tap_990

579..586

Therapeutic Apheresis and Dialysis 15(6):579586 doi: 10.1111/j.1744-9987.2011.00990.x 2011 The Authors Therapeutic Apheresis and Dialysis 2011 International Society for Apheresis

Mobilization and Harvesting of Peripheral Blood Stem Cells in Pediatric Patients With Solid Tumors
Dobrila Veljkovic, Dragana Vujic, Olivera Serbic Nonkovic, Dragana Jevtic, Zeljko Zecevic, and Emilija Lazic
Department of Transfusion Medicine and Bone Marrow Transplantation Unit, Institute for Mother and Child upic Health Care of Serbia Dr Vukan C , Belgrade, Serbia

Abstract: Survival of patients with high-risk pediatric solid tumors has improved with the introduction of a high-dose chemotherapy regimen and autologous stem cell rescue. Here, we present our data regarding the evaluation of the efcacy and safety of hematopoietic stem cell mobilization and harvesting in children with solid tumors. From November 2002 to March 2010, 85 children underwent autologous peripheral blood stem cell collection; 35 (41.1%) of them weighed less than 20 kg and were diagnosed with neuroblastoma, Wilms tumor, medulloblastoma, yolk sac sarcoma, or non-Hodgkins lymphoma. The mobilization regimens included disease-specic chemotherapy plus granulocyte colony-stimulating factor in most of the patients. The median age and weight at the time of apheresis was 36 months and 13.5 kg, respectively. Large-volume leukapheresis was performed with the aim of reducing the psychological and nancial impact of leukapheresis by

reducing the number of procedures while collecting a large number of cells. The median number of mobilization and leukapheresis procedures per case was one. The preapheresis CD34+ cell count ranged from 2 to 845 mL, with a median of 24 mL. A median of four patient blood volumes was processed per procedure, lasting 279 min (range, 113 420 min). A radial catheter was used for harvesting in 35 procedures (71.4%). The median yield of CD34+ cells was 6.6 106/kg per patient. The targeted dose of 5 106/kg CD34+ cells was realized in 80% of patients. The tolerance of peripheral blood stem cell collection in our patients was good. In conclusion, the collection of peripheral blood stem cells is an effective and safe procedure, even when conducted on the youngest children. Key Words: Children, Harvesting, Mobilization, Pediatric, Peripheral blood stem cell, Tumor.

High-dose chemotherapy with stem cell rescue has been shown to improve event-free survival for children with solid tumors, including high-risk neuroblastoma, Wilms tumor, relapsed or refractory lymphoma, retinoblastoma, high-risk Ewings sarcoma, and medulloblastoma (13). In addition, peripheral blood stem cells (PBSC) have become the preferred source of cells for autologous transplantation because they are easily accessible with a shortened time to engraftment and immunological reconstitution, have a lower tumor contamination

Received March 2011; revised May 2011. Address correspondence and reprint requests to Dr Dobrila Veljkovic, Department of Transfusion Medicine, Mother and Child upic Health Care Institute of Serbia Dr Vukan C , Radoja Dakica 6-8, 11070 Novi Beograd, Srbija. Email: imdtransfuzija@yahoo.com

risk, and there is no need for surgical procedures to be performed (4,5). PBSC collection using an automated or semiautomated blood cell separator in low body weight pediatric patients is much more complicated and risky than collection in older children and adults. The difference involves technical problems, such as hemodynamic and metabolic disturbances due to an extracorporeal volume of blood that is greater than 25% of the total blood of the patient because of the low body mass of these children. However, the maintenance of adequate vascular access providing a sufcient blood ow rate is difcult because of the small size of their blood vessels. The ow capacity of the permanent implanted central venous catheter (CVC) is usually not sufcient for leukapheresis and successful collection. Systemic anticoagulation with citrate is problematic because small children are very sensitive to hypocalcemia. In addition, compliance of the child 579

