Infusion of Culture Positive Infusion of Culture Positive Stem Cell ProductsStem Cell Products

FDA Liaison MeetingFDA Liaison Meeting June 16, 2006 June 16, 2006

D. A. Gastineau

Mayo College of MedicineMayo College of Medicine

OverviewOverview• Background-historical perspectiveBackground-historical perspective

• Questions to be addressedQuestions to be addressed

• Specific Experience at Mayo Specific Experience at Mayo

• ISCT SurveyISCT Survey

• Changes to practice of respondentsChanges to practice of respondents• influence on sterility resultsinfluence on sterility results

• Closed SystemsClosed Systems

• ConclusionsConclusions

BackgroundBackground

• Human bone marrow and PBPC transplant Human bone marrow and PBPC transplant products have traditionally been sampled products have traditionally been sampled for bacterial contaminationfor bacterial contamination

• A small percentage of these products have A small percentage of these products have positive culturespositive cultures

• Known culture positive products have Known culture positive products have commonly been infused commonly been infused

• However, strict interpretation of the new However, strict interpretation of the new GTP regulations prohibit this practiceGTP regulations prohibit this practice

Experience at MayoExperience at Mayo

• 7233 HPC products collected from 7233 HPC products collected from January 1998 through March 2006 January 1998 through March 2006

• 2118 transplants performed2118 transplants performed

• Review of medical records for Review of medical records for evidence of adverse reactions, evidence of adverse reactions, toxicity and post-transplant blood toxicity and post-transplant blood cultures for the 69 patients cultures for the 69 patients (3% of (3% of total)total) who received positive products. who received positive products.

Collection and Transplant ActivityCollection and Transplant Activity

Total Collections 7233

Auto BM 131

Allo BM 127

PBPC 6838

DLI 137

Transplants Auto 1764

Transplants Allo 354

Sterility Testing at MayoSterility Testing at Mayo

• Before 3/04 1 ml was injected in an Before 3/04 1 ml was injected in an isolator tube and incubated for 28 isolator tube and incubated for 28 daysdays

• After 3/04 1 ml was injected in peds After 3/04 1 ml was injected in peds Bactec bottle and incubated for 5 Bactec bottle and incubated for 5 days.days.

• Samples were taken from the product Samples were taken from the product upon arrival in the laboratory and upon arrival in the laboratory and after all processing before freezingafter all processing before freezing

Frequency of Positive ProductsFrequency of Positive ProductsTotal Products Apheresis Bone

Marrow

All Collections 7233 6975 258

Culture positive (%) 119 (1.6%) 111 (1.6%) 8 (3.1%)

Excluding Culture Positive Donors

7201 6944 257

Culture positive (%) 87 (1.2%) 80 (1.2%) 7 (2.7%)

Sources of Bacteria in Product Sources of Bacteria in Product CulturesCultures

• Donor (patient)Donor (patient)

• Introduced during collectionIntroduced during collection

• Introduced during processingIntroduced during processing

• Introduced during samplingIntroduced during sampling

• Introduction during thawing processIntroduction during thawing process• Did not examineDid not examine

Post Thaw Culture Concordance Rate(n=67)

0

25

50

75

100

% C

ult

ure

Po

sit

ive

Donor/Patient as the Source of Donor/Patient as the Source of contaminationcontamination

Preprocess Preprocess sample=Asample=A

Postprocess Postprocess sample=Bsample=B

Positive Positive ProductsProducts

Products Products from Culture from Culture Positive Positive PatientsPatients

TotalTotal 119119 32 (27%)32 (27%)

Sample ASample A 4343 2 (5%)2 (5%)

Sample A&BSample A&B 2929 21 (72%)21 (72%)

Sample BSample B 4747 9 (19%)9 (19%)

Culture Positive ProductsCulture Positive Products

• When two cultures are positive (the When two cultures are positive (the “A” and “B” cultures), 21 of 29 or “A” and “B” cultures), 21 of 29 or 72% of patients had concomitant 72% of patients had concomitant bacteremias documented by blood bacteremias documented by blood culture.culture.

