow cells following incubation with CD11b showing the position of nonmyeloid cells. The red circle in 1C and 1D represents ghost cells and/or debris.

Gating of whole rat bone marrow was divided into 4 regions (Figure 1B, regions A, B, C, and D) based on cell populations purified by magnetic bead isolation of myeloid and lymphoid cells, which was in agreement with the gating of major cell types in peripheral blood of rats. Importantly, the pattern of separation of various populations was consistent in untreated rats. Rat bone marrow myeloid cells sorted with CD11b resided in regions A (maturing myeloid) and B (proliferating myeloid) of the Sysmex scattergram (Figure 1C). Myeloid cells have lower cell and nuclear size with increasing degrees of maturity. Therefore, myeloblasts, promyelocytes, and myelocytes are positioned above the mature myeloid cells in the scattergram (Figure 1C, Region B); they represent the proliferating myeloid region. The discriminating line between proliferating and maturing myeloid cells such as mature neutrophils, band neutrophils, and metamyelocytes was set by comparison with microscopic differential counts in untreated rats.

The CD11b antibody also binds to bone marrow eosinophils (Figure 1C, E, yellow circle) and macrophages (Figure 1C, M, white circle). The noncaptured or flow-through population consisting of nonmyeloid cells, including predominantly lymphocytes and erythroid cells, co-locates to regions C and D of the cytogram (Figure 1D). A small additional population of cells (< 2% of the entire population) located near the base of the cytogram of both magnetically captured and flow-through aliquots probably represents a population of ghost cells and cellular debris (Figure 1C, D, red circle).

In contrast, >99% of the purified lymphoid cells locate in region C (Figure 2B), and large numbers of lymphocytes co-locate with the erythroid population in unpurified whole bone marrow (Figure 2A). This was confirmed by microscopy, where cells corresponding to the population in region C consisted of a mixed population of polychromatophilic rubricytes, metarubricytes, and lymphoid cells. It was therefore defined as a mixed region containing both maturing erythroid and lymphoid cells. In contrast, proliferating erythroid cells such as prorubricytes and basophilic rubricytes were located in Region D (Figure 2A). A small number of erythroid cells was also present in Region A and was considered to represent < 2% ghost cells (Figure 2B).

Figure 2.

Definition of lymphoid cell gating of Wistar Han rat bone marrow samples before and after magnetic field separation of lymphoid cells, analyzed on a Sysmex XT-2000iV. (A) Whole washed bone marrow cells distributed in 4 distinct regions (A, B, C, and D) of the Sysmex cytogram (B) Rat bone marrow cells incubated with CD45RA and anti-T-lymphocyte microbeads followed by magnetic field separation provided evidence that lymphocytes are located nearly exclusively in Region C of the cytogram. The cells inside the white circle are ghost cells.

Intra-sample precision

The intra-sample precision of the 5-part bone marrow differential counts and the calculated M:E ratios were excellent, with CV in one representative single animal ranging from 1.1-4.0% for all components of the 5-part differential count (Table 2). The inter-animal CV in 10 rats ranged from 1.6-17.1% for all components of the 5-part differential count, reflecting the expected range of biologic variation in 10 different animals. The minimum, median (center line), maximum, 25% and 75% quartiles obtained in each of 10 rats for 10 replicated analyses are shown in Figure 3. The maximum difference for any of the 5-part differential components within any of the 10 rats ranged from 0.8-1.8%, whereas the maximum difference for M:E ratios within the same sample was 0.11 units.

Table 2. Intra-sample precision and inter-animal variance of Sysmex XT-2000iV marrow differential counts in 10 control male rats
Analysis No.Relative 5-Part Bone Marrow Differential Count (%)
MyeloidErythroidLymphoidM:E
ProliferatingMaturingProliferatingMaturing
  1. SD indicates standard deviation; CV, coefficient of variation.

  2. a

    Percent variance within when assessed across 10 individual rats.

19.439.48.033.010.21.19
28.839.98.831.710.81.20
39.640.27.931.211.11.27
49.539.08.432.210.91.19
59.039.58.433.010.11.17
68.839.98.532.810.01.18
78.940.48.831.410.51.23
88.640.28.931.810.51.20
99.039.98.232.310.61.21
109.239.58.432.710.21.18
Mean ± SD9.08 ± 0.33339.79 ± 0.4338.43 ± 0.33732.21 ± 0.66210.49 ± 0.3671.20 ± 0.029
Intra-Animal CV (%)3.71.14.02.13.52.4
Inter-Animal CV (%)a3.52.217.19.61.68.4
Max Difference (%)a0.81.41.01.80.90.11
Figure 3.