580

D Veljkovic et al. Hematopoietic stem cell mobilization In total, 41 stem cell mobilization procedures were performed on 35 patients. For 29 patients with highrisk neuroblastoma, we performed 35 mobilizations; for 27 of these mobilization procedures, we used the regular cycle of the COJEC protocol (vincristine 1.5 mg/m2 13 day, etoposide 175 mg/m2 13 days and cyclophosphamide 1050 mg/m2 12 days or vincristine 1.5 mg/m2 and cisplatin 80 mg/m2 on day 1). Following disease-based chemotherapy, once-daily subcutaneous injections of recombinant granulocytecolony stimulating factor (G-CSF) (Neupogen; Amgen, Thousand Oaks, CA, USA) at a dose of 10 mg/kg/day were continued through to the last day of PBSC collection. For additional stem cell mobilization in three patients, we used G-CSF at a dose of 10 mg/kg/day. In two patients with unsuccessful mobilization after chemotherapy and G-CSF at a dose of 10 mg/kg/day, a second mobilization was performed with G-CSF at a dose of 10 mg/kg twice per day. For one patient, we used cyclophosphamide 4000 mg/m2 and G-CSF, and for two patients, a G-CSF dose of 10 mg/kg/day was used. The ICE protocol (ifosfamide 1200 mg/m2 on days 15, carboplatin 400 mg/m2 on days 12, and etoposide 1000 mg/m2 on days 15) and G-CSF were used in children with yolk sac sarcoma and Wilms tumor (four mobilizations), and for patients with B-cell non-Hodgkins lymphoma (dexamethasone 10 mg/m2 15 days, cyclophosphamide 200 mg/m2/ day 15, adriamycin 25 mg/m2 on days 4 and 5, and vincristine 1.5 mg/m2 for one day) and medulloblastoma (carboplatin 200 mg/m2 14 days and etoposide 100 mg/m2 14 days) (2). These mobilization protocols lead to an increase in leukocytes after a passing aplasia on approximately 10 day after the start of therapy. The time from the beginning of mobilization with chemotherapy and G-CSF to PBSC collection for our patients was between 8 and 21 days (median 13 days) and between 4 and 6 days with G-CSF. In the hematopoietic recovery phase, the complete blood count and the number of CD34+ cells in the peripheral blood (PB) were determined, and PBSC collections were performed when the number of CD34+ cells in the peripheral blood was 20 mL in most patients or 10 mL in poor mobilizers. If the targeted dose of CD34+ cells was not collected, we would repeat the collection procedures the next day. Hematopoietic stem cell collection All of the procedures were performed using the regular program for mononuclear cell collection on the semi-automated separator with continual blood ow (Cobe Spectra, version 6.1; Gambro BCT, Lake 2011 The Authors Therapeutic Apheresis and Dialysis 2011 International Society for Apheresis

during the procedure is usually limited (6). Despite these limitations, several groups have concluded that PBSC collection can be performed in small children with low body weight when the necessary precautions are taken (79). In this study, we present our data regarding the evaluation of the feasibility, efcacy, and safety of PBSC collection in 35 pediatric oncology patients at our center, who weighed less than 20 kg. PATIENTS AND METHODS Patients The patients included 35 children (26 boys and nine girls) who weighed 20 kg or less when they underwent leukapheresis for the harvesting of mobilized PBSC. The median age of the patients was 36 months (range, 675 months), and the median body weight was 13.5 kg (range, 719 kg). The children were treated for neuroblastoma (29 patients), yolk sac sarcoma (two patients), Wilms tumor (two patients), medulloblastoma (one patient), and B-cell non-Hodgkins lymphoma (one patient). The time between diagnosis to hematopoietic stem cell (HSC) mobilization and collection was 325 months (median 5 months). The youngest patient was a 182day-old infant with a body weight of 7 kg who had high-risk neuroblastoma. The diagnosis and primary treatment of solid tumors was performed in the pediatric hematooncology centers in Serbia: Mother and Child upic Health Care Institute of Serbia Dr Vukan C in Belgrade, University Childrens Hospital in Belgrade, Institute for Child and Youth Health Care in Novi Sad, University Childrens Clinic in Nis, and the Childrens Department of the Institute for Oncology and Radiology of Serbia. The mobilizations and collections of PBSC were conducted between November 2002 and March 2010. Leukapheresis was performed at the section for apheresis within the Department for Transfusion Medicine, Mother and Child Health Care Institute of Serbia upic Dr Vukan C . The patients were sorted in accordance with internationally accepted criteria into a high-risk group of either progressing or recurring diseases. The mobilization of the HSC was started at one of the regional centers or the children were referred to the National Center for Pediatric HSC Transplantation in our hospital for HSC mobilization, collection, cryopreservation, storage, and reinfusion after high-dose chemotherapy. The risk of the procedure was explained to the parents before obtaining approval in the form of informed consent for the leukapheresis procedures.

