CD34(+) progenitors are reproducibly recovered in thawed umbilical grafts, and positively influence haematopoietic reconstitution after transplantation.

Cord blood (CB) units are increasingly used for allogeneic transplantation. Cell dose, a major factor for CB selection, is evaluated before freezing by each CB bank, using various techniques. This may introduce variability and affect the prediction of cell recovery after thawing, or haematopoietic reconstitution. Forty-two children were transplanted at the same institution with unrelated CB units. All units were thawed and evaluated at the same cell therapy facility, using standard procedures. We investigated: (i) factors that affect cell loss after thawing, and (ii) the importance of CD34(+) cell doses. Prefreeze and post-thaw CD34(+) cell doses were statistically correlated, thus suggesting that variability in numeration techniques used by different CB banks does not compromise the biological and clinical value of these figures. CD34(+) cell recovery appeared to be correlated with the absolute number of CD34(+) cells per frozen bag. Infused CD34(+) is the cell dose that better correlates with platelet reconstitution delay; in addition, when using a quartile comparison, haematopoietic recovery appeared to be related with prefreeze and post-thaw CD34(+) cell doses. We conclude that enumeration of CD34(+) cells in CB units is of biological significance, and may help select CB units and identify patients at risk of delayed recovery.

A quality system for placental blood banking.

A Quality System for Placental Blood Banking aimed at the transplantation of haematopoietic stem cells to related and unrelated allogeneic recipients is described. It includes the organizational structure, procedures, processes and resources needed to implement quality management. The Quality System described in this article is based on ISO 9002, a model for quality assurance in production, installation and servicing developed in 1987 and revised in 1994 by the International Organization for Standardization. ISO 9002 includes 20 clauses that provide guidance for the implementation of the Quality System. The development of the Quality System is started by the Placental Blood Bank Medical Director with the definition of a General Quality Plan including: (1) the written description of the Mission, Objectives, Technical and Organizational Policies, and Staff Organization Chart; (2) the definition and acquisition of adequate financial, human and structural resources; (3) the appointment of a Quality System Head, who must identify the Placental Blood Banking process together with the Placental Blood Bank personnel; implement a documentation plan; identify quality indicators; start regular internal audit; report audit results to the Medical Director for review. Following staff training and qualification, the Quality System is launched. The Placental Blood Bank can then undergo audit by an external inspector and be finally certified for compliance to ISO 9002. The Quality System must be maintained and subjected to external audit at regular intervals so that certification is confirmed.

Ultrastructural features of CD34+ hematopoietic progenitor cells from bone marrow, peripheral blood and umbilical cord blood.

Hematopoietic progenitor cells from different sources have been widely characterized, but their ultrastructural morphology has never been described in detail. In this study, imunomagnetically separated CD34+ cells from normal bone marrow (BM), mobilized peripheral blood (PBSC) and human umbilical cord blood (CB) were studied by transmission electron microscopy (TEM) using a cytochemical method which reveals endogenous myelo-peroxidase (MPO) activity. This technique is particularly suited for detecting early signs of the myeloid commitment. The CD34+ cells from PBSC were morphologically very homogeneous and 94.7+/-4.5% of these cells were MPO-: these ultrastructural features are generally considered typical of immature cells. The CD34+ BM cells were instead more heterogeneous, with 24.6+/-7.4% showing intense MPO activity. The ultrastructural characteristics of CB cells fell between those observed in PBSC and BM, but there was a high percentage of morphologically immature cells with no evidence of MPO activity (about 83%). The number of apoptotic cells within samples from different sources was also examined both by TEM and flow cytometry. The percentage of apoptotic cells was 0.7% in PBSC, 2.3% in BM, 2.9% in CB from vaginal delivery and 11.6% in CB from cesarean section. These observations confirm the relative phenotypic immaturity of CB in comparison with BM cells; they also suggest that CB collected after cesarean section may be associated with reduced stem cells viability.

Human lymphocyte antigens in Graves' disease: correlation with persistent course of disease.