Intra-sample precision for Sysmex XT-2000iV 5-part bone marrow differential count and M:E ratio in male Wistar Han washed bone marrow samples from 10 rats analyzed in 10 successive runs. Boxes show median (center line), lower and upper quartiles (box), total range (whiskers), and outliers (·).

Right-to-left femur comparability

The group mean M:E for the 5 left femurs was 1.18 vs 1.17 for the 5 right femurs, demonstrating essentially identical means between the 2 sets of femurs in male control rats (Table 3). The correlation coefficients were 0.671 for proliferating myeloid, 0.976 for maturing myeloid cell, 0.916 for proliferating erythroid cells, 0.996 for maturing erythroid cells, 0.881 for lymphoid cells, and 0.983 for the M:E ratio. The maximum difference between any pairs of right and left femur was 2.0% for proliferating myeloid, 2.1% for maturing myeloid, 1.4% for proliferating erythroid, 1.7% for maturing erythroid, 1.0% for lymphocytes, and 0.08 units for M:E ratio.

Table 3. Comparison of paired right and left femur bone marrow differential counts in healthy control male rats determined by a Sysmex 2000iV
Animal No./SideRelative 5-Part Bone Marrow Differential Count (%)
MyeloidErythroidLymphoidM:E
ProliferatingMaturingProliferatingMaturing
1 Left9.337.48.235.59.61.07
2 Left8.634.611.333.711.80.96
3 Left9.040.19.130.111.71.25
4 Left7.646.98.327.110.11.54
5 Left9.337.87.436.19.41.08
Mean ± SD8.76 ± 0.70939.36 ± 4.6468.86 ± 1.49132.50 ± 3.81810.52 ± 1.1521.18 ± 0.226
1 Right9.535.87.736.910.11.02
2 Right8.033.99.935.412.80.92
3 Right10.140.68.529.711.11.33
4 Right7.644.88.626.410.61.55
5 Right8.437.57.437.49.31.02
Mean ± SD9.12 ± 0.88138.52 ± 4.2888.42 ± 0.97333.16 ± 4.86410.78 ± 1.3101.17 ± 0.263
Maximum Difference2.0%2.1%1.4%1.7%1.0%0.08
Correlation Coefficient (r)0.6710.9760.9160.9960.8810.983

Comparison of Sysmex-generated and microscopic bone marrow differential counts in untreated Wistar rats

The mean ± 2SD for the Sysmex XT-2000iV automated 5-part bone marrow differential components and accompanying mean microscopic differential counts for a total of 50 male and female rats are provided in Table 4. Mean Sysmex M:E ratios and 5-part differentials in males and females were comparable to those obtained in microscopic myelograms. The maximum difference between any Sysmex M:E value and its corresponding microscopic M:E value in 50 rats evaluated was < 0.3 units, affirming the close approximation of methodologies. Maximum difference in 5-part differential between methods also remained < 5%, showing that differentials, as well as M:E ratios, could be interchangeably used (Table 4). All of the differences observed were considered acceptable when comparing an automated with a manual methodology.

Table 4. Comparison of Sysmex 2000iV and microscopic bone marrow differential counts in healthy control male and female Wistar Rats
Group (n = 25)Relative 5-Part Bone Marrow Differential Counts (Mean ± SD)
MyeloidErythroidLymphocytes (%)M:E (%)
Proliferating (%)Maturing (%)Proliferating (%)Maturing (%)
SysmexMicroSysmexMicroSysmexMicroSysmexMicroSysmexMicroSysmexMicro
  1. Micro indicates microscopic; UAL, upper agreement limit between methods (+2SD); LAL, lower agreement limit between methods (-2SD).

  2. a

    Data expressed as mean ± standard deviation (SD) n = 25 rats/sex.

  3. b

    Maximum difference between any Sysmex and microscopic value.

