microbial testing of cell therapy products summary of nih clinical center studies elizabeth read md...

Microbial Testing of Cell Therapy Products

Summary of NIH Clinical Center Studies

Elizabeth Read MDChief, Cell Processing Section

Department of Transfusion MedicineNIH Clinical Center

Bethesda, MD

6/16/06

Study Design & Goals

Parallel Study

CFR vs BacT/AlertCFR vs Bactec

Using actual cell therapy products,compare use of automated

culture methods with CFR method

Khuu et at. Transfusion 2006, in press

Seeded Study

CFRvs BacT/Alert

vs Bactec

Using mock MNC products,demonstrate that automated

culture methods areequivalent to CFR method

Khuu et al. Cytotherapy 2004;6:183-195

Seeded Study: Design• Goal: Evaluate organism

detection and time to detection

• Mock mononuclear cell products from leukapheresis

• 6 commonly used product media– Citrated autologous plasma– PlasmaLyte A + HSA– Freeze mix (DMSO/Pentastarch)– RPMI 1640– X-VIVO 20 (contains gentamycin)– RPMI 1640 w/ multiple antibiotics

• Each sample seeded with 10 and 50 CFU of 10 different organisms

• CFR vs BacT/Alert vs Bactec

0

20

40

60

80

100

SA ML KP PG YE BS CS PB CA AN

per

cen

t p

osi

tive

CFR/USP BacT/Alert Bactec

Both BacT/Alert and Bactec were superior to CFR in overall organism

detection

N=6x3x2=36; except AN, n=28

A nigerC albicans

P acnesC sporogenes

B subtilisY enterocolitica

P aeruginosaK pneumoniae

M luteusS aureus

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336

Time to detection (hours)

A nigerC albicans

P acnesC sporogenes



M luteusS aureus

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336


A nigerC albicans

P acnesC sporogenes



M luteusS aureus

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336


CFR/USP

BacT/Alert

Bactec

Both BacT/Alert and Bactec were superior to CFR in time to detection

Even for inocula of 10 CFU, time to detection was < 7 days for both BacT/Alert and Bactec (but not for CFR)

0

50

100

P lasma Infusion Freeze RP MI XVIVO ABX

per

cen

t p

osi

tive


Multiple antibiotics in product medium impaired detection of organisms in all

systems

N=10x3x2=60

0

20

40

60

80

100


% p

ositi

ve

SA

ML

KP

PG

YE

BS

CS

PB

CA

AN

In medium with multiple antibiotics, impaired detection was variable from one organism to the

next

No Growth

SA, YE, BS, AN

No Growth

SA, ML, BS, PB

Parallel Study: Design

• Goal: evaluate field performance, false positives (true pos evaluation is best done by seeded study)

• Tested in process and final product samples from real cell therapy products

• Timeframes– 12/1/02 - 5/16/04

• BacT/Alert vs CFR1125 samples– 5/17/04 – 12/31/05

• Bactec vs CFR 492 samples

Definition of positive results

• Positive results expressed as– True positive = detection by system +

confirmation by gram stain and/or subculture

– False positive = detection by system, but could not confirm presence of organisms by gram stain or subculture

Parallel Study: Results

• True positive– Rates comparable for all systems– Time to detection: Automated systems were

equivalent to, or faster than, CFR/USP

• False positive– High rates (7.1%) with CFR method vs almost

none with automated methods (0.2%)– Most related to high cell (RBC or WBC) counts

in product

Parallel Study: Results by Product CategoryProduct Category

Donor Types

Collection Methods

Sampled Product Types

Processing Methods

Containers Microbial Culture Results

Apheresis Group A

N=514

Living, autologous or allogeneic

Apheresis in hospital apheresis unit

PBSC

MNC

Minimal manipulation:

RBC reduction,

plasma reduction, cryopreservation

Bags True pos

0.4%

False pos

0.2%

Apheresis Group B

N=446

Living, autologous or allogeneic, some pd research donors


PBSC

MNC

Minimal manipulation:

immunomagnetic selection or elutriation in semiclosed systems

Bags, vials True pos

1.6%

False pos

0.2%

Apheresis Group C

N=385

Living, autologous or allogeneic, some pd research donors


PBSC

MNC

Cultured for 3-15 days, some open processing steps, gentamycin used in most cultures

Product pooling in some cases

Bags, tubes, vials, flasks

True pos

0.5%

False pos

0.0%

Parallel Study: Results by Product Category

Product Category

Donor Types

Collection Methods


Processing Methods


Bone marrow

N=20


Percutaneous needle aspiration of posterior iliac crests in operating room

Bone marrow

Minimal manipulation: automated cell concentration and washing in closed systems

Bags True pos

5.0%

False pos

0.0%

Umbilical cord blood

N=72

Living, full term neonate

Post partum umbilical venipuncture in room adjacent to delivery room

Umbilical cord blood

Minimal manipulation: RBC reduction and WBC concentration with some open processing steps

Bags, tubes, vials

True pos

1.4%

False pos

0.0%

Pancreas Harvest

N=52

Cadaveric Whole organ removed from abdomen in operating room

Pancreas transport medium

Addition of transport medium (UW solution with penicillin) to, and packaging of, organ

Bags True pos

36.5%

False pos

0.0%

Parallel Study: Results by Product CategoryProduct Category

Donor Types

Collection Methods


Processing Methods


Processed tissue

N=107

Cadaveric or living, autologous

Organ or tissue harvested in operating room

Pancreatic islets, tumor infiltrating lymphs

Extensive manipulation may include dissection, enzyme & mechanical digestion, washing, density gradient separation and culture, with many open steps

Bags, tubes, flasks

True pos

2.8%

False pos

1.9%

Other

N=21


Various PBSC, MNC

Various, may include samples taken after container failures

Bags, vials True pos

9.5%

False pos

0.0%

Organisms Detected in Cell Therapy Products*

(not including pancreas and processed tissue)*most common organisms

Gram Positive• Staphylococcus epidermidis• Staphylococcus capitis• Staphylococcus, coag

negative• Proprionobacterium acnes

• Actinomyces spp• Bacillus spp• Corynebacterium spp• Coryneform bacterium• Enterococcus faecalis• Enterococcus spp• Peptostreptococcus• Rothia spp• Staphylococcus aureus• Streptococcus mits• Streptococcus, alpha hemolytic• Streptococcus, Group B

Gram Negative

• Pseudomonas fluorescens• Pseudomonas putida• Stenotrophomonas maltophilia• Brevundimonas dimunuta• Bacteroides spp• Proteus mirabilis

Do all true positives represent actual product contamination?

• Highly unlikely, because we are frequently unable to demonstrate organism growth in samples from same product or product derived from same parent product and processed in parallel

• Given limited volume and number of samples available for a given cell therapy product, this is a problem that is not easily resolved

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