Vascular access for autologous peripheral blood stem cells collection by large volume leukapheresis: Single center experience.

INTRODUCTION: Peripheral blood stem cell (PBSC) harvesting requires reliable and safe vascular access. In our institution, a change of practice was implemented and the central venous catheter (CVC) placement for all autologous PBSC collections was abandoned in favor of a careful evaluation of peripheral venous access (PVA) for each individual patient. The aim of this prospective study was to evaluate the rate of patients with adequate peripheral veins for autologous PBSC collection and compare patient characteristics, collection efficacy, and complication rate between patients with PVA and CVC. METHOD: Peripheral veins were assessed by the apheresis nurse team in all patients referred between January 2020 and July 2021 to autologous PBSC collection. Only in case of difficult venous access, CVC was inserted. Large volume leukapheresis (LVL) procedures, which processed ≥3 total blood volumes, were performed. RESULTS: In 65 (57%) patients PVA was used, while 49 (43%) patients required placement of short-term CVC. Peripheral venous access was successfully used significantly more often in males (69.8%) (P = 0.010), and patients with multiple myeloma (71.0%) than in patients with non-Hodgkin's lymphoma (35.9%) and Hodgkin's lymphoma patients (33.3%) (P < 0.001). There was a significant difference in the type of prior administered chemotherapy; in the patients who received cytostatics free chemotherapy, PVA was used more often (75.0%) (P = 0.007). In terms of the efficacy and safety of LVLs, there were no differences between procedures performed using PVA and CVCs. CONCLUSION: Peripheral venous access is feasible for autologous PBSC collection in more than a half of patients, in particular in those with multiple myeloma. Changes in the treatment of multiple myeloma, using new proteasome inhibitors-based and immunomodulatory agents that do not adversely affect peripheral veins, have enabled the use of PVA even at the high blood flow rates required by LVL. Peripheral venous access is not associated with safety issues or with a lesser collection efficiency, and it is cost-effective as well. Each patient referred to autologous PBSC collection needs to be evaluated individually by the experienced apheresis team for the most appropriate venous access.

The impact of cryoprotectant exposure time on post-thaw viability of autologous and allogeneic hematopoietic stem cells and leukocyte subpopulations.

Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one-and three-hours post-thaw. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+ (p < 0.001), CD14+ (p < 0.001), and CD19+ cells (p < 0.001). No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N = 20), and those stored < 24 hours (N = 10), except for viable CD3+/CD4+ cells after three hours post-thaw (p = 0.028). In conclusion, DMSO had different effects on leukocyte subpopulations in cryopre-served PBSC samples. The type of donors and the length of storage before cryopreservation did not affect the post-thaw stability of cryopreserved PBSC samples.

The world apheresis association registry, 2023 update.

The WAA apheresis registry contains data on more than 140,000 apheresis procedures conducted in 12 different countries. The aim is to give an update of indications, type and number of procedures and adverse events (AEs). MATERIAL AND METHODS: The WAA-registry is used for registration of apheresis procedures and is free of charge. The responsible person for a center can apply at the site www.waa-registry.org RESULTS: Data includes reported AEs from 2012 and various procedures and diagnoses during the years 2018-2022; the latter in total from 27 centers registered a total of 9500 patients (41% women) that began therapeutic apheresis (TA) during the period. A total of 58,355 apheresis procedures were performed. The mean age was 50 years (range 0-94). The most common apheresis procedure was stem cell collection for which multiple myeloma was the most frequent diagnosis (51%). Donor cell collection was done in 14% and plasma exchange (PEX) in 28% of patients; In relation to all performed procedures PEX, using a centrifuge (35%) and LDL-apheresis (20%) were the most common. The main indication for PEX was TTP (17%). Peripheral veins were used in 56% as the vascular access. The preferred anticoagulant was ACD. AEs occurred in 2.7% of all procedures and were mostly mild (1%) and moderate 1.5% (needed supportive medication) and, only rarely, severe (0.15%). CONCLUSION: The data showed a wide range of indications and variability in apheresis procedures with low AE frequency.

Implementation and operational management of marketed chimeric antigen receptor T cell (CAR-T Cell) therapy-a guidance by the GoCART Coalition Pharmacist Working Group.

