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    Dose-response curve for a flow cytometric assay developed to detect the extent of IgG binding to RBCs. For assay evaluation, DEA1-positive RBCs from hematologically healthy dogs were incubated with doubling dilutions of anti-DEA1 blood-typing serum and then assayed, yielding a curvilinear reduction in signal (MdFI) with a reduction in the amount of IgG bound to cells.

  • View in gallery

    Representative flow cytometry dot plots of canine bone marrow cells in top (A), middle (B), and bottom (C) density-gradient fractions after labeling cells for CD18 (x-axis) and staining them for nucleic acids with LDS 751 (y-axis). Nucleated RBCs, reticulocytes, and mature RBCs in gated regions R1, R2, and R3, respectively, were CD18 negative and LDS 751 positive, CD18 negative and LDS 751 moderate, and CD18 negative and LDS 751 negative, respectively.

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    Representative gated regions and cytologic findings for flow cytometer-sorted canine bone marrow aspirate cells after density gradient separation into top, middle, and bottom cell fractions. A—Dot plot of bone marrow cells in the middle density gradient fraction after cell labeling for CD18 (x-axis) and staining of nucleic acids with LDS 751 (y-axis) showing the location of nRBCs, reticulocytes, and mature RBCs in gated regions R1, R2, and R3, respectively. B through D—Photomicrographs of cells from the top, middle, and bottom R1 fractions, showing a predominance of early- (B), mid- (C), and late-(D)stage nRBCs, respectively, with little contamination by other cell types. E and F—Photomicrographs of the R2- and R3-gated cells depicted in panel A and consisting mostly of reticulocytes (E) and mature RBCs (F), respectively, as was true for R2- and R3-gated cells in the top and bottom fractions as well.

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Analytic characterization of flow cytometric assays for detection of immunoglobulin G on canine erythroid cells, including detection of dog erythrocyte antigen 1 on erythroid precursors

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  • 1 Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.
  • | 2 Department of Medicine, College of Human Medicine, Michigan State University, East Lansing, MI 48824.
  • | 3 Department of Medicine, College of Human Medicine, Michigan State University, East Lansing, MI 48824.
  • | 4 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.
  • | 5 Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.

Abstract

OBJECTIVE To develop and characterize flow cytometric assays for detecting IgG bound to canine erythrocytes and bone marrow erythroid precursors.

SAMPLE Blood samples from 20 healthy and 61 sick dogs with (n = 33) or without (28) immune-mediated hemolytic anemia (IMHA) and bone marrow samples from 14 healthy dogs.

PROCEDURES A flow cytometric assay for measurement of IgG on RBCs was developed, and appropriate positive control cells were generated. Analytic and diagnostic performance were characterized. The RBC IgG assay was then combined with density-gradient fractionation of aspirated bone marrow cells and a 2-color process to yield an assay for detecting IgG on nucleated RBCs (nRBCs). Cell sorting and cytologic examination confirmed target cell populations, and anti–dog erythrocyte antigen 1 (DEA1) blood-typing serum was used to generate IgG-positive nRBCs.

RESULTS Within- and between-run coefficients of variation for the RBC IgG assay were 0.1% to 13.9%, and > 90% of spiked IgG-positive RBCs were detected. Diagnostic sensitivity and specificity of the assay for detection of IMHA were 88% and 93%, respectively. Cytologic findings for sorted bone marrow fractions rich in early-, mid-, and late-stage nRBCs from 3 healthy dogs indicated 89% to 98% nRBC purity. After IgG coating with anti-DEA1 blood-typing serum, IgG was detected on nRBCs from DEA1-positive, but not DEA1-negative, healthy dogs.

CONCLUSIONS AND CLINICAL RELEVANCE The developed RBC IgG assay had favorable analytic and diagnostic performance for detection of IMHA in dogs and was successfully adapted to detect IgG on canine nRBCs of various maturation stages. The findings supported the presence of DEA1 on canine nRBCs.

Precursor-targeted immune-mediated anemia has been described in dogs in various studies1–8 and is the clinical diagnosis for approximately 25% of dogs undergoing bone marrow biopsies at the Michigan State University Veterinary Medical Center. However, the pathogenesis of PIMA is unclear. The condition is characterized by nonregenerative anemia, ineffective erythropoiesis, and, in most affected dogs evaluated at the authors’ institution, rubriphagocytosis, which is the phagocytosis of intact erythroid precursors (ie, nRBCs). Rubriphagocytosis in dogs with PIMA appears to be quite stage selective, with the maturation stage of the phagocytized nRBCs (ie, early, mid, or late) aligning with a spectrum of cytologic and histologic bone marrow patterns that commonly include mild to severe myelofibrosis.8

An immune-mediated mechanism for the pathogenesis of PIMA has been proposed, but not established, on the basis of rubriphagocytosis,1,3,4 apparent responses to immunosuppressive treatment,1–6 apparent relapses after withdrawal of immunosuppressive treatment,1,2,5,6 and evidence of concurrent IMHA in some affected dogs.3–7 However, responses to immunosuppressive drugs are slow, relapses are common, and many affected dogs die or are euthanized because of clinical pessimism or financial constraints.

