Collection Performance Metrics in MNC collection Dr. Rasika Setia Associate Director & HOD Department of Transfusion Medicine, BLK Super Speciality Hospital, New Delhi

Success of HSCT Successful peripheral blood stem cell (PBSC) transplantation depends on the infusion of an adequate number of hematopoietic stem cells to produce a rapid and durable hematological recovery. Adequate mobilization and optimal MNC collection Patient’s diagnosis, chemotherapy regimen and number of previous chemotherapy cycles or radiation. 

Monitoring Peripheral Blood CD34+

Defining a Target for Successful Outcome? ≥ 2 x 106 CD34+ cells/kg hematopoietic engraftment in 95% of patients Patients receiving > 2.5 x 106 CD34+ cells/kg experience durable neutrophil engraftment by day 18 (Haas et al ) 5 x 106/kg may be threshold for rapid platelet engraftment Unclear if > 5 x 106/kg will result in any better engraftment, may be associated with improved outcome Klumpp et al (8.0 × 106CD34+ cells/kg). To LB, et al. Blood. 1997;89:2233-58; 2Schiller G, et al. Blood. 1995;86:390-7; 3Kiss JE, et al.  Bone Marrow Transplant. 1997;19:303–10; Weaver CH, et al. Blood 1995;86:3961–9; 5Dercksen MW, et al. J Clin Oncol. 1995;13:1922–32.

Collection Performance Metrics in MNC collection

Definition Performance metrics describe the characteristics the procedure and characteristics of the final product ( Stem Cells) which affect the final outcome of the procedure on the donor (in allogeneic HSCT) and transplant in the recipient (both in autologous and allogeneic HSCT). .

Factors Affecting Collection Metrics Adequate mobilization Pre-apheresis counting Collection efficiency Product Purity and Quality Viability of CD 34+ cells Final Product volume Mobilization of stem Collection efficiency of the procedure Target Dose

HPC-Apheresis

Pre-apheresis cell counts CD34+ cells: Looking For A Needle In A Haystack? Pre-apheresis cell counts RBC 4.00 x 10^12/L PLT 200 x 10^9/L WBC 50 x 10^9/L MNC% 10% of WBC CD34+ 100/µL Proportion of CD34+ In MNC: 1 : 50 In WBC: 1 : 500 In total cells: 1 : 42551

Prediction It is possible to predict accurately the hematopoietic progenitor cell (HPC) dose that will be collected in an apheresis session depending on: Peripheral CD 34 concentration at the start of collection Patient weight Volume of blood to be processed (from predicted end-run results), & benchmark mean “CE2” Collection Efficiency of the cell separator In addition, a prediction tool estimates blood volumes required to obtain target dose2 It may allow collecting the HPC transplant in fewer procedures or shorter procedure times 1.Antony F. Cousins et al., HPC-A Dose Prediction on the OptiaVR Cell Separator Based on a Benchmark CE2 Collection Efficiency:Promoting Clinical Efficiency, Minimizing Toxicity, and Allowing Quality Control, Journal of Clinical Apheresis 00:00–00 (2015) 2.Daniel L. Leberfinger,’ Improved planning of leukapheresis endpoint with customized prediction algorithm: minimizing collection days, volume of blood processed, procedure time, and citrate toxicity’, TRANSFUSION Volume 00, Month 2016

Performance metrics – what do they mean Efficiency (CE%): the higher the efficiency, the less blood needs to be processed to obtain a desired cell yield. Throughput: the higher the throughput (cells/min), the less time it takes to process a given volume of blood. Platelet loss: the lower the platelet loss, the lower the bleeding risk of the patient/donor, and the less likely a platelet transfusion will be required post-collection. Product volume: cell processing labs like smaller products; they require less cryo-protectant DMSO (it is toxic for patients) and take less space in the nitrogen tanks. Product purity: granulocyte contamination in products generally causes problems during the freezing & thawing process, and granulocytes are toxic for the patient.