Malea9.4 ± 2.0710.4 ± 1.7040.8 ± 7.0341.5 ± 6.788.6 ± 1.928.3 ± 2.0630.5 ± 3.8729.7 ± 3.3910.7 ± 2.5610.1 ± 3.211.32 ± 0.331.36 ± 0.32
Femalea9.7 ± 1.0410.1 ± 1.3942.3 ± 4.9442.5 ± 5.438.6 ± 1.428.7 ± 1.5829.6 ± 4.8329.0 ± 4.649.8 ± 1.779.0 ± 2.021.38 ± 0.291.42 ± 0.32
Maximum Differenceb
Male3.0%4.5%2.2%4.7%3.6%0.26 units (ratio)
Female2.4%4.5%2.4%2.7%3.0%0.29 units (ratio)
Bland–Altman Agreement Assessment - Upper and Lower Limits of Agreement (± 2SD difference in methods)
 UALLALUALLALUALLALUALLALUALLALUALLAL
Male+1.48%−3.44%+4.03%−5.4%+2.93%−2.36%+6.03%−4.35%+4.06%−2.84%+0.26−0.34
Female+2.2%−3.08%+4.59%−5.0%+3.14%−3.35%+3.83%−3.93%+3.07%−1.45%+0.27−0.35

The level of agreement for M:E ratios for males and females was excellent (Figure 4 A and B, Table 4). M:E values ranged from 0.8 to 2.0 across the 50 rats tested, a range consistent with previous historical ranges obtained either by flow cytometry or microscopic differential counts. The small difference in M:E ratio denoted in the Bland–Altman plots (< 0.3) for both males (Figure 4A) and females (Figure 4B) further demonstrates the comparability of M:E ratios generated with the Sysmex XT-2000iV automated method or by microscopic evaluation. Sysmex XT-2000iV-generated 5-part differentials were also consistent with historical reference values generated by flow cytometry. Across the cohort of control rats tests, the proportion of maturing myeloid cells ranged from 30 to 55% with a mean difference in methods of ± 5%, demonstrating good comparability between the 2 methods (Figure S1). Numbers of proliferating myeloid cells were also similar between methods and to previously established historical values (Figure S1). Variability between methods was higher in males than in females for maturing erythroid cells, but the method comparison was considered acceptable for maturing erythroid cells in both sexes with a mean difference of <6% (Figure S2). Proliferating erythroid proportions were equivalent and the good agreement between methods for these variables was evident by a low difference in percentage between the 2 methods (Figure S2). Differences in bone marrow lymphocyte proportions were similar in magnetically separated samples analyzed with the Sysmex XT-2000iV analyzer and in microscopic evaluation. Additionally, there was no evidence for a bias between the 2 methods as shown by similar numbers of values both above and below the line of true agreement. Methods were also considered interchangeable for lymphocyte determination as percent difference between methods remained small (Figure 5, Table 4).

Figure 4.

Bland–Altman plots for bone marrow M:E ratio in normal Wistar Han rats generated by Sysmex XT-2000iV compared with microscopic evaluation in (A) males and (B) females. Mean ideal agreement between methods represented by the dotted blue line. Mean actual agreement between methods represented by solid black line. UAL = upper limit of agreement (+2 SD). LAL = lower limit of agreement (-2 SD). n = 25 rats/sex.

Figure 5.

Bland–Altman plots for bone marrow lymphoid cell counts in Wistar Han rats generated by Sysmex XT-2000iV compared with microscopic evaluation in (A) males and (B) females. Bone marrow lymphocytes were captured by magnetic separation following incubation with anti-rat T-cell CD3 and CD45RA. Mean ideal agreement between methods represented by the dotted blue line. Mean actual agreement between methods represented by solid black line. UAL = upper limit of agreement (+2 SD). LAL = lower limit of agreement (−2 SD). n = 25 rats/sex.