Graves' disease is a thyroid autoimmune disorder associated with specific human lymphocyte antigen (HLA) alleles, characterized by an unpredictable long-term course. To investigate possible relations between HLA phenotype and outcome of the disease, the authors typed for HLA antigens in 105 patients with Graves' disease with different course of disease. All patients were treated with antithyroid drugs for at least 12 months; 29 patients had stable remission 24 or more months after withdrawal of treatment; 76 patients had persistent disease--66 unremitting/relapsing hyperthyroidism, 10 stable hypothyroidism--36 or more months after onset of disease. The following findings emerged from this study: 1) HLA B8 and DR3 were increased significantly in Graves' patients versus 6,682 control subjects from the same geographic area (23.80% vs 12.01%, odds ratio [OR] 1.98, and 31.43% vs 18.00%, OR 1.75, respectively); the antigen combinations B8-DR3, B8-Cw7-DR3, and A1-B8-Cw7-DR3 were significantly more frequent in Graves' patients vs control subjects; in addition, these combinations were present exclusively in patients with persistent disease (B8-DR3 28.95%, OR 7.14, B8-Cw7-DR3 27.63%, OR 11.24, and A1-B8-Cw7-DR3 18.42%, OR 11.29). These data provide evidence that not only susceptibility to Graves' disease, but also persistent activity of the autoimmune process, producing either hyperthyroidism or stable hypothyroidism, is associated with specific HLA antigen phenotypes.

Towards responsible cord blood banking models.

On 31 May 2010, 14 072 567 bone marrow/apheresis donors registered in 44 countries and 426 501 cord blood units banked in 26 countries for public use were available to treat candidates to haemopoietic stem cell transplant lacking a family related compatible donor. Despite these impressive numbers, additional efforts are required to ensure that all patients, including those from ethnic minorities, can promptly find a suitable donor. Governments, clinicians, scientists, patients and stakeholders should share the responsibility to develop haemopoietic stem cell donation and cord blood banking models able to fully match all patient needs. In this regard, current scientific evidence and prevalent opinions among expert clinicians support solidaristic cord blood donation for public use against the alternative option of commercial autologous cord blood storage.

KIR-ligand incompatibility in the graft-versus-host direction improves outcomes after umbilical cord blood transplantation for acute leukemia.

Donor killer cell immunoglobulin-like receptor (KIR)-ligand incompatibility is associated with decreased relapse incidence (RI) and improved leukemia-free survival (LFS) after haploidentical and HLA-mismatched unrelated hematopoietic stem cell transplantation. We assessed outcomes of 218 patients with acute myeloid leukemia (AML n=94) or acute lymphoblastic leukemia (n=124) in complete remission (CR) who had received a single-unit unrelated cord blood transplant (UCBT) from a KIR-ligand-compatible or -incompatible donor. Grafts were HLA-A, -B or -DRB1 matched (n=21) or mismatched (n=197). Patients and donors were categorized according to their degree of KIR-ligand compatibility in the graft-versus-host direction by determining whether or not they expressed HLA-C group 1 or 2, HLA-Bw4 or HLA-A3/-A11. Both HLA-C/-B KIR-ligand- and HLA-A-A3/-A11 KIR-ligand-incompatible UCBT showed a trend to improved LFS (P=0.09 and P=0.13, respectively). Sixty-nine donor-patient pairs were HLA-A, -B or -C KIR-ligand incompatible and 149 compatible. KIR-ligand-incompatible UCBT showed improved LFS (hazards ratio=2.05, P=0.0016) and overall survival (OS) (hazards ratio=2.0, P=0.004) and decreased RI (hazards ratio=0.53, P=0.05). These results were more evident for AML transplant recipients (2-year LFS and RI with or without KIR-ligand incompatibility 73 versus 38% (P=0.012), and 5 versus 36% (P=0.005), respectively). UCBT for acute leukemia in CR from KIR-ligand-incompatible donors is associated with decreased RI and improved LFS and OS.

Cost of umbilical cord blood units released for transplantation.

