Filgrastim, fibrinolysis, and neovascularization
Data files
Jun 19, 2025 version files 11.42 MB
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2025_06_10_DRYAD_Eton_APPENDIX_TABLES.xlsx
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2026_10_Dryad_Tables_from_TERM_publication_by_Eton.xlsx
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FACS.xlsx
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README.md
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Abstract
Objective: Segmental recanalization of chronically occluded arteries was observed in patients with chronic limb-threatening ischemia (CLTI) treated with Filgrastim, a granulocyte colony stimulating factor, every 72 hours for up to a month, and an infra-geniculate programmed compression pump (PCP) for 3 hours daily. Molecular evidence for fibrinolysis and neovascularization was sought.
Methods: CLTI patients were treated with PCP alone (N=19), or with Filgrastim and PCP (N=8 and N=6, at 2 institutions). ELISA was used to measure the plasma concentration of Plasmin and of Fibrin Degradation Products (FDP), and the serum concentration of proteins associated with neovascularization. In the PCP alone group blood was sampled on day 1 (baseline) and after 30 days of daily PCP. In the Filgrastim and PCP group blood was drawn on day 1, and one day after the 5th and the 10th Filgrastim doses. Each blood draw occurred before and after 2 hours of supervised PCP.
Results: Significant (p<0.01) PCP independent increases in the plasma concentration of plasmin (>10 fold) and FDP (>5 fold) were observed one day after both the 5th and the 10th Filgrastim doses, compared to day 1. Significant (p<0.05) increases in the concentration of pro-angiogenic proteins (e.g., HGF, MMP-9, VEGF A) were also observed.
Conclusion: Filgrastim at this novel dosimetry induced fibrinolysis without causing acute hemorrhage, in addition to inducing a pro-angiogenic milieu conducive to NV. Further clinical testing is warranted at this novel dosimetry in CLTI, as well as in other chronically ischemic tissue beds.
Dataset DOI: 10.5061/dryad.b2rbnzsgw
Description of the data and file structure
The key hematologic and ELISA data were obtained in patients treated with Filgrastim and the ArtAssist Device together, and were derived from 2 independent university-affiliated institutions from patients with Chronic Limb Threatening Ischemia (CLTI) recruited from the Vascular Surgery practices at each institution. The control group of hematologic and ELISA data was obtained from patients treated with the ArtAssist Device alone. The goal was to measure hematologic (CD34, VEGFR2) and ELISA evidence supporting clinical evidence of neovascularization (VEGF, HGF, MMP9, PDGF…) in patient serum and of fibrinolysis (plasmin, Fibrin Degradation Products) in patient plasma and serum. Also, in the control group, the goal was to measure evidence of endothelial cell activation by the ArtAssist device, including MCP-1 serum level (ELISA), upregulation of nitric oxide synthase (serum nitrite level), and CD31 (Cytometry). Throughout the datasets, PCP refers to Programmed Compression Pump; the one used was the ArtAssist device
Files and variables
File: 2026_10_Dryad_Tables_from_TERM_publication_by_Eton.xlsx
Description:
Table 1- Rationale for a Novel Neovascularization Strategy. Neovascularization is a natural response to arterial occlusive disease. CLTI arises when neovascularization fails. Our strategy (Column 3) was developed to overcome the obstacles listed in this table. The goal of this strategy is to help restore this natural process.
Table 2- Patient Demographics. Column 1 is a 62 patient cohort treated with PCP alone at UC and does not include the 14 patients treated with PCP and Filgrastim in this study. Columns 2 to 4 represent the patients in whom the assays were performed: PCP alone (N=19), PCP and Filgrastim (N=14). Numbers in parentheses are standard deviation from the mean.
Table 3- Effect of Filgrastim (paired to control data). The relative percent change in concentration was calculated using data from Day 1 (before Filgrastim and PCP) as baseline for each patient. Paired T-Tests using the concentrations were used to derive the P Values. [Plasmin] and [FDP] were measured in plasma at UIC and in serum at UC. The other proteins were measured in serum. P values <0.05 are in bold. Despite the magnitude of increase, some did not reach significance due to the wide standard deviation (SD) in this small population.