Ther Apher Dial, Vol. 15, No. 6, 2011

PBSC Mobilization and Harvesting in Children wood, CO, USA). The median value for blood volume in these patients was 1125 mL (range, 516 1740 mL) according to the Nadler and Allen formula used by Cobe Spectra, which considers weight, height, and gender (6). The median for the expected loss of blood volume at the beginning of the procedure was 25% (range, 1655%), and that during diverting prime saline was 13.3% (range, 8.629%); the median for excess liquid at the end of the procedure was 366 mL (range, 252671 mL), which was 35% (range, 2064%) of the total blood volume of our patients. The median for the shortage of erythrocyte volume at the end of the procedures was 39 mL (range, 3056 mL), which was 10.7% (range, 6.4 22%) of the total erythrocyte volume. The collection rate range was 0.81.0 mL/min. The same team performed all of the procedures. Interface adjustment was optimized and manually adjusted to obtain a nal product with an hematocrit of 6%. Anticoagulation was accomplished with acid citrate dextrose-A (ACD-A) solution (Haemonetics, Braintree, MA, USA). The blood-to-ACD-A ratio was 12:1 to 16:1 (median, 14:1), depending on the number of platelets. For children with a normal or increased platelet count, unfractionated heparin was added to the ACD-A bag (3000 IU). Calcium was provided orally during the procedure at a dose of 0.5 g/10 kg body weight. Vital signs were measured noninvasively every 30 min and blood pressure was measured on the hour. We did not use any other medications, and the patients were not sedated during the procedures. Blood was withdrawn through a temporary catheter inserted into the radial artery before leukapheresis (2024 G; Terumo, Tokyo, Japan) in 35 procedures (71.4%), the femoral artery in 11 procedures (22.4%), the femoral vein in two procedures (4.2%) and the cubital vein in one procedure (2.0%). In 19 of 49 procedures (39%), the extracorporeal line was primed with leukodepleted and 25 Gy-irradiated ABO/rhesus D compatible erythrocytes (510 mL/ kg), following regular priming with physiological solution. The processed blood was returned through a central venous catheter in 25 cases (51%), and a temporary 1822 G catheter was inserted into the femoral vein in 14 cases (28.6%), the jugular vein in six cases (12.2%), and the cubital vein in four cases (8.2%). When patients were undergoing leukapheresis on a daily basis, the catheters were not removed, and ushes with heparin solution were used to prevent occlusion of the catheter. At the beginning of the procedure, the ow was maintained at minimal values. Depending on the patients ability to tolerate the procedure, we increased the blood inow, but rarely to the maximum value. Inlet ow was from 8 to
2011 The Authors Therapeutic Apheresis and Dialysis 2011 International Society for Apheresis

581

28 mL/min (median, 16 mL/min). The collection procedure was repeated until the targeted dose of 5 106 CD34+ cells/kg body weight was achieved, or in cases in which the yield was so low that the continuation of leukapheresis was pointless. The outcomes of complications from leukapheresis were observed 24 h after collection. Processing and storage of PBSC Cells collected during leukapheresis in 1000 mL collection bags were cryopreserved using a conventional procedure in a control rate freezer (IceCube 1800D; Sy-Lab, Neupurkersdorf, Austria). The cryopreservation solution consisted of 10% dimethylsulfoxide (CryoSure-DMSO; WAK-Chemie Medical, Steinbach [Taunus], Germany) and autologous plasma (10). Blood cell count and CD34+ cell enumeration A total cell count, including the differential cell count in the peripheral blood prior to apheresis and the apheresis product, was determined on an automatic hematological analyzer. The CD34+ cell concentrations in both peripheral blood and apheresis product were assessed using ow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA, USA) after double staining with a phycoerythrinconjugated anti-CD34 monoclonal antibody (HPCA-2; Becton Dickinson) and a uoresceinisothiocyanate-conjugated anti-CD45 monoclonal antibody (Becton Dickinson) according to the International Society of Hematotherapy and Graft Engineering (ISHAGE) protocol (11). Statistical analysis Statistical evaluation of the data was performed using SPSS software, version 17 (SPSS, Chicago, IL, USA). The patients, leukapheresis procedures, and the peripheral blood and apheresis product characteristics were described using appropriate summary statistics, such as the average, median, and minimal and maximal values; these statistics are shown in Tables 1 and 2. Correlations were assayed using linear regression or nonparametric tests. The correlation tool determines whether large values of one set are associated with large values of the other sets (positive correlation), whether small values of one set are associated with large values of the other sets (negative correlation), or whether the values in the two sets are unrelated. The correlation of the number of CD34+ cells with the number of CD34+ cells collected in a single leukapheresis procedure and the total number per patients were examined and are

Ther Apher Dial, Vol. 15, No. 6, 2011

582

D Veljkovic et al.
20

TABLE 1. Summary of patient data and technical peripheral blood stem cell data

No. of patients

No. of patients No. of leukapheresis procedures Gender (male/female) Age (months) Body weight (kg) No. of months pre-Tx No. of days from the beginning of mobilization Mean number of mobilizations Mean number of procedures (range) Mean volume processed blood (mL/kg/session) Mean ow rate (mL/min) Mean ACD : blood ratio Volume of ACD-A infused (mL) Volume AP collected per procedure (mL) Time of procedure (min) Expected blood loss Volume shift during diverting prime saline (-) Volume shift at the end procedure (+) RBC shift at the end of procedure (-) Priming with RBC component

35 49 26/9 36 (675) 13.5 (719) 5 (325) 13.5 (821) 1 (12) 1 (13) 320 (141594) 16 (8-28) 1:14 (1:121:16) 330 (121733) 250 (98350) 279 (113420) 25% (1655%)* 13% (929%)* 35% (2064%)* 11% (622%)* 19/49 (38.7%)

15 10 5 0

<10

10-19

20-49

50

CD34+ cells in peripheral blood on the harvest day (cells/mL)

FIG. 1. Mobilization success in 35 children of low body weight.

