- Email: [email protected]
Acute anaphylaxis study of mesenchymal stem cell-derived exosomes in Guinea pigs
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Poster Abstract Presentations
buffer media of different pH-values ranging from pH 8.0 to pH 4.8. Upon processing supernatants of mesenchymal stem/stromal cells (MSCs) cultured in the presence of 10% human platelet lysate, EVs are recovered in a small number of FFE fractions lacking most proteins of the conditioned media. Currently, we characterize the identified fractions in more detail. Prepared EVs are quantified by Nano Particle Tracking Analysis (NTA) and imaging flow cytometry. Furthermore, the presence of several EV markers and the absence of contaminants are analyzed by Western Blot and mass spectrometry-based proteomics. We conclude FFE is a feasible and quick method to highly purify EVs in an accurate manner. 199 INCREASING YIELD OF MSC-EVS IN SCALABLE XENO-FREE MANUFACTURING K. Adlerz, M. Trempel, J.A. Rowley & T. Ahsan RoosterBio, Inc., Frederick, Maryland, United States Background & Aim: Extracellular vesicles (EVs) are increasingly being investigated as both drug delivery vehicles and as a cell-free therapy. EVs from mesenchymal stem/stromal cells (MSC-EVs), for example, have been shown to recapitulate many of the therapeutic effects of the cells themselves. A critical barrier in the development of MSC-EVs as a commercial therapy, however, is generating the large amount of EVs that will be required per dose. In a recent survey, the majority of those working with EVs were working with less than 100mL of sample (ISEV 2016). In this study, two strategies were investigated to maximize EV yield. First, timing parameters for culturing human bonemarrow derived MSCs and collecting MSC-EVs were evaluated to increase EV yield. Second, conditioning the cells was investigated by adding an EV activator during the EV collection period. Methods, Results & Conclusion: MSCs were first cultured in an expansion media (RoosterNourishTM, RoosterBio) in a fed-batch system. The cultures were then switched to a collection media (RoosterCollectTM-EV, RoosterBio) with or without an EV activator (EV BoostTM, RoosterBio). After two days, the conditioned media was harvested. The EVs in the collected conditioned media were analyzed for particle concentration and particle size distributions by nanoparticle tracking analysis, protein expression, RNA expression, and wound healing capability. Particle concentration (particles per L of media) was used as a measure of EV yield. Particle concentration increased with increased culture time in expansion media, with confluent cultures generating the most EVs. The EV activator at a low concentration increased EV yield about 2-fold compared to without the activator after 24hrs. The EV yield from the low EV activator culture at 24hrs was still about 45% greater than the yield of the culture without the activator after 48hrs. After 6hrs of collection, the high EV activator concentration increased EV yield to about the same level as without the activator after 48hrs of collection. Optimizing protocols for EV yield will become increasingly important as MSC-EVs move towards the clinic and lot sizes of clinically-relevant doses are required. Pre-conditioning of cells or conditioning cells during EV collection offers one possibility to further increase EV yield and decrease process time.
their potential as a key bioactive agent in regenerative medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly being investigated as a clinical therapy. It was recently found that the number of exosomes released from 2 million MSCs in 48 hours is equivalent to a single dose for a rodent (Phinney, Pittenger 2017). Hence, most indications would require an MSC production lot size that is hardly achievable in 2D planar cell culture; larger scalable bioreactor systems will be crucial to generate enough EVs for clinical doses. Therefore, this study compared the yield and characteristics of MSC-derived EVs generated in typical 2D culture with those from scalable 3D bioreactor systems. Methods, Results & Conclusion: Human bone marrow-derived MSCs were cultured on microcarriers in 3L and 15L bioreactors. After initial cell inoculation, a bioreactor feed was added on Day 3, and cultures were switched to an EV collection media on Day 4, then cultured for 3 additional days. Samples of the conditioned media were analyzed for particle size and concentration. The collected MSC-EVs were evaluated for protein and RNA expression and wound healing capability. MSC-EVs were compared between bioreactor scales and with a 2D control cultured in a 10-layer cell stack. Higher cell densities were achieved in 3D compared to 2D culture, with similar cell densities in the 3L and 15L bioreactors. The conditioned media was analyzed after the 3-day EV collection period. Particle size distributions were similar across culture systems. The particle concentration in the conditioned media (particles/L media) was similar between the 3L and 15L bioreactors, with the 3D cultures about 3x higher than 2D culture. When particle concentration was normalized to cell number, the productivity (particles/cell) was comparable in the two bioreactor cultures, with 3D cultures greater than 2D. Optimizing EV yield will become increasingly important as EVs are used in the clinic. We have developed a scalable process for MSC-EV generation in a bioreactor. The comparison between 2D and bioreactor cultures demonstrates that a greater EV yield can be accomplished in 3D culture. We intend to continue to scale this process up to production scale (80L) bioreactors, which is critical to generating lot sizes for clinically relevant doses of MSC-EVs.