Chimeric Antigen Receptor T cells (CAR-T cells) are a type of Advanced Therapy Medicinal Product (ATMP) classified as ex-vivo (cell-based) gene therapy. CAR-T cells constitute an immunotherapy that works by enabling T cells to specifically recognise cancer cells and destroy them [1]. CAR-T cells are currently licensed to treat certain blood cancers including relapsed or refractory lymphomas, B-cell acute lymphoblastic leukaemia or multiple myeloma [2]. The indications for their use are expanding and are expected to encompass other therapeutic areas. CAR-T cells are used both in children and adults [2]. CAR-T cells are biologic drugs and are therefore more complex than traditional medicinal products. T cells collected from the patient (or donor) are sent to a Good manufacturing Practice (GMP) manufacturing facility where they are genetically modified to contain a chimeric antigen receptor (CAR). This receptor is designed to recognise and target a specific protein on cancer cells. Once manufactured, they are delivered to the hospital where they are administered to the designated patient. Hospital pharmacies are central in the process of ensuring appropriate organisational governance, operational handling, clinical suitability, and pharmacovigilance [1, 3]. The GoCART Coalition Pharmacist working group's mission was to develop standards of care to advance the field of cellular therapies in Europe. The purpose of this document is to provide practical guidance on the implementation and safe operational use of marketed CAR-T cell products within hospital pharmacies primarily throughout Europe. This document outlines the key areas where pharmaceutical expertise should focus and the key considerations for the hospital pharmacy. Countries may have different requirements and there may be variation in practice between hospitals. This document is intended as a guide and the recommendations should be adapted to meet local requirements. This document does not provide clinical information relating to the use of CAR-T cell products. The Summary of medicinal Product Characteristics (SmPC) [4, 5], and national and international clinical guidelines (where in place) should be followed for the most up-to-date clinical management of CAR-T cell patients. An example is the UK "institutional readiness documents" for pharmacy which includes detailed checklists for each stage of the pathway [6]. Spain developed the Plan of Advanced Therapies in the National Health System: CAR medicines published in November 2018 [7], the CAR-T Medicines Management Procedure of the Spanish Society of Hospital Pharmacy [8] or the Hospital pharmacist's roles and responsibilities with CAR-T medicines article published also by the Spanish Oncology group of the Spanish Society of Pharmacy [9]. This guide has been designed to support the implementation of marketed CAR-T products; however, the principles may also be applicable to clinical trials. For CAR-T cell products being used in clinical trials, additional trial regulation and clinical trial protocols must be followed. This document is divided into two sections. Section 1 outlines considerations for hospital pharmacies during the implementation of a CAR-T cell service. Section 2 outlines the key operational considerations for hospital pharmacies in the patient and product pathway.

Variable recovery of cryopreserved hematopoietic stem cells and leukocyte subpopulations in leukapheresis products.

INTRODUCTION: Due to the expansion of cell therapy using not only haematopoietic stem cells (HSC) but also other leukocyte subpopulations, the loss of these cells in cryopreserved apheresis products needs to be evaluated. Various factors that could negatively affect post-thaw recovery, such as leukapheresis product characteristics, storage time and cryopreservation protocols have been identified. METHODS: The post-thaw recovery of HSCs, lymphocytes, NK cells and monocytes, as well as the factors that could adversely affect it were analysed in autologous and allogeneic leukapheresis products. RESULTS: The lowest post-thaw recovery was observed in autologous and allogeneic CD34+ cells, with the median of 73.7% and 68.1%, respectively. In leukocyte subpopulation, the lowest post-thaw recovery was observed for CD14+ cells, both autologous and allogeneic. The highest post-thaw recovery was observed for CD3+/CD8+ cells in autologous, and for CD19+ cells in allogeneic samples. The statistically significant difference was observed between autologous and allogeneic PBSC products for CD3+ cell recovery (P = 0.031) and CD3+/CD8+ cell recovery (P = 0.009). The evaluation of factors that could adversely affect the post-thaw recovery in autologous samples showed weak negative correlations between platelet concentration and CD3+ recovery, as well as between storage time and CD3+CD8+ recovery. In allogeneic samples, a strong negative correlation was observed only between the percentage of granulocytes and CD3+, CD3+/CD8+ and CD3+/CD4+ cell recoveries. CONCLUSION: Since various post-thaw recoveries of leukocyte subpopulations were observed, the cell therapy manufacturing centers should evaluate how their cryopreservation method and other factors affect the recovery of cell population of interest in their settings.