Table 4- Effect of PCP alone. The relative percent change in concentration was calculated using data from Day 1, T=0 (before onset of PCP) as baseline for each patient. Paired T-Tests using the concentrations were used to derive the P Values. P values <0.05 are in bold.
Table 5- Filgrastim effect in CLTI (unpaired Group analysis). This is a summary comparison of ELISA concentrations aggregated into 2 groups. Group A: no Filgrastim exposure (includes all Day 1 data on all patients, and Day 30 Data in the PCP alone group). Group B: all data obtained one day after both the 5th and 10th Filgrastim doses. P Values are derived from unpaired T Test of ELISA data. N is the number of ELISA results.
Table 6- Filgrastim effect on circulatory ells in CLTI patients treated with Filgrastim and PCP. Cytometry confirmed the significant percentage increase in CD34+ progenitor and VEGFR2+ endothelial progenitor cells one day after each Filgrastim dose. The Differential blood cell count also shows the percentage increase in white blood cell count (WBC) and neutrophils the day after the 5th Filgrastim dose.
Table 7- Assays and Equipment
File: 2025_06_10_DRYAD_Eton_APPENDIX_TABLES.xlsx
Description:
Table A1- ELISA results for Plasmin in plasma and in serum. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [Plasmin] one day after the 5th and 10th doses (referenced to Day 1) at T=0 and T=2 hours (P values highlighted). Two hours of PCP had no significant impact. P008, 1403, and 1406 had recent thromboses, with an active fibrinolytic cascade on Day 1.
Table A2- ELISA results for FDP in plasma and in serum. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [FDP] one day after the 5th and 10th doses (referenced to Day 1) at T=0 and T=2 hours (P values highlighted). Two hours of PCP had no significant impact. P008, 1403, and 1406 had recent thromboses, with an active fibrinolytic cascade on Day 1.
Table A3- ELISA results for serum Hepatocyte Growth Factor (HGF). P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [HGF] one day after the 5th and 10th doses (referenced to Day 1) at T=0 and T=2 hours (P values highlighted). Two hours of PCP had no significant impact.
Table A4- ELISA results for serum [MMP-9], diluted 100-fold. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [MMP-9] one day after the 5th and 10th doses (referenced to Day 1) at T=0 and T=2 hours (P values highlighted). Two hours of PCP had no significant impact.
Table A5- ELISA results for serum VEGF-A. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [VEGF=A] one day after the 5th and 10th doses (referenced to Day 1) at T=0 and T=2 hours (P values highlighted) in the UIC group. Two hours of PCP had no significant impact. Patient 1406 had the lowest Filgrastim dose (6.6 mcg/kg), and 1407 had significantly elevated [VEGF-A] on Day 1, dampening the percent change after Filgrastim.
Table A6- ELISA results for serum Angiopoietin-1. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [Angiopoietin-1] by the 10th dose (referenced to Day 1) at T=0 and T=2 hours (P values highlighted). The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A7- ELISA results for serum PDGF-AA. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [PDGF-AA] by the 10th dose (referenced to Day 1) at T=0 and T=2 hours (P values highlighted). The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A8- ELISA results for serum PDGF-BB. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [PDGF-BB] by the 10th dose (referenced to Day 1) at T=0 and T=2 hours (P values highlighted). The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A9- ELISA results for serum PDGF-AB. P values (paired TTest) were derived from the ELISA concentrations. [PDGF-AB] increased after the 10th Filgrastim dose (referenced to Day 1) at T=0 (p=0.08) and T=2 hours (p=0.058), but the increase did not reach the significance threshold, likely due to the large SD and small N. The trend, however, is evident. The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A10- ELISA results for serum TNF-alpha. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [TNF-alpha] by the 10th dose (referenced to Day 1) at T=0 and T=2 hours (P values highlighted). The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A11- ELISA results for serum TGF-beta. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [TGF-beta] by the 10th dose (referenced to Day 1) at T=2 hours (P value highlighted). The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A12- ELISA results for serum IGF-1. P values (paired TTest) were derived from the ELISA concentrations. Filgrastim significantly elevated [IGF-1] by the 10th dose (referenced to Day 1) at T=0 in the UC dataset only (P value highlighted). The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A-13- ELISA results for serum PLGF. P values (paired TTest) were derived from the ELISA concentrations. [PLGF] increased after the 10th Filgrastim dose (referenced to Day 1) at T=0 (p=0.1) and T=2 hours (p=0.5), but the increase did not reach the significance threshold. The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A-14- ELISA results for serum IL-6. P values (paired TTest) were derived from the ELISA concentrations. [IL-6] increased after the 10th Filgrastim dose (referenced to Day 1) at T=0 (p=0.1) and T=2 hours (p=0.5), but the increase did not reach the significance threshold. The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A-15- ELISA results for MCP-1. P values (paired TTest) were derived from the ELISA concentrations. [MCP-1] decreased after the 10th Filgrastim dose (referenced to Day 1) at T=0 (p=0.1) and T=2 hours but the decrease did not reach the significance threshold. The percent change from 2 hours of PCP was not significant on any of the days (P>0.05).