*Data are presented as median (range). ACD(-A), acid citrate dextrose solution (formula A); AP, apheresis product; RBC, red blood cell; Tx, treatment (number days from beginning of mobilization to the day of harvesting).

shown as a graph. The results were considered signicant if the P-value was <0.01. RESULTS A total of 49 PBSC collections were performed in 35 children weighing less than 20 kg. Summary data regarding the patients and technical PBSC data are presented in Table 1, and data on peripheral blood on
TABLE 2. Summary data on peripheral blood on the day of harvesting peripheral blood stem cells, apheresis product and cell yield
Parameter Peripheral blood White blood cells (109/L) Hematocrit (L/L) Platelets (109/L) CD34+ (/mL) Total cell count/apheresis product White blood cells (109) Mononuclear cells (109) Hematocrit (L/L) Platelets (109) CD34+ (/mL) Yield of CD34+ cells/procedure Yield of mononuclear cells (108/kg) Yield of CD34+ cells (106/kg) Yield of CD34+ cells/patient Yield of mononuclear cells (108/kg) Yield of CD34+ cells (106/kg) Median 21.8 0.031 127 24 28 7.5 0.006 427 68 5.5 5.0 7.5 6.6 Range 3.083.1 0.0230.037 29483 2845 482 1.857.0 0.0030.015 2901830 32119 1.725.3 0.2117.7 1.728.4 1.4117.7

the day of harvesting, APs and yields are shown in Table 2. A wide variation in the timing of the harvests and the mobilization success was observed. The median number of days from the beginning of chemotherapy to the day of the initial leukapheresis was 13.5 days (range, 821 days). We found a high variability in the number of cells among patients on the day of PBSC collection; the median value for leukocytes was 21.8 109/L (range, 3.083.1 109/L) and CD34+ cells was 24 mL (range, 2845 mL) with a CD34+ cell representation percentage of 0.36% (range, 0.013.6%) and 127 109/L platelets (range, 29483 109/L). In two of the 35 patients (5.7%), the number of CD34+ cells in the peripheral blood was <10 cells/mL, between 10 and 19 cells/mL in six patients (17.1%), between 20 and 49 cells/mL in 11 patients (31.4%), and 50 cells/mL in 16 patients (45.7%) (Fig. 1). No signicant correlation was observed between peripheral blood CD34+ concentration on the day of collection and the patient age, body weight, gender, duration of disease, duration of pre-collection chemotherapy, or the day of the collection. No correlation was present between the CD34+ cell concentration in the peripheral blood on the harvest day and WBC, platelet or hematocrit in the same samples. A median of 4.0 volumes of patient blood, corresponding to 320 mL/kg (range, 141 594 mL/kg), was processed per procedure, which lasted 279 min (range, 113420 min). The median number of large volume leukapheresis procedures per patient was 1 (range, 13). The median for the volume of ACD-A used was 333 mL (range, 121 733 mL) per procedure. The median number of cells collected per apheresis procedure was 5.5 108/kg (range, 1.725.3 108/kg) for mononuclear cells and 5.0 106/kg (range, 0.2117.7 106/kg) for CD34+ cells. The total number of cells collected per patient was 7.5 108/kg (range, 1.728.4 108/kg) for mono 2011 The Authors Therapeutic Apheresis and Dialysis 2011 International Society for Apheresis

Ther Apher Dial, Vol. 15, No. 6, 2011

PBSC Mobilization and Harvesting in Children

583

CD34+ cell count in the peripheral blood (/mL)

Spearmens rho 0.850 P < 0.01

tions occurred. A reduction in the initial number of thrombocytes by 3050% was noticed in all patients, with only one child having less than 20 109/L after ending leukapheresis and needing a platelet transfusion. We used a variety of artery catheters: 24 G in four patients, 22 G in 16 patients, 20 G in 10 patients, and 18 G in ve patients. DISCUSSION

CD34+ cells ( X 106/kg body mass)

FIG. 2. Correlation between the CD34+ cell count in peripheral blood and the yield of CD34+ cells 106/kg body mass.