Flow cytometry has been used to diagnose and characterize IMHA in dogs9–12 and humans.13–15 This method appears more sensitive than standard agglutination tests for IgG detection.9–11,13–15 A potential PIMA counterpart that may be mediated by IgG directed against nRBCs has been identified in humans,16–18 and flow cytometry was used to identify IgG on bone marrow–derived nRBCs in 1 instance.18 These findings suggest that flow cytometry could also be used to assess for increases in the amounts of IgG bound to nRBCs in dogs with PIMA.

The purpose of the study reported here was to develop a method that would allow isolation and flow cytometric assessment of IgG on the surface of various maturation stages of nRBCs from canine bone marrow samples. The initial goal was to develop an assay for measurement of IgG bound to RBCs and then apply the assay to marrow-derived nRBCs. Our intention was that these assays would facilitate various blood and stage-selective marrow assessments for dogs with PIMA as well as other disorders and help determine whether IgG has a role in the pathogenesis of PIMA in dogs. A better understanding of the pathophysiologic mechanisms underlying PIMA may lead to improvements in management strategies and treatment responses for affected dogs.

Materials and Methods

Ethics statements

The study protocol was approved by the Institutional Animal Care and Use Committee of Michigan State University. The Institutional Animal Care and Use Committee at MPI Research also gave approval for the collection of blood and bone marrow from healthy dogs used for assay evaluation.

Dogs

For assessment of IgG on RBCs, blood samples were collected from 20 dogs enrolled in the Michigan State University Veterinary Medical Center Blood Donor Program via routine venipuncture into potassium-EDTA tubes.a These dogs had been deemed clinically and hematologically healthy through annual physical examination, CBC, serum biochemical analysis, and infectious disease (heartworm disease, babesiosis, ehrlichiosis, leishmaniasis, and hemotropic mycoplasmosis) screening.

Blood samples were similarly collected from 61 canine patients (28 unhealthy dogs with a non-IMHA condition and 33 dogs with IMHA) of the same medical center as part of their routine diagnostic evaluation. Dogs with IMHA were classified as such if they had a clinical diagnosis of IMHA associated with regenerative anemia (Hct in each was < 33%) and at least 2 of the following criteria: spherocytosis, erythrocyte agglutination, or a positive Coombs test result, each with no other apparent cause. These dogs with IMHA were identified prospectively and consecutively from May 2012 through November 2014 by laboratory and hospital personnel who were in communication with the investigative team. Dogs for which a sufficient amount of blood sample remained following CBC at the initial visit were used in the assessment of the diagnostic performance of the RBC IgG assay, provided diagnostic criteria for IMHA were met and samples could be tested within 24 hours after collection. Most days on which a dog with suspected IMHA was selected, a paired unhealthy dog with a non-IMHA condition was selected by reviewing the clinical history, physical examination findings, and laboratory data of other dogs that had a CBC performed that day. Any sick dog with no clinical suspicion or hematologic evidence of immune-mediated anemia (ie, spherocytosis, erythrocyte agglutination, a positive Coombs test result, unexplained regenerative anemia, or clinical or hematologic findings suggestive of PIMA) was considered for this group, and the first dog encountered on a given day was selected.

Dogs included in the IMHA group had a mean age of 6.8 years (range, 2 to 13 years) and consisted of 14 spayed females, 2 sexually intact females, 15 castrated males, and 2 sexually intact males of various breeds. Dogs included in the unhealthy non-IMHA group had a mean age of 8 years (range, 0.5 to 13 years) and consisted of 16 spayed females, 11 castrated males, and 1 sexually intact male of various breeds. Conditions of dogs in this group included various cancers (n = 9); inflammatory or infectious disease (6); disease of the liver, heart, kidney, or nervous system (6); postoperative recovery for parathyroidectomy, gastric volvulus surgery, or nephrectomy and adrenalectomy (3); endocrine disease (2); immune-mediated neutropenia (1); and fever of unknown origin (1). Six dogs were tested but excluded from the study because of sample age (n = 3), unclear diagnosis (2), or unclear interpretation of IgG results (1; Supplementary Appendix S1, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.11.1123).

Dogs in the unhealthy non-IMHA group were additionally classified by whether they were anemic (n = 18) or nonanemic (10). Anemic dogs in this group included postoperative, hemorrhagic, inflammatory, and chronic renal disease patients with a mean Hct of 32% (range, 19% to 40%). Nonanemic dogs were included to increase the spectrum of assessed disorders and because increased RBC-bound IgG has been detected via flow cytometry in blood samples from nonanemic dogs.12 No bone marrow samples were collected from any of these dogs because bone marrow collection was not clinically indicated; they were used only to develop and evaluate the RBC IgG assay.

For evaluation of a flow cytometric assay for detection of IgG bound to nRBCs, bone marrow samples were collected from 14 healthy research dogs, and blood samples from 9 of these dogs were used for DEA blood typing. Bone marrow samples from 10 of these dogs were used to determine baseline amounts of IgG bound to nRBCs. Samples from 2 of these 10 dogs and the other 4 dogs were used for incubation of nRBCs with polyclonal anti-DEA1 blood-typing serumb to generate IgG-positive or -negative control nRBCs. Bone marrow samples from 3 of these healthy dogs were also used to confirm which cell-sorted fractions contained nRBCs. All 14 dogs received either 1 to 2 physical examinations/mo or 2 physical examinations/y. Hematologic health of these dogs was confirmed through bone marrow aspirate evaluation, semiannual CBCs (some dogs), and CBCs performed at the time of marrow collection (other dogs).

