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G-CSF is used for increasing cocentrations of circulating HSC. In 1995, 3 pivotal studies demonstrated the safety and feasibility of using G-CSF mobilized PB allografts. Patients experienced prompt engraftment with an incidence of GvHD similar to that of BM recipients. In addition, no serious short-term complication of G-CSF mobilized PB harvesting were observed in the donors.
During MNC procedure, anticoagulated whole blood enters the inlet chamber through the inlet tube. As the blood flows through the channel, the system separates it into three layers: RBC on the outside, buffy coat containing WBC in the center, and platelet-rich plasma on the inside. The system establishes the RBC plasma interface during Quick Start. After Quick Start, the operator adjusts the plasma pump flow rate to hold the interface in a constant position. The system draws the MNC from the channel through the WBC collect tube, while the platelet-rich plasma exists through the plasma tube. The RBC exit through the RBC tube. During Spectra AutoPBSC procedure, anticoagulated whole blood enters the first stage of the channel through the inlet tube. In the first stage, the system separates the RBC and WBC from the platelet-rich plasma. The RBC and granulocytes exit the channel through the RBC tube. Platelet-rich plasma flows over the dam into the second stage where the system concentrates the platelets in the plasma. The plasma exit through the collect tube to return to the donor, and the remaining plasma flows through the channel to the plasma tube. MNC accumulate above the layer of RBC. During the Harvest phase, the MNC flow over the dam into the second stage. Once the collect concentration monitor (CCM) detects cells in the collect line, the collect valve opens and the MNC flow to the collection bag. The Chase phase follows the Harvest phase during which plasma "chaces" the MNC in the collect line up to the collection bag. Concurrent collection of a specific plasma volume is optional. The system determines the plasma and collect pump flow rate based on the donor/ patient hematocrit, and maintains the interface postion. A small volume of plasma and RBC flow into the control tube to help maintain the interface. Common apheresis complications: citrate toxicity (hypocalcemia, hypomagnesaemia, hypokalaemia, metabolic alkalosis), thrombocytopenia, hypovolemia, catheter malfunction, infection. The collected stem cell products are maintaining in liquid nitrogen at -196ËšC until the time of patients transplantation.
-- DMSO 10% + Albumine 4% or HES 6% for programmable freezing;
-- DMSO 5% + HES 6% for freezing at -80ËšC; -- DMSO 10% + NaCl + autoFFP/ACD.
Reactions to DMSO:
-- Common: nausea, vomiting, abdominal cramping, headache, garlic aftertaste;
-- Rare: hypotension, rapid heart rate, shortness of breath, fever, neurologic complications.
There are 2 possibilities of thawing: with flushing out or without flushing out. The flushing out procedure must be done immediately after thawing, without any delay, for the purpose of limiting the toxicity of DMSO. This procedure consists of: spin over (2000 rpm), removal of the supernatant liquid and admixture to the remaining cells of a solution composed of 10% ACD + 2% Albumine. The tawing procedure without flushing out: we out each frozen bag into a bath of water at 37ËšC and we shake it with gentile movements, 2 minutes until it is completely defrosen. A number of 86 procedures of hematopoietic stem cells (HSC) harvest and cryopreservation from 64 volunteer donors, 54 adults (28 women and 26 men) and 10 children (5 girls and 5 boys) with ages between 6-66 years (on average 30.5) were carried out in the Bone Marrow Transplant Center from Clinical Institute Fundeni, Bucharest. HSC mobilization from the bone marrow into the peripheral blood was realized by the subcutaneous administration of Neupogen (Filgastrim, G-CSF). We counted the WBC and the number of CD34+ cells from the peripheral blood, beginning the day +4 from the mobilization regimen. HSC from peripheral blood were harvested by leukapheresis procedure with the help of discontinuous flow separators (Haemonetics MCS plus) and continuous flow separators (Cobe Spectra), autoPBSC procedure and MNC procedure, by treating several total blood volumes (SVL method - Standard Volume Leukapheresis and LVL method - Large Volume Leukapheresis). A number of 24 procedures were carried out with Haemonetics separator (16 donors) and 62 procedures with Cobe Spectra separator (48 donors). ACD-A was the anticoagulant agent that we used with 1:9 ratio (Haemonetics separator) and 1:12 ratio respectively (Cobe Spectra separator). HSC harvested were than combined with a cryopreservation solution (DMSO) at a final concentration of 10% (DMSO 10%). The freezing procedure was realized with the help of nitrogen liquid programmable freezer MiniDigitCool. The graft quality control was done as well before freezing procedure (native product) as after the freezing procedure (cyiotubes and cryocites bags). There have been done the following tests: total blood count, blood smear, total number of CD34+ cells, cells viability (with tripan blue), number of CFU-GM). The HSC graft was thawed in a water bath at +37ËšC by smooth movements, immediately followed by the infusion to the patient. HSC mobilization was achieved for all the 64 volunteer donors by administration of Filgastrim, on an average 8.4 mcg/donor weight (limits: 5-16.64 mcg/donor weight), leukapheresis procedure being realized in day +5 of Filgastrim administration. We started leukapheresis procedure at a WBC level in peripheral blood of 45.9 Ã- 109/L (limits 5.1-72.1 Ã- 109/L) and a CD34+ cells level in peripheral blood of 101.5 Ã- 106/L (limits 16.2-112.8 Ã- 106/L). The optimal dose of stem cells CD34+ was achieved at 45 donors by a single leukapheresis procedure and at 18 donors by two leukapheresis procedures. There have been achieved 59 SVL procedures and 27 LVL procedures; we treated on average 2.98 total blood volumes (limits 1.8-4.2) in 331.4 minutes (limits 159- 865 minutes). The medium number of CD34+ cells harvested/procedure was 7.5 Ã- 106/L (limits 0.29-52.1 Ã- 106/L) and the medium number of CD34+ cells harvested/patient was 9.5 Ã- 106/L (limits 1.3-52.1 Ã- 106/L). Cells viability obtained with the direct test was 97.6% (limits 87-100%) and cells viability after the mixing with DMSO solution was 71.4% (limits 30-100%). In vitro testing of clonogenic capacity of progenitor cells showed on average 529.9 Ã- 104 CFU-GM/body weight/sample (limits 34-3435 Ã- 104). The tests performed after the graft thawing showed the following results: cells viability 55.8% (limits 10-84%) in 71 collections; number of CD34+ cells 4.4 Ã- 106/ patient body weight (limits 0.66-21.98) in 12 collections; number of CFU-GM: 98.5 Ã- 104/patient body weight (limits 0-635) in 54 collections. We performed allogeneic transplant with the source of stem cells from the peripheral blood in 50 patients; the graft that was administrated was formed on average by 5.6x106 CD34+ cells/patient body weight (limits 2.11-15.09 x106/patient body weight). In conclusion, a healthy volunteer donor, will undergo in most cases 4 or 5 days of Filgastrim administration. The WBC and the number of CD34+ cells from the peripheral blood will be counted beginning with the 4th day. When the number of CD34+ cells from peripheral blood will reach a certain level (usually on the 4th or 5th day), the volunteer donor will be sent to the apheresis unit for harvesting stem cells. 1 or 2 sessions of apheresis will be enough for collecting the total amount of stem cells for a graft. The apheresis session will take on average 4 hours (if it will be used MNC procedure) or 5 hours (if it will be used auto PBSC procedure). HSC will be combined with a cryopreservation solution; then the graft will be stored in liquid nitrogen at -196ËšC degree until the patient will be transplanted. When it will be needed, the graft will be thawed in a water at +37ËšC by smooth movements, immediately followed by its infusion to the patient.