Comparison of three mobilization protocols for peripheral blood stem cell apheresis with Spectra Optia continuous mononuclear cell protocol in healthy dogs.

Peripheral blood stem cell (PBSC) transplantation following consolidation therapy is a feasible treatment option for canine haematological malignancies. In veterinary medicine, haematopoietic stem cells are generally mobilized into peripheral circulation using a granulocyte colony-stimulating factor (G-CSF). This pilot study aimed to evaluate the haematopoietic stem cell mobilization effect of three different regimens for PBSC apheresis with Spectra Optia continuous mononuclear cell (CMNC) protocol in healthy dogs. Stem cell mobilization was performed using high-dose plerixafor (CXCR-4 inhibitor) alone, a G-CSF alone, or a combination of the low-dose plerixafor and G-CSF. Three dogs were assigned to each mobilization protocol. Regardless of the mobilization protocol, the total blood volume processed was uniformly set as 270 mL/kg and many PBSCs, defined as CD34+/CD45dim cells, within the apheresis product were compared. Changes in complete blood count, PBSC counts, and blood chemistry analysis were monitored before, during, and after apheresis. All dogs tolerated the apheresis procedure using the Spectra Optia system with minimal adverse effects. The mean PBSC counts of the apheresis products for plerixafor, G-CSF, and the combination groups were 1.3 ± 0.24, 4.2 ± 0.47, and 6.4 ± 0.9 × 106 cells/kg, respectively. The apheresis procedure using Spectra Optia CMNC protocol in dogs is safe and feasible. Furthermore, PBSC mobilization with a combination of G-CSF and plerixafor appeared more effective than either compound alone in mobilizing PBSC to the peripheral blood in dogs.

Coleta de células progenitoras periféricas por aférese automatizada em equino: relato de procedimento / Viability of automated collection of peripheral blood progenitor cells in a horse: report of procedure

The biotechnology used in tendon, bone and joint recoveries in Equine Medicine has improved in recent years. The most used are platelet rich plasma and stem cells from adipose tissue and bone marrow. However, recent studies have shown that stem cells can be found in the bloodstream, also named peripheral blood progenitor cells (CPP). This note aims at reporting the feasibility of automated collection of CPP in horses. The procedure was conducted in an equine, female, Quarter Horses, 2 years old, 385kg. The automated collection of CPP was conducted using apheresis equipment Fresenius- Kabi coupled to C4Y kit. The procedure lasted two hours and 30 minutes without complications, processing 5054mL of whole blood and obtaining 351mL of CPP. Upon completion of the collection, the content of CPP was separated into 10 ml aliquots and immediately stored at -18°C. The automated technique proved to be feasible for horses, but needs improvement in order to achieve greater efficiency and reduce procedure time.(AU)

Collection of hematopoietic CD34 stem cells in rhesus macaques using Spectra Optia.

BACKGROUND: Nonhuman primates, particularly rhesus macaques, are ideal preclinical large animal models to investigate organ tolerance induction protocols using donor hematopoietic stem cells (HSCs) to induce chimerism. Their relatively small size poses some challenges for the safe and effective collection of peripheral blood HSCs through apheresis procedures. We describe our experiences using the Spectra Optia apheresis unit to successfully obtain HSCs from mobilized peripheral blood of rhesus macaques. METHOD: Mobilization of peripheral blood HSCs was induced using granulocyte stimulating factor (G-CSF) and Mozobil. The Spectra Optia unit was used in 18 apheresis procedures in 13 animals (4.9-10 kg). Animal health was carefully monitored during and after the procedure. Changes in peripheral blood cells before, during and after procedure were determined by complete blood count and flow cytometry. RESULTS: The automatic settings of the Spectra Optia unit were applied successfully to the procedures on the rhesus macaque. All animals tolerated the procedure well with no mortality. Mobilization of HSCs were most consistently achieved using 50 µg/kg of G-CSF for 5 days and a single dose of Mozobil on the 5th day, followed by collection of cells 3 h after Mozobil injection. The final apheresis product contained an average of 23 billion total nucleated cells with 47% granulocytes, 3,871 million total CD3 cells and 77 million CD34 cells which resulted in an average of 10 million CD34+ cells/kg of donor weight. CONCLUSION: Apheresis of peripheral blood mobilized HSCs in rhesus macaques using Spectra Optia is a safe and effective procedure.