Histograms, normalized to concentration, show EVs maintained similar size profiles across culture systems. The productivity of MSCs, measured as particles/cell, was higher in bioreactor systems than in 2D culture.
201 ACUTE ANAPHYLAXIS STUDY OF MESENCHYMAL STEM CELL-DERIVED EXOSOMES IN GUINEA PIGS G. Yoo1, A. Lee1, S. Park1, S. Han1, J. Kim1, S. Lee1, S. Na1, M. Yoo1, D. Kim1, Y. Cho2 & K. Moon1 1 Korea Institute of Toxicology, Daejeon, the Republic of Korea, 2Hanyang University ERICA, Ansan, the Republic of Korea
A Parameters for MSC expansion time and EV collection time were investigated in two donors. B The addition of an EV activator to the collection media increased EV productivity, similar particle level to 48hrs without EV activator was achieved at 24 hours with low EV activator and 6hrs with high EV activator. 200 COMPARISON OF MSC-EVS MANUFATURED IN 2D VERSUS SCALABLE 3D BIOREACTOR SYSTEMS K. Adlerz, M. Trempel, D. Wang, R.D. Kirian, J.A. Rowley & T. Ahsan RoosterBio, Inc., Frederick, Maryland, United States Background & Aim: There have been over 800 registered clinical trials using mesenchymal stem/stromal cells (MSCs) for therapeutic applications. Due to
Background & Aim: Mesenchymal stem cells (MSC) therapy in global regenerative medicine market has grown rapidly in the last decade. However, there are still limitations of MSC-based therapy, such as limited survival of stem cells and delivery approach to clinics. Recently, exosomes of MSC are proposed to be potential alternative to MSC therapy. MSC-derived exosomes are known to have similar therapeutic effects as its parental cells, while equipped with convenience of long term storage and easier laboratory manipulation. On the other hand, toxicological assessments of MSC-derived exosomes remain scarce. Methods, Results & Conclusion: Here, we used mesenchymal stem cell derived exosomes of defined quality to assess acute anaphylactic response in guinea pigs. For sensitization, guinea pigs were subcutaneously administered with low dosage (1 £ 108 particles/head) or high dosage (1 £ 109 particles/head) of MSC-derived exosome for thrice in the period of two weeks. Two weeks after the last exosome administration, animals were challenged by intravenous injection of the exosomes with high dosage (1 £ 109 particles/head) and then their anaphylactic responses were scored. We found
Poster Abstract Presentations that compared to ovalbumin injected positive control group, both low and high dosage injected experimental groups showed mild symptoms of anaphylactic response. We propose that MSC-derived exosomes has little antigenicity at least in guinea pig, which are considered to be biologically safe in humans. 202 FDP-2B:THE NEW GENERATION OF CGMP COMPLIANT LYOPHILIZED PLATELET RICH PLASMA. S. RAJINDRA1 & S. CHOON2 1 Laboratory, StemTech International Sdn.Bhd, Cyberjaya, Selangor Darul Ehsan, Malaysia, 2Department of Orthopaedic Surgery, National Orthopaedic Centre of Excellence for Research and Learning (NOCERAL), University of Malaya, Kuala Lumpur, Malaysia Background & Aim: Platelet Rich Plasma (PRP) is a fraction of peripheral blood by concentration of platelets that generates clinically useful levels of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factors (FGF) and epithelial growth factor (EGF). However, due to intensive time and labour, it can be difficult to prepare an adequate and consistent amount of platelets. Thus, we have generated a new method of PRP which could be isolated before hand and stored for further use. Methods, Results & Conclusion: Fresh PRP with 2 billion platelets/vial and freeze-dried PRP (FDP-2B) which were also stored in 2 billion platelets/vial has been prepared from peripheral blood of 8 healthy human volunteers (300 mL/person) ages 20-50 years old. Every vial of the fresh PRP from each donor were sent for growth factor studies right after the processing from the whole blood while the rest of the vials from each donor were freeze-dried