Extracorporeal photopheresis as an immunomodulatory treatment modality for chronic GvHD and the importance of emerging biomarkers.

Haematopoietic stem cell transplantation (HSCT) is the treatment of choice for malignant haematological diseases. Despite continuous improvements in pre- and post-transplantation procedures, the applicability of allo-HSCT is limited by life-threatening complications such as graft-versus-host disease (GvHD), engraftment failure, and opportunistic infections. Extracorporeal photopheresis (ECP) is used to treat steroid resistant GvHD with significant success. However, the molecular mechanisms driving its immunomodulatory action, whilst preserving immune function, require further understanding. As ECP is safe to administer with few significant adverse effects, it has the potential for earlier use in the post-HSCT treatment of GvHD. Thus, further understanding the immunomodulatory mechanisms of ECP action may justify more timely use in clinical practice, as well as identify biomarkers for using ECP as first line or pre-emptive GvHD therapy. This review aims to discuss technical aspects and response to ECP, review ECP as an immunomodulatory treatment modality for chronic GvHD including the effect on regulatory T cells and circulating vs. tissue-resident immune cells and consider the importance of emerging biomarkers for ECP response.

The appropriateness of platelet transfusions in hematological patients and the potential for improvement.

BACKGROUND: Hematology patients are intensive platelet users. In clinical practice, a substantial proportion of platelet (PLT) transfusions are routinely administered outside the guidelines despite compelling evidence for recommendations. Those unnecessary PLT transfusions are an unjustified extra burden on a scarce healthcare resource and may also be detrimental to the patients. This study aims to evaluate indications and assess the appropriateness of PLT transfusion, as well as to identify common discrepancies and propose modalities for better compliance with guidelines. MATERIAL AND METHODS: The audit of all PLT orders for adult hematological inpatients was conducted over 2 months. The assessment was performed using guidelines for PLT transfusion. Patient demographic, clinical, and transfusion data were collected from hospital electronic medical records. RESULTS: Based on 286 PLT orders, 344 PCs were transfused to 67 patients: 235 (82.2%) prophylactical due to low PLT count, 34 (11.9%) preprocedural and 17 (5.9%) therapeutic. Overall, 105 (36.77%) PLT transfusions were inappropriate: 78 (33.2%) of all prophylactic PLT transfusions due to low PLT count, 17 (50%) off all preprocedural and 10 (58.8%) of all therapeutical transfusion. The major reason for PLT transfusion inappropriateness was transfusion above the recommended threshold. Double units of PCs were transfused in 36.7% of all PLT transfusions and 32.4% of them were considered inappropriate. CONCLUSION: Our audit of PLT transfusion practice found a large proportion of inappropriate PLT transfusions. Based on the most common deviations from the guidelines a variety of targeted measures for improvement are proposed.

Role of flow cytometry in evaluation of the cellular therapy products used in haematopoietic stem cell transplantation.

Cellular therapy nowadays includes various products from haematopoietic stem cells (HSC) collected from bone marrow, peripheral blood, and umbilical cord blood to more complex adoptive immune therapy for the treatment of malignant diseases, and gene therapy for inherited immune deficiencies. Broader utilization of cellular therapy requires extensive quality testing of these products that should fulfil the same requirements regarding composition, purity, and potency nevertheless they are manufactured in various centres. Technical improvements of the flow cytometers accompanied by the increased number of available reagents and fluorochromes used to conjugate monoclonal antibodies, enable detailed and precise insight into the function of the immune system and other areas of cell biology, and allows cell evaluation based on size, shape, and morphology or assessment of cell surface markers, as well as cell purity and viability, which greatly contributes to the development and progress of the cell therapy. The aim of this paper is to give an overview of the current use and challenges of flow cytometry analysis in quality assessment of cellular therapy products, with regard to basic principles of determining HSC and leukocyte subpopulation, assessment of cells viability and quality of thawed cryopreserved HSC as well as the importance of validation and quality control of flow cytometry methods according to good laboratory practice.

Changes in the incidence of transfusion reactions in hematological patients over the past 30 years.