nuclear cells and 6.6 106/kg CD34+ cells (range, 1.4 117.7 106/kg). For 24 patients (68.6%), the target dose of 5 106/kg CD34+ cells was collected by one leukapheresis procedure, and for four patients (11.4%), the target dose was collected in two procedures. In six patients (17.1%), the yield ranged from 2.8 to 4.9 106 CD34+ cells/kg, and in one patient, the yield was 1.4 106 CD34+ cells/kg. In 24 patients, one leukapheresis procedure was performed, in 8 patients two, and in 3 patients three procedures were performed. For one patient, the yield was only 1.4 106/kg CD34+ cells/kg after two mobilizations and three leukapheresis procedures. The median volume of apheresis product used was 250 mL (range, 98350 mL), and the median for hematocrit was 6% (range, 315%). A signicant positive correlation (r = 0.850, P < 0.01) between the yield of CD34+ cells and the number of CD34+ cells in the peripheral blood before leukapheresis (Fig. 2) and a negative correlation between the volume of processed blood and the yield of CD34+/kg (r = -0.647, P < 0.01) were found. The target dose of 5 106/kg CD34+ cells was realized in 80% of patients. Morbidity related to PBSC collection in low body weight children was low. Transient and reversible cyanosis or hypotensive episodes developed at the beginning of leukapheresis in six patients (17.1%). A temporary reduction in the rate of blood withdrawal induced a prompt recovery in all cases. None of the patients developed post-apheresis fever, and none of the patients were readmitted for neutropenia with or without infectious complications. Pallor was present in all patients, and tachycardia, with a pulse rate up to 140/min, arose in 19 of the procedures (38.8%). Seven children (20%) were particularly anxious, despite the continual presence of their mothers during the procedure. Switching the return catheters to different blood vessels was necessary in ve procedures (10%). No bleeding or thrombotic complica 2011 The Authors Therapeutic Apheresis and Dialysis 2011 International Society for Apheresis

Apheresis procedures in children, especially in children with a small body mass, are difcult because no method exists to secure the optimal amount of blood inow into the separator (8), a high extracorporeal blood volume is present in respect to the blood volume of patients, and the cooperation of the patient is either absent or challenging (6). Neither the mobilization technique nor the collection procedure are totally optimized. With the aim being to optimize the PBSC collection in low body weight patients with malignant disease, large volume leukapheresis was performed to collect a target number of 5 106/kg CD34+ cells using a reduced number of procedures (1214). A dilemma exists in relation to the ease of HSC mobilization in the youngest pediatric patients with solid tumors compared to older children and adults (15). Our results show that through cancer-specic chemotherapy and G-CSF, we achieved satisfactory mobilization of CD34+ cells (>20 mL) in the majority of patients (77%), which coincides with the results of other authors (1517). We also used higher doses of G-CSF for successful mobilization in two children who failed to mobilize HSC with disease-specic chemotherapy and standard dose G-CSF (18,19). Pediatric patients have smaller blood and erythrocyte volumes than adults because of their smaller body masses. To avoid acute hypovolemia and hypoxemia at the start of a stem cell procedure, most authors would routinely ll the system with blood, especially in children with a body mass <15 kg (12 17). In 19 of 49 procedures (39%), which represents 14 out of 27 children (52%) with a body mass <15 kg, we lled the extracorporeal line with leukodepleted and 25 Gy-irradiated ABO/rhesus D compatible erythrocytes (510 mL/kg), following regular priming of the extracorporeal line with physiological solution. The priming of the extracorporeal line with erythrocytes can be avoided if the patients hematocrit levels are >35% and by adjusting the inow of blood into the separator to the minimum rate (17). The downside of this adjustment is an increased procedure time and decreased work efciency, such that we could only accommodate one patient during normal