and stored at three different temperatures of 4,-20 and -80°C. The growth factors (PDGF, FGF and EGF) concentration were examined after freezing and tested on day 10th, 1 month, 3 month, 6 month and 1 year post freeze drying. The successful samples processed for storage is 100%. All the three growth factors studied were present in both the fresh PRP(2B) and FDP-2B. However, it was observed that for every sample studied, the lyophilized PRP (FDP2B) shows a significant increase (p<0.05) in all growth factors as compared to the fresh PRP. The results of our study suggest that freeze-drying is an effective technique in preserving PRP bioactivity. Furthermore, we have developed the product in a cGMP environment, taking into account the bacterial and fungal culture testing for each donor in order to improve the safety of the procedure and to ensure it is free from contamination. In conclusion, our results suggest that freeze-dried human PRP maintains the growth factor levels that are comparable to those of fresh PRP(2B), even after the storage with potential clinical value. 205 G-CSF CAUSES DECREASE IN PERIPHERAL BLOOD PLATELET COUNTS UNRELATED TO LEUKAPHERESIS DURING AUTOLOGOUS STEM CELL MOBILIZATION M.S. Bakeer3,2, A. Zubair2 & V. Roy1 1 Hematology-Oncology, Mayo Clinic, Jacksonville, Florida, United States, 2 Mayo Clinic, Jacksonville, Florida, United States, 3Internal Medicine, AlAzhar university, Cairo, Egypt Background & Aim: Autologous transplant utilizing G-CSF mobilized peripheral blood stem cells (PBSC) is an established treatment for various conditions. PBSC collection has been reported to be associated with decrease in platelet counts, attributed to leukapheresis. Whether G-CSF directly affects platelet counts is unclear and studies show conflicting findings. We studied the change in platelet count, prior to start of leukapheresis, in patients receiving G-CSF for PBSC mobilization for autologous stem cell transplant Methods, Results & Conclusion: Myeloma patients undergoing PBSC collection in our institution were retrospectively studied. Patients received 10 mg/kg G-CSF SQ daily. Stem cell collection started on day 4 (after 3 doses of G-CSF) if blood CD34 count on day 4 was at least 10 cells/mL (Good mobilizer). If not (Poor mobilizer), patients received plerixafor and another dose of G-CSF on day 4 followed by collection starting day 5. Complete blood count was measured before G-CSF on day 0 and on days 2,3,and 4 prior to start of leukapheresis. 163 patients were studied, median age 61 yr (39-74), 99(61%) were men. Platelet count (Mean § SE) on day 0 was 230§5.9, which dropped to 192§4.4 (p<0.0001) on day 2, 190§4.6 on day 3, and 163§3.8 on day 4. Platelets decreased to a greater extent in poor mobilizers (n=74) compared to good mobilizers (n=89) (17.9%v11.9% on day 2, p=0.016; 18.2v12.3% on day 3, p=0.034). Platelet decrease was weakly correlated with CD34 count on day 3 (p=0.06). There was no correlation of age, sex, race, BMI or day 0 neutrophil count with day 0 platelet count or decrease in platelet count after G-CSF.
S59
There was no correlation of platelet decline with transplant outcomes - time to neutrophil or platelet engraftment, transfusion requirement, or infections. We show a direct effect of G-CSF on platelet counts unrelated to leukapheresis. The decline is more severe in poor mobilizers. This together with known association of thrombocytopenia with poor mobilization, and expected further drop after leukapheresis is an important consideration when assessing safety and efficacy of G-CSF based mobilization in patient with low platelet count. The mechanism of G-CSF induced thrombocytopenia is likely multifactorial. Possibilities include G-CSF induced differentiation and proliferation of myeloid progenitors at the expense of megakaryocytic lineage, G-CSF induced activation and consumption (Platelets are known to have functional G-CSF receptors), or sequestration from G-CSF induced splenomegaly.