• The PATIENT is a major source of The PATIENT is a major source of bacteriabacteria

Organisms from HPCsOrganisms from HPCs

Organisms Isolates Infused

Coagulase negative Staphylococci 73 57

Staphylococcus aureus 8 5

Corynebacterium sp. 7 5

Enterococcus faecalis 6 6

Acinetobacter sp. 3 1

Moraxella sp 2 0

Micrococcus sp. 3 2

Gram negative bacillus 3 3

Stenotrophomonas maltophilia 3

Organisms from HPCsOrganisms from HPCsOrganisms Isolates Infused

Pseudomonas aeruginosa 2 2

Acid fast bacilli 1 1

Escherechia coli 1 1

Enterobacter cloacae 1 1

Filamentus fungus 1 1

Propionibacterium sp. 1 1

Ralstonia pickettii 1 1

Staphylococcus haemolyticus 1 1

Staphylococcus lugdunensis 1 1

Group A streptococcus 1 1

Chaetomium sp. 1

Chryseobacterium sp 1

Clostridium perfringens 1

Methylobacterium sp. 1

Pseudomonas fluorescens/putida 1

Infusion of Positive ProductsInfusion of Positive ProductsTotal Number of Total Number of Patients with positive Patients with positive products collectedproducts collected

9595

Total Positive Total Positive Products collectedProducts collected

119119

Total Patients Total Patients Receiving Positive Receiving Positive ProductsProducts

6969

Total Positive Total Positive Products InfusedProducts Infused

8888

100 Day Survival Following Infusion of Culture Positive Products

0 10 20 30 40 50 60 70 80 90 1000

25

50

75

100

Culture Pos n = 69

Controls n = 2046p = 0.419

Days

Per

cen

t S

urv

ivin

g

Figure 2Figure 2

ConclusionConclusion

• Clinical Significance of infusion of Clinical Significance of infusion of positive productspositive products

• Minimal acute toxicity or adverse Minimal acute toxicity or adverse reactionsreactions

• Equivalent short-term and long-Equivalent short-term and long-term survivalterm survival

Survey Sponsored by ISCTSurvey Sponsored by ISCT• GoalsGoals

• Obtain general information about Obtain general information about the current practice of the current practice of hematopoietic stem cell therapyhematopoietic stem cell therapy

• Obtain preliminary information Obtain preliminary information concerning experience with safety concerning experience with safety of culture-positive productsof culture-positive products

Overall Summary: 177 Reporting InstitutionsOverall Summary: 177 Reporting Institutions

Institution/Organizational Unit

3

29 26

119

0

20

40

60

80

100

120

140

Stand alonecollection facility

Stand aloneprocessing facility

Clinical transplantprogram

Combinedclinical,

collection,processing facility

90%

9% 1%

Yes

No

Not sure

Survey Question: Does your institution have a policy for handling culture-positive products?

Policies encompass these elementsPolicies encompass these elements

Investigation of the positive culture, 146

Involvement of the cell therapy lab director, 143

Notification of the patient's physician, 150

Infusion of the product w ith positive culture , 118

Informed consent of the patient if product is used, 76

Evaluation of the patient for current antibiotic use and/or

use of prophylactic antibiotics if product is used,

120

0 20 40 60 80 100 120 140 160

Types of Products Types of Products (92 Respondents)(92 Respondents)

48372

125475005

65366

0

10000

20000

30000

40000

50000

60000

70000

Auto PBSC

Related PBSC/DLI

URD PBSC/DLI

Total

Products

Frequency of Positive ProductsFrequency of Positive Products

26296

464

36736

5120

5000

10000

15000

20000

25000

30000

35000

40000

- Cult Infused

+ Cult Infused

- Products

+ Products

1.4%1.7%

Infusion of positive products: Infusion of positive products: Institutional ResponsesInstitutional Responses

Has your institution ever infused or released for infusion a PBPC or DLI product:

18

71

64

5

53

117

0 20 40 60 80 100 120 140

Not Sure

No

Yes

That was known to be culture-positive before the infusion?

That became culture-positive afterthe infusion?

(67%)

Test Methods for SterilityTest Methods for Sterility

37%

40%

8%

15%

BacT/Alert

Bactec

CFR/USP compliantmethod

Other (Please specify)

Distribution of Organisms FoundDistribution of Organisms Found

46%

5%4%0%

12%

3%

2%

1%

0%

2%

1%

0%

1%6%

1%

2%

15%

Coagulase-negative staphylococcus Staphylococcus aureus

Streptococcus species Acinetobacter

Propionobacterium species Corynebacterium

Micrococcus Klebsiella species

Clostridium species E. coli

Enterobacter Stenotrophomonas maltophilia

Pseudomonas aeruginosa Other gram negative rods

Candida species Aspergillus species

Other

Association With Donor Association With Donor BacteremiaBacteremia

• 32% of positive cultures reported to 32% of positive cultures reported to be associated with a positive culture be associated with a positive culture in the patient/donor within 5 days in the patient/donor within 5 days before or after collectionbefore or after collection

