• View in gallery

    Outer sheath (left) and inner needle (right) of a 15-gauge harvesting needle used for insertion into the shaft of the humerus and femur in dogs that underwent collection of BMCs via a perfusion method. This needle is currently not commercially available.

  • 1.

    Thomas ED. Bone marrow transplantation: a review. Semin Hematol 1999; 36: 95103.

  • 2.

    Goldman JM, Horowitz MM. The international bone marrow transplant registry. Int J Hematol 2002; 76 (suppl 1:) 393397.

  • 3.

    Appelbaum FR, Deeg HJ, Storb R, et al. Marrow transplant studies in dogs with malignant lymphoma. Transplantation 1985; 39: 499504.

  • 4.

    Frimberger AE, Moore AS, Rassnick KM, et al. A combination chemotherapy protocol with dose intensification and autologous bone marrow transplant (VELCAP-HDC) for canine lymphoma. J Vet Intern Med 2006; 20: 355364.

    • Crossref
    • Search Google Scholar
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  • 5.

    Weiden PL, Storb R, Lerner KG, et al. Treatment of canine malignancies by 1200 R total body irradiation and autologous marrow grafts. Exp Hematol 1975; 3: 124134.

    • Search Google Scholar
    • Export Citation
  • 6.

    Weiden PL, Storb R, Sale GE, et al. Allogeneic hematopoietic grafts after total-body irradiation in dogs with spontaneous tumors. J Natl Cancer Inst 1978; 61: 353357.

    • Search Google Scholar
    • Export Citation
  • 7.

    Weiden PL, Storb R, Deeg HJ, et al. Prolonged disease-free survival in dogs with lymphoma after total-body irradiation and autologous marrow transplantation consolidation of combination-chemotherapy-induced remissions. Blood 1979; 54: 10391049.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Kushida T, Inaba M, Ikebukuro K, et al. A new method for bone marrow cell harvesting. Stem Cells 2000; 18: 453456.

  • 9.

    Blazar BR, Taylor PA, Snover DC, et al. Nonmitogenic anti-CD3F(ab')2 fragments inhibit lethal murine graft-versus-host disease induced across the major histocompatibility barrier. J Immunol 1993; 150: 265277.

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    • Export Citation
  • 10.

    Kataoka Y, Iwasaki T, Kuroiwa T, et al. The role of donor T cells for target organ injuries in acute and chronic graft-versus-host disease. Immunology 2001; 103: 310318.

    • Crossref
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    • Export Citation
  • 11.

    Ichiki Y, Bowlus CL, Shimoda S, et al. T cell immunity and graft-versus-host disease (GVHD). Autoimmun Rev 2006; 5: 19.

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    Lupu M, Sullivan EW, Westfall TE, et al. Use of multigeneration-family molecular dog leukocyte antigen typing to select a hematopoietic cell transplant donor for a dog with T-cell lymphoma. J Am Vet Med Assoc 2006; 228: 728732.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Appelbaum FR, Deeg HJ, Storb R, et al. Cure of malignant lymphoma in dogs with peripheral blood stem cell transplantation. Transplantation 1986; 42: 1922.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Ide K, Matsuura S, Fujino Y, et al. Investigation of various methods for the cryopreservation of canine bone marrow-derived CD34(+) cells. J Vet Med Sci 2008; 70: 12111217.

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    • Export Citation
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    Bodenberger U, Kolb HJ, Rieder I, et al. Fractionated total body irradiation and autologous bone marrow transplantation in dogs: hemopoietic recovery after various marrow cell doses. Exp Hematol 1980; 8: 384394.

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    Epstein RB, Sarpel SC. Autologous bone marrow infusion following high dose chemotherapy of the canine transmissible venereal tumor (TVT). Exp Hematol 1980; 8: 683689.

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    Schuening FG, Kawahara K, Miller AD, et al. Retrovirus-mediated gene transduction into long-term repopulating marrow cells of dogs. Blood 1991; 78: 25682576.

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    Thomas ED, Storb R, Epstein RB, et al. Symposium on bone marrow transplantation: experimental aspects in canines. Transplant Proc 1969; 1: 3133.

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    Vriesendorp HM, Klapwyk WM, Heidt PJ, et al. Factors controlling the engraftment of transplanted dog bone marrow cells. Tissue Antigens 1982; 20: 6380.

