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autologous transplantation; CD34; blood substitutes. In order to achieve total cure of solid tumours in children, treatment may require high-dose chemotherapy ...

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Peripheral blood stem cells Peripheral blood stem cell collection in 24 low-weight infants: experience of a single centre D Orbach1, S Hojjat-Assari2, F Doz1, H Pacquement1, A Guillaume3, C Mathiot4, J-M Zucker1 and J Michon1 1 Department of Paediatrics, Curie Institute, Paris; 2Transfusion Unit (Etablissement Franc¸ais du Sang – Ile de France), Curie Institute, Paris; 3Anaesthetic Department, Curie Institute, Paris; and 4Haematology Unit, Curie Institute, Paris

Summary: Peripheral blood stem cells (PBSC) harvest may be difficult in young children. Extracorporeal separator line priming by red blood cells is usually required to improve haemodynamic tolerance and efficacy of collection. We present our experience with 24 children weighing less than 15 kg treated between January 1997 and September 1999, in whom we tried to avoid systematic blood priming. The median age and weight at the time of apheresis were 2.4 years and 12 kg, respectively. A total of 48 PBSC were performed. When haemoglobin was less than 12 g/dl, packed red cells were transfused before collection (40% of aphereses). The median cell yield per apheresis was 7.1 (2.2–30.6)  106/kg CD34+ cells and 16.0 (3.3–44.3)  105 CFU-GM/kg. Initial collection failed in three cases. Four children required an additional haematopoietic progenitor mobilization. This procedure allowed PBSC collection without transfusion in 37.5% of children, and was safe (two serious and five mild transient side effects) and effective (median CD34+ cells collected per child: 7.1  106/kg (4.6–30.6) and CFU-GM: 15.1  105/kg (4.7–44.3)). Despite their low weight, insertion of a femoral catheter was avoided in 43% of children. Bone Marrow Transplantation (2003) 31, 171–174. doi:10.1038/sj.bmt.1703825 Keywords: peripheral blood stem cell collection; infants; autologous transplantation; CD34; blood substitutes

In order to achieve total cure of solid tumours in children, treatment may require high-dose chemotherapy (HDC), for example, in stage 4 neuroblastoma,1 refractory or recurrent lymphoma, retinoblastoma with extraocular involvement,2 high-risk Ewing’s tumour and brain tumour.3,4 Doselimiting toxicity is commonly haematological. To overcome prolonged and life-threatening neutropenia and thrombocytopenia following HDC, haematopoietic stem cells are transfused to reconstitute the child’s bone marrow.

Correspondence: Dr D Orbach, De´partement de Pe´diatrie, Institut Curie, 26 rue d’Ulm, Paris 75005, France Received 7 May 2002; accepted 17 July 2002

Following HDC, rescue with peripheral blood stem cells (PBSC) presents certain advantages over autologous bone marrow transplantation in children treated for solid tumours, particularly the rapidity of haematological recovery after myeloablative treatment. PBSC mobilization can be obtained by injection of granulocyte colonystimulating factor (G-CSF) either alone, when the white cell count is stable, or following appropriate chemotherapy. This technique is now widely used.5–8 In children weighing less than 15 kg, PBSC harvest may be difficult, as the extracorporeal separator has a minimal circuit volume of 280 ml (ie 425% of the child’s blood volume BV). During the procedure, the child may also develop various other problems, such as metabolic disorders (hypocalcaemia, hypoglycaemia) and venous access may be difficult because of the small size of the vessels. PBSC can be nevertheless be successfully performed with caution6,9 even in small children.10–12 Red blood cell priming of the extracorporeal circuit (alone or with albumin) is frequently required in small children to improve haemodynamic tolerance and efficacy.6,11 We try to avoid systematic transfusion when possible because of the risks of infection (viral and other) and immunological problems related to multiple transfusions. The haemodynamic status during the initial collection phase may remain satisfactory when high molecular weight Hydroxyethyl Starch (Elohess) or albumin alone are used as priming solutions. We present our experience of 24 children weighing less than 15 kg treated in a single centre according to a procedure designed to avoid blood transfusion for priming.

Patients and methods Patients From January 1997 to September 1999, 74 children underwent PBSC in our paediatric department. Of these, 24 (32.4%) children weighed less than 15 kg. The median age at the time of apheresis was 2.4 years (range: 1–4 years), median weight was 12 kg (range: 9–14 kg). There were 11 boys and 13 girls and they presented the following tumours: neuroblastoma (17 cases), extraocular retinoblastoma (four cases), brain tumours (two cases) and non-Hodgkin’s lymphoma (one case). All children had a single-lumen

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superior vena cava central line for chemotherapy. Blood processed during apheresis was returned to the child either via a catheter in the femoral vein or a peripheral vein, depending on the state of the peripheral veins in each case. Femoral catheters (4.5 F Hemoclav, length 10 cm, Vygons, France) were inserted under general anaesthesia.