Ther Apher Dial, Vol. 15, No. 6, 2011

584

D Veljkovic et al. approach is a dialysis catheter (22) or provisional catheter in the jugular, femoral, or peripheral artery/ vein (17). For returning blood, we used CVCs and provisional catheters in larger blood vessels and peripheral veins. No complications with blood clots through the catheter occurred in children with a high count of leukocytes in the cases of mobilization with G-CSF. Switching the catheter to different blood vessels during the procedure was necessary in ve procedures (10%). The yield of mononuclear and CD34+ cells in our patients was in accordance with the results of other authors; these yields were 7.5 108/kg body mass (range, 1.728.4 108/kg) and 6.6 106/kg (range, 1.41117.7 106/kg), respectively (1520,2326). In 24 patients (68.6%), the target dose of 5 106/kg CD34+ cells was collected from only one leukapheresis procedure, and in four patients (11.4%) the target dose was collected through two leukapheresis procedures. In six patients (17.1%), the yield was in a range from 2.8 to 4.9 106 CD34+ cells/kg, and in one patient, the yield was 1.41 106 CD34+ cells/kg. In 80% of patients, the yield of CD34+ cells was 5 106/kg. We could achieve this kind of yield through large volume leukapheresis procedures. Namely, in only eight patients, we processed 3 blood volumes, and the median value for the processed volumes was 4.0 (range, 1.477.5) per procedure and 4.1 (range, 1.4113.7) per patient. In our opinion, large volume leukapheresis reduces the level of stress for the child, their parents, and our personnel in the apheresis unit, and also reduces the risks in connection with the placement of provisional catheters as well as the price per procedure, without having a signicant effect or presenting a risk for the patient. Other authors have taken a similar approach (27,28). A signicant correlation (P < 0.01) was present between the number of the CD34+ cells in the peripheral blood before leukapheresis and the number of CD34+ cells collected during each procedure. However, no signicant correlation was observed between the yield of CD34+ cells and white blood cells, hematocrit or platelet count on the day of leukapheresis. Therefore, the only predictor for successful collection of CD34+ cells in our group of patients with small body mass was the number of CD34+ cells in the peripheral blood before apheresis (29,24). The age of the patients and duration of their disease had no impact on the yield of CD34+ cells. Similar to other studies, we used ACD-A for anticoagulation in our patients during the collection. The administration of citrate in the form of infusion through the line for blood collection prevents blood
2011 The Authors Therapeutic Apheresis and Dialysis 2011 International Society for Apheresis

working hours. The risk involved in transfusion of allogeneic erythrocytes is not affected by whether the patient had received a transfusion before collection or the blood had been used for lling the extracorporeal line during leukapheresis. The median value of hematocrit for our patients was 31% (range, 23-37%), the speed of blood inow was 728 mL/ min, with the median being 16 mL/min, and the median for the volume of processed blood per procedure was 320 mL in 279 min. Because the risks of hypovolemia and metabolic complications are present in children with a small body mass, Korstek et al. have suggested a manual technique for hematopoietic cell collection from the peripheral blood (20). In our experience, volume and red blood cells lost at the beginning of leukapheresis as a volume excess are not problems at the end of the procedure if the child has a good hematocrit, and heart and kidney function before collection. Except in three harvesting procedures, all blood from the extracorporeal line was returned to the patients slowly and carefully during rinseback. The second specicity of PBSC collection in children with small body mass is the difculty in the vascular approach (6,21,22). Maintaining an adequate blood inow during apheresis is necessary to achieve efcient collection and a satisfactory yield of targeted cells. Based on the experience of Takaue et al. (15), we used an arterial catheter inserted in the radial artery by the pediatric anesthesiologist the night before or immediately before apheresis. Removal of the catheter after the completion of the procedure took 45 h, and in cases in which the procedure was repeated the next day, the same catheter was used. In our experience, the use of the temporary catheter in the radial artery increases the inow and reduces the collection time. However, most pediatric transplantation centers use tunneled double-volume central venous catheters (CVCs) (21), which are routinely inserted in the majority of hemato-oncological patients for implementing chemotherapy and other vein therapies and for collecting samples for blood analysis. In our group of patients, 17 of 35 (48.6%) had CVCs. Long CVCs usually cause resistance that slows the blood ow. In cases in which the placement of the radial catheter was unsuccessful, we used an alternative venous access, such as the femoral, jugular, or cubital vein. The placement of a radial catheter is a routine procedure in neonatology and pediatric cardiology. Our institution has so far conducted 167 apheresis procedures in pediatric patients through catheters inserted in the radial artery, and no direct or indirect complications have been observed in these procedures. The second most common