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    Weiden PL, Storb R, Shulman H, et al. Dimethyl myleran and autologous marrow grafting for the treatment of spontaneous canine lymphoma. Eur J Cancer 1977; 13: 14111415.

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    Sorror ML, Leisenring W, Mielcarek M, et al. Intensified postgrafting immunosuppression failed to assure long-term engraftment of dog leukocyte antigen-identical canine marrow grafts after 1 gray total body irradiation. Transplantation 2008; 85: 10231029.

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  • 22.

    Storb R, Rudolph RH, Kolb HJ, et al. Marrow grafts between DL-A-matched canine littermates. Transplantation 1973; 15: 92100.

  • 23.

    Kushida T, Inaba M, Ikebukuro K, et al. Comparison of bone marrow cells harvested from various bones of cynomolgus monkeys at various ages by perfusion or aspiration methods: a pre-clinical study for human BMT. Stem Cells 2002; 20: 155162.

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    Storb R, Prentice RL, Buckner CD, et al. Graft-versus-host disease and survival in patients with aplastic anemia treated by marrow grafts from HLA-identical siblings. Beneficial effect of a protective environment. N Engl J Med 1983; 308: 302307.

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    Thomas ED, Storb R, Clift RA, et al. Bone-marrow transplantation (first of two parts). N Engl J Med 1975; 292: 832843.

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    Thomas ED, Storb R, Clift RA, et al. Bone-marrow transplantation (second of two parts). N Engl J Med 1975; 292: 895902.

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Perfusion method for harvesting bone marrow cells from dogs

Masahiko Sato DVM, PhD1, Yuko Goto-Koshino DVM, PhD2, Hiroyuki Mochizuki DVM3, Yasuhito Fujino DVM, PhD4, Koichi Ohno DVM, PhD5, and Hajime Tsujimoto DVM, PhD6
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  • 1 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, Japan.
  • | 2 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, Japan.
  • | 3 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, Japan.
  • | 4 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, Japan.
  • | 5 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, Japan.
  • | 6 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, Japan.

Abstract

Objective—To compare composition and colony formation of bone marrow mononuclear cells (BMMCs) harvested from dogs by means of a new perfusion method and the conventional aspiration method.

Animals—7 healthy adult Beagles.

Procedures—BMMCs were collected from the humeri and femurs of Beagles via perfusion and aspiration methods. Flow cytometric analysis was performed to quantify the presence of contaminant cells from the peripheral blood and the percentage of CD34+ progenitor cells in the BMMCs. A CFU assay was conducted to determine the number of progenitor cells in the BMMCs.

Results—The perfusion method was safely performed in all 7 dogs. Flow cytometric analysis revealed that the percentages of contaminant CD3+CD4+, CD3+CD8+, and CD21 + lymphocytes in BMMCs obtained via perfusion were significantly lower than percentages obtained via aspiration. The percentage of CD34+ cells obtained via perfusion was significantly higher than that obtained via aspiration. In addition, perfusion yielded a significantly higher CFU count than did aspiration.

Conclusions and Clinical Relevance—The perfusion method used in this study can minimize the contamination of bone marrow samples with peripheral blood and was a more efficient means for collecting canine bone marrow progenitor cells than the conventional aspiration method. Therefore, the perfusion method can be more suitable than aspiration for harvesting bone marrow cells for transplantation in dogs.

Abstract

Objective—To compare composition and colony formation of bone marrow mononuclear cells (BMMCs) harvested from dogs by means of a new perfusion method and the conventional aspiration method.

Animals—7 healthy adult Beagles.

Procedures—BMMCs were collected from the humeri and femurs of Beagles via perfusion and aspiration methods. Flow cytometric analysis was performed to quantify the presence of contaminant cells from the peripheral blood and the percentage of CD34+ progenitor cells in the BMMCs. A CFU assay was conducted to determine the number of progenitor cells in the BMMCs.

Results—The perfusion method was safely performed in all 7 dogs. Flow cytometric analysis revealed that the percentages of contaminant CD3+CD4+, CD3+CD8+, and CD21 + lymphocytes in BMMCs obtained via perfusion were significantly lower than percentages obtained via aspiration. The percentage of CD34+ cells obtained via perfusion was significantly higher than that obtained via aspiration. In addition, perfusion yielded a significantly higher CFU count than did aspiration.

Conclusions and Clinical Relevance—The perfusion method used in this study can minimize the contamination of bone marrow samples with peripheral blood and was a more efficient means for collecting canine bone marrow progenitor cells than the conventional aspiration method. Therefore, the perfusion method can be more suitable than aspiration for harvesting bone marrow cells for transplantation in dogs.