Evaluation of two platelet-rich plasma processing methods and two platelet-activation techniques for use in llamas and alpacas.

OBJECTIVE To evaluate 2 processing methods (commercial kit vs conical tube centrifugation) for preparing platelet rich plasma (PRP) for use in llamas and alpacas. SAMPLES Blood samples (30 mL each) aseptically collected from 6 healthy llamas and 6 healthy alpacas. PROCEDURES PRP was prepared from blood samples by use of a commercial kit and by double-step conical tube centrifugation. A CBC was performed for blood and PRP samples. Platelets in PRP samples were activated by means of a freeze-thaw method with or without 23mM CaCl2, and concentrations of platelet-derived growth factor-BB and transforming growth factor-ß1 were measured. Values were compared between processing methods and camelid species. RESULTS Blood CBC values for llamas and alpacas were similar. The commercial kit yielded a significantly greater degree of platelet enrichment (mean increase, 8.5 fold vs 2.8 fold) and WBC enrichment (mean increase, 3.7 fold vs 1.9 fold) than did conical tube centrifugation. Llamas had a significantly greater degree of platelet enrichment than alpacas by either processing method. No difference in WBC enrichment was identified between species. Concentrations of both growth factors were significantly greater in PRP samples obtained by use of the commercial kit versus those obtained by conical tube centrifugation. CONCLUSIONS AND CLINICAL RELEVANCE For blood samples from camelids, the commercial kit yielded a PRP product with a higher platelet and WBC concentration than achieved by conical tube centrifugation. Optimal PRP platelet and WBC concentrations for various applications need to be determined for llamas and alpacas.

Evaluation of canine red blood cell quality after processing with an automated cell salvage device.

OBJECTIVE: To evaluate the properties of RBC concentrate harvested after processing fresh whole blood units from healthy dogs with an automated cell salvage device. DESIGN: Prospective, in vitro, experimental study. SETTING: University teaching hospital. ANIMALS: Sixteen healthy, privately owned dogs of various breeds. INTERVENTIONS: Fresh canine whole blood collected in bags with citrate phosphate dextrose adenine solution was processed with an automated cell salvage device and analyzed in vitro. Laboratory values determined before (baseline, from a catheter sample) and after processing RBCs (procRBCs) included a complete blood count, selected blood chemistry analytes, erythrocyte osmotic resistance, whole blood viscosity, RBC aggregation, and RBC deformability. MEASUREMENTS AND MAIN RESULTS: Total recovery of RBCs was 80% ± 12%. Hematocrit of the procRBCs yielded by the device was 77% ± 3.7% (mean ± standard deviation). Gross morphology of the RBCs remained unchanged. The mean corpuscular volume, erythrocyte osmotic resistance, RBC deformability, RBC aggregation, and the activity of lactate dehydrogenase showed minor but statistically significant changes from baseline. No differences in the concentrations of free hemoglobin were observed. Whole blood viscosity was less in the procRBCs. Seventy-seven percent (mean) of the platelets were washed out, while a mean of 57% of the leukocytes remained in the procRBCs. CONCLUSIONS: Although processing canine blood with this automated cell salvage device leads to slight changes in some properties of RBCs, most of these changes are comparable to changes seen in human blood after processing. Present data indicate that the use of this cell salvage device does not induce changes in canine RBC concentrate that would preclude its use for transfusion.

Autologous peripheral blood hematopoietic cell transplantation in dogs with T-cell lymphoma.