Ther Apher Dial, Vol. 15, No. 6, 2011

PBSC Mobilization and Harvesting in Children clots by reducing the concentration of ionized calcium. The side effects of citrate anticoagulation can occur in the form of hypocalcemia, as has been described by many authors (25). Infusion of ACD-A at scales of 1:8 to 1:12 represents a cumulative dose of 0.61.0 mg/kg/min that leads to a 2030% reduction in ionized calcium. This level of reduction is enough to cause mild symptoms of hypocalcemia in almost one-quarter of adults and adolescents undergoing apheresis therapy, who are then treated with oral calcium replacement (6). We used scales of 1 : 12 to 1 : 16 ACD-A to patient blood (1.19 mL ACD/min/ 1.14 mL patient blood) with additional oral replacement of calcium, and observed no signs of hypocalcemia, blood coagulation in the system or aggregation of thrombocytes in the apheresis product. None of the patients had liver or bladder insufciencies, and none received fresh frozen plasma. An alternative approach would be a combination of citrate and heparin (26). Citrate poisoning is manifested by anxiety, pallor, sweating and nausea, which can lead to tachycardia or hypotension. In the youngest children, hypotension can be considered to be the only sign of hypocalcemia. The tolerance of PBSC collection in our patients was good. Some of patients developed cyanosis and tremors at the beginning of the rst procedure. Pallor was present in all patients. Seven of the children had severe anxiety during the procedure, even with the presence of a parent by their side. The exchange of blood vessels was necessary in ve procedures. None of the patients had a fever at the end of the procedure, and no infective complications occurred. No bleeding or thrombotic complications occurred. We found a reduction in the initial number of platelets by 3050% in all patients (unpublished observation), except in one child in which the number of thrombocytes was <20 109/L. The majority of our patients were pale before, during and after the procedure, which was not surprising if we take into consideration that the average hematocrit was 31% and that the young children had an active malignant disease. The patients fear of coming into contact with unfamiliar people, the separator, and the surroundings contributed to their dejection, paleness, and increased heartbeat. Therefore, it is important that people who perform apheresis procedures get a chance to meet and talk to the children and their parents before the procedure. The child must carefully and continually examined. The presence of a parent, familiarity and unconventional atmosphere all contribute to better tolerance to the procedures by animating the child through watching cartoon lms, or telling or reading stories to them. The continual presence of the doctor
2011 The Authors Therapeutic Apheresis and Dialysis 2011 International Society for Apheresis

585

is essential because it gives a sense of security to the parent and child. The care for our young patients and the dedication and composure of the personnel has a positive effect, but the shortcoming of this dedication is that we are unable to collect HSC in more than one patient per day. If a problem occurs in the ow, an alternative vascular access (usually a peripheral vein) should always be an option, which parents and the children (where possible) should understand. In cases in which hypocalcemia symptoms arise, the procedure should be slowed in order to identify the concentration of ionized calcium, and calcium gluconate should be introduced if the concentration of calcium is low (6). CONCLUSION Our results show that collection of an optimal dose of stem cells for autologous transplantation is possible, with certain adjustments, in children with a body mass <20 kg. Filling of the extracorporeal blood circulation system with compatible, leukodepleted, and irradiated erythrocytes combined with minimal blood inow into the separator prevents hypovolemia in patients, while large-volume leukapheresis increases collection efciency without increasing the risk of any complications. The number of CD34+ cells in the peripheral blood before leukapheresis is the best predictor of a successful HSC collection in children with a small body mass. Continual blood inow during the procedure is enabled by using a provisional catheter inserted into the radial artery. REFERENCES
1. Ceschel S, Cassoto V, Valsecchi GM et al. Survival after relapse in children with solid tumors: a follow up study from Italian off-therapy registry. Pediatr Blood Cancer 2006;47: 5606. 2. Landenstein R, Ptschger U, Hartman O et al. 28 years of high-dose therapy and SCT for neuroblastoma in Europe: lessons from more than 4000 procedures. Bone Marrow Transplant 2008;41:S11827. 3. Grupp AS, Cohn LS, Wall D. Reynolds PC for the Hematopoietic Stem Cell Transplant Discipline and the Neuroblastoma Disease Committee, Childrens Oncology Group. Collection, storage and infusion of stem cells in children with high-risk neuroblastoma: saving for a rainy day. Pediatr Blood Cancer 2006;24:28916. 4. Hale GA. Autologous hematopoietic stem cell transplantation for pediatric solid tumors. Expert Rev Anticancer Ther 2005;5: 83546. 5. Diaz MA, Kanold J, Vincent MG, Halle P, Madero L, Demeocq F. Using peripheral blood progenitor cells (PBPC) for transplantation in pediatric patients: a state of the art review. Bone Marrow Transplant 2000;26:12918. 6. Kim CH. Therapeutic pediatric apheresis. J Clin Apher 2000; 15:12957.