Platelet count at baseline (Day 0) and after treatment with G-CSF
206 FACTORS PREDICTING PERIPHERAL BLOOD STEM CELL (PBSC) MOBILIZATION WITH PLERIXAFOR IN MULTIPLE MYELOMA PATIENTS WHO HAD INADEQUATE MOBILIZATION WITH G-CSF. M.S. Bakeer3,2, A. Zubair2 & V. Roy1 1 Hematology-Oncology, Mayo Clinic, Jacksonville, Florida, United States, 2 Mayo Clinic, Jacksonville, Florida, United States, 3Internal Medicine, Al-Azhar University, Cairo, Egypt Background & Aim: Autologous transplant utilizing G-CSF mobilized peripheral blood stem cells (PBSC) is considered standard practice for eligible multiple myeloma (MM) patients. G-CSF is often utilized as the preferred agent and Plerixafor added if there is inadequate PBSC mobilization with G-CSF alone. To the best of our knowledge, there are no studies predicting the response of the poor mobilizers to plerixafor. Methods, Results & Conclusion METHODS: MM patients undergoing PBSC collection in our institution were retrospectively studied. Patients received 10 mg/kg G-CSF SQ daily. Stem cell collection started on day 4 (after 3 doses of G-CSF) if blood CD34 count on day 4 was at least 10 /mL (Good mobilizer). If not (Poor mobilizer), patients received 240 mg/kg plerixafor SQ and another dose of G-CSF on day 4 followed by collection starting day 5. Complete blood count was measured before G-CSF (day 0) and on days +2, +3, and +4, . and CD34 count on day +5 . Based on day +5 CD34 count patients were categorized into two groups; Above Median (AM) with CD34 count above median or Below Median (BM; CD34 =< median). We analyzed associations of various variables with BM versus AM CD34 category using JMP statistical software. Results: 73 patients were studied. Median age 61 yr, (39-74), 44 (61%) were men. Median CD34 count on day +5 was 41.9/mL, 37 were in BM and 36 in AM groups. Disease duration prior to mobilization strongly correlated with plerixafor response (25.9m v 15.1m in BM v AM, p=0.02) . Mean day 0, day 2 and day 3 platelet count was higher in AM compared to BM (236 v 192,P =0.0075 on day 0, 182 v 148, p=0.0003 on day 2, and 195 v 152 p=0.0001 on day 3). Platelet count dropped from day 0 to day 2 in the whole cohort but
Poster Abstract Presentations
buffer media of different pH-values ranging from pH 8.0 to pH 4.8. Upon processing supernatants of mesenchymal stem/stromal cells (MSCs) cultured in the presence of 10% human platelet lysate, EVs are recovered in a small number of FFE fractions lacking most proteins of the conditioned media. Currently, we characterize the identified fractions in more detail. Prepared EVs are quantified by Nano Particle Tracking Analysis (NTA) and imaging flow cytometry. Furthermore, the presence of several EV markers and the absence of contaminants are analyzed by Western Blot and mass spectrometry-based proteomics. We conclude FFE is a feasible and quick method to highly purify EVs in an accurate manner. 199 INCREASING YIELD OF MSC-EVS IN SCALABLE XENO-FREE MANUFACTURING K. Adlerz, M. Trempel, J.A. Rowley & T. Ahsan RoosterBio, Inc., Frederick, Maryland, United States Background & Aim: Extracellular vesicles (EVs) are increasingly being investigated as both drug delivery vehicles and as a cell-free therapy. EVs from mesenchymal stem/stromal cells (MSC-EVs), for example, have been shown to recapitulate many of the therapeutic effects of the cells themselves. A critical barrier in the development of MSC-EVs as a commercial therapy, however, is generating the large amount of EVs that will be required per dose. In a recent survey, the majority of those working with EVs were working with less than 100mL of sample (ISEV 2016). In this study, two strategies were investigated to maximize EV yield. First, timing parameters for culturing human bonemarrow derived MSCs and collecting MSC-EVs were evaluated to increase EV yield. Second, conditioning the cells was investigated by adding an EV activator during the EV collection period. Methods, Results & Conclusion: MSCs were first cultured in an expansion media (RoosterNourishTM, RoosterBio) in a fed-batch system. The cultures were then switched to a collection media (RoosterCollectTM-EV, RoosterBio) with or without an EV activator (EV BoostTM, RoosterBio). After two days, the conditioned media was harvested. The EVs in the collected conditioned media were analyzed for particle concentration and particle size distributions by nanoparticle tracking analysis, protein expression, RNA expression, and wound healing capability. Particle concentration (particles per L of media) was used as a measure of EV yield. Particle concentration increased with increased culture time in expansion media, with confluent cultures generating the most EVs. The EV activator at a low concentration increased EV yield about 2-fold compared to without the activator after 24hrs. The EV yield from the low EV activator culture at 24hrs was still about 45% greater than the yield of the culture without the activator after 48hrs. After 6hrs of collection, the high EV activator concentration increased EV yield to about the same level as without the activator after 48hrs of collection. Optimizing protocols for EV yield will become increasingly important as MSC-EVs move towards the clinic and lot sizes of clinically-relevant doses are required. Pre-conditioning of cells or conditioning cells during EV collection offers one possibility to further increase EV yield and decrease process time.