BACKGROUND: Peripheral blood hematopoietic cell transplantation (PBHCT) is a feasible treatment option for dogs with B-cell lymphoma. OBJECTIVE: To examine apheresis and PBHCT outcomes in dogs diagnosed with T-cell lymphoma (TCL). ANIMALS: Fifteen client-owned dogs diagnosed with high-grade TCL. METHODS: After high-dose cyclophosphamide and rhG-colony-stimulating (rhG-CSF) factor treatment, peripheral blood mononuclear cells were collected using cell separators. The harvested cells then were infused after varying doses of total body irradiation (TBI). Postirradiation adverse effects were managed symptomatically and dogs were discharged upon evidence of hematopoietic engraftment. RESULTS: More than 2 × 10(6) CD34+ cells/kg were harvested from 15/15 dogs. Thirteen of 15 (87%) dogs engrafted appropriately, whereas 2 (13%) of the dogs died in the hospital. One dog developed cutaneous B-cell lymphoma 120 days post-PBHCT. The median disease-free interval and overall survival (OS) of the 13 dogs transplanted in first remission from the time of PBHCT were 184 and 240 days, respectively. Stage and substage of disease at diagnosis had no effect on OS. Two of 13 (15%) dogs were alive 741 and 772 days post-PBHCT. CONCLUSIONS AND CLINICAL IMPORTANCE: PBHCT may be considered as a treatment option for dogs with TCL.

Apheresis in three dogs weighing <14 kg.

HISTORY: CaridianBCT apheresis machines require a ~285 mL priming volume (extracorporeal blood) that is withdrawn from the patient in ~10 minutes. Therefore, apheresis in dogs has generally been limited to dogs > ~20 kg to assure <20% of the blood volume is removed in the priming phase. ANIMALS/PHYSICAL EXAMINATION: Three dogs weighing <14 kg (13.6, 10.5, and 9.9 kg) with lymphoma that underwent apheresis. MANAGEMENT: The dogs were premedicated for placement of apheresis catheters with hydromorphone (0.1 mg kg(-1) ) IM. Anesthesia was induced with propofol, to effect, intravenously and general anesthesia was maintained with isoflurane in oxygen. Following catheter placement, dogs were allowed to recover from isoflurane but were kept sedated with either a dexmedetomidine constant rate infusion (CRI) or a propofol CRI. Real time autologous blood priming was not performed in any of the dogs. Instead, priming solutions were composed of a combination of hetastarch, lactated Ringer's solution, and/or autologous blood that was harvested 4 days before the procedure. During apheresis, dogs received anticoagulant citrate-dextrose, solution-A (ACD-A) to prevent clotting and 10% calcium gluconate as needed to maintain normal ionized calcium concentrations. Dogs were monitored for cardiovascular and cardiopulmonary stability, anemia and lactic acidosis. FOLLOW-UP: All of the dogs had cardiovascular and cardiopulmonary values within clinically acceptable ranges. Immediately following apheresis all of the dogs were mildly to moderately anemic (PCV; 17-35%) although none of the dogs required a transfusion or had an increased lactate concentration. CONCLUSIONS: Dogs as small as 9.9 kg can successfully undergo apheresis with a variety of priming solutions. Dexmedetomidine or propofol given as a CRI provides sufficient sedation for this procedure.

Evaluation of equine peripheral blood apheresis product, bone marrow, and adipose tissue as sources of mesenchymal stem cells and their differentation potential.

OBJECTIVE: To evaluate effects of apheresis on mesenchymal stem cells (MSCs) and compare those MSCs with MSCs obtained from adipose tissue or bone marrow (BM). SAMPLE POPULATION: Samples obtained from 6 adult horses. PROCEDURES: Samples of blood from a peripheral vein, adipose tissue, and BM aspirate were obtained from each horse. Samples were processed via apheresis of blood and techniques reported elsewhere for adipose tissue and BM. Cultures were maintained until adherence and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteoblastogenic, and chondrogenic potential. RESULTS: Apheresis product had a significantly higher mononuclear percentage, higher platelet count, and lower RBC count, compared with values for peripheral blood. No cell adherence to the tissue culture plates was detected for the apheresis product. Adherence was detected for 6 of 6 adipose-derived and 4 of 6 BM-derived samples. Variations in efficiency were detected for differentiation of adipose- and BM-derived cells into adipocytes, chondrocytes, and osteoblasts. CONCLUSIONS AND CLINICAL RELEVANCE: Apheresis was able to concentrate mononuclear cells and reduce RBC contamination. However, the apheresis product was unable to adhere to the tissue culture plates. In matched horses, adipose- and BM-derived MSCs were capable of producing lipids, glycosaminoglycan, and mineral. The BM was vastly superior to adipose tissue as a source of MSCs with osteoblastogenic potential in matched horses. Additional studies will be necessary to optimize apheresis techniques for horses before peripheral blood can be considered a suitable source for multipotential cells for use in cell-based treatments.