Haematopoietic progenitor mobilization All PBSC collections were performed after initial conventional chemotherapy and in complete bone marrow remission when initially metastatic. Stem cell mobilization was obtained in ‘haematological steady state’ by once-daily subcutaneous injections of recombinant G-CSF after 5 days (10 mg/kg once daily) in 15 cases or following chemotherapy (with G-CSF 5 mg/kg once daily) in nine cases: C.A.V (cyclophosphamide 70 mg/kg once daily on days 1 and 2, doxorubicin 75 mg/m2, oncovin 0.1 mg/kg by continuous i.v. infusion over 72 h) in four cases, (cyclophosphamide 1 g/m2 on days 1–3, etoposide 150 mg/m2 on days 1–3) in two cases, (carboplatin 160 mg/m2 on days 1–5, etoposide 100 mg/m2 on days 1–5) in one case, CAdO (cyclophosphamide 300 mg/m2 on days 1–5, doxorubicin 30 mg/m2 on days 4 and 5, oncovin 1.5 mg/m2 on days 1 and 5) in one case and (cyclophosphamide 4 g/m2) in one case. In these children, collections started during the haematological recovery phase, as soon as peripheral blood CD34+ cell counts exceeded 10  106/ml measured by FACS-analysis, as described elsewhere.13 Depending on the high-dose chemotherapy programme, PBSC collection was repeated, if necessary, on three consecutive days until at least 5  106 CD34+/kg cells were obtained.

Priming of the separator The extracorporeal line was primed with high molecular weight hydroxyethyl starch (Elohess) or 4% albumin, using a priming volume of about 100–150 ml. Blood priming was not systematically performed. Transfusion of leukocyte-depleted phenotyped irradiated packed red blood cells was performed either before the first harvest, if haemoglobin was less than 12 g/dl, or before the other harvests, if necessary, when haemoglobin fell below 12 g/dl.

Stem cell collection PBSC were collected in the Paediatric Oncology Department using a continuous flow blood cell separator (COBE spectras; Cobe Lakewood, CO, USA). The procedure was performed under manual control. The collection rate calculated by the machine was 1 ml/min. Autologous platelet-rich plasma was automatically returned to the patient via the inlet line. The same operator (SHA) performed all aphereses. Adjustment of the interface was optimized and corrected under visual control. BV was estimated at 80 ml/kg. Acid–citrate–dextrose was used as anticoagulant. Patients received citrate : dextrose with an inlet anticoagulant ratio of 1/12 (during first passage of the processed blood equal to the value of the child’s BV) to 1/14 (during the second passage). To prevent hypocalcaemia, 10% calcium gluconate was given intraveBone Marrow Transplantation

nously: 3 ml at the beginning and at the end of each volume equal to the BV. Fluid balance was 100%. Vital signs were monitored noninvasively at 15 min intervals during cell harvesting. No systematic calcium assay was performed. The children did not receive any systematic sedation, but was entertained by the presence of a parent in the room during collection. Total cell count of the apheresis product was determined by an automatic cell counter and the differential cell count was determined manually. CD34+ cell concentrations were measured by flow cytometry after double staining using phycoerythrin-conjugated CD34 and FITC-conjugated CD 45 antibodies.13 Granulocyte–macrophage progenitor cells (colony-forming unit, CFU-GM) were measured as previously described.13,14 The cell volume was adjusted, distributed into an appropriate number of plastic bags according to the HDC programme, frozen according to standard methods in a controlled rate freezer and stored in liquid nitrogen at 1961C.

Results A total of 48 PBSC collections were performed in 24 children: a single apheresis was sufficient to obtain the required cell number in nine cases. Two aphereses were performed on consecutive days in seven cases, three aphereses were performed in seven cases and four aphereses were performed in one case. Red blood cells were transfused before the first collection in eight patients to ensure a haemoglobin level greater than 12 g/dl, and before subsequent aphereses in nine patients (40% of all aphereses). Three children had two repeat aphereses and two children underwent three repeat aphereses without transfusion. However, blood line priming was performed in two children. One girl presented haemodynamic abnormalities with faintness during the initial harvest despite previous Table 1

Technical details of PBSC collection

Volume of 10% calcium gluconate injected Volume of citrate:dextrose injected Number of blood volumes processed Duration of the procedure

Median

Range

7 ml 194 ml 2.66 173 min

3.5–11 108–300 1.86–4.25 130–240

Table 2 Results of the collections in the 24 children (per apheresis), with or without transfusion before apheresis regardless of haemoglobin level

Total group (48 aphereses) CD34+/kg CFU-GM/kg Non-transfused children (17 aphereses): CD34+/kg CFU-GM/kg Transfused children (31 aphereses): CD34+/kg CFU-GM/kg

Median

Range

7.1  106 16.0  105

2.2–30.6  106 3.0–44.3  105

7.7  106 15.1  105

2.2–30.6  106 3.0–26.0  105

6.2  106 16.0  105

4.2–17.7  106 4.7–44.3  105

Apheresis in young children D Orbach et al

transfusion and the next apheresis was performed with blood priming. In another one, the third apheresis was performed with blood line priming because of blood loss following early thrombus formation in the collection line, which required a complete change of the extracorporeal line. In summary, 9/24 children (37.5%) did not receive blood transfusion during the entire procedure.