Procurement-Collection Efficiency It describes the number of target cells collected as a proportion of the total number processed. CE1 % is defined as the target cells collected as a percentage of target cells available in the TBV processed based on the average of the pre and post collection estimates of the target cells number CD34+ cells collected CD34+ cell average concentration x blood processed Collection efficiency CE1% = CE 2% is defined as the target cells collected as a percentage of the target cells available in the TBV processed based on the pre collection estimates of the target cell numbers CD34+ cells collected CD34+ cell initial concentration x blood processed Collection efficiency CE2% =

Affects of Centrifugation at the Interface Mononuclear cells are quickly and efficiently expelled from the denser red cell layer due to the red cells zeta potential Granulocytes are less dense and more pliable than mononuclear cells, thus resides between the mononuclear and red cell interface. Depending on the number of granulocytes, some may rise into the buffy coat layer, but most can be found embedded in the upper red cell layer.

Circulating CD34+ Cells During Apheresis Intra Procedural Mobilization is variable

CE% Changes According To Recruitment

Type of Collection Intermittent flow optical/manual input from the operator Continuous flow method controlled by computer software consisting of double chamber designed to collect PBSC- collection occurs in phases. Automated , continuous flow process using single stage separation chambers and cyclic MNC collection with a software application.

Type of Collection

Performance : Donor Collection Continuous CD341 cell collection by a new device is safe and more efficient than by a standard collection procedure: results of a two-center, crossover, randomized trial Cancelas et al; Transfusion Volume 56; November 2016

Are There enough CD 34+ Cells Performance – Hematopoietic Cells In the Bag Determined by (cell count * volume) Accurate cell counting? Accurate volume measure? Accurate flow cytometry? Small errors here or with the pre-apheresis counting can have a profound effect on other metrics like collection efficiency Are There enough CD 34+ Cells In the Bag?

Product Purity Things that do not contribute to engraftment and/or can harm the patient Volume High Product Volume = more toxic dimethyl sulfoxide (DMSO) and more freezer storage space Red cells ABO mismatched allografts Red Cell Lysis Frozen thawed products Platelets Excess loss in patient/donor Granulocytes Clumping after freezing and thawing and during extra processing Peri-infusional toxicities during re-infusion

Product Purity Things that do not contribute to engraftment and/or can harm the patient or donor Platelets Donor/patient may loose excess platelets during the procedure These platelets end up in collection bag May be reason for clumping after freezing and thawing and during extra processing Scott D. Rowley et al., “Effect of Cell Concentration on Bone Marrow and Peripheral Blood Stem Cell Cryopreservation’’

Product Purity Red cells1 Acceptable range: Haematocrit of 2-5 % Higher haematocrit not desirable for : ABO mismatched allografts – RBC lysis Frozen thawed products – RBC lysis Gravity Sedimentation Centrifugation Ficoll-Hypaque High Hematocrit Low Hematocrit Techniques For Reduction of RBCs /HCT Gravity Sedimentation Centrifugation Ficoll-Hypaque 1.Georg Stussi et al 2006

Product Purity Granulocytes Literature describes four factors which may trigger adverse events during the infusion of the autografts1 Intrinsic factors Patient age Sex Extrinsic factors Number of granulocytes Clumping 6.065 x 109 granulocytes is possibly best cut off to predict the occurrence of adverse events1 When clumping is present in the cryocyte bags, the infusion filter is replaced after the infusion of each bag1 1.R Cordoba et al., ‘The occurrence of adverse events during the infusion of autologous peripheral blood stem cells is related to the number of granulocytes in the leukapheresis product’, Bone Marrow Transplantation (2007), 1–5