Ther Apher Dial, Vol. 15, No. 6, 2011

586

D Veljkovic et al.
19. Merlin E, Piguet C, Auvrignon A, Rubie H, Demeocq F, Kanold J. The pro and cons of split-dose granulocyte colonystimulating factor alone rather than a single high dose for hematopoietic progenitor cell mobilization in small children (<15 kg) with solid tumors. Haematologica 2006;91:10045. 20. Koristek Z, terba J, Havranova D, Mayer J. Technique for PBSC harvesting in children of weight under 10 kg. Bone Marrow Transplant 2002;29:5761. 21. Biffy R, Pozzi S, Agazzi A et al. Use of totally implantable venous access ports for high-dose chemotherapy and peripheral blood stem cell transplantation: results of a monocentre series of 376 patients. Ann Oncol 2004;15:296300. 22. Johansson E, Sollen Hansson A, Nilsson AS, Engervall P. Vascular access devices used during harvest of peripheral blood stem cells: high complication rate in patients with long-term dialysis central venous catheter. Bone Marrow Transplant 1999;24:7937. 23. Cecyn ZK, Seber A, Ginani CV et al. Large volume leukapheresis for peripheral blood progenitor cell collection in low body weight pediatric patients: a single center experience. Transf Apher Sci 2005;32:26974. 24. Berger M, Kanold J, Repatel C. Feasibility of PB CD34+ cell transplantation procedure using standard leukapheresis products in very small children. Bone Marrow Transplant 1997;20: 1918. 25. Urban C, Schwinger W, Benesch M. Feasibility of peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMNC) separation in children with body weight below 20 kg. Med Pediatr Oncol 1997;29:11520. 26. Sevilla J, Vincent-Gonzalez M, Plaza-Fernandez S, Madero L, Diaz AM. Heparin-based anticoagulation during peripheral blood stem cell collection may increase the CD34+ cell yield. Haematologica 2004;89:24951. 27. Ikeda K, Kozuka T, Harada M. Factors for PBSC collection efciency and collection predictors. Transf Apher Sci 2004;31: 24559. 28. Diaz AM, Sevilla J, de la Rubia J et al. Factors predicting peripheral blood progenitor cell collection from pediatric patients. Haematologica 2004;89:24951. 29. Schroeder H. Preharvest CD34+ count correlates with total number of collected CD34+ cells/kg. Pediatr Blood Cancer 2006;46:78692. 30. Sevilla J, Gonzalez-Vicent M, Madero L, Garcia-Sanchez F, Diaz MA. Large volume leukapheresis in small children: safety prole and variables affecting peripheral blood progenitor cell collection. Bone Marrow Transplant 2003;31:2637.

7. Worel N, Peters C, Gerhart K, Wagner A, Jurco S, Hocker P. Collection of peripheral blood stem cells (PBSC) after chemotherapy and administration of the rhGM-CSF in children weighing less than 17 kg. Transf Sci 1996;6:6016. 8. Shimizu N, Asai T. Analysis for the optimal blood draw speed to collect sufcient peripheral blood mononuclear cells by Cobe Spectra. Ther Apher Dial 2004;8:1903. 9. Kanold J, Halle P, Berger M et al. Large volume leukapheresis procedure for peripheral blood progenitor cell collection in children weighting 15 kg or less: efcacy and safety evaluation. Med Ped Oncol 1999;32:710. 10. Rowley SD. Techniques of bone marrow and stem cell cryopreservation and storage. In: Sacher RA, AuBuchon JP, eds. Marrow Transplantation: Practical Aspects of Stem Cell Reconstitution. Bethesda, MD: AABB, 1992; 105. 11. Sutherland DR, Anderson L, Keenez L, Nayer R, Chin-Yee I. The ISHAGE guidelines for CD34+ cell determination by ow cytometry. J Hematother 1996;5:21326. 12. Dolgopolov I, Yankelevich M, Andreeva L, Mscheidye D, Ljoguine-Dsiegel S, Mentkevich G. Feasibility and safety of peripheral blood stem cell collection in children with poorprognosis solid tumors: a single center experience. Pediatr Hematol Oncol 1999;16:2918. 13. Cho JH, Jung KH, Sung WK, Ku HH, Lee HS, Kim WD. Autologous peripheral blood stem cell collection in children weighting less than 10 kg with solid tumors: experience of single center. J Clin Apher 2005;20:6571. 14. Diaz MA, Alegre A, Villa M et al. Pediatric experience with autologous peripheral blood progenitor cell transplantation: inuence of the CD34+ cell dose in engraftment kinetics. Bone Marrow Transplant 1997;19:197204. 15. Takaue Y, Kawano Y, Abe T et al. Collection and transplantation of peripheral blood stem cells in very small children weighting 20 kg or less. Blood 1995;86:37280. 16. Ravagnani F, Coluccia P, Notti P et al. Peripheral blood stem cell collection in pediatric patients: feasibility of leukapheresis under anesthesia in uncompliant small children with solid tumors. J Clin Apher 2006;21:8591. 17. Orbach D, Hojjat-Assari S, Diaz F et al. Peripheral blood stem cell collection in 24 low-weight infants: experience of a single centre. Bone Marrow Transplant 2003;31: 1714. 18. Perez-Duenas B, Alcorta I, Estella J, Rives S, Toll T, Tuset E. Safety and efcacy of high-dose G-CSF (24mg/kg) alone for PBSC mobilization in children. Bone Marrow Transplant 2002; 30:9878.

Ther Apher Dial, Vol. 15, No. 6, 2011

2011 The Authors Therapeutic Apheresis and Dialysis 2011 International Society for Apheresis