Bone marrow collection

Approximately 2 to 5 mL of bone marrow was aspirated from the proximal aspect of the humerus or wing of the ilium of anesthetized dogs by use of a 15-or 18-gauge Illinois bone marrow needle.c Bone marrow was collected into a syringe containing 0.5 mL of 3% potassium-EDTA in saline (0.9% NaCl) solution, then dispensed into a Petri dish for particle harvesting and preparation of cytologic smears; the remaining sample was used for flow cytometric analysis.

RBC sample preparation

Collected blood samples were used on the day of collection or stored at 4°C and used the next day. All samples were processed at room temperature (20° to 22°C). Each sample was centrifuged at 1,200 × g for 5 minutes, and plasma and buffy coat were removed. Then, 10 μL of packed RBCs were withdrawn, washed twice by resuspension in 1.5 mL of a PBSS–0.3% BSA mixture (137 mM NaCl, 2.7mM KCl, 9.6mM NaH2PO4, 1.5 mM KH2PO4, 0.3% BSA,d and 0.025% sodium azide; overall pH, 7.3), and then centrifuged at 1,200 × g for 1 minute. The washed pellet was resuspended in 800 μL of the PBSS–0.3% BSA mixture.

Flow cytometric assay for RBC IgG

The following procedure was applied after initial optimization steps that included assessment of signal-to-noise ratios and the number of wash steps. For each dog, 50 μL of washed RBC suspension was incubated with 15 μL of fluorescein isothiocyanate–conjugated caprine anti–dog IgGγ IgGe (IgG test antibody) in the dark for 30 minutes. The RBCs were washed once as described previously, resuspended in 1.5 mL of PBSS–0.3% BSA mixture, and analyzed with a flow cytometerf by acquisition of 10,000 events and use of associated cytometric software.g The electronic settings for RBC analysis were as follows: FSC voltage = E00 and AmpGain = 2.25, SSC voltage = 304 and AmpGain = 1.32, FL1 voltage = 703, and FSC threshold = 100. The RBCs were recognized and gated on the basis of FSC and SSC, as described elsewhere.19

Positive control samples were developed, and negative and positive control samples were assessed with each test sample. Negative control samples were created by incubation of washed RBCs from healthy dogs with IgG test antibody (negative control sample for IgG on RBCs) and by incubation of test RBCs with fluorescein isothiocyanate–conjugated caprine anti–chicken IgG heavy- and light-chain IgGe (nonsense antibody; control sample for nonspecific binding). Positive control samples consisted of DEA1-positive RBCs treated with anti-DEA1 blood-typing serum to yield IgG-coated RBCs. Specifically, positive control cells were generated by 30 minutes of room-temperature incubation of an 800-μL washed RBC suspension containing DEA1-positive RBCs with 800 μL of anti-DEA1 blood-typing serum diluted with PBSS–0.3% BSA mixture. Cells were washed twice by resuspension in 1.5 mL of PBSS-BSA mixture and then centrifuged at 1,200 × g for 1 minute. The washed pellet was resuspended in 800 μL of PBSS–0.3% BSA mixture. This positive control sample was assessed for stability, and new batches were made approximately once per month, as needed. The assay signal for IgG bound to positive control RBCs varied from moderate to strong, depending on the cells (more or less DEA1 expression) and dilution of typing serum (ie, more or less anti-DEA1 antibody) used.

After careful assessment of various methods and cutoffs for determining and reporting sample positivity for cell-bound IgG, results were reported as the percentage of positive events (RBCs). The cutoff for positively fluorescing cells in each test sample was set at the fluorescence level attained by only 2% of that sample's cells when aliquots were incubated with the nonsense antibody (nonspecific binding). When RBC agglutinates were identified on flow plots on the basis of RBC FSC and SSC (Supplementary Appendix S2, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.11.1123), they were gated out and excluded from the analysis. This was done because agglutinated RBCs may have an increase in nonspecific fluorescence with nonspecific control antibodies, so their inclusion may increase the cutoff for designating positive fluorescence and therefore falsely decrease the measured IgG positivity (ie, percentage of positive events) of tested samples.20

Analytic precision of negative and positive samples was assessed. The final CVs were calculated on the basis of the percentage of positive events (ie, events above the individual cutoff based on nonspecific binding for each sample). For within-run precision, 20 RBC suspensions were prepared from a single blood sample collected from a healthy dog, and 40 μL of each suspension were spiked with 10 μL of positive control cells to yield 20 samples concocted to contain 20% IgG-positive cells (analogous to a relatively weak-positive result from patients with IMHA). Washed RBCs from the same healthy dog were tested in parallel as negative control cells. For assessment of between-run precision and stability over time, IgG-negative and positive control RBCs were stored in Alsever solutionh with 0.5% BSA at 4°C, and RBCs were tested for 10 consecutive weekdays, followed by weekly testing for 3 weeks. Prior to each assessment, 50 μL of RBC suspension were washed with 1.5 mL of PBSS–0.3% BSA mixture, resuspended in 50 μL of the same buffer, and tested for IgG positivity with test and nonsense antibodies as described above.

Analytic accuracy was assessed in triplicate by a mixing experiment in which IgG-negative control RBCs were spiked with IgG-positive control RBCs to target 0%, 20%, 40%, 60%, 80%, and 100% positive events. The percentage of targeted positivity that was detected was determined. To assess changes in the IgG signal (MdFI of test sample minus nonsense antibody) with decreasing amounts of IgG per RBC, DEA1-positive RBCs incubated with doubling dilutions of anti-DEA1 blood-typing serum from 1:16 to 1:512 in PBSS–0.3% BSA mixture were assessed.