their potential as a key bioactive agent in regenerative medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly being investigated as a clinical therapy. It was recently found that the number of exosomes released from 2 million MSCs in 48 hours is equivalent to a single dose for a rodent (Phinney, Pittenger 2017). Hence, most indications would require an MSC production lot size that is hardly achievable in 2D planar cell culture; larger scalable bioreactor systems will be crucial to generate enough EVs for clinical doses. Therefore, this study compared the yield and characteristics of MSC-derived EVs generated in typical 2D culture with those from scalable 3D bioreactor systems. Methods, Results & Conclusion: Human bone marrow-derived MSCs were cultured on microcarriers in 3L and 15L bioreactors. After initial cell inoculation, a bioreactor feed was added on Day 3, and cultures were switched to an EV collection media on Day 4, then cultured for 3 additional days. Samples of the conditioned media were analyzed for particle size and concentration. The collected MSC-EVs were evaluated for protein and RNA expression and wound healing capability. MSC-EVs were compared between bioreactor scales and with a 2D control cultured in a 10-layer cell stack. Higher cell densities were achieved in 3D compared to 2D culture, with similar cell densities in the 3L and 15L bioreactors. The conditioned media was analyzed after the 3-day EV collection period. Particle size distributions were similar across culture systems. The particle concentration in the conditioned media (particles/L media) was similar between the 3L and 15L bioreactors, with the 3D cultures about 3x higher than 2D culture. When particle concentration was normalized to cell number, the productivity (particles/cell) was comparable in the two bioreactor cultures, with 3D cultures greater than 2D. Optimizing EV yield will become increasingly important as EVs are used in the clinic. We have developed a scalable process for MSC-EV generation in a bioreactor. The comparison between 2D and bioreactor cultures demonstrates that a greater EV yield can be accomplished in 3D culture. We intend to continue to scale this process up to production scale (80L) bioreactors, which is critical to generating lot sizes for clinically relevant doses of MSC-EVs.
Histograms, normalized to concentration, show EVs maintained similar size profiles across culture systems. The productivity of MSCs, measured as particles/cell, was higher in bioreactor systems than in 2D culture.
201 ACUTE ANAPHYLAXIS STUDY OF MESENCHYMAL STEM CELL-DERIVED EXOSOMES IN GUINEA PIGS G. Yoo1, A. Lee1, S. Park1, S. Han1, J. Kim1, S. Lee1, S. Na1, M. Yoo1, D. Kim1, Y. Cho2 & K. Moon1 1 Korea Institute of Toxicology, Daejeon, the Republic of Korea, 2Hanyang University ERICA, Ansan, the Republic of Korea
A Parameters for MSC expansion time and EV collection time were investigated in two donors. B The addition of an EV activator to the collection media increased EV productivity, similar particle level to 48hrs without EV activator was achieved at 24 hours with low EV activator and 6hrs with high EV activator. 200 COMPARISON OF MSC-EVS MANUFATURED IN 2D VERSUS SCALABLE 3D BIOREACTOR SYSTEMS K. Adlerz, M. Trempel, D. Wang, R.D. Kirian, J.A. Rowley & T. Ahsan RoosterBio, Inc., Frederick, Maryland, United States Background & Aim: There have been over 800 registered clinical trials using mesenchymal stem/stromal cells (MSCs) for therapeutic applications. Due to
Background & Aim: Mesenchymal stem cells (MSC) therapy in global regenerative medicine market has grown rapidly in the last decade. However, there are still limitations of MSC-based therapy, such as limited survival of stem cells and delivery approach to clinics. Recently, exosomes of MSC are proposed to be potential alternative to MSC therapy. MSC-derived exosomes are known to have similar therapeutic effects as its parental cells, while equipped with convenience of long term storage and easier laboratory manipulation. On the other hand, toxicological assessments of MSC-derived exosomes remain scarce. Methods, Results & Conclusion: Here, we used mesenchymal stem cell derived exosomes of defined quality to assess acute anaphylactic response in guinea pigs. For sensitization, guinea pigs were subcutaneously administered with low dosage (1 £ 108 particles/head) or high dosage (1 £ 109 particles/head) of MSC-derived exosome for thrice in the period of two weeks. Two weeks after the last exosome administration, animals were challenged by intravenous injection of the exosomes with high dosage (1 £ 109 particles/head) and then their anaphylactic responses were scored. We found
