Testing for bacterial contamination in platelets

Septic reaction from transfusions of bacterially contaminated platelets represents the greatest current patient risk of contamination in transfusion medicine. The risk of severe septic reaction from contaminated platelets in the United States is approximately 1:