Clinical assessment of the RBC IgG assay

Samples from healthy dogs, dogs with IMHA, and unhealthy dogs without IMHA were tested (Supplementary Appendix S1) with the developed RBC IgG assay to determine the diagnostic sensitivity, specificity, and accuracy of this assay for detection of IMHA. The mean percentage of positive events plus 3 SDs for samples obtained from the 20 healthy dogs was used as a cutoff to classify IMHA and unhealthy non-IMHA dogs into IgG-negative (below cutoff) and IgG-positive (above cutoff) groups. To assess the effects of testing day-old samples and to generate comparison values if they differed, the values for healthy dogs were determined for fresh and day-old samples. Pertinent clinical and laboratory information were gathered for each unhealthy dog.

Fractionation of bone marrow cells

Bone marrow samples were transported to the laboratory in a cooled container and processed at room temperature within 30 minutes after collection to minimize potential changes in cell membrane or density that might promote nonspecific binding or affect gradient density separation, respectively. Samples were combined with 40 mL of PBSS–1% BSA mixture (PBSS containing 1% BSA and 0.025% sodium azide; overall pH, 7.3) and centrifuged at 500 × g for 10 minutes before supernatant was removed and cell pellets were resuspended in 5 mL of PBSS-BSA mixture. Cell suspensions were applied to a 3-layer density gradienti (3 mL each of 52%, 64%, and 68% Percoll in PBSS-BSA mixture) on the basis of findings in a previous study.21 After centrifugation at 500 × g for 30 minutes, the 3 cell fractions (referred to as top, middle, and bottom fractions) were harvested from top to bottom by use of plastic Pasteur pipettes. Each fraction was washed twice with PBSS–1% BSA mixture (first with 10 mL and then with 3 mL) and centrifuged at 500 × g. Washed pellets were resuspended in PBSS–1% BSA mixture (0.5 to 3 mL, depending on pellet size), and nucleated cell concentrations were determined with a hemocytometer. Desired cell separation was confirmed by microscopic examination of stained (Wright stain), air-dried, flow-back line smear preparations of cells from each layer.

Preparation of bone marrow fractions for flow cytometric analysis

A 2-color assay was developed by use of the nucleic acid stain LDS 751j in combination with labeling for CD18 on nonerythroid cells. The following processing procedure was adopted after trial assessments and optimization steps. For each sample, 106 bone marrow cells in 100 μL of PBSS–1% BSA mixture were incubated with 2 μL of murine anti–dog CD18 antibodyk during mechanical rotation for 30 minutes, washed twice with 1.5 mL of PBSS–0.3% BSA mixture, and resuspended in 100 μL of the same buffer. Cells were then incubated with 5 μL of phycoerythrin-conjugated goat anti–mouse IgG IgGl during mechanical rotation for 30 minutes in the dark, washed twice with 1.5 mL of PBSS–0.3% BSA mixture, resuspended in 400 μL of the same buffer, and transferred to a flow cytometer analysis tube. These cells were then incubated for at least 20 minutes (progressively longer for samples analyzed later in a batch) in the dark with 160 μL of LDS 751 working solution prepared by diluting 2 μL of stock LDS 751 solution (ie, as provided by manufacturer) into 1.5 mL of PBSS–0.3% BSA mixture.22

Flow cytometric identification of bone marrow nRBCs

The nRBCs in the top, middle, and bottom fractions were expected to stain strongly with LDS 751 but have no labeling for CD18. This was assessed by processing bone marrow samples as described previously and then sorting them with a cell sorterm and cell sorter software.n Sorted fractions of interest were collected into buffered saline solution, to which bovine fetal serumo was immediately added at a ratio of 50 μL of serum for every 1 mL of saline solution. The cell content of each erythroid fraction of 3 dogs was assessed by differential cell counts performed on modified Wright-stained cytocentrifuge preparations.

IgG detection on nRBCs

Top, middle, and bottom fractions of bone marrow from 10 healthy dogs were assessed for IgG-positive nRBCs by essentially combining the assay for RBC IgG detection with the described 2-color bone marrow assay to yield a 3-color assay for detection of IgG bound to nRBCs. For each fraction, 106 nucleated cells were processed as described for the 2-color assay with the addition of test anti-IgG and nonsense antibodies at the time of incubation with phycoerythrin-conjugated goat anti–mouse IgG. Cells were analyzed in a flow cytometerf by acquiring 10,000 events processed by use of cytometric software.g The electronic settings for analysis of erythroid populations were as follows: FSC voltage = E00 and AmpGain = 2.0, SSC voltage = 400 and AmpGain = 1.0, FL1 voltage = 673, FL2 voltage = 610, FL3 voltage = 540, FSC threshold = 100, FL1 – %FL2 = 15, FL2 – %FL1 = 21, FL2 – %FL3 = 6.7, and FL3 – % FL2 = 6.7%. As for the RBC assay, results of the nRBC IgG assay were reported as the percentage of positive events in the gates of interest.