Discussion

Although the Sysmex XT-2000iV has been broadly validated for the assessment of peripheral blood hematology of rats,[11, 12] this is the first report demonstrating that the Sysmex XT-2000iV is capable of producing reliable, reproducible, and high-quality bone marrow analysis in untreated Wistar rats when coupled with a magnetic bead isolation, purification, and sequestration of bone marrow lymphoid cells. Although the magnetic separation requires additional time for the overall procedure, the application is still more efficient than our previous flow cytometric application. Specifically, with the protocol presented in this study, bone marrow populations are distinct, and once gating has been defined for the 5-part bone marrow differential components (proliferating and maturing myeloid, proliferating and maturing erythroid, and lymphoid), there is no need for further adjustment. This eliminates operator bias in setting gates during analysis. The precision, reproducibility, and accuracy when compared with microscopic bone marrow differential counts as the gold standard make this method a real alternative that can be implemented for studies conducted under Good Laboratory Practice conditions. To date, up to 80 rat bone marrow samples have been processed in a single day. Although microscopic bone marrow differential counts yield comparable results, they require more time, which can result in delays of days or weeks depending on the number of animals and the availability of trained staff. Potential disadvantages of automated bone marrow evaluation by the Sysmex XT-2000iV are the inability to archive the sample (unless cytocentrifuge preparations are available), and the customized generic gating procedure of the instrument, which requires a certain expertise with handling and processing of cell suspensions.

Bone marrow evaluation in rodent nonclinical studies is required to determine potential primary hematotoxicity of new compounds.[13] In many cases, histologic evaluation coupled with peripheral blood hematology end points may be adequate. Earlier reports suggested that the need for cytologic evaluation of bone marrow smears should be decided on a case-by-case basis, and cited multiple treatment-related changes requiring a more in-depth bone marrow evaluation.[14] Examples include characterization of changes in bone marrow cellularity relative to peripheral blood cell counts, differentiation of the involved cell series, specifically the distinction between erythroid and lymphoid populations (as these are difficult to discern by histopathology alone), abnormalities in RBC indices, significant decrease in blood neutrophil counts with no evidence of an underlying response to acute tissue inflammation, and known antiproliferative effects of the compound, marked increases in peripheral eosinophil counts, or significant increases in RBC counts that are not accounted for by dehydration or hemoconcentration.[14] This includes also the evaluation of abnormal or atypical cells in peripheral blood, which would, however, probably require a microscopic evaluation or flow cytometric phenotyping, depending on the extent of the changes. The same report also pointed out that, “Flow cytometry when appropriately executed, may be used in place of manual differential counts of bone marrow hematopoietic cells and thus can be applied to many of the situations outlined above for cytological assessment.”[14] This also applies to the automated Sysmex quantitative bone marrow assay; however, in either flow cytometric or the Sysmex applications, bone marrow smears or cytocentrifuge preparations of bone marrow cell suspensions should always be collected in case cellular morphologic evaluation is necessary.

We have utilized a flow cytometric bone marrow differential procedure to evaluate hematotoxicity in nonclinical studies for the past 10 years,[7, 8] and have found that this methodology eliminates > 90% of rat microscopic bone marrow evaluations (unpublished observation). In addition, during the past year, we have replaced the flow cytometric procedure with the Sysmex application for bone marrow assessments. Importantly, we are not recommending that either a flow cytometric or Sysmex methodology be performed in isolation. Instead, quantitative bone marrow results should always be conducted in conjunction with a histopathologic review. This allows the clinical pathologist to compare quantitative results with histologic findings and determine the need for further cytologic review. Neither the Sysmex nor flow cytometric methods provide cytomorphological characteristics. Importantly, cytologic review remains critical when a complete classification of all cell types or lineage maturation is required, for instance, for the assessment of morphologic changes in any lineage, the confirmation of suspect megaloblastic changes, or the evaluation of megakaryocyte-related effects. A thorough understanding of hematology, cytology, and histology is essential in appropriately evaluating treatment-related disturbances of hematopoietic physiology and in putting quantitative findings into proper context.

The Sysmex XT-2000iV methodology is an advancement compared with our previous flow cytometric method, as bone marrow cytograms remain stable throughout the analytic process. Therefore, results are available at the time of sample analysis. This is in contrast to our previous flow cytometric method, where time-consuming manual gating was required for final results of each sample. Another major advantage of the Sysmex methodology compared with our previous flow cytometric method is the assessment of the differentiation/maturation process. The DIFF channel of the Sysmex XT-2000iV platform measures the intensity of staining of DNA/RNA with the dye polymethine on the y-axis and the measurement of side scatter light (cell content) on the x-axis. In contrast, our flow cytometric procedure relied on peroxidase staining.[7, 8] Importantly, the level of peroxidase activity is species-dependent. Therefore, although our previous flow cytometric application works well in rats, it can be problematic in mice and rabbits due to species-specific differences in bone marrow cell peroxidase activity (unpublished observation). For completeness, it should be noted that flow cytometric bone marrow analysis methods that are not peroxidase-dependent may not have the same limitations. Nevertheless, as gating in the Sysmex system is dependent on DNA staining, bone marrow evaluation can be more readily applied in multiple species. Recent studies have demonstrated that the Sysmex-generated 5-part differential is also applicable in mice, dog, and cynomolgus monkey (unpublished observation).