BACKGROUND: Patients with hematological diseases are polytransfused and often immunocompromised, therefore susceptible to transfusion reactions (TR). This study aims to document the incidence of TRs in adult hematological patients and assess the effect of changes in the production of blood components and transfusion practice on their occurrence. STUDY DESIGN AND METHODS: Retrospective observational analysis of TRs reported from 1993 to 2019 was performed. For the analysis of the effect of changes on the incidence of TRs, the evaluated time was divided into two periods: the 1st period before the introduction of changes in production, when leukoreduced blood components were used only selectively, and the 2nd period, when semi-automated method of production and universal leukoreduction was introduced. RESULTS: The decrease in the incidence of TRs was observed for both red blood cell (RBC) and platelet concentrate (PC) transfusions in the 2nd period. Since platelet additive solution has been used, a further decrease in the incidence was reported. The decrease in incidence was also observed for delayed hemolytic/serological transfusion reactions and for transfusion-transmitted bacterial infections. Four cases of incorrect blood transfusions were uniquely related to the hematological patients, caused by antigen loss and transfusion ordering after ABO-incompatible hematopoietic stem cell transplantation. DISCUSSION: Our results provided evidence that the introduction of tools offered by modern transfusion medicine: universal leukodepletion, plasma replacement with additive solutions, sensitive laboratory techniques, prophylactic antigen matching policy, informatization, and automatization, decreased the incidence of TRs and improved transfusion safety.

Using the World Apheresis Association Registry Helps to Improve the Treatment Quality of Therapeutic Apheresis.

Therapeutic apheresis (TA) is prescribed to patients that suffer from a severe progressive disease that is not sufficiently treated by conventional medications. A way to gain more knowledge about this treatment is usually by the local analysis of data. However, the use of large quality assessment registries enables analyses of even rare findings. Here, we report some of the recent data from the World Apheresis Association (WAA) registry. Data from >104,000 procedures were documented, and TA was performed on >15,000 patients. The main indication for TA was the collection of autologous stem cells (45% of patients) as part of therapy for therapy. Collection of stem cells from donors for allogeneic transplantation was performed in 11% of patients. Patients with indications such as neurological diseases underwent plasma exchange (28%). Extracorporeal photochemotherapy, lipid apheresis, and antibody removal were other indications. Side effects recorded in the registry have decreased significantly over the years, with approximately only 10/10,000 procedures being interrupted for medical reasons. CONCLUSION: Collection of data from TA procedures within a multinational and multicenter concept facilitates the improvement of treatment by enabling the analysis of and feedback on indications, procedures, effects, and side effects.

B regulatory cells and monocyte subpopulations in patients with chronic graft-vs-host disease.

AIM: To assess the correlations of B regulatory cells (Bregs) and monocyte subsets in peripheral blood with the National Institutes of Health (NIH)-consensus-defined clinical manifestations of chronic graft-vs-host disease (cGvHD), in an attempt to establish their role as cellular biomarkers. METHODS: This multidisciplinary prospective study enrolled adult cGVHD patients treated in the University Hospital Center Zagreb and University of Zagreb School of Medicine. Immunophenotypic subpopulations of CD24highCD38high Bregs (CD27-, CD27+, and total) and monocyte (classical, intermediate, and non-classical) counts were correlated with demographic, transplant, and cGVHD-related data. Bivariate correlation analysis was performed to evaluate the correlations between Bregs and monocytes subsets and cGVHD organ involvement, as well as cGVHD severity and immunosuppression intensity. RESULTS: Twenty-two adult patients (54.5% female) with cGVHD were enrolled. The median (range) age was 44.5 years (24-65). All patients were transplanted for hematologic malignancies and 40.9% had severe NIH cGVHD global score. The median time from cGVHD diagnosis to the analysis was 16.6 months (0-176). The organ most frequently affected with cGVHD were the eyes (68.2%), skin (45.5%), lungs (45.5%), and liver (40.9%). Lower total and CD27-Bregs counts were correlated with worse cGVHD severity, higher immunosuppression intensity, and lung cGVHD, in terms of cell count, but also with skin cGVHD, in terms of percentages. Patients with liver and joint/fascia cGVHD had a lower percentage of non-classical monocytes and patients with more severe global NIH score had a higher classical monocytes count. CONCLUSION: Different organs affected by cGVHD are differently associated with different subpopulations of Bregs and monocytes.

