# Presentation: 893893

## BacTxTM Kit for Detection of Bacterial Contamination of Platelets

- Andrew E. Levin, Ph.D.Immunetics, Inc.Blood Products Advisory Council MeetingMarch 9, 2006

## BacTxTM Kit for Detection of Bacterial Contamination of Platelet Units

## BacTxTM Kit Features

- R&D supported by NHLBI grant HL065877

- < 1 hour turnaround time
- 1 ml sample volume
- Premeasured vial reagent dispensing
- Detects both gram positives and gram negatives
- 103 – 104 CFU/ml assay sensitivity
- < 10 CFU/ml detected at 24 hrs post platelet collection
- Absorbance readout = result record

## Bacterial Peptidoglycan Cell Wall 

- NAM

- NAG

- Peptide bridge

- NAG = N-Acetyl GlucosamineNAM = N-Acetyl Muramic Acid

## BacTxTM Assay Target: Peptidoglycan

-  Peptidoglycan –         universal component        of bacterial cell wall    – both gram positive    and gram negative    species
-  Common molecular       structure recognized    by peptidoglycan    binding protein

## BacTxTM Assay Technology

- BacTxTM Assay Technology

- Peptidoglycan

- Peptidoglycan binding protein (PGBP)

- Enzyme

- Chromogen

-  Binding of PGBP to peptidoglycan triggers enzymatic conversion of chromogenic   substrate to visible product with measurable absorbance

## BacTxTM Assay Format

- Spin down, extract

- Incubate

- 30 min

**Platelet Unit **

** **

**Results**

- 1 ml

- 10 min

- Read Absorbance

- Sample

## Assay dose-response curve:Bacteria spiked into platelets 

- Assay dose-response curve:Bacteria spiked into platelets

- 35,000

- 18,000

- 3,500

- 1,800

- 350

- 180

- 35

- 18

- 0

- 0

- CFU/ml

**Notes:**
S. marcescens or E. coli spiked into platelets and assayed immediately.

## BacTxTM Sensitivity for Gram Positive and Negative Bacteria

- ng/ml

- BacTxTM Sensitivity for Gram Positive and Negative Bacteria

**Notes:**
Bacteria spiked at indicated CFU/ml and platelet samples assayed immediately.

## Time course of detection of Staph. epidermidis spiked at 10 CFU/ml into platelet bag

- AABB 2004 Abstract #SP47

- Earliest detection < 7 hours post-inoculation

**Notes:**
A-500 assay result increases in parallel with bacterial concentration (CFU/ml) in growing cultures in platelet bags.

## Time to detection 103-104 CFU/ml BacTxTM vs. BacT/ALERT

- Time to detection 103-104 CFU/ml BacTxTM vs. BacT/ALERT

- AABB 2005 Abstract #SP74

- BacTxTM < 1 hour  vs.  BacT/ALERT > 8 hours

## Time to Detection: BacTxTM vs. BacT/ALERT

- AABB 2005 Abstract #SP74

- 100% concordance

**Inoculum**

**BacT/ALERT**

**BacTx****TM**

**BacTx****TM**

**BacTx****TM**

**Notes:**
Bacteria were spiked into apheresis platelet units at 3 levels: 1, 10 and 100 CFU/ml.  Actual spiked concentrations were verified by plating, as shown above.  Bact/ALERT culture bottles were inoculated simultaneously with the same levels of bacteria.  Samples from platelet bags were assayed at 24 and 48 hours post-spiking.  At 24 hours, bacteria are detected by BacTx in all but one platelet unit (S. aureus at 200 CFU/ml); at 48 hours, all are detected in agreement with Bact/ALERT results.  A-500 values interpreted vs. cut-off = 0.190 (A-500 > 0.190 = positive, A-500 < 0.190 = negative).

## BacTxTM vs. Culture: Testing Pre-screened Negative Units

| 0% | 0% |
| --- | --- |
| 0% | 100% |

- Automated culturePall BDS or Bact/ALERT

- BacTx **TM**

- n=45 13 SDP, 25 RDP, 7 Non-LR RDP

- Pos

- Neg

- Pos

- Neg

- 100% Concordance

- BacTxTM vs. Culture: Testing Pre-screened Negative Units

## Apheresis vs Random Donor units

- Units inoculated at ~10 CFU/ml, sampled for assay at 48 hours

| CFU/ml @ 48 hrs | 0 | 0 |  | 3.6x108 | 6x107 |  | 8x107 | 7x108 |  | 5x107 | 9x107 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

- Equivalent performance in BacTxTM assay

**Notes:**
Bacteria are detected similarly in apheresis platelet units and random donor platelet units.  Three bacterial strains were spiked in parallel into both AP (SDP) and RDP units and samples tested 24 and 48 hours later.  CFU/ml in each platelet bag were determined by plating.  Bacteria failed to grow in one SDP unit spiked with S. marcescens.

## R&D supported by NHLBI grant HL065877