Poster Abstract Presentations that compared to ovalbumin injected positive control group, both low and high dosage injected experimental groups showed mild symptoms of anaphylactic response. We propose that MSC-derived exosomes has little antigenicity at least in guinea pig, which are considered to be biologically safe in humans. 202 FDP-2B:THE NEW GENERATION OF CGMP COMPLIANT LYOPHILIZED PLATELET RICH PLASMA. S. RAJINDRA1 & S. CHOON2 1 Laboratory, StemTech International Sdn.Bhd, Cyberjaya, Selangor Darul Ehsan, Malaysia, 2Department of Orthopaedic Surgery, National Orthopaedic Centre of Excellence for Research and Learning (NOCERAL), University of Malaya, Kuala Lumpur, Malaysia Background & Aim: Platelet Rich Plasma (PRP) is a fraction of peripheral blood by concentration of platelets that generates clinically useful levels of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factors (FGF) and epithelial growth factor (EGF). However, due to intensive time and labour, it can be difficult to prepare an adequate and consistent amount of platelets. Thus, we have generated a new method of PRP which could be isolated before hand and stored for further use. Methods, Results & Conclusion: Fresh PRP with 2 billion platelets/vial and freeze-dried PRP (FDP-2B) which were also stored in 2 billion platelets/vial has been prepared from peripheral blood of 8 healthy human volunteers (300 mL/person) ages 20-50 years old. Every vial of the fresh PRP from each donor were sent for growth factor studies right after the processing from the whole blood while the rest of the vials from each donor were freeze-dried and stored at three different temperatures of 4,-20 and -80°C. The growth factors (PDGF, FGF and EGF) concentration were examined after freezing and tested on day 10th, 1 month, 3 month, 6 month and 1 year post freeze drying. The successful samples processed for storage is 100%. All the three growth factors studied were present in both the fresh PRP(2B) and FDP-2B. However, it was observed that for every sample studied, the lyophilized PRP (FDP2B) shows a significant increase (p<0.05) in all growth factors as compared to the fresh PRP. The results of our study suggest that freeze-drying is an effective technique in preserving PRP bioactivity. Furthermore, we have developed the product in a cGMP environment, taking into account the bacterial and fungal culture testing for each donor in order to improve the safety of the procedure and to ensure it is free from contamination. In conclusion, our results suggest that freeze-dried human PRP maintains the growth factor levels that are comparable to those of fresh PRP(2B), even after the storage with potential clinical value. 205 G-CSF CAUSES DECREASE IN PERIPHERAL BLOOD PLATELET COUNTS UNRELATED TO LEUKAPHERESIS DURING AUTOLOGOUS STEM CELL MOBILIZATION M.S. Bakeer3,2, A. Zubair2 & V. Roy1 1 Hematology-Oncology, Mayo Clinic, Jacksonville, Florida, United States, 2 Mayo Clinic, Jacksonville, Florida, United States, 3Internal Medicine, AlAzhar university, Cairo, Egypt Background & Aim: Autologous transplant utilizing G-CSF mobilized peripheral blood stem cells (PBSC) is an established treatment for various conditions. PBSC collection has been reported to be associated with decrease in platelet counts, attributed to leukapheresis. Whether G-CSF directly affects platelet counts is unclear and studies show conflicting findings. We studied the change in platelet count, prior to start of leukapheresis, in patients receiving G-CSF for PBSC mobilization for autologous stem cell transplant Methods, Results & Conclusion: Myeloma patients undergoing PBSC collection in our institution were retrospectively studied. Patients received 10 mg/kg G-CSF SQ daily. Stem cell collection started on day 4 (after 3 doses of G-CSF) if blood CD34 count on day 4 was at least 10 cells/mL (Good mobilizer). If not (Poor mobilizer), patients received plerixafor and another dose of G-CSF on day 4 followed by collection starting day 5. Complete blood count was measured before G-CSF on day 0 and on days 2,3,and 4 prior to start of leukapheresis. 163 patients were studied, median age 61 yr (39-74), 99(61%) were men. Platelet count (Mean § SE) on day 0 was 230§5.9, which dropped to 192§4.4 (p<0.0001) on day 2, 190§4.6 on day 3, and 163§3.8 on day 4. Platelets decreased to a greater extent in poor mobilizers (n=74) compared to good mobilizers (n=89) (17.9%v11.9% on day 2, p=0.016; 18.2v12.3% on day 3, p=0.034). Platelet decrease was weakly correlated with CD34 count on day 3 (p=0.06). There was no correlation of age, sex, race, BMI or day 0 neutrophil count with day 0 platelet count or decrease in platelet count after G-CSF.