Evaluation of the BD Stem Cell Enumeration Kit on the BD FACSCanto II flow cytometer using bd facscanto clinical and bd facsdiva software.

INTRODUCTION: CD34+ hematopoietic stem cell (HSC) enumeration by cell flow cytometry is routinely used in clinical laboratories for monitoring of HSC mobilization into peripheral blood and assessment of the quality of HSC products. The modified ISHAGE protocol is the most often used procedure for determination of CD34+ cells using flow cytometry. The aim of this study was to evaluate BD Enumeration stem cell kit on flow cytometer BD facscanto II, using facscanto clinical and facsdiva softwares. METHODS: Validation study included determination of within-run and between-run precision, trueness (bias), comparison of the test results analyzed on facscanto clinical and facsdiva softwares, assessment of linearity, specimen stability, and carryover. RESULTS: For between-run precision, coefficients of variation (CVs) were all <10%, except for low control level on facsdiva software. CVs for within-run precision were <10%, except for high absolute count of CD34+ cells on facsdiva software. Comparison of data showed no statistically significant differences between facscanto clinical and facsdiva software (Spearman's rank correlation coefficients were .993 for % of CD34+ cells and 0.983 for absolute count of CD34+ cells). In linearity study, bias for all dilutions was < 20%, and carryover assessment cannot be considered significant on both softwares. There was a statistically significant difference (P = .044) in absolute count of CD34+ cells after 24 hours of storage, when using facscanto clinical software. CONCLUSION: BD Stem Cell Enumeration Kit can be used in routine laboratory work on BD FACSCanto II instrument, whereas facscanto clinical and facsdiva software were used for acquisition and data analysis.

An automated mini buffy coat preparation method for use in mini extracorporeal photopheresis treatment of graft-vs-host-disease in a low body weight pediatric patient.

A mini extracorporeal photopheresis (mini-ECP) "off line" technique has been developed for use in the treatment of small children and patients with apheresis contraindications. Until now various methods have been used for buffy coat separation from whole blood. In this report we describe a protocol for mini buffy coat preparation using the automated Sepax laboratory separator for "off line" ECP treatment in a low body weight child with graft-vs-host-disease. According to our results this alternative method has been proven feasible and tolerable.

Performance prediction algorithm for autologous PBSC collection in adults and pediatric patients using large volume leukapheresis.

BACKGROUND AND OBJECTIVES: The number of CD34+ cells collected in apheresis procedures depends mainly on the collection efficiency of the device and the blood volume processed. Large volume leukapheresis (LVL) can improve CD34+ cell yield and has previously been investigated using the COBE Spectra device (Terumo BCT, USA). MATERIALS AND METHODS: This was a retrospective analysis of LVL performance in patients undergoing continuous mononuclear cell collection (CMNC) using the new Spectra Optia apheresis system (Terumo BCT, USA) at the University Hospital Center, Zagreb, from March 2016 to September 2016. CD34+ cell yield predictability, determined using a customized algorithm, was also assessed. RESULTS: In total, 67 procedures performed in 46 adults and 14 performed in 11 children were included in the analysis. In adults, 30 (65.2%) patients successfully reached their target preapheresis CD34+ cell count on day 1, with a median (interquartile range [IQR]) CD34+ collected cell dose of 4.8 × 106 /kg (2.3-10.6 × 106 /kg). In the pediatric group, 81.8% successfully collected the target CD34+ cell dose on the first day, with a median (IQR) CD34+ collected cell dose of 11.1 × 106 /kg (3.2-16.3 × 106 /kg). The customized algorithm showed a strong and significant linear correlation with actual CD34+ cell dose (P < 0.0001). CONCLUSION: The results of this study support the use of LVL and the customized prediction algorithm in apheresis procedures. The ability to tailor the procedure to meet the needs of the individual patient may help to minimize the blood volume processed, shorten the duration, reduce the volume of infused anticoagulants, and improve patient comfort.

LEUKOCYTAPHERESIS IN THE MANAGEMENT OF SEVERE STEROID-DEPENDENT ULCERATIVE COLITIS.

