Sterility Testing of Peripheral Blood Stem cell (PBSC) harvests in a Tertiary Oncology Setup
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1 Sterility Testing of Peripheral Blood Stem cell (PBSC) harvests in a Tertiary Oncology Setup Bankar S 1, Tirlotkar A 1, Ojha S 1, Bhat V 2,Kannan S 3, Rajadhyaksha S 1 1. Department of Transfusion Medicine, Tata Memorial Centre 2. Department of Microbiology- Tata Memorial Centre-ACTREC 3. Department of Biostatistics- Tata Memorial Centre-ACTREC
2 Introduction Peripheral blood stem cells (PBSC) are extensively used as a source of hematopoietic progenitor cells for autologous and allogeneic transplantation in patients with a wide range of malignant diseases To ensure the availability of an adequate product for transplantation, a policy of strict quality control of the collected product is essential
3 Stem Cell Transplant Process Administration of mobilization agent Administration of preparative regimen Stem cell transplantation (product issue & QC) Mobilization (CD34 evaluation) Cryopreservation Engraftment and recovery Collection (and Product quality check) Preparation of product for storage Follow up
4 Aim 1. To know the rate of microbiological contamination of PBSC harvests in a Tertiary Oncology Setup 2. To highlight the Importance of proper microbiological analysis during cryopreservation and thawing as an important part of quality control procedure
5 Material and methods Retrospective analysis of microbiological cultures done in 482 PBSC harvests (autologous and allogeneic) undergoing cryopreservation in our laboratory, from 1 st January 2011 to 31 st July 2015
6 336 Patients & Donors 220 Males 116 Females 336 Procedures 192 Autologous Procedures 144 Allogeneic procedures 482 Harvests 303 Autologous harvests 179 Allogeneic harvests
7 Type of venous access 336 patients & donors 156(46%) Central venous access 180(54%) Peripheral venous access
8 COBE Spectra PBSC collections Fresenius COM.TEC Cryopreservation Done within 24 hours of collection DMSO(8.7%); at a final concentration of 4.35% Storage at C mechanical freezer Thawing in 37 0 C waterbath Testing for product viability by Trypan blue dye exclusion test Infusion under sterile conditions and strict patient monitoring
9 Cryopreservation and storage of stem cells product as per department SOP
10 Bacterial cultures were performed on Sample 1 After collection Sample 2 Immediately before freezing (After adding DMSO) Sample 3 At the time of thawing
11 STERILITY TESTING For microbiological cultures, the segment of cryopreserved bag is cleaned with 70% alcohol and sample is drawn using aseptic techniques Sample is plated on McConkeys agar and 5% sheep blood agar following an overnight incubation in Tryptic soy broth Any growth observed is further isolated and identified using gram stain and biochemical tests
12 Statistical analysis Any correlation of contamination among samples at different stages was determined by the McNemar test, a non-parametric test for two related ordinal variables. Correlation between culture positive units between post collection and post DMSO addition samples, autologous and allogeneic collections and central and peripheral access was determined Calculations were performed using the Statistical Package for Social Science (SPSS; v 8.0).