BACKGROUND: A large number of institutions have started programs banking umbilical cord blood (UCB) for allogeneic unrelated-donor and related-donor transplantation. However, limited information is available on the financial issues surrounding these activities. STUDY DESIGN AND METHODS: The aim of this study was to determine the fee per UCB unit released for transplantation that would allow cost recovery after 10 years. Three organizational models were considered suitable to provide units for five UCB transplants per 1 million population per year, a figure that would translate into an annual need for 280 units in Italy. Models A, B, and C included, respectively, seven networked banks, each with an inventory of 1,500 units; two networked banks, each with an inventory of 5,000 units; and one bank with an inventory of 10,000 units. It was estimated that it would take 3 years to develop the cryopreserved inventory and that approximately 3 percent of the inventory could be released and replaced each year during the 7-year interval between the fourth and tenth years of activity. The data on the costs of labor, reagents and diagnostics, disposables, depreciation and maintenance, laboratory tests, and overhead, as well as the operational data used in the analysis were collected at the Milano Cord Blood Bank in 1996. RESULTS: Fees of US $15,061, $12,666, and $11,602 per unit released during the fourth through the tenth years of activity allow full cost recovery (principle and interest) under Models A, B, and C, respectively. CONCLUSION: Although UCB procurement costs compare favorably with those of other hematopoietic cell sources, these results and the current fee of US $15,300 used in some institutions show that UCB is an expensive resource. Therefore, judicious planning of banking programs with high quality standards is necessary to prevent economic losses. The advantages of lower fees associated with the centralized banking approach of Model C should be balanced with the more flexible collection offered by Model A.

Long-term expansion and maintenance of cord blood haematopoietic stem cells using thrombopoietin, Flt3-ligand, interleukin (IL)-6 and IL-11 in a serum-free and stroma-free culture system.

Although cord blood (CB) compares favourably with other haematopoietic stem cell (HSCs) sources, its use in large patients is limited by the low number of cells available. Ex vivo expansion of CB HSCs has been used to overcome this limitation. In this study, we investigated the effect of different cytokine cocktails, including interleukin (IL)-6, IL-11, Flt3-ligand (FL) and thrombopoietin (TPO) combined with serum or serum-free medium on the ex vivo expansion of CD34+ cells from CB. Initial experiments showed that expansion could be slightly improved using serum, but we chose to use serum-free medium in the subsequent investigations to apply good medical practice (GMP) conditions suitable for clinical use. The highest expansion of CD34+ cells was obtained with a cocktail containing FL + TPO + IL-6 + IL-11. The median (range) fold expansions of CD34+ cells at 5 and 10 weeks with serum-free medium were 235.6 (131.3-340) and 5205.6 (4736.6-5674.7) respectively. The absence of IL-11 was associated with a similar fold expansion after 5 weeks (median 215.6, range 149.8-281.5), but after 10 weeks expansion was slightly lower (median 1314.7, range 645-1984.4). Our data support the possibility of maintaining long-term expansion of CB HSCs in a simple stroma- and serum-free system.

Interleukin-6 and interleukin-11 act synergistically with thrombopoietin and stem cell factor to modulate ex vivo expansion of human CD41+ and CD61+ megakaryocytic cells.

BACKGROUND AND OBJECTIVE: Thrombopoietin (TPO), the ligand for the c-mpl receptor, regulates in vivo platelet production and increases the number of colony-forming unit megakaryocytes (CFU-MK). Other cytokines including interleukin (IL) -3, IL-6, IL-11 and stem cell factor (SCF) can stimulate megakaryopoiesis. The aim of this study was to evaluate the effects of different combinations of cytokines involved in megakaryocytopoiesis on stroma-free liquid cultures of purified human CD34+ cells. DESIGN AND METHODS: Peripheral blood cells were collected after mobilization with granulocyte colony-stimulating factor (G-CSF). Purified CD34+ cells were then cultured with different combinations of TPO, SCF, IL-3, IL-6 and IL-11. RESULTS: The addition of TPO and SCF alone generated a population positive for the antigens CD41 (5.5+/-2.9%) and CD61 (6. 1+/-2.2%) but induced a low amplification of cell number (8.1+/-0.9 fold expansion). The presence of IL-6 or IL-11 was associated with MK progenitor cell expansion, and up to 7-10% of cultured cells were found to be CD41 and CD61 positive by flow cytometry. Conversely, the addition of IL-3 to this cytokine combination was associated with a prominent expansion of the myeloid lineage (70+/-10% of CD33+ cells) but only 0.9% and 2% of cultured cells were positive for CD61 and CD41 respectively. INTERPRETATION AND CONCLUSIONS: Our study supports the idea that IL-6 and IL-11 play crucial roles in the proliferation of MK progenitors and the use of SCF, TPO, IL-6 and IL-11 for ex vivo expansion of this cell population.

Implementation of a quality system (ISO 9000 series) for placental blood banking.