Ulcerative colitis (UC) is a multifactorial disease of unknown precise etiology and immunopathogenesis. Peripheral blood granulocytes and monocytes/macrophages are the major sources of cytokines, which regulate inflammation. Leukocytapheresis (LCAP) is a method where blood is processed by apheresis system that removes lymphocytes and plasma before being returned to the body. We report the first case in Croatia where we used LCAP in the treatment of a patient with severe steroid-dependent UC. After 12 LCAP procedures, good clinical response was obtained and there were no significant adverse side effects noticed. The patient remained in clinical remission over two years in which he underwent regular follow ups at outpatient clinic. Over a 10-year follow-up period after LCAP, the patient had only occasional clinical symptoms of disease activity. The clinical course was complicated with the development of metastatic colorectal carcinoma, which points to the importance of regular disease monitoring rather than the increased risk of malignant disease after LCAP. Patients with UC are a demanding group of patients that warrant the search for novel treatment strategies other than conventional pharmacological therapies. Although LCAP is still not a common treatment modality in our daily practice, data from recent studies suggest it to be an effective and safe procedure in the management of active UC patients.

Autologous blood as a source of platelet gel for the effective and safe treatment of oral chronic graft-versus-host disease.

BACKGROUND: Oral chronic graft-versus-host disease (cGvHD) impairs oral function and patients' quality of life. Some lesions are refractory to local and systemic immunosuppressive therapy, and new therapeutic modalities are required. The aim of the study was to assess the efficacy and safety of topical application of autologous platelet gel (PG) in patients with oral cGvHD. STUDY DESIGN AND METHODS: PG was prepared from autologous blood and applied on ulcerous lesions using an automated system. The oral cGvHD was assessed using the 273-point Oral Mucositis Rating Scale (OMRS) prior and after completion of the PG treatment. The overall response to treatment of particular topography expressed as the total score on OMRS was compared to total score on National Institutes of Health cGvHD Oral Mucosal Score (NIH OMS). The pain intensity was measured by the Numeric Pain Rating Scale (NRS). RESULTS: In five patients, 12 autologous blood collections were performed; median 3 (range 1-3) per patient, and 26 PG applications were performed; median 6 (range 2-8) per patient. PG applications reduced lesions in oral cGvHD: median OMRS total score was reduced for 43.2% (range 9.6%-47.3%), and median NIH OMS total score for 27.3% (range 20.0%-50.0%) from baseline values. Median of pain intensity reduction on NRS scale was 57.1% (range 50%-100%). No side effects were observed. CONCLUSION: Application of autologous PG in oral cGvHD showed as an efficient and safe treatment option for patients who do not respond to standard local treatment.

Haemolysis, pure red cell aplasia and red cell antibody formation associated with major and bidirectional ABO incompatible haematopoietic stem cell transplantation.

BACKGROUND: Acute and delayed haemolysis, alloimmunisation and pure red cell aplasia (PRCA) are potential complications after ABO incompatible haematopoietic stem cell transplantation (HSCT). The aims of this study were to investigate acute and delayed red blood cell (RBC) antibody-associated complications, including haemolysis, PRCA and alloimmunisation in major and bidirectional ABO incompatible HSCT. MATERIALS AND METHODS: We retrospectively examined the transplant courses of 36 recipients of bone marrow or peripheral blood stem cells from ABO incompatible donors and evaluated the current practice of performing plasmapheresis in patients with higher isoagglutinin titres. We investigated the role of ABO incompatibility in haematopoietic recovery, transfusion requirements, alloimmunisation and PRCA. RESULTS: Laboratory signs of acute haemolysis were noted in five (14%) patients, one (3%) of whom had clinically overt haemolysis. Patients with haemolysis had IgM titres ≥1:8 and received >16 mL of RBC in the HSCT. In patients with higher titres, plasmapheresis performed prior to the transplant prevented acute haemolysis. Delayed haemolysis was not recorded in the follow up. Haematopoietic recovery and transfusion requirements did not differ notably between patients with and without haemolysis. De novo RBC antibodies were detected in two (5.5%) patients after HSCT, and PRCA was noted in one (3%) patient. DISCUSSION: Carried out with adequate graft processing, plasmapheresis and blood component support, haemolysis is not a common complication after HSCT. Our results confirm that the occurrence of haemolysis depends on larger RBC volumes and higher isoagglutinin titres. Despite the reduction of patients' isoagglutinin titres by plasmapheresis, we still noted a critical combination for the development of laboratory signs of haemolysis (IgM titre ≥1:8 and RBC volume >16 mL). De novo immunisation to RBC antigens and PRCA are rare events following ABO incompatible HSCT.

