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

2. 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. 

3. Monitoring Peripheral Blood CD34+

4. 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: ; 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.

6. Collection Performance Metrics in MNC collection

7. 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). .

8. 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

9. HPC-Apheresis

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

12. 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

13. 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.

14. 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% =

15. 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.

16. Circulating CD34+ Cells During Apheresis
Intra Procedural Mobilization is variable

17. CE% Changes According To Recruitment

18. 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.

19. Type of Collection

20. 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

21. 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?

22. 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

23. 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’’

24. 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

25. 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

26. 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 Mar 16. doi: /trf [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], ; p < 0.0001), diagnosis other than multiple myeloma (OR, 1.44; 95% CI, ; p = 0.004), larger volume of infusion per body weight (OR, 1.66; 95% CI, ; p < 0.0001), and number of granulocytes infused per body weight (OR, 1.30; 95% CI, ; 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:

27. Product Volume
Product Volume
Shorter procedure time is desirable

28. 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)

29. 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

30. Thank You