To assess the ability of the assay to detect IgG bound to nRBCs, we explored the possibility of generating IgG-positive, DEA1-positive nRBCs by incubation of nRBCs with anti-DEA1 blood-typing serum, in much the same way as the positive control for the RBC assay was generated. For 3 DEA1-positive dogs, 100 μL of cell suspension containing 106 cells from each bone marrow fraction was incubated with 100 μL of anti-DEA1 blood-typing serum at low (1:16) or high (1:128) dilutions (vol/vol) in PBSS–0.3% BSA mixture for 30 minutes. Low and high dilutions were used to assess detection of stronger and weaker signals. Cells were washed twice with 1.5 mL of PBSS–0.3% BSA mixture and resuspended in 100 μL of the same buffer prior to 3-color testing as previously described.

Bone marrow fractions of 3 DEA1-negative dogs were processed similarly, and samples from 10 healthy dogs were also assessed for IgG bound to nRBCs (without the DEA antiserum steps). The same RBC control samples used for the RBC IgG assay were used for each bone marrow sample. The DEA1 status was determined via flow cytometric blood typing.19

Statistical analysis

Significant (P < 0.05) differences between groups in RBC and nRBC IgG signal (determined by percentage positive events) were assessed with the Kruskal-Wallis and Dunn multiple comparison post hoc tests by use of statistical software.p Confidence limits for diagnostic sensitivity and specificity of the RBC IgG assay for the detection of dogs with a diagnosis of IMHA were determined by computation of the Wilson score interval.q

Results

RBC IgG assay characterization

For the RBC IgG assay, the CV for within-run precision was 6.8% for a modestly positive sample with a mean of 20.9% positive events and was 13.4% for negative control cells with a mean of only 6.8% positive events. Between-run precision analysis of negative (mean of 4.4% positive events) and strongly positive (mean of 99.9% positive events) control cells yielded 10-day CVs of 13.9% and 0.1%, respectively. Results for a 10-day assessment period are reported because mild shifts in positivity were apparent beyond 10 days, but control cells were clearly negative and positive for a period of at least 1 month, with the IgG positivity for the negative control cells varying from 4.9% (day 1) to 2.8% (day 34), and that for the positive control cells varying from 100% (day 1) to 99.8% (day 34). Progressive hemolysis impeded testing for a longer duration.

The IgG-uncoated RBCs spiked with IgG-coated cells targeting 0%, 20%, 40%, 60%, 80%, and 100% positive events yielded 0%, 18%, 37%, 56%, 78%, and 100% positive cells, with the percentage of detected to targeted cells varying from 90% to 100%. For samples with 100% positive cells but decreasing amounts of IgG per RBC, MdFI decreased curvilinearly with the IgG signal, ranging from 2,372 to 190 (Figure 1).

Figure 1—
Figure 1—

Dose-response curve for a flow cytometric assay developed to detect the extent of IgG binding to RBCs. For assay evaluation, DEA1-positive RBCs from hematologically healthy dogs were incubated with doubling dilutions of anti-DEA1 blood-typing serum and then assayed, yielding a curvilinear reduction in signal (MdFI) with a reduction in the amount of IgG bound to cells.

Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1123

Clinical assessment of the RBC IgG assay

Mean IgG positivity for fresh and day-old samples from 20 healthy dogs, reported as the percentage of positive events, was 4.8% and 5.2%, respectively, and the cutoffs for IgG positivity based on the mean plus 3 SDs were 9.4% and 8.9% for fresh and day-old samples, respectively. Of the 64 samples tested within 24 hours of collection, 3 were excluded from calculations of diagnostic performance. One of these 3 was excluded because of an unclear IgG result caused by high nonspecific binding of the control antibody, and the other 2 (1 positive and 1 negative for RBC IgG) were excluded because of unclear diagnoses (Supplementary Appendix S1).

Results of the RBC IgG assay were positive for 29 of the 33 (88%) dogs with IMHA and for 2 of the 28 (7%) unhealthy dogs without IMHA, and results were the same regardless of whether cutoffs designating positive and negative results for fresh or day-old samples were used. Median (range) percentages of positive events for dogs in the IMHA group with negative and positive results were 3.2% (2.6% to 6.7%) and 82.5% (10.4% to 100%), respectively, and for dogs in the non-IMHA group were 4.9% (1.2% to 8.2%) and 17.4% (10.5% to 24.2%), respectively. Differences in IgG positivity were significant between the IMHA group and each other group (healthy group and unhealthy non-IMHA group), but not between the unhealthy non-IMHA and healthy groups. On the basis of these results, the diagnostic sensitivity, specificity, and accuracy of the RBC IgG assay for detecting IMHA were 88% (95% confidence interval, 73% to 95%), 93% (95% confidence interval, 77% to 98%), and 90%, respectively.

Bone marrow fractions and detection of nRBCs

Density-gradient separation yielded a top fraction containing an increased proportion of early-stage nRBCs, with fewer midstage nRBCs, many early to midstage myeloid cells, and few megakaryocytes, macrophages, and lymphocytes. The middle fraction contained an increased proportion of midstage (with few late-stage) nRBCs and also contained a mixture of mid- to late-stage granulocytes, few lymphocytes, and rare plasma cells. The bottom fraction contained mostly late-stage (with few midstage) nRBCs and segmented neutrophils (Supplementary Figure S1, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.11.1123). All 3 fractions also contained low to moderate numbers of mature RBCs and reticulocytes, with higher numbers in the bottom fraction.

Gating on the CD18-negative (FL2 channel) and LDS 751–positive (FL3 channel) events allowed the assessment of erythroid populations (Figure 2), which were shown by cell sorting and cytologic examination to have little contamination with other cell types (Figure 3). Cytologic assessment of cell-sorted nRBC regions from 3 healthy dogs revealed ≥ 89% nRBCs in all samples, with 89% to 99% in the early-stage nRBC region and 98% to 100% nRBCs in the mid- and late-stage nRBC regions. Additionally, populations that were CD18 negative and LDS 751 moderate and populations that were CD18 negative and LDS 751 negative consisted of reticulocytes and mature RBCs, respectively, making it possible to assess nRBCs, reticulocytes, and RBCs simultaneously if enough cells were acquired.

Figure 2—
Figure 2—

Representative flow cytometry dot plots of canine bone marrow cells in top (A), middle (B), and bottom (C) density-gradient fractions after labeling cells for CD18 (x-axis) and staining them for nucleic acids with LDS 751 (y-axis). Nucleated RBCs, reticulocytes, and mature RBCs in gated regions R1, R2, and R3, respectively, were CD18 negative and LDS 751 positive, CD18 negative and LDS 751 moderate, and CD18 negative and LDS 751 negative, respectively.

Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1123

Figure 3—
Figure 3—

Representative gated regions and cytologic findings for flow cytometer-sorted canine bone marrow aspirate cells after density gradient separation into top, middle, and bottom cell fractions. A—Dot plot of bone marrow cells in the middle density gradient fraction after cell labeling for CD18 (x-axis) and staining of nucleic acids with LDS 751 (y-axis) showing the location of nRBCs, reticulocytes, and mature RBCs in gated regions R1, R2, and R3, respectively. B through D—Photomicrographs of cells from the top, middle, and bottom R1 fractions, showing a predominance of early- (B), mid- (C), and late-(D)stage nRBCs, respectively, with little contamination by other cell types. E and F—Photomicrographs of the R2- and R3-gated cells depicted in panel A and consisting mostly of reticulocytes (E) and mature RBCs (F), respectively, as was true for R2- and R3-gated cells in the top and bottom fractions as well.

Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1123

Most of the contaminating cells in the nRBC regions appeared lymphoid, and because of the greater contamination in the nRBC gate of top fractions, this region was further assessed by cell sorting and cytologic evaluation after splitting it into top and bottom sections. The top and bottom sections had similar numbers of lymphocytes, and the bottom section also included many midstage nRBCs in mitosis. Consequently, to maximize assessment of early-stage nRBCs, only the top section of the nRBC region from the top fraction was used to assess for IgG on nRBCs.

IgG detection on nRBCs

Median percentages of IgG-positive events in the nRBC regions of fractionated bone marrow samples from 10 healthy dogs were 5.7% (range, 1.6% to 12.3%) for early nRBCs, 8.6% (range, 2.5% to 15.2%) for midstage nRBCs, and 6.8% (range, 4.2% to 17.5%) for the late-stage nRBCs. Reticulocytes were sufficiently abundant in the bottom fractions of each sample to allow assessment, revealing a median of 5% IgG-positive events (range, 2.1% to 15.9%). Mature RBCs were generally not acquired in sufficient concentration to allow assessment for IgG because that could be performed by use of blood samples rather than bone marrow samples.

Median percentage of IgG-positive events for nRBCs from the top, middle, and bottom fractions of the 3 DEA1-negative dogs following incubation of cells with anti-DEA1 blood-typing serum was 4.2% (range, 3.6% to 7.3%), 6.0% (range, 5.3% to 10.2%), and 16.1% (range, 13.1% to 16.3%), respectively. Reticulocytes from the bottom fractions had a median value of 12.6% (range, 11.8% to 25.6%). In contrast, after sample incubation with anti-DEA1 blood-typing serum, the top, middle, and bottom nRBC populations from the 3 DEA1-positive dogs yielded much greater median values of 71.1% (range, 49.1% to 90.1%), 98.1% (range, 95.7% to 99.3%), and 99.4% (range, 99.3% to 99.5%), respectively, as did the reticulocyte population from bottom fractions, which yielded 100% positive events. Compared with values for healthy dogs, the marked increases in percentage of IgG-positive cells from each nRBC fraction for DEA1-positive dogs were significant, and they were similar in magnitude to the marked increases over DEA1-negative cells (Supplementary Figure S2, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.11.1123). This concurrently demonstrated successful IgG detection and DEA1 expression on nRBCs of each bone marrow fraction.

Discussion

To investigate the role of IgG in canine immune-mediated anemias, including IMHA and PIMA, we first developed and then characterized a flow cytometric assay for detection of IgG bound to RBCs by use of a heavy chain–specific antiglobulin reagent and positive and negative control RBCs. The assay had good analytic precision and accuracy, and diagnostic accuracy was good when applied to a group of unhealthy dogs with or without IMHA. This RBC IgG assay was then applied to early-, mid-, and late-stage nRBCs after developing a process to harvest the cells from bone marrow and identify them with a 2-color flow cytometric assay. This nRBC assay reliably detected IgG on early-, mid-, and late-stage nRBCs. Additionally, we identified, for the first time, the expression of DEA1 on early- to late-stage canine nRBCs on the basis of immunologic detection.

During initial stages of assay development, we attempted to detect IgM and C3 on erythroid cells, in addition to IgG, because IgM and C3 may also play roles in mediating cell destruction. However, the IgM assay was hindered by difficulty in developing a reliable positive control substance solely for verifying detection of IgM, and despite development of reliable positive control RBCs for verifying detection of canine complement, extensive testing of dogs with IMHA yielded only 1 questionable positive result for canine C3. Therefore, assay development was limited to detection of IgG, further ensured by use of a heavy-chain specific antiglobulin reagent, which was selected to minimize cross-reactivity with other classes of immunoglobulins.

Although different approaches exist to define positive results of flow cytometric assays for cell-associated antibody,9–12,19 we used the percentage of positive events after accounting for nonspecific binding of a nonsense antibody because this approach appeared to be most sensitive and reliable. The approach was more sensitive than use of MdFI or mean fluorescence intensity because many dogs with IMHA had only a subpopulation of detectably positive cells, and the percentage of positive events could be clearly increased without significantly shifting the MdFI and mean fluorescence intensity (Supplementary Figure S3, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.11.1123).

page). Regarding nonspecific binding, amounts varied among dogs, and increasing amounts of autologous canine IgG on the RBCs were associated with increasing nonspecific binding of the nonsense antibody and thus presumably the test antibody. Therefore, use of the nonsense antibody binding to set the cutoff for defining increased cell fluorescence for each sample was important.

In the present study, a particularly noteworthy example of the importance of using the nonsense antibody was observed in a dog that ultimately received a diagnosis of zinc toxicosis but was initially evaluated as a candidate for the IMHA group. The RBCs from this dog had nonspecific binding with the nonsense antibody that was as great as a strong positive reaction, so it was impossible to assess IgG positivity in this sample, and the dog was therefore excluded. Similar to other dogs with zinc toxicosis,23 this dog had a severe regenerative anemia (Hct, 13%) associated with marked hemolysis, Heinz bodies, moderate spherocytosis (not pyknocytosis), many ghost cells, and schizocytosis. Although this dog may truly have had RBCs with increased amounts of surface-bound IgG, the strong nonspecific signal made analysis impossible, perhaps because of high autofluorescence or membrane changes that caused a high amount of nonspecific binding (ie, nonimmune protein adhesion) of nonsense and test antibodies.

Clinical evaluation of the RBC IgG assay for dogs with and without IMHA yielded favorable results, with few (4/33) negative results for dogs with IMHA and few (2/28) positive results for unhealthy dogs without a clinical diagnosis of IMHA. The negative results for the 4 dogs with IMHA may have been related to mediation by IgM (ie, C3) or IgA without involvement of IgG9,24–26 or, in 1 dog, to 4 days of immunosuppressive treatment prior to testing, given that the detection of RBC-bound IgG by flow cytometric assay can decrease with treatment.9 Of the other 11 dogs already receiving immunosuppressive treatment at the time of sample collection, 9 had clearly positive results (17% to 100% positive events); the 2 with very weak results (11% positive events) included the only other dog treated for > 2 days (8 days). Insufficient assay sensitivity may have also yielded negative results, but flow cytometric assays reportedly have greater diagnostic sensitivity for detection of IMHA than the Coombs test for both humans and dogs.9–11,13–15 Regarding the positive results for 2 unhealthy dogs in the non-IMHA group, one was a very weak positive result and pertained to a nonanemic dog with a pheochromocytoma, and the other was only mildly high and pertained to an anemic dog (Hct, 33%) with hypoadrenocorticism and hypothyroidism; both conditions may have had an immune-mediated pathogenesis in the second dog. The anemia in this dog was not evaluated further, and an immunologic component to the anemia could not be excluded. Testing a greater number of dogs with and without IMHA would have provided a more accurate assessment of diagnostic performance, but enough dogs were tested to suggest the assay had moderate to high diagnostic accuracy.

In a previous study,27 storage of blood samples for up to 7 days had no effect on Coombs test results, compared with results for fresh samples, but effects on results of a flow cytometric assay, which may be more sensitive, were not assessed. Findings reported here for 20 healthy dogs indicated that testing of blood-derived RBCs for IgG on the day of sample collection versus the day after sample collection following storage at 4°C had no effect on results or clinical interpretation, and we have seen no clinically important differences with 1 to 2 days of storage in sporadic paired assessments performed outside of this study. However, we have not systematically assessed effects of storage over longer periods or on samples from unhealthy dogs, including those with increased RBC-bound IgG.

Favorable analytic and diagnostic performance of the RBC IgG assay justified attempts to apply it to bone marrow–derived nRBCs. Initial attempts involved lysis of RBCs to remove them as interfering events from bone marrow samples. However, pre- and postlysis cytologic preparations indicated that Tris ammonium chloride, ammonium chloride, and a commercial erythrocyte lysing bufferr all resulted in lysis of nRBCs as well as mature RBCs (data not shown). By combining a density gradient separation method with a 2-color flow cytometric assay, concentrations of mature RBCs and reticulocytes were markedly reduced and easily discriminated from nRBCs, and omission of a lysis step decreased potential cell damage and loss. If anti–dog nRBC antibodies (eg, against dog transferrin receptor or dog glycophorin) become available, they should be explored as better labor- and time-saving options that would mirror human methods for nRBC analysis.28–31

As finally developed, the CD18-negative and LDS 751–positive populations identified by the assay contained very few nonerythroid cells, and the combined use of density-gradient fractionation and 2-color separation allowed independent analysis of largely early-, mid-, and late-stage nRBCs. All 3 of these nRBC fractions from DEA1-positive dogs had strong signals for IgG after incubation with anti-DEA1 blood-typing serum. This was in contrast to the nRBC fractions from DEA1-negative dogs subjected to the same treatment, all of which had nRBC IgG results similar to those for nRBCs from the 10 healthy dogs. Detection of this IgG on the DEA1-positive nRBCs demonstrated the ability of the assay to detect IgG bound to nRBCs and supported application of this assay for dogs with PIMA. As such, we believe our study is the first in which a method was developed to assess the amount of IgG binding in canine bone marrow nRBC fractions by flow cytometry. Our study also yielded new evidence that DEA1 is expressed on early through late stages of nRBCs in DEA1-positive dogs. Additional evidence of DEA1 expression on canine nRBCs could be sought through nonimmunologic approaches such as mass spectrometry of RBC membranes or detection of DEA1 mRNA.

The presence of DEA1 on precursors could be of interest given that certain blood group antigens are expressed on human nRBCs31–33 and human RBC–incompatible allogeneic bone marrow transplantations can have deleterious consequences, such as immediate and delayed hemolytic transfusion reactions, delayed RBC recovery, and pure red cell aplasia.32,34 Although current donor bone marrow conditioning and immunosuppressive regimens appear to overcome these deleterious effects, crossmatching is still warranted, and RBC-compatible bone marrow transplants are still preferred.34 Expression of DEA1 on canine nRBCs may be relevant to allogeneic bone marrow transplantations in dogs.

In the study reported here, a new method was developed for isolation and identification of early-, mid-, and late-stage nRBCs from the bone marrow of dogs, revealing that IgG could be detected on all erythroid populations by applying the fundamentals of a flow cytometric assay for measurement of IgG bound to RBCs. In the process, we obtained evidence that DEA1 is expressed on rubriblasts through mature RBCs of DEA1-positive dogs. We believe these assays will allow detection of IgG on all erythroid populations from the bone marrow and blood of dogs with PIMA and that the assays could be used to help clarify the underlying mechanism and appropriate management for this hematologic disease.

Acknowledgments

This manuscript represents a portion of a dissertation submitted by Dr. Lucidi to the Department of Pathobiology and Diagnostic Investigation, Michigan State University, as partial fulfillment of the requirements for a Doctor of Philosophy degree.

Supported in part by the Immunology and Serology Laboratory, Michigan State University.

Presented in part in abstract form at the 65th Annual Meeting of the American College of Veterinary Pathologists and the 49th Annual Meeting of the American Society for Veterinary Clinical Pathology, Atlanta, November 2014.

The authors thank Dr. Adam Aulbach, Rose Wahl, Kristin Koehl, and Dr. Andras Komaromy for technical assistance with dogs and samples, and Dr. William Jackson for his help with statistical analyses.

ABBREVIATIONS

AmpGain

Amplifier gain

BSA

Bovine serum albumin

C3

Complement component 3

CD

Cluster of differentiation

CV

Coefficient of variation

DEA1

Dog erythrocyte antigen 1

FSC

Forward scatter

IMHA

Immune-mediated hemolytic anemia

MdFI

Median fluorescence intensity

nRBC

Nucleated RBC

PIMA

Precursor-targeted immune-mediated anemia

SSC

Side scatter

Footnotes

a.

BD K2-EDTA tubes, Becton-Dickinson, San Jose, Calif.

b.

Animal Blood Resources International, Stockbridge, Mich.

c.

Becton-Dickinson, San Jose, Calif.

d.

BSA (heat shock fraction, protease free, essentially globulin free, pH 7, ≥ 98%), Sigma-Aldrich, St Louis, Mo.

e.

Kirkegaard & Perry Laboratories Inc, Gaithersburg, Md.

f.

FACSCalibur, Becton-Dickinson, San Jose, Calif.

g.

CellQuest Pro software, Becton-Dickinson, San Jose, Calif.

h.

Sigma-Aldrich Corp, St Louis, Mo.

i.

Percoll (pH, 8.5 to 9.5; 25°C; cell culture tested), Sigma-Aldrich Corp, St Louis, Mo.

j.

Life Technologies, Grand Island, NY.

k.

Mouse anti–dog CD18 monoclonal antibody, clone CA1.4E9, isotype IgG1, AbD Serotec, Raleigh, NC.

l.

Goat anti–mouse IgG:RPE (rat adsorbed) polyclonal IgG antibody, AbD Serotec Ltd, Raleigh, NC.

m.

BD Influx, Becton-Dickinson, San Jose, Calif.

n.

BD FACS Sortware sorter software, Becton-Dickinson, San Jose, Calif.

o.

BSA solution (22% in saline solution), Sigma-Aldrich Corp, St Louis, Mo.

p.

GraphPad Prism, GraphPad Software Inc, San Diego, Calif.

q.

Analyse-it for Microsoft Excel, version 4.65.2, build 6012.38758, Analyse-it Software Ltd, Leeds, England.

r.

Erythrolyse RBC lysing buffer (10X), diluted in ddH2O, AbD Serotec Ltd, Raleigh, NC.

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Contributor Notes

Dr. Lucidi's present address is Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706.

Address correspondence to Dr. Lucidi (lucidi@wisc.edu).