Product Volume Product Volume Dimethyl sulfoxide (DMSO) is known to induce toxic effects when used in clinical settings: infusion of cryopreserved HPCs has always been associated with a variety of adverse events (AEs) The infusion of DMSO gives rise to dose dependent symptoms: Gastrointestinal-Abdominal cramps, nausea ,diarrhoea Cardiovascular- hypo- or hypertension, brady- or tachycardia Neurological toxicity Skin flushing Dyspnoea Even though patients received less than the maximum recommended DMSO, one dose (i.e., 1 mg/kg body weight/day) more than 1/3 experience AEs DMSO is notorious for toxicities even in lower than 10% concentrationsIn the univariate analysis, factors showing a statistically significant association with onset of adverse events total volume infused, amount of DMSO, total nucleated cells/kg, total number of granulocytes and clumping1 Otrock et. al. Transfusion. 2017 Adverse events of cryopreserved hematopoietic stem cell infusions in adults: a single-center observational study Otrock ZR Cordoba et al., ‘The occurrence of adverse events during the infusion of autologous peripheral blood stem cells is related to the number of granulocytes in the leukapheresis product’, Bone Marrow Transplantation (2007), 1–5 Transfusion. 2017 Mar 16. doi: 10.1111/trf.14072. [Epub ahead of print] 2. Adverse events of cryopreserved hematopoietic stem cell infusions in adults: a single-center observational study. Otrock ZK1, Sempek DS1, Carey S1, Grossman BJ1. Author information: 1 Department of Pathology and Immunology, Barnes-Jewish Hospital, Washington University, St Louis, Missouri. Abstract BACKGROUND: Autologous hematopoietic stem cell (HSC) transplantation has been used for almost three decades for the management of malignant hematologic diseases and some solid tumors. Dimethyl sulfoxide (DMSO) is used as a cryoprotective agent for hematopoietic progenitor cells (HPCs) collected by apheresis (HPC-A). We evaluated the factors contributing to the occurrence of adverse events (AEs) of cryopreserved HPC-A infusion. STUDY DESIGN AND METHODS: Between January 2009 and June 2014, a total of 1269 (1191 patients) consecutive HPC-A infusions were given to adult patients undergoing autologous HSC transplantation at Barnes-Jewish Hospital. Only infusions on the first day of transplant were included in the analysis. RESULTS: AEs were reported in 480 (37.8%) infusions. The most common AEs were facial flushing in 189 (39.4%) infusions, nausea and/or vomiting in 183 (38.1%) infusions, hypoxia requiring oxygen in 139 (29%) infusions, and chest tightness in 80 (16.7%) infusions. Multivariate analysis using logistic regression showed that female sex (odds ratio [OR], 1.78; 95% confidence interval [CI], 1.40-2.26; p < 0.0001), diagnosis other than multiple myeloma (OR, 1.44; 95% CI, 1.12-1.84; p = 0.004), larger volume of infusion per body weight (OR, 1.66; 95% CI, 1.29-2.15; p < 0.0001), and number of granulocytes infused per body weight (OR, 1.30; 95% CI, 1.01-1.67; p = 0.042) were significant predictors of occurrence of AEs during infusion. CONCLUSION: AEs due to HPC-A infusion occurred in more than one-third of patients. Interventions need to be instituted to reduce AEs and thus improve the safety of HPC-A infusion. Many of these toxicities can be attributed to DMSO, and this is reflected in the volume of infusion. It might be warranted to consider implementing DMSO-reducing protocols before infusion. © 2017 AABB. PMID: 28301051

Product Volume Product Volume Shorter procedure time is desirable

Laboratory Processing Cell processing lab needs: Timely and predictable delivery of products for processing Small product volumes for cryopreservation and storage As few non-target cells as possible May need additional plasma for processing and/or storage Cell processing lab activities may include Product quality measurements Volume reduction Red cell depletion Cryopreservation, storage and transport Secondary processing (CD34+ selection, ECP)

Collection Performance Efficiency Stem Cell Mobilization Easy operating the system Accuracy In reporting Collection efficiency and yield Product purity Viability of CD 34+ cells Product Volume Collection Performance Efficiency

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