13 RESULTS
14 Rate of culture positivity SAMPLE 1 SAMPLE 2 SAMPLE 3 POST COLLECTION AFTER ADDING DMSO (BEFORE FREEZING) POST THAW No. % No. % No. % POSITIVE NEGATIVE TOTAL
15 Comparison of Autologous Vs Allogeneic Autologous harvests Allogeneic harvests Total No Culture positive postcollection Culture positive after adding DMSO(before freezing) Total 30(9.9%) 13(7.26%)
16 Bacterial contamination in Autologous collection Post Collection After addition of DMSO( before freezing) Acinetobacter - 1 Coagulase Neg Staph aureus (CoNS) 1 6 Contaminants 2 4 E coli - 2 Enterococcous fetalis 1 1 K pneumoniae 3 3 K pneumoniae & Pseudomonas - 3 P aeruginosa - 1 Vancomycin resistant enterococi - 2 Total 7 23
17 Bacterial contamination in Allogeneic collection Post collection After addition of DMSO( before freezing) Acinetobacter 2 - CoNS - 1 Contaminants 1 - E coli 1 2 K oxytoca 1 1 K pneumoniae 1 - P aeruginosa 1 - Staph aureus 2 - Total 9 4
18 Bacterial contamination in post thaw PBSC samples Microorganisms isolated Post thaw Bacillus 1 CoNS (Coagulase neg Staph aureus) 3 Contaminants 1 Enterobacter agglomerans 2 Flavobacterium 1 Total 8
19 Comparison of isolates at sequential points of the process for autologous harvests Sample 2 Negative Positive Total Sample 1 Negative Positive Total p= P value < 0.05,it is statistically significant
20 Comparison of isolates at sequential points of the process for allogeneic harvests Sample 2 Negative Positive Total Sample 1 Negative Positive Total p value=0.227 P value > 0.05,which means it is not statistically significant
21 Correlation of venous access with culture positivity Central venous access Peripheral venous access Total Negative Positive p value=0.984 Total p value =0.984 which is > 0.05 so not statistically significant
22 Salient features Most common microorganism found in Autologous harvests are CoNS followed by Klebsiella pneumoniae Most common microorganism found in Allogeneic harvests are E coli followed by Klebsiella species Most common microorganism found at the time of thawing is CoNS
23 Comparison of isolates of autologous PBSC harvest at post collection and pre freezing was found to be statistically significant which suggest there may be contamination during handling of bag and during addition of DMSO Comparison of isolates of allogeneic PBSC harvests at post collection and pre-freezing was not statistically significant
24 The correlation of venous access and culture positivity was not statistically significant that means whether it is central or peripheral access, rate of bacterial contamination is not dependent on venous access
25 What should we do with the culture positive products??
26 Dilemma Possible side effects Precious stem cells
27 All the culture positive PBSC were infused under antibiotic coverage. The patients didn t have any significant side-effects
28 Conclusion We found that bacterial contamination affects 3.31% in post collection, 5.6% in post DMSO,1.96% in post thawing period Most of the positive cultures were due to skin flora or environmental micro-organisms including Coagulase negative Staphylococcus aureus (CoNS), Klebsiella pneumoniae, E coli Although bacteria are said to survive cryopreservation poorly,we observed culture positive units post thaw, indicating that in most cases,the source of bacterial contamination could result during thawing or improper handling techniques during microbiological testing
29 At our laboratory, the rate of contamination of autologous PBSC was higher. Possible reasons may be a More patients with indwelling catheters Higher no. of previously infected patients However, none of the patients were septic or had signs of catheter infection at the time of PBSC harvesting
30 Comparison of current study with previous studies Sr No. Year and study group Period of study Type of study Total No. of PBSC units 1 Attarian H et al years Prospective % % of culture positive units 2 M J Majado et al years Prospective % 3 Mark A Klein et al years Prospective % 4 Donmez et al years Retrospective % in post DMSO 3.66% in post thawing period 5 Present study /2 years Retrospective % in post collection 5.6% in post DMSO 1.96% in post thawing period
31 References Quality control of bacterial contamination in autologous peripheral blood stem cells for transplantation LUIS LARREA et al haematologica 2004; 89(10): Risk factors for microbial contamination of peripheral blood stem cell products Ayhan Donmez et al Transfusion Volume 52, Issue 4: , April 2012 Microbial contamination of peripheral blood stem cell collections Attarian HB WM Bensinger C D Buckner D L McDonald S D Rowley Bone Marrow Transplantation 06/1996; 17(5): Microbial Contamination of Hematopoietic Stem Cell Products: Incidence and Clinical Sequelae Mark A.Klein 1,Diane Kadidlo 3,Jeffrey McCullough 2,David H.McKenna 2,Linda J.Burns 1 Biology of Blood and Marrow Transplantation Volume 12,November 2006,Pages Influence of harvest bacterial contamination on autologous peripheral blood progenitor cells post-transplant M J Majado et al Bone Marrow Transplantation (2007) 39,
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