Bone marrow transplantation for the treatment of lymphoma, leukemia, solid tumors, and some non-malignant diseases is an important therapeutic option in human medicine.1,2 Although the clinical application of BMT in veterinary medicine is still less common than in humans, several studies3–7 have been conducted to evaluate BMT for the treatment of dogs with spontaneously developing lymphoma. Engraftment and prolonged survival were observed in several dogs that underwent BMT, yet 12% to 81% of dogs died from transplant complications such as failure to recover BMCs and GVHD.3,5–7

Bone marrow cells are usually obtained via bone marrow aspiration. However, BMCs become contaminated with cells from peripheral blood during conventional aspiration.8 In a study8 involving cynomolgus monkeys, the percentage of T cells from the peripheral blood in the bone marrow exceeded 20%. Because blood contamination of bone marrow specimens reduces the proportion of pure bone marrow progenitor cells, it may cause unsuccessful engraftment of the bone marrow. Furthermore, the presence of T cells from peripheral blood may trigger GVHD because these contaminant cells can immunologically attack the host, resulting in the need for immunosuppressive treatment.9–11

To reduce the contamination by peripheral blood, a new means of BMC harvesting, the perfusion method, was developed in monkeys for possible use in humans.8 This novel method can markedly reduce blood contamination of BMCs and the time during which a donor is anesthetized for the collection procedure.8

With advancements in supportive care, donor-recipient matching, and transplant preservation,7,12–14 hematopoietic stem cell transplantation is expected to become increasingly common in small animal practice to improve treatment results for various diseases. The purpose of the study reported here was to compare the perfusion method with the aspiration method in dogs with respect to peripheral blood contamination.

Materials and Methods

Animals—Seven healthy adult Beagles aged 3 to 5 years (body weight, 9.3 to 15.0 kg; 2 males and 5 females) were included in this study. Bone marrow cells were first collected from both humeri of all of the dogs, with the perfusion method used for one humerus and the aspiration method used for the other. After a period of 2 months, BMCs were collected from both femurs of the same dogs in a similar manner.

All dogs received sedatives and analgesics before each procedure (medetomidine [20 μg/kg, IM], butorphanol [0.2 mg/kg, IM], and meloxicam [0.2 mg/kg, SC]). Anesthesia was maintained with isoflurane (approx 1.5% in oxygen) for bone marrow collection. A peripheral blood sample was concurrently collected via a jugular vein by use of a 21-gauge needle for each experiment. After each procedure, dogs received meloxicam (0.1 to 0.2 mg/kg, PO, q 24 h for 3 days). The study was conducted in accordance with the guidelines of the Animal Care Committee of the Graduate School of Agricultural and Life Sciences of the University of Tokyo.

BMC harvesting via the perfusion method—Bone marrow fluid was collected by means of the perfusion method reported for cynomolgus monkeys,8 with slight modifications. A 15-gauge bone marrow–harvesting needlea was inserted into the humeral or femoral head following routine veterinary practices. Another 15-gauge harvesting needle (Figure 1),b which was engineered specifically for the perfusion method, was inserted into the bone shaft by use of an electric drill at the distal aspect of humerus or femur. After insertion was completed, the inner needle was removed from the bone head and a 50-mL syringe containing 30 U of dalteparin sodiumc/mL of bone marrow fluid was connected to the outer sheath of the bone-head needle. The inner needle inserted into bone shaft was removed, and a 50-mL syringe containing 50 mL of saline (0.9% NaCl) solution was connected to the outer sheath of the bone-shaft needle. Saline solution was gently injected via the bone-shaft needle as bone marrow fluid was aspirated via the bone-head needle. This procedure was repeated to obtain 100 mL of bone marrow fluid from each bone. One, 7, and 28 days after each harvesting procedure, dogs underwent a complete physical examination, hematologic analysis, and radiographic examination of the thorax and the treated bone for detection of adverse events.

Figure 1—
Figure 1—

Outer sheath (left) and inner needle (right) of a 15-gauge harvesting needle used for insertion into the shaft of the humerus and femur in dogs that underwent collection of BMCs via a perfusion method. This needle is currently not commercially available.

Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1344

BMC harvesting via the aspiration method—Bone marrow fluid was collected from 1 humerus or femur into saline solution containing 30 U of heparin/mL at a volume ratio of 2 mL of saline solution to 1 mL of marrow as described elsewhere.4 A total of 30 mL of bone marrow fluid was collected from each bone.

Preparation of BMMCs and PBMCs—Bone marrow fluid was strained through 100-μm mesh strainers to remove fat and other tissue particles. Erythrocytes in marrow suspensions were lysed by adding hemolytic solution (8.26 g of NH4Cl, 1 g of KHCO3, and 0.037 g of Na4EDTA/L of distilled water), and the quantity of BMCs was assessed with a hemacytometer. Bone mar row mononuclear cells and PBMCs were collected via density gradient centrifugation.d

Analysis of expression of cell surface antigens on BMMCs and PBMCs—The expression of cell surface antigens on BMMCs and PBMCs was analyzed by use of monoclonal antibodiese–g (Appendix). Bone marrow mononuclear cells and PBMCs were incubated with each antibody (CD3-biotin, 10 μg/mL; CD4-fluorescein isothiocyanate, 100 μg/mL; CD8-PE, 10 μg/mL; CD21-PE, 10 μg/mL; CD34-PE, 2 μg/mL) in a plastic tube at 4°C for 60 minutes. Biotin-conjugated mouse anti-canine CD3 monoclonal antibody was added, followed by streptavidin conjugateh (20 μg/mL) at 4°C for 30 minutes. Cell quantification was performed by use of a flow cytometer and associated software.g

CFU assay—The colony-forming ability of the BMMCs was assayed as described elsewhere.14 Briefly, phytohemagglutinini-stimulated lymphocyte-conditioned medium was prepared by culturing canine PBMCs (2 × 106 cells/mL) in Iscove modified Dulbecco mediumh containing 15% fetal bovine serum and 10 μg of phytohemagglutinin/mL for 1 week. The culture supernatant was collected as conditioned medium and stored at −80°C until used. The BMMCs (2.2 × 105 cells) were resuspended in 250 μL of conditioned medium and then transferred into 2.5 mL of methylcellulose mediumj containing a mixture of recombinant human cytokines (50 ng of stem cell factor/mL, 20 ng of granulocyte-macrophage colony-stimulating factor/mL, 20 ng of granulocyte colony stimulating factor/mL, 20 ng of interleukin-3/mL, 20 ng of interleukin-6/mL, and 3 U of erythropoietin/mL). The cells were vigorously mixed with the methylcellulose medium, plated in 35-mm plates, and incubated at 37°C in a humidified atmosphere of 5% CO2. After cells had been cultured for 2 weeks, the number of CFUs was counted.

Statistical analysis—Data are reported as median and IQR. The Wilcoxon signed rank test was used to assess statistical differences between the perfusion and aspiration methods with respect to the percentages of CD3+CD4+, CD3+CD8+, CD21+, and CD34+ cells in BMMCs and the CFU count. This test was also used to evaluate differences between humeri and femurs in the number of BMCs harvested. Values of P < 0.05 were considered significant.

Results

Animals—Physical examination, hematologic analysis, and radiography performed after the harvesting procedures had been completed revealed that no severe complications such as bone fracture, paralysis, or pulmonary infarction had developed in any of the 7 Beagles. Slight lameness persisting for several minutes was observed in 4 of 7 dogs after recovery from anesthesia (2 after BMC collection from the humerus, 1 after collection from the femur, and 1 after collection from both bone types); however, this lameness was brief, and dogs recovered within 2 hours.

Perfusion method—The entire perfusion procedure was completed within 20 minutes for all dogs. Median (IQR) numbers of BMCs collected by use of the perfusion method from 1 humerus and 1 femur from each dog (7 of each bone in total) were 7.8 × 108 cells (6.8 × 108 cells to 8.1 × 108 cells) and 2.4 × 108 cells (1.7 × 108 cells to 4.0 × 108 cells), respectively. On a dog body weight basis, median values were 6.2 × 107 cells/kg (5.4 × 107 cells/kg to 7.7 × 107 cells/kg) and 2.1 × 107 cells/kg (1.4 × 107 cells/kg to 3.5 × 107 cells/kg), respectively. The number of BMCs harvested from the humerus was significantly (P = 0.008) greater than from the femur.