Although placental blood has recently become a new source of hematopoietic progenitors for marrow replacement, limited attention has been given to systems suitable to ensure the short-term and long-term quality of placental blood units used for transplantation. In this article, we describe a quality system for placental blood banking developed in accord with ISO 9002 norms at Milano Cord Blood Bank. The quality system is the organizational structure, procedures, processes, and resources needed to implement quality management. ISO 9002 is a model for quality assurance in production, installation, and servicing, which includes a number of clauses providing guidance for the implementation of the quality system. The quality system was started by the bank medical director with step 1: the general quality plan, which included (a) the written description of mission, objectives, technical and organizational policies, and staff organization chart of the placental blood bank, (b) the definition and acquisition of adequate financial, human, and structural resources, (c) the appointment of a quality system head independent from the production laboratory and reporting directly to the medical director. Tasks of the quality system head were (a) to identify the placental blood banking process together with the placental blood bank personnel, (b) to implement a documentation plan finalized at the production and maintenance of (i) the quality manual, which provides a summary on how the bank operates with a quality system in compliance with the ISO 9002 clauses, (ii) the general procedures (or quality system procedures), which provide more detail on selected clauses, including at least those prescribed by the ISO 9002 standard, (iii) the operative procedures (or process procedures), which describe in detail the process of placental blood banking and how technical activities must be performed, (iv) the work instructions, which provide stepwise descriptions of individual activities, (v) records/forms for data collection and storage, (c) to identify quality indicators, (d) to start a regular internal audit, (e) to report audit results to the medical director for review. This was followed by step 2: the job descriptions, staff training, and qualification; step 3: the documentation plan; step 4: the internal audit plan; step 5: the launch of the quality system, and step 6: the assessment by an external team from an accredited third-party organization and final certification for compliance to ISO 9002. The quality system, which must be maintained and undergo external audit at regular intervals so that certification is confirmed, ensures the high probability that placental blood units provided to clinicians conform regularly to predefined levels of quality.

The Milan Cord Blood Bank and the Italian Cord Blood Network.

We describe the activities of the Milan Cord Blood (CB) Bank and of the Italian Cord Blood Network. By October 31, 1995, 763 units were banked in Milan. Of these, 8 units were used to perform 4 related and 4 unrelated transplants in 5 children and 3 adult patients. Early cord clamping after delivery was found to be crucial to increase the volume of CB collected. This procedure does not seem to be detrimental to the newborn. Of various red cell sedimenting agents used to reduce the unit volume and concentrate progenitor cells, 3% gelatin seems to be associated with the best yields. After a preliminary experience of 2 years, the Italian Cord Blood Network (Gruppo Italiano Amplificazione Cellule Emopoietiche, GRACE) was founded in 1995. The initial activities of GRACE are aimed at the development at the national level of CB banking standard operative procedures in agreement with the draft issued by The North American Task Force for The Development of Standards for Hematopoietic Cell Transplantation. Moreover, a wet workshop has been organized to standardize colony-forming unit (CFU) evaluation. The main goal in Milan is the collection of 5000 CB units. Other issues of interest include CB volume reduction, hematopoietic progenitor purification, ex vivo expansion prior to transplantation, and experimental protocols for gene transfer, such as those related to the multidrug resistance (MDR) gene.

Iron status in red cell pyruvate kinase deficiency: study of Italian cases.

We investigated the iron status of 33 pyruvate kinase (PK) deficient patients, most of the cases reported in Italy. Serum ferritin (SF) was higher than the upper limit of the range of matched controls in 15/25 (60%) non-transfused patients (median 228 micrograms/l, range 58-3160 v 43, 22-310). Liver siderosis and fibrosis were found in 8/9, and cirrhosis in two who died at age 39 and 42 of complications of iron overload. SF was independent of age, sex, or severity of haemolysis. The prevalence of HLA-A3 antigen in PK deficient patients was not significantly different from that of our healthy population (29.6% v 23%). The HLA-A3 positive, non-transfused patients had significantly higher SF values than the HLA-A3 negative ones (median 675 micrograms/l, range 340-3160 v 145, 58-400). A pedigree study of six high SF-probands indicated that iron overload has a multifactorial pathogenesis. In particular, the association of PK deficiency-induced haemolysis, splenectomy and an additional factor (heterozygosity for idiopathic haemochromatosis, ineffective erythropoiesis) leads to severe iron accumulation. We suggest that monitoring iron status would be useful in PK deficient patients, particularly in splenectomized and HLA-A3 positive ones, to identify those at risk of iron overload and prevent the clinical consequences of iron accumulation.