Deaths Associated with InfusionsDeaths Associated with Infusions

• Of 91 respondentsOf 91 respondents• 4 reported deaths likely related to 4 reported deaths likely related to

infusioninfusion• 0 were felt to be due to microbial 0 were felt to be due to microbial

contaminationcontamination• 1 response was excluded as it 1 response was excluded as it

reported 6 deaths but fewer than reported 6 deaths but fewer than 100 infusions and was otherwise 100 infusions and was otherwise very incompletevery incomplete

ISCT Survey Respondents ISCT Survey Respondents Collection/processing changes introduced Collection/processing changes introduced

to reduce contaminationto reduce contamination• Clean room facilities introducedClean room facilities introduced

• No effectNo effect• Second cultures sent from frozen samplesSecond cultures sent from frozen samples

• If negative, presumed to be false If negative, presumed to be false positivepositive

• All products since April 2003 have been in cGMP All products since April 2003 have been in cGMP facility, Grade A critical area, Grade B backgroundfacility, Grade A critical area, Grade B background

• No change in ratesNo change in rates• Validated blood culture process, discontinued use Validated blood culture process, discontinued use

of multi-use heparin vialsof multi-use heparin vials• Reduction from 4% to 1.9% between Reduction from 4% to 1.9% between

2003 and 20052003 and 2005

• Implemented new cleaning of BSCs, “robust” Implemented new cleaning of BSCs, “robust” changeover procedures, increased BSC changeover procedures, increased BSC preventive maintenancepreventive maintenance

• Process in BSC, clean with 70% sterile alcohol between each product Process in BSC, clean with 70% sterile alcohol between each product processed. processed.

• More strict gowning procedures, including use of sterile gloves. More strict gowning procedures, including use of sterile gloves. • Perform EM dailyPerform EM daily• Innoculate cultures for microbial testing in BSC in cell processing lab Innoculate cultures for microbial testing in BSC in cell processing lab

and then transport to microbiology.and then transport to microbiology.• GMP/GTP training performed for all staff in Cell Processing and GMP/GTP training performed for all staff in Cell Processing and

Microbiology departments including using sterile technique. Microbiology departments including using sterile technique. • Rearrangement of microbiology laboratory to accommodate microbial Rearrangement of microbiology laboratory to accommodate microbial

cultures for cellular products and to incubate all of these cultures in cultures for cellular products and to incubate all of these cultures in an isolated incubator.an isolated incubator.

• Perform tracking and trending of all positive cultures.Perform tracking and trending of all positive cultures.

• No effect on rates seenNo effect on rates seen

ISCT Survey Respondents ISCT Survey Respondents Collection/processing changes introduced Collection/processing changes introduced

to reduce contaminationto reduce contamination

• We used to culture every product immediately We used to culture every product immediately after collection and after processing. We reduced after collection and after processing. We reduced cost of culture and removed an unnecessary step. cost of culture and removed an unnecessary step. We have implemented additional training We have implemented additional training regarding the line connections to reduce possible regarding the line connections to reduce possible contamination. We had our infection control contamination. We had our infection control department observe all steps of the process to department observe all steps of the process to make suggestions to improve our handling and make suggestions to improve our handling and reduce possible contamination in the laboratory. reduce possible contamination in the laboratory. Overall our number of positive cultures has Overall our number of positive cultures has dropped from approximately 5.5% to less than 4%. dropped from approximately 5.5% to less than 4%. In 2005 we did not have any contaminated In 2005 we did not have any contaminated products. (?)products. (?)

ISCT Survey Respondents ISCT Survey Respondents Collection/processing changes introduced Collection/processing changes introduced

to reduce contaminationto reduce contamination

Changes Introduced to Reduce Infection Risk and Changes Introduced to Reduce Infection Risk and EffectsEffects

• Introduction of sterile sleevesIntroduction of sterile sleeves

• Sterile glovesSterile gloves

• No longer perform separate fungus No longer perform separate fungus culture. Reduced the number of culture. Reduced the number of positive cultures related to positive cultures related to contamination by reference lab. (no contamination by reference lab. (no numbers given)numbers given)

What is a Closed System?What is a Closed System?• ClosedClosed

• “ “ . . . an isolated system having no . . . an isolated system having no interaction with an environment.” interaction with an environment.” Dictionary of Cybernetics and SystemsDictionary of Cybernetics and Systems

• To be completely closed a product should To be completely closed a product should not be exposed to the external not be exposed to the external environment from beginning to end of the environment from beginning to end of the processing.processing.

• Is this feasible?Is this feasible?• Collection: VenipunctureCollection: Venipuncture• Sterile docking—adding reagentsSterile docking—adding reagents• SamplingSampling• InfusionInfusion

What is a Closed System?What is a Closed System?