Aspiration method—The entire perfusion procedure was completed within 40 minutes for all dogs and bones. The median number of BMCs collected by use of the aspiration method from 1 humerus and 1 femur (7 of each bone in total) was 3.2 × 108 cells (1.9 × 108 cells to 8.0 × 108 cells) and 1.1 × 108 cells (1.0 × 108 cells to 1.9 × 108 cells), respectively. On a dog body weight basis, median values were 2.4 × 107 cells/kg (1.8 × 107 cells/kg to 6.7 × 107 cells/kg) and 1.1 × 107 cells/kg (1.0 × 107 cells/kg to 1.9 × 107 cells/kg), respectively. The number of BMCs harvested from the humerus was significantly (P = 0.025) larger than that from the femur. The total number of BMCs harvested by the aspiration method was significantly (P = 0.015) smaller than that harvested by the perfusion method.

Cell surface antigens of BMMCs and PBMCs—The percentages of differentiated lymphocytes (CD3+CD4+, CD3+CD8+, and CD21+ cells) were significantly lower in BMMC samples obtained by the perfusion method than in those obtained by the aspiration method (Table 1). The percentage of undifferentiated CD34+ cells was significantly higher in BMMC samples obtained via perfusion than via aspiration. Furthermore, the percentages of T cells (CD3+CD4+ and CD3+CD8+ cells) were > 20% in the BMMC samples collected via aspiration, whereas < 10% of cells in the BMCC samples obtained via perfusion consisted of T cells.

Table 1—

Median (IQR) numbers of T cells (CD3+CD4+, CD3+CD8+, and CD21+) and progenitor cells (CD34+) identified by surface antigens in PBMC and BMMC samples harvested from the humeri (n = 7) and femurs (7) of 7 healthy Beagles by means of a perfusion method and the conventional bone marrow aspiration method.

Cell typeCD3+CD4+CD3+CD8+CD21+CD34+
PBMCs26.6 (24.4–29.9)12.1 (10.4–14.6)14.7 (12.1–16.6)
BMMCs via perfusion3.4 (2.9–5.7)*3.4 (2.8–5.3)*7.9 (6.0–12.9)*5.5 (4.2–7.1)
BMMCs via aspiration13.7 (8.2–21.9)9.2 (6.7–12.2)12.2 (10.2–14.0)4.5 (3.1–5.0)

Value is significantly (P < 0.02) lower than the corresponding value for BMMCs harvested via aspiration.

Value is significantly (P = 0.006) higher than the corresponding value for BMMCs harvested via aspiration.

— = Not applicable.

CFU assay—The median CFU count generated from 2.2 × 105 cells was 62.0 CFUs (46.3 to 70.0 CFUs) in BMMC samples obtained via the perfusion method, which was significantly (P = 0.001) higher than the count in BMMCs obtained via aspiration (20.5 CFUs [17.3 to 28.0 CFUs]).

Discussion

The primary goal of the present study was to investigate whether a perfusion method for harvesting BMCs from dogs would have advantages over the conventional bone marrow aspiration method. Findings suggested that the perfusion method reduced the intermixing of CD3+CD4+ and CD3+CD8+ T cells with bone marrow fluid, thereby enabling collection of more progenitor cells than could be obtained via aspiration. These results are similar to those of the previous study8 in cynomolgus monkeys. The perfusion method was conducted safely, and none of the study dogs developed any physical, hematologic, or radiographic signs of clinically important adverse effects.

For successful autologous BMT in dogs, > 5.0 × 107 BMCs/kg are required.5,7,15–20 In the present study, the perfusion method, which includes the infusion of 100 mL of saline solution (50 mL × 2) into the bone marrow, enabled the collection of a median of 6.2 × 107 cells/kg from a single humerus, thus meeting this requirement. The time required to collect enough BMCs for BMT via the perfusion method was considerably shorter (< 20 minutes) than via the conventional method (mean, < 60 minutes4). Furthermore, the CFU assay revealed that the BMCs collected via perfusion had a higher progenitor cell count than those collected via aspiration. Thus, a large number of unadulterated BMCs can be harvested with the new method and the number of BMCs required for BMT may be reduced because of the high percentage of hematopoietic progenitor cells obtained.

To the authors' knowledge, the required minimal number of BMCs for allogeneic transplantation in dogs remains uncertain. Recipient dogs were given > 1.0 × 108 donor BMCs/kg in previous reports.6,21,22 Therefore, it is possible that harvesting from ≥ 2 bones would be necessary to ensure a sufficient quantity of BMCs when the perfusion method is used for allogeneic transplantation. Use of granulocyte colony-stimulating factor might increase the yield of the required number of BMCs for BMT, although this was not investigated in the present study.