Central lines Insertion of a femoral central venous line was necessary in 13 children (mean age: 1.8 year; mean weight: 11.6 kg). In the other 11 children (mean age: 3.1 years and mean weight: 12.5 kg), an additional peripheral venous line was used.

Complications related to apheresis Morbidity related to PBSC collections was low. Transiently catheter occlusion occurred in four cases and transient hypotension during the procedure required intravenous fluids in two cases. In three cases, the procedure failed because of haemodynamic abnormalities or severe vomiting. In these cases, cells were collected on the following days without any problem. One child developed symptomatic hypocalcaemia (tremor) corrected by calcium injection. No child experienced any collection-related infection. A transient femoral arteriovenous fistula (AVF) was observed in one child after femoral catheter removal and resolved spontaneously over several weeks. The technical characteristics are presented in Table 1. The numbers of cells obtained by apheresis are indicated in Table 2. Comparison of the children who never received blood products during the procedure and the children who required blood transfusion (before or during the collection) did not reveal any difference in terms of the number of cells collected. Four children (17%) required two haematopoietic progenitor mobilizations because of insufficient CD34+ harvest on the first harvest in three cases and for a sequential HDC programme in one case. The median CD34+ cell count collected per child was 7.1  106/kg (4.6–30.6) and the median CFU-GM was 15.1  105/kg (4.7–44.3).

Discussion PBSC harvest may be difficult in small children owing to the large extracorporeal separator volume compared to the Table 3

child’s weight. This can lead to various problems, such as metabolic or haemodynamic disorders. Venous access may also be difficult because of the small size of the vessels at this age. Nevertheless, PBSC can be successfully performed in small children. The tolerance of the extracorporeal circuit, even in very small children, can be good. In our series, as in series reported by other authors,15,16 apheresis induced few side effects: two serious (femoral AVF and haemodynamic disorders) and five mild transient side effects. To avoid acute BV loss during initiation of the stem cell separation procedure, most authors systematically use red blood cells for separator priming, especially for small children.10,12,14–16 However, in many cases, this initial systematic transfusion can be avoided even in low-weight children. With reference to initial haemoglobin values, fewer children will be transfused with this method, even when repeated collections are required, than if each apheresis is systematically primed with red blood cells. This can also reduce the number of donors. This procedure facilitates apheresis and presents several advantages: no need to prepare and wait for irradiated blood product, possibility of pretransfusion on the day before apheresis, lower risk of thrombus formation in the line when it contains high molecular weight solutions when preparation of the child is prolonged. There is also no risk of bacterial contamination or viral transmission. Finally, when blood is not used for priming, all of the blood still present in the line at the end of the procedure can be returned to the child, together with the child’s own platelets, thereby decreasing the risk of postapheresis peripheral thrombocytopenia. The results of the two nonrandomized groups (with or without transfusion before collection according to the initial haemoglobin level) appear to be comparable (Table 2). Nevertheless, four children required two progenitor mobilizations to collect the number of PBSC necessary for transplantation and blood priming had to be performed in two cases. The mean values of CFU-GM cell collection in small children are comparable to those reported by other authors (Table 3) according to various apheresis techniques. Our results show that a normal volume apheresis procedure (ie o3  BV) can be successfully used, while some authors prefer large-volume apheresis (6  BV) to increase absolute yields of CD34+.16–19 In our experience, despite their low weight, insertion of a femoral catheter can be avoided in 43% of children and use of a peripheral vein was sufficient. Some authors12 use a similar technique but, in most of their cases, regardless of

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Comparison of the results reported in the literature

Authors

Date

Weight (Kg)

Number of children

CD 34  106/kg per apheresis Median (range)

CFU-GM  105/kg per apheresis Median (range)

Deme´ocq11 Takaue10 Urban16 Kanold19 Orbach

1994 1995 1997 1998 2002

o25 o20 o20 o15 o15

20 38 7 14 24

NSa 15 (2–71) 3.6 (0.08–24) 8.2 (1.3–31.7)b 7.1 (2.2–30.6)

2.3 (0.1–88.2) 3.4 (0.2–153) NSa 7.66 (0.07–121.7)b 16.0 (3.0–44.3)

Technical data

Large volume apheresis

a

NS: not specified. Per child (17 aphereses).

b

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the child’s low weight, a transient second central line is inserted either into the radial artery10 or femoral vein.11,20 The morbidity following catheter insertion is low,20 but our results show that the child’s low weight does not systematically indicate the need for central venous catheter and, with experience, an arm or forearm vein can be used for return of processed blood, even in small children. In summary, apheresis can be performed in low-weight children under safe and effective conditions even when systematic priming by blood is avoided. This procedure allows PBSC collection in small children without transfusion. Insertion of a femoral catheter was avoided in almost half of these children despite their low weight.

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