• What would be a closed system?What would be a closed system?• A container with all necessary A container with all necessary

additives and vessels for additives and vessels for processing attached to an processing attached to an original collection containeroriginal collection container

• Final vessel disconnected from Final vessel disconnected from original set of containersoriginal set of containers• BUT: How to sample in BUT: How to sample in

process (also separate process (also separate sample bags?)sample bags?)

What is a Semi-closed System?What is a Semi-closed System?

• Definition varies widelyDefinition varies widely• Generally refers to processes Generally refers to processes

which have been adapted to bags which have been adapted to bags from “open” processes such as from “open” processes such as ficoll-hypaqueficoll-hypaque• Tsang, Transfusion, 2001Tsang, Transfusion, 2001

What is a Functionally-closed What is a Functionally-closed System?System?

• Functionally-closed systemsFunctionally-closed systems• Varies as wellVaries as well• Processing cord blood with bags Processing cord blood with bags

attached to central processing kit, attached to central processing kit, valves to open/close for each process.valves to open/close for each process.• Sepax-S100, Biosafe S.A.Sepax-S100, Biosafe S.A.• Zingsem, Transfusion, 2003Zingsem, Transfusion, 2003

• Product positive rate—7.5%, similar to that Product positive rate—7.5%, similar to that of literatureof literature

• Unable to demonstrate superiority of Unable to demonstrate superiority of “functionally-closed system”“functionally-closed system”

Functionally Closed SystemFunctionally Closed System

• Fluids intrinsic to sterilized kitFluids intrinsic to sterilized kit• No flexibility of anticoagulationNo flexibility of anticoagulation

• Sterile filters used for added reagentsSterile filters used for added reagents

How Closed is Closed?How Closed is Closed?

• A matter of degree, with nothing A matter of degree, with nothing completely closed as some air and completely closed as some air and starting material (blood) enter the starting material (blood) enter the system and may not be sterile.system and may not be sterile.

• Closed remains a relative termClosed remains a relative term

Clinical BalanceClinical Balance

• Risk of Infusion of Positive ProductsRisk of Infusion of Positive Products• Very low with the current clinical Very low with the current clinical

practice of often giving antibiotics, but practice of often giving antibiotics, but even without antibiotics, symptoms are even without antibiotics, symptoms are fewfew

• Vast majority of programs have SOPs Vast majority of programs have SOPs addressing culture-positive productsaddressing culture-positive products

• Alternatives to use of positive productsAlternatives to use of positive products• No treatmentNo treatment• RecollectionRecollection

Risk of RecollectionRisk of Recollection

• Healthy donor may need catheter re-Healthy donor may need catheter re-insertedinserted

• Autologous donor may have Autologous donor may have underlying disease increasing underlying disease increasing donation riskdonation risk

Risks of CollectionRisks of Collection

• During period of reportDuring period of report• Two patients with amyloid died Two patients with amyloid died

during mobilization/collection (not during mobilization/collection (not while on machine)while on machine)

• One central catheter migrated to One central catheter migrated to pericardial spacepericardial space

• One central catheter created AV One central catheter created AV fistula in thoraxfistula in thorax

Strategies for Reducing Positive Strategies for Reducing Positive Culture RatesCulture Rates

• Improved screening of donorsImproved screening of donors• Drawing blood cultures 48 hours prior Drawing blood cultures 48 hours prior

to collectionto collection• Cost: 100-200 blood cultures to Cost: 100-200 blood cultures to

detect a bacteremia, and difficult to detect a bacteremia, and difficult to measure benefitmeasure benefit

• Some bacteremias are transient Some bacteremias are transient (clostridium) and won’t be detected(clostridium) and won’t be detected

• Clean room environment does not have a Clean room environment does not have a clear benefitclear benefit

• ““Closed” systems-some additional Closed” systems-some additional protection for post-donor sourcesprotection for post-donor sources

SummarySummary

• Infusion of culture-positive HPC products appears Infusion of culture-positive HPC products appears to be associated with minimal toxicity when to be associated with minimal toxicity when associated with usual clinical practice and associated with usual clinical practice and precautionsprecautions

• The type of bacterium detected may affect the The type of bacterium detected may affect the clinical decision whether or not to use the productclinical decision whether or not to use the product

• HPCs are not analogous to blood products in HPCs are not analogous to blood products in ease or risk of replacementease or risk of replacement

• Overall risk to the patient AND donor for Overall risk to the patient AND donor for recollection must be balanced against the recollection must be balanced against the assessed risk of infusion of culture-positive assessed risk of infusion of culture-positive productproduct