Table A-16- PCP alone for 2 hours on Day 1 led to a 19%+32% increase (p=0.006) in serum [MCP-1] measured by ELISA (pg/ml). Values are listed prior to PCP (T=0) and after 2 hours of supervised PCP (T=2HR). (SD=Standard deviation)
Table A-17- Baseline cytometry on Day 1 for the 19 patients treated with pump alone.
Table A-18- CD31+, CD34+, and VEGFR2+ Cell Fractions (10,000 cells) in each patient treated with Filgrastim and PCP. The percentage increase due to PCP (T=2HR vs T=0 on each Day) and to Filgrastim (Referenced to Day 1 data) is tabulated. SD=Standard Deviation
Table A-19- Nitrite (nM/ml) progressively increased during 30 days of PCP alone, 3 hours daily. On Days 1 and 30, nitrite increased after 2 hour PCP. This assay does not distinguish between NO generation in patients produced by inflammatory cells (iNOS) versus NO produced from the endothelium (eNOS).
Table A-20- Nitrite (nM/ml) progressively increased in patients receiving Filgrastim over the 10 dose course, and after 2 hour PCP sessions. [Nitrite] for each patient are listed prior to PCP (T=0) and after 2 hours of supervised PCP (T=2HR). Note the significant P values. (SD=Standard deviation)
Table A-21- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [Plasmin]
Table A-22- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [FDP]
Table A-23- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [HGF]
Table A-24- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [MMP-9]
Table A-25- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [VEGF-A]
Table A-26- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [Angiopoietin-1]
Table A-27- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [IGF-1]
Table A-28- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [PDGF-AA]
Table A-29- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [PDGF-BB]
Table A-30- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [PDGF-AB]
Table A-31- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [MCP-1]
Table A-32- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [TGF-beta]
Table A-33- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [TNF-alpha]
Table A-34- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [PLGF]
Table A-35- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [IL-6]
File: FACS.xlsx
Description:
Sheet 1: Appendix
TABLE 1- EFFECT OF 2 HOURS OF PCP ON SERUM PROTEINS ON DAY 1 AND ON DAY 30. Each entry is the percent increase in the serum concentration of a given protein after 2 hours of PCP on the specific day. Statistically significant elevation in MCP-1 and IL-6 were obseserved after two hours of PCP on Day 1 only.
TABLE 2- EFFECT OF 30 DAYS OF PCP (THREE HOURS DAILY).The percent increase in the serum concentration of a given protein at 30 days (referenced to Day 1) is tabulated before (T=0) and after 2 hours of PCP (T=2hrs). Statistically significant elevation in TNF-alpha were observed on Day 30 compared to Day 1 before starting the 2 hour PCP session.
TABLE 3- EFFECT OF 2 HOURS OF PCP ON SERUM PROTEINS IN THE FILGRASTIM+PCP group (UC data). Column headings “DAY 1”: Prior to Filgrastim, “5th dose”:1 day after the 5th dose of Filgrastim, “10th dose”: 1 day after the 10th dose of Filgrastim. Each entry is the percent increase in the serum concentration of a given protein after 2 hours of PCP on the specific day. Two hours of observed PCP did not statistically influence the concentration of these proteins.