Cryopreservation of canine platelets.

BACKGROUND: Platelet cryopreservation allows long-term storage and immediate availability of transfusion products. HYPOTHESIS: The addition of a preparation inhibiting platelet activation (Thrombosol, in 2% dimethyl sulfoxide [DMSO]) will enhance in vitro function and prolong in vivo survival of cryopreserved platelets compared with those preserved in 6% DMSO. ANIMALS: Thirty-three research dogs. METHODS: Prospective study. Eleven fresh canine apheresis platelet concentrates (PCs) were each split into 3 units: fresh and cryopreserved in 6% DMSO or Thrombosol. Platelet analysis, performed 1-10 weeks postfreezing, included in vitro functional testing and in vivo survival assessed by administration of biotinylated platelets. RESULTS: Platelet aggregation was diminished in cryopreserved PC. Cryopreserved platelets could be activated, as based on mean thrombin-stimulated P-selectin expression (6% DMSO, 23.0%; Thrombosol, 18.4%), although to a lesser extent than fresh PC (49.1%) (P < .0001). The mean maximum in vivo platelet recovery for fresh PC was 80.3%, significantly greater than recovery for 6% DMSO (49.2%) and Thrombosol PC (43.7%) (P< or = .001). The half-life (days) of fresh PC (3.8 +/- 0.4) was significantly (P < .002) greater than that of 6% DMSO (1.9 +/- 1.0) and Thrombosol (2.4 +/- 1.1) PC, with no difference (P= .3) between cryopreserved PC. CONCLUSIONS AND CLINICAL IMPORTANCE: Cryopreservation of canine platelets using Thrombosol did not provide any advantage over preservation using 6% DMSO. Cryopreserved platelets can be activated in vitro and provide therapeutic benefit when fresh platelets are unavailable. Further studies are needed to assess their in vivo hemostatic function.

Use of a centrifugation-based, point-of-care device for production of canine autologous bone marrow and platelet concentrates.

OBJECTIVE: To analyze a centrifugation-based, point-of-care device that concentrates canine platelets and bone marrow-derived cells. ANIMALS: 19 adult sexually intact dogs. PROCEDURES: Anticoagulated peripheral blood (60 mL) and 60 mL of anticoagulated bone marrow aspirate (BMA) were concentrated by centrifugation with the centrifugation-based, point-of-care device to form a platelet and a bone marrow concentrate (BMC) from 11 dogs. Blood samples were analyzed on the basis of hemograms, platelet count, and PCV. The BMA and BMC were analyzed to determine PCV, total nucleated cell count, RBC count, and differential cell counts. The BMC stromal cells were cultured in an osteoinductive medium. Eight additional dogs were used to compare the BMC yield with that in which heparin was infused into the bone marrow before aspiration. RESULTS: The centrifugation-based, point-of-care device concentrated platelets by 6-fold over baseline (median recovery, 63.1%) with a median of 1,336 x 10(3) platelets/microL in the 7-mL concentrate. The nucleated cells in BMCs increased 7-fold (median recovery, 42.9%) with a median of 720 x 10(3) cells/microL in the 4-mL concentrate. The myeloid nucleated cells and mononuclear cells increased significantly in BMCs with a significant decrease in PCV, compared with that of BMAs. Stromal cell cultures expressed an osteoblastic phenotype in culture. Infusion of heparin into the bone marrow eliminated clot formation and created less variation in the yield (median recovery, 61.9%). CONCLUSIONS AND CLINICAL RELEVANCE: Bone marrow-derived cell and platelet-rich concentrates may form bone if delivered in an engineered graft, thus decreasing the need for cancellous bone grafts.