Large volume leukapheresis is efficient and safe even in small children up to 15 kg body weight.

BACKGROUND: The collection of peripheral blood stem cells, although now a routine procedure, is still a challenge in low body weight children because of specific technical and clinical issues. For paediatric patients it is crucial to obtain an adequate number of CD34+ cells with the minimum number of procedures: this can be done using large volume leukapheresis (LVL). MATERIALS AND METHODS: We analysed the efficacy and safety of 54 autologous LVL performed in 50 children (33 [66%] males and 17 [34%] females), median age 2 years (range, 1-5) and median body weight 12 kg (range, 6-15). The procedures were performed with a COBE Spectra previously primed with red blood cells; ACD-A solution and heparin were used as anticoagulants. RESULTS: The target CD34+ cell dose (≥5×10/kg body weight) were collected with one LVL in 46 (92%) patients, while four (8%) patients needed another procedure. All our LVL were well tolerated. Side effects were observed in five (9.2%) patients and one procedure had to be discontinued because of catheter-related haemorrhage. The platelet count decreased significantly (p<0.001) after each procedure but without bleeding or need for transfusion support. DISCUSSION: Our experience confirms that LVL is efficient and safe even in small children, if the procedure is adjusted considering the weight and age of child. The most important factors are good venous access, adequate preparation of the child's electrolyte status, and surroundings in which the small child as well as parents feel comfortable, and can tolerate the procedure better. Although a median platelet loss of 50% can be expected, LVL is safe and reduces the overall number of procedures required. It can be recommended for peripheral blood stem cell collection even in small body weight children with malignant diseases, particularly those who mobilise low numbers of CD34+ cells.

[Extracorporeal photopheresis in treatment of chronic graft versus host disease]. / Ekstrakorporalna fotofereza u lijecenju kronicne reakcije transplantata protiv primatelja.

Extracorporeal photopheresis (ECP) is an immunomodulatory therapy which has been used in the treatment of chronic GVHD (cGVHD). ECP involves separation of the mononuclear cells with leukapheresis, followed by ex vivo administration of 8-methoxypsoralen and UV-A radiation and reinfusion to the patient. Aim of the study was to evaluate clinical and immunomodulatory effect of ECP procedures in patients with cGVHD. We analyzed 341 ECP procedures performed in 7 patients with cGVHD; median ECP per patient was 37 (range 13-131). All patients suffered from skin changes in combination with impaired joint mobility and symptoms of oral disease. ECP procedures were performed for two consecutive days: in initial phase weekly, followed by every two weeks and than monthly according to clinical response. Median of ECP treatment duration was 10 months (range 2-58). The effect of ECP in patients with cGVHD with skin andjoint involvement was mostly beneficial: 6 patients experienced either improvement or stabilization in skin changes and joint mobility. In 2 patients who suffered from oral disease, the total recovery was observed. Clinical response was typically delayed until 2 to 3 months, and reduction in glucocorticoid dose was observed. Adverse reactions were observed in 4.9% procedures. In patients who responded to ECP treatment, CD4+/CD8+ ratio and number of NK cells were normalized. ECP proved to be an efficient and safe procedure that may be recommended for patients with cGVHD who do not respond to conventional therapy.

Large volume leukapheresis: Efficacy and safety of processing patient's total blood volume six times.

Large-volume leukapheresis (LVL) differs from standard leukapheresis by increased blood flow and an altered anticoagulation regimen. An open issue is to what degree a further increase in processed blood volume is reasonable in terms of higher yields and safety. In 30 LVL performed in patients with hematologic malignancies, 6 total blood volumes were processed. LVL resulted in a higher CD34+ cell yield without a change in graft quality. Although a marked platelet decrease can be expected, LVL is safe and can be recommended as the standard procedure for patients who mobilize low numbers of CD34+ cells and when high number of CD34+ cells are required.