TABLE 4- EFFECT OF FILGRASTIM ON SERUM PROTEINS IN THE FILGRASTIM+PCP group (UC data). Column headings: “5th dose”: 1 day after the 5th dose of Filgrastim, “10th dose”: 1 day after the 10th dose of Filgrastim. Each entry is the percent increase in the serum concentration of a given protein at start (T=0), and at end of 2 hours of PCP (T=2hrs) on each of those days. Percent in Columns B and C are relative to T=0 and T=2hrs on Day 1. The P values were derived from paired TTest comparing the ELISA concentrations before and after PCP use one day after the 5th FIlgrastim Dose (“B”) and one day after the 10th Filgrastim dose (“C”) to the Day 1 ELISA concentrations before and after PCP use.. Statistically significant elevation in Plasmin, FDP, HGF,MMP-9 were observed on both days. Statistically significant elevation in PDGF-AA, PDGF-BB, IGF-1, TGFbeta, and TNF-alpha were observed at one time point. Statistically significant drop in MCP-1 was observed at one time point prior to starting the 2 hour PCP session.
TABLE 5- EFFECT OF 2 HOURS OF PCP ON SERUM PROTEINS IN THE FILGRASTIM+PCP group (UIC data) ON DAY 1, 14 AND 29. Each entry is the percent increase in the serum concentration of a given protein after 2 hours of PCP on the specific day. Two hours of observed PCP did not statistically influence the concentration of these proteins. Pvalues were derived from the the paired TTest comparing ELISA concentrations before and after PCP use on each Day.
TABLE 6- EFFECT OF FILGRASTIM ON SERUM PROTEINS IN THE FILGRASTIM+PCP group (UIC data). Each entry is the percent increase in the serum concentration of a given protein at start (T=0), and at end of 2 hours of PCP (T=2hrs) on each of those days. Percent on Days 14 and 29 are relative toDay 1, at T=0 and T=2hrs. The P values were derived from paired TTest comparing the ELISA concentrations before and after PCP use on Day 14, and Day 29, to Day 1 ELISA concentrations before and after PCP use. Statistically significant elevation in Plasmin, FDP, HGF,VEGF-A, and MMP-9 were observed on both days.
TABLE 7- EFFECT OF PCP ON SERUM NITRITE. The first 2 columns are percent nitrite increases on Day 1 and Day 30 before and after 2 hours of PCP derived from the ELISA concentrations before and after 2 hours of PCP on each day. The Pvalues are derived from the T-test comparing the ELISA concentrations. The 3rd and 4th columns show the cumulative effect of 30 days of PCP. Each entry is the percent increase in serum nitrite before and after 2 hours on PCP on Day 30 relative to before after 2 hours on PCP on Day 1. The Pvalues are derived from the T-test comparing the ELISA concentrations.
TABLE 8- SERUM NITRITE IN PATIENTS TREATED WITH FILGRASTIM AND PCP. The first 3 columns show the effect of 2 hours of PCP 1)prior to Filgrastim (“DAY 1”), 2) One day after the 5th dose of Filgrastim (“B”), and 3) One day after the 10th dose of Filgrastim (“C”). Each entry in the first 3 columns is the percent increase in serum nitrite after 2 hours of PCP. The P values are derived from a paired TTest of the serum nitrate values from blood drawn before and immediately after 2 hours of PCP. The last 4 columns show the effect of FIlgrastim relative to Day 1, both before and after 2 hours of PCP. The P values are derived from a paired TTest of the serum nitrate values from blood drawn on Day 1 and on Day 30 before and immediately after 2 hours of PCP.
TABLE 9-CYTOMETRY IN PATIENTS TREATED WITH PCP ALONE
TABLE 10-CYTOMETRY IN PATIENTS TREATED WITH FILGRASTIM AND PCP
TABLE 11-CYTOMETRY IN PATIENTS TREATED WITH FILGRASTIM AND PCP. Percent increase in CD31, CD34, and VEGFR2 cell counts after 2 hours of PCP use on Day 1, and 24 hours after the 5th and 10th doses of Filgrastim. The pValues are derived from the paired TTests compairing cell counts at T=0 to those at T=2 hours). The results show small but significant increases in circulating CD31, CD34 and VEGFR2.