In addition, the versatile gating capability of the Sysmex XT-2000iV allowed accurate assessment of TNCC, maturing and proliferating myeloid cell populations, maturing and proliferating erythroid cell populations, lymphocytes, and M:E ratios in rat bone marrow. Because bone marrow lymphocytes co-locate within the same gated region as erythroid precursors, the analysis of purified erythroid and lymphoid cell populations allowed validation of the automated Sysmex method generating lymphocyte results comparable to microscopic differential counts. In addition, the overall relative rat bone marrow differential counts obtained in the studies here are comparable to ranges established over the past 10 years using the antibody-specific flow cytometric method. Also, gating of blasts within the bone marrow 5-part differential count was consistent with a previous report of blast identification using the Sysmex platform in peripheral blood of dogs with leukemia.[15] Most importantly, once established, gating can be consistently used without further adjustment, although a certain expertise and training with the Sysmex instrument is required.

Bland–Altman plots have been used extensively to evaluate the agreement of 2 measurement techniques.[19, 20] The Bland–Altman comparison between the Sysmex XT-2000iV and the microscopic myelogram data revealed no random or systematic error between the 2 methods. Specifically, the Bland–Altman upper and lower limits demonstrated acceptable agreements between Sysmex and microscopic bone marrow differential counts and suggest that the assays can be used interchangeably.

In addition, Sysmex XT-2000iV bone marrow ranges in rats were nearly identical to published reference ranges.[7, 8, 13, 16-18] Mean M:E ratios in mature healthy rats ranged from 1.07 to 1.93 as reported by a variety of investigators,[13] and these values are consistent with M:E ratios generated in mature rats by the Sysmex methodology. The proportion of proliferating to maturing marrow cells in mammals typically is about 1:4, as defined in people[21] and in rats.[22]

Bone marrow lymphocyte identification is highly dependent on the training of the microscopist as well as the quality of the smears, which in turn is dependent on stain quality and degree of autolysis of cells. Lymphocytes possess some morphologic characteristics similar to those found in polychromatophilic rubricytes and metarubricytes, which may account for some of the broad variability in the reported lymphocyte range in rats (4–63%),[2] although strain differences may also contribute to varying bone marrow lymphocyte counts in rats. In a more contemporary publication, lymphocytes in healthy rat bone marrow ranged from 7.34 to 21.0%,[13] the range of which was met with the Sysmex generated data. The major advantage of using lymphocyte-specific antibodies for gating purposes is better data consistency between multiple operators without need for morphologic training.

Implementation of automated bone marrow analysis in rodent safety studies can greatly improve consistency and timeliness of data generation compared with microscopic analysis. Automated quantitative bone marrow differential counts have a clear advantage of increased throughput and precision due to the analysis of higher cell numbers. The Sysmex quantitative bone marrow differential assay is not dependent on the training of the microscopist, nor is it dependent on the quality of the slide. However, to safely and reliably practice bone marrow analysis, Sysmex operators still need to be adequately trained to identify abnormal cytograms, and a histopathologic examination needs to be performed on all animals so that samples that require further cytologic review by a clinical pathologist are identified prior to reporting. Clinical pathologists should be closely connected to the interpretation of quantitative bone marrow data and to the drug development project so that any other additional needs including microscopic slide review for morphologic assessment are also granted. Although quantitative bone marrow differential counts in nonclinical studies may not be required for all new compound evaluations, the sensitivity and precision of the Sysmex XT-2000iV bone marrow differential count could be a valuable tool in selection of the right target or compound for further development.

Acknowledgments

Thanks to Walt Bobrowski, Principal Scientist Investigative Pathology, Pfizer (Groton, CT) for assistance in preparation of figures for this publication.

Disclosure. The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.

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