S59
There was no correlation of platelet decline with transplant outcomes - time to neutrophil or platelet engraftment, transfusion requirement, or infections. We show a direct effect of G-CSF on platelet counts unrelated to leukapheresis. The decline is more severe in poor mobilizers. This together with known association of thrombocytopenia with poor mobilization, and expected further drop after leukapheresis is an important consideration when assessing safety and efficacy of G-CSF based mobilization in patient with low platelet count. The mechanism of G-CSF induced thrombocytopenia is likely multifactorial. Possibilities include G-CSF induced differentiation and proliferation of myeloid progenitors at the expense of megakaryocytic lineage, G-CSF induced activation and consumption (Platelets are known to have functional G-CSF receptors), or sequestration from G-CSF induced splenomegaly.
Platelet count at baseline (Day 0) and after treatment with G-CSF
206 FACTORS PREDICTING PERIPHERAL BLOOD STEM CELL (PBSC) MOBILIZATION WITH PLERIXAFOR IN MULTIPLE MYELOMA PATIENTS WHO HAD INADEQUATE MOBILIZATION WITH G-CSF. M.S. Bakeer3,2, A. Zubair2 & V. Roy1 1 Hematology-Oncology, Mayo Clinic, Jacksonville, Florida, United States, 2 Mayo Clinic, Jacksonville, Florida, United States, 3Internal Medicine, Al-Azhar University, Cairo, Egypt Background & Aim: Autologous transplant utilizing G-CSF mobilized peripheral blood stem cells (PBSC) is considered standard practice for eligible multiple myeloma (MM) patients. G-CSF is often utilized as the preferred agent and Plerixafor added if there is inadequate PBSC mobilization with G-CSF alone. To the best of our knowledge, there are no studies predicting the response of the poor mobilizers to plerixafor. Methods, Results & Conclusion METHODS: MM patients undergoing PBSC collection in our institution were retrospectively studied. Patients received 10 mg/kg G-CSF SQ daily. Stem cell collection started on day 4 (after 3 doses of G-CSF) if blood CD34 count on day 4 was at least 10 /mL (Good mobilizer). If not (Poor mobilizer), patients received 240 mg/kg plerixafor SQ and another dose of G-CSF on day 4 followed by collection starting day 5. Complete blood count was measured before G-CSF (day 0) and on days +2, +3, and +4, . and CD34 count on day +5 . Based on day +5 CD34 count patients were categorized into two groups; Above Median (AM) with CD34 count above median or Below Median (BM; CD34 =< median). We analyzed associations of various variables with BM versus AM CD34 category using JMP statistical software. Results: 73 patients were studied. Median age 61 yr, (39-74), 44 (61%) were men. Median CD34 count on day +5 was 41.9/mL, 37 were in BM and 36 in AM groups. Disease duration prior to mobilization strongly correlated with plerixafor response (25.9m v 15.1m in BM v AM, p=0.02) . Mean day 0, day 2 and day 3 platelet count was higher in AM compared to BM (236 v 192,P =0.0075 on day 0, 182 v 148, p=0.0003 on day 2, and 195 v 152 p=0.0001 on day 3). Platelet count dropped from day 0 to day 2 in the whole cohort but