TABLE 12-CYTOMETRY IN PATIENTS TREATED WITH FILGRASTIM AND PCP. The influence of Filgastim on the percent increase in CD31, CD34, and VEGFR2 cell counts 24 hours after the 5th (Column B) and 10th (Column C) doses of Filgrastimare tabulated (relative to Day 1 prior to Filgrastim dose). Entries were derived from the nitrite in blood drawn before (T=0) and then after 2 hours of PCP (T= 2 hrs) on each day and compared to the same times on Day 1. The pValues are derived from paired TTests compairing cell counts at T=0 to those at T=2 hours on each day. Significant increases in circulating CD31, CD34 and VEGFR2 were identified.
Sheet 2: APPENDIX TABLES
Tabbles: PCP ALONE ELISA TABLES, FILGRASTIM + PCP ELISA TABLES, CYTOMETRY PCP ALONE, CYTOMETRY FILGRASTIM + PCP, NITRITE: PCP ALONE, NITRITE (nM) FILGRASTIM + PCP, and PCP alone for 30 days.
Sheet 3: FACS
Fluorescence Activated Cell Sorting plots for CD31, CD34, VEGFR2 for each patient at enrollment and then one day after the fifth and one day after the 10th Filgrastim dose. These data are from the university-affiliated institution in patients who received Filgrastim.
Human subjects data
The human data obtained from the University affiliated hospitals were obtained and published with each patient’s consent.
Population: CLTI patients who were not candidates for, or had failed, previous invasive revascularization procedures.
Inclusion criteria: Patients complaining of ischemic forefoot rest pain, gangrene, and/or ischemic ulceration were enrolled if the ankle brachial arterial index was <0.45.
Exclusion criteria: Acute limb ischemia, non-salvageable extremity, untreated hypercoagulable disorder, sickle cell disease, myeloproliferative disorder, dialysis, creatinine above 3.5 mg/dl, active cancer, dementia, non-compliance, intolerance of PCP, body mass index over 34, venous stasis ulcer, history of lymphoma or leukemia, uncorrected symptomatic coronary artery disease, severe carotid stenosis, sepsis proximal to the forefoot, allergy to Filgrastim.
Patient groups: Table 2 lists the patient characteristics. Blood assays were performed on 2 patient groups. The first group was treated at a university-affiliated institution (UC, 2012-13) with PCP alone (N=19). The second group (N=14) was treated with PCP and Filgrastim: six at another independent university-affiliated institution (UIC, 2016-19) and eight at a hospital (2014-15).
Filgrastim: Each patient was to receive Filgrastim subcutaneous injections, one every 72 hours at approximately 10 mcg/kg for up to ten doses. Filgrastim (Neupogen) was purchased from Amgen Inc (Thousand Oaks, CA) in 480 and 300 mcg vials. These were kept refrigerated between 2°C and 8°C and stored in the dark. Small losses of agent occurred during administration, which typically required two separate injections in the subcutaneous tissue in the lower abdomen. Fourteen patients received 5 doses. Ten of the 14 received all 10 doses. Mobilized progenitor cells circulated until either being engrafted or otherwise cleared from the circulation. The leukocyte count returned to normal approximately every 72 hours. The product label for Filgrastim lists progenitor cell mobilization as an indication (not CLTI). The FDA granted a waiver to use Filgrastim at the novel dosimetry in CLTI patients at both institutions. Filgrastim has been safely used in healthy patients for the purpose of bone marrow harvest of mobilized progenitor cells.[i] Oncologists have nearly 3 decades of experience with Filgrastim. Initial concerns over promoting tumor angiogenesis were not substantiated [ii]. While severe complications are uncommon, they can be significant (e.g., capillary leak syndrome, myocardial infarction, and splenic rupture). Hemorrhage is not a reported side effect; fibrinolysis and NV are not listed on the Filgrastim label.
PCP: The ArtAssist device (ACI Medical LLC, San Marcos CA) applies sequential pressure to the calf, and foot. PCP was used at home on both legs in the seated position for 3 hours daily. Rapid inflation of pneumatic cuffs (0 to 120 mmHg in <0.3 sec) provides an endothelial shear stress stimulus, while at the same time driving in oxygenated nutritive blood flow and facilitating venous return (Table 1). The pressure is held in each of the cuffs for three seconds. Rapid deflation follows. Three cycles occur per minute.