The use of CD 34(+) mobilized peripheral blood as a donor cell source does not improve chimerism after in utero hematopoietic stem cell transplantation in non-human primates.

In utero hematopoietic stem cell transplantation is a therapeutic procedure that could potentially cure many developmental diseases affecting the immune and hematopoietic systems. In most clinical and experimental settings of fetal hematopoietic transplantation the level of donor cell engraftment has been low, suggesting that even in the fetus there are significant barriers to donor cell engraftment. In postnatal hematopoietic transplantation donor cells obtained from mobilized peripheral blood engraft more rapidly than cells derived from marrow. We tested the hypothesis that use of donor hematopoietic/stem cells obtained from mobilized peripheral blood would improve engraftment and the level of chimerism after in utero transplantation in non-human primates. Despite the potential competitive advantage from the use of CD 34(+) from mobilized peripheral blood, the level of chimerism was not appreciably different from a group of animals receiving marrow-derived CD 34(+) donor cells. Based on these results, it is unlikely that this single change in cell source will influence the clinical outcome of fetal hematopoietic transplantation.

Uso del pollo como biomodelo experimental en arteriosclerosis / Use of the chicken as experimental biomodel in atherosclerosis

En este artículo se describen los principales trabajos que justifican el interés del pollo en el estudio de la arteriosclerosis. Se comentan estudios de arteriosclerosis espontánea y arteriosclerosis experimental o inducida, así como los modelos mutantes. Se detallan los más importantes métodos experimentales aplicados, diferenciando los estudios en animales intactos, con y sin denudación, los estudios en arterias aisladas, los llevados a cabo e cultivos celulares y los que emplean la aféresis (AU)

Preparation of blood components.

The processing of blood into various components and the knowledge of component usage enables veterinarians to support and beneficially treat more animals. Blood products include packed red blood cells; fresh frozen, fresh, and modified plasma; cryoprecipitate; platelets; and concentrates. Some methods of preparation of blood products and storage are presented.

Evaluation of leukapheresis and thrombocytapheresis in the horse.

Continuous-flow centrifugation leukapheresis techniques were used to collect 300-ml volumes of leukocyte-rich plasma from 5 nonmedicated horses and from 5 corticosteroid-stimulated horses. White blood cell counts and differential counts were performed on the horses before (base line) and up to 48 hours after leukapheresis. Systemic administration of hydrocortisone increased numbers of total WBC and neutrophils and improved harvest of these cells. Nonmedicated horses had a mean yield of 3.38 X 10(10) leukocytes in the 300-ml volume. Stimulated horses yielded a mean of 6.88 X 10(10) leukocytes. After leukapheresis, WBC counts decreased a mean of 38% in nonstimulated horses and decreased a mean of 30% in stimulated horses. By 6 hours after leukapheresis, circulating WBC counts of horses in both groups had returned to preleukapheresis values. The relationship between neutrophil yield and the 4 variables (preleukapheresis WBC count, preleukapheresis neutrophil count, preleukapheresis lymphocyte count, and the PCV of the leukocyte-rich plasma) were examined, using simple (pair-wise) correlation and multiple linear regression. A significant positive correlation was found between neutrophil yield and preleukapheresis WBC and neutrophil counts. Because sodium citrate was used in the collection system to prevent extracorporeal blood coagulation, ionized and total serum calcium concentrations were monitored before and after leukapheresis. Although total serum calcium concentrations remained unchanged, ionized calcium concentrations decreased approximately 33% from base-line values during the 2-hour leukapheresis procedures. Occasional mild muscle fasciculations were the only adverse clinical signs of citrate toxicity exhibited by the horses.(ABSTRACT TRUNCATED AT 250 WORDS)