The quantity of BMCs harvested from the femur was much less than the quantity obtained from the humerus for both methods in our study. This is consistent with findings in monkeys.23 Additionally, approaching the femur for BMC collection in large or overweight dogs can be challenging. Therefore, the humerus is recommended as an acceptable site for the perfusion method of harvesting.

A needle engineered specifically for the perfusion method was connected to an electric drill to facilitate access to the bone marrow. This needle is not commercially available. Bone marrow biopsy or other needles can be used as substitutes for this technique, but such a substitution will likely make the approach to the bone marrow from the shaft of a long bone more difficult because such instruments are not designed for use with electric drills.

The percentages of CD3+CD4+ and CD3+CD8+ subsets in BMMCs obtained with the aspiration method were nearly 3 to 4 times as high as those obtained with the perfusion method. However, the percentage of CD21+ cells was only 1.5 times as high. The proportions of mature cell subsets obtained with the 2 methods differed, suggesting factors other than peripheral blood contamination of BMC samples may have been involved. One possible explanation is that mature lymphoid cells partially exist in bone marrow, which results in alterations of subset proportions. For example, CD21+ cells might reside in bone marrow more abundantly than mature T cells.

In humans, BMT is used in the treatment of various diseases, ranging from leukemia to autoimmune disorders.1,2 Associated with a considerable mortality rate, GVHD is a severe complication of BMT. Donor T cells are considered instrumental in the development of GVHD because removal of T cells from donor inocula effectively prevents GVHD.9–11 Although administration of immunosuppressants can reduce the undesirable consequences of GVHD, such treatments do not completely prevent it. Interestingly, 35% to 45% of humans given human leukocyte antigen–identical marrow grafts from siblings still develop acute GVHD.24–26 A similar complication of BMT occurs in dogs. Fifty-five percent of dogs given dog leukocyte antigen–identical marrow grafts from littermates reportedly developed GVHD when they did not receive immunosuppressive treatment after grafting.24 In another report,6 13 of 16 dogs with lymphoma or leukemia that received dog leukocyte antigen–identical allogeneic marrow transplantation after total-body irradiation developed GVHD, even though immunosuppressants were administered. As shown in the present study, the perfusion method may minimize T-cell contamination of BMC samples in dogs. Contamination of BMCs with peripheral blood, as determined by the percentages of CD3+CD4+ and CD3+CD8+ T cells, was significantly lower for the perfusion method (< 10%) than for the aspiration method (> 20%).

Another severe problem associated with BMT is unsuccessful engraftment. Death from subsequent transplant complications can be caused by a failure of BMCs to recover after total-body irradiation. Failure of BMC recovery can occur in 15% to 45% of dogs that undergo BMT.3 Because the quantities of progenitor cells harvested through the perfusion method were greater than the quantities obtained with the aspiration method in our study, the perfusion method should prevent such complications.

One limitation of the present study was that the subjects were limited to healthy Beagles aged 3 to 5 years. The safety of electrical drill assistance to perform the perfusion method should be evaluated in dogs of various sizes, particularly small dogs, before clinical application is attempted. We performed the perfusion method with only humeri and femurs because BMC are usually obtained from long bones for transplantation in dogs.3–7,15–22 However, the quantity of collectable BMCs from long bones is expected to decrease with age. In human medicine, BMCs are typically collected from the sternum or iliac crest, and harvesting from in the iliac crest in cynomolgus monkeys can be safely and effectively achieved via perfusion.23 Additional studies are needed to identify the optimal site from which BMCs can be harvested in dogs.

The perfusion method for harvesting BMCs from dogs was considered technically feasible because it was associated with no severe complications and a shorter period of anesthesia, compared with the aspiration method. Although further development of the perfusion method is required prior to application in clinical trials, we believe that this new technique will contribute to the advancement of the use of BMT in veterinary medicine.

ABBREVIATIONS

BMC

Bone marrow cell

BMMC

Bone marrow mononuclear cell

BMT

Bone marrow transplantation

GVHD

Graft-versus-host disease

IQR

Interquartile range

PBMC

Peripheral blood mononuclear cell

PE

Phycoerythrin

a.

Medical Device Technologies Inc, Gainesville, Fla.

b.

Provided by Japan Immunoresearch Laboratories Co Ltd, Gunma, Japan.

c.

Pfizer, New York, NY.

d.

Ficoll-hypaque gradient media, GE Healthcare Bioscience Corp, Piscataway, NJ.

e.

Leukocyte Antigen Biology Laboratory, Davis, Calif.

f.

Serotec Ltd, Oxford, England.

g.

BD Bioscience, San Diego, Calif.

h.

Streptavidin-PerCP-Cy 5.5 conjugate, eBioscience Inc, San Diego, Calif.

i.

Sigma-Aldrich, St Louis, Mo.

j.

Stem Cell Technologies Inc, Vancouver, BC, Canada.

Appendix

Monoclonal antibodies used to detect specific cell types in bone marrow samples.

Antibody cloneCell specificity-labelCell phenotype
CA17.2A12eCD3-biotinT lymphocyte
YKIX302.9fCD4-FITCT helper
YCATE55.9fCD8-PET cytotoxic
CA2.1DfCD21-PEMature B lymphocyte
1H6gCD34-PEProgenitor cell

FITC = Fluorescein isothiocyanate.

References

  • 1.

    Thomas ED. Bone marrow transplantation: a review. Semin Hematol 1999; 36: 95103.

  • 2.

    Goldman JM, Horowitz MM. The international bone marrow transplant registry. Int J Hematol 2002; 76 (suppl 1:) 393397.

  • 3.

    Appelbaum FR, Deeg HJ, Storb R, et al. Marrow transplant studies in dogs with malignant lymphoma. Transplantation 1985; 39: 499504.

  • 4.

    Frimberger AE, Moore AS, Rassnick KM, et al. A combination chemotherapy protocol with dose intensification and autologous bone marrow transplant (VELCAP-HDC) for canine lymphoma. J Vet Intern Med 2006; 20: 355364.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Weiden PL, Storb R, Lerner KG, et al. Treatment of canine malignancies by 1200 R total body irradiation and autologous marrow grafts. Exp Hematol 1975; 3: 124134.

    • Search Google Scholar
    • Export Citation
  • 6.

    Weiden PL, Storb R, Sale GE, et al. Allogeneic hematopoietic grafts after total-body irradiation in dogs with spontaneous tumors. J Natl Cancer Inst 1978; 61: 353357.

    • Search Google Scholar
    • Export Citation
  • 7.

    Weiden PL, Storb R, Deeg HJ, et al. Prolonged disease-free survival in dogs with lymphoma after total-body irradiation and autologous marrow transplantation consolidation of combination-chemotherapy-induced remissions. Blood 1979; 54: 10391049.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Kushida T, Inaba M, Ikebukuro K, et al. A new method for bone marrow cell harvesting. Stem Cells 2000; 18: 453456.

  • 9.

    Blazar BR, Taylor PA, Snover DC, et al. Nonmitogenic anti-CD3F(ab')2 fragments inhibit lethal murine graft-versus-host disease induced across the major histocompatibility barrier. J Immunol 1993; 150: 265277.

    • Search Google Scholar
    • Export Citation
  • 10.

    Kataoka Y, Iwasaki T, Kuroiwa T, et al. The role of donor T cells for target organ injuries in acute and chronic graft-versus-host disease. Immunology 2001; 103: 310318.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Ichiki Y, Bowlus CL, Shimoda S, et al. T cell immunity and graft-versus-host disease (GVHD). Autoimmun Rev 2006; 5: 19.

  • 12.

    Lupu M, Sullivan EW, Westfall TE, et al. Use of multigeneration-family molecular dog leukocyte antigen typing to select a hematopoietic cell transplant donor for a dog with T-cell lymphoma. J Am Vet Med Assoc 2006; 228: 728732.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Appelbaum FR, Deeg HJ, Storb R, et al. Cure of malignant lymphoma in dogs with peripheral blood stem cell transplantation. Transplantation 1986; 42: 1922.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Ide K, Matsuura S, Fujino Y, et al. Investigation of various methods for the cryopreservation of canine bone marrow-derived CD34(+) cells. J Vet Med Sci 2008; 70: 12111217.

    • Crossref
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Contributor Notes

Presented in abstract form at the American College of Veterinary Internal Medicine Forum, Anaheim, Calif, June 2010.

The authors thank Dr. Shuji Nakamura and Katsuyuki Sado for technical assistance.

Address correspondence to Dr. Tsujimoto (atsuji@mail.ecc.u-tokyo.ac.jp).