Phlebotomy: Group 1 patients were treated with PCP alone, and had blood drawn on Day 1 and Day 30. Group 2 patients were treated with Filgrastim and PCP and had three blood samples drawn: on Day 1, on the day after the 5th Filgrastim dose, and on the day after the 10th Filgrastim dose. In both Groups, on each day, blood was drawn using a 21- gauge butterfly inserted before and again after 2 hours of observed PCP. Blood was collected into serum separator and EDTA plasma separation tubes. Specimens were immediately transported on ice to the independent laboratories at the university-affiliated institutions.
Serum separation: Whole blood was collected in serum separator tubes. Following clot formation (30-60 min) each tube was centrifuged for 15 min at 1000g. Serum aliquots were stored at -80°C in labeled cryovials (to avoid repetitive freeze/thaw) until batch analysis.
Plasma separation was used for preparation of samples for ELISA, and for cytometry. Whole blood was collected in EDTA-treated tubes. Red cells and platelets were removed after centrifugation at 2,000g for 20 minutes at 4oC. Plasma aliquots were stored at -80°C.
Enzyme-linked immunosorbent assay (ELISA): Protein concentrations were measured in duplicate in plasma or in serum using Human ELISA Kits according to each kit’s instructions (Table 7). Plasmin and Fibrin Degradation Products (FDP) were measured in plasma at UIC and in serum at UC to assess fibrinolysis. Proteins associated with NV included HGF[iii], VEGF-A[iv], MMP-9[v], Angipoietin-1 [vi],PDGF-AA, PDGF-BB, PDGF-AB [vii],TNF-a[viii], MCP-1[ix] and TGF-b[x], PLGF[xi], IL-6[xii], and IGF-1 [xiii]. All patient-specific samples were analyzed together using a single ELISA kit.
Cytometry: Mature endothelial CD31+ (PECAM-1), progenitor CD34+, and endothelial progenitor CD309+ (VEGFR2+) cell populations were measured (Table 7). White Blood Cell (WBC) and differential counts were obtained on plasma in the hospital hematology laboratories.
Serum nitrite: Serum nitrite, a breakdown product of nitric oxide (NO), reflects nitric oxide synthase (NOS) activity. It was measured before and after 2 hours of supervised PCP with a quantitative fluorometric assay based on the reaction of nitrite with 2,3- diaminonaphthalene under acidic conditions to form fluorescent 1-(H)-naphthotriazole (Table 7).
Statistics: Each patient had 2 internal controls. The first internal control was obtained on Day 1, prior to PCP or Filgrastim. Changes resulting from daily PCP use for 30 days (Group 1) or caused by Filgrastim (Group 2) were referenced to data from Day 1. The second control was obtained on arrival (at T=0) to each 2 hour observed PCP session, and was used to ascertain the effect of PCP (at T=2HR) on Day 1 for all patients, on Day 30 for the PCP alone group, and on the day after the 5th and the 10th doses of Filgrastim in the Filgrastim+ PCP group. Percent changes are reported relative to each of these two controls (change from control divided by the control). The paired Student t-test was used to estimate a P value directly from the ELISA data (not from the percent changes). An unpaired T-Test was used when comparing the aggregated ELISA data in all patients obtained before any Filgrastim exposure to all data obtained after Filgrastim was given. P values <0.05 are considered significant. The significant findings in the UC study led to the confirmatory laboratory study at UIC in 6 CLTI patients. The UIC sample size was calculated from the UC data to provide 80% power to detect changes in protein levels of 0.66 standard deviations assuming a two-sided significance level of 0.05.
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Guide
- Brackets: “[protein]” mean “concentration of” that protein
- T=0: first blood draw on a particular day
- T=2HR: blood draw after 2 hours of PCP
- Patients 1201-1219: PCP alone at a university-affiliated institution: Table Gaps: two patients stopped PCP before 30 days.
- Patients 1401 to 1408: Filgrastim+PCP at a hospital, with all assays done at a university-affiliated institution. Table Gaps: two patients stopped Filgrastim at 5 doses (1404 due to transfer out of state, and 1408 due to resolution of rest pain), and one at 7 doses (1406 due to intolerance of the PCP); one did not store the filgrastim correctly (1401) and had to start over, and one stopped at 5 doses only to resume several months later (1407).
- Patients P003-P009: Filgrastim+PCP at a university-affiliated institution: Table Gap: P009 had only one blood draw on last day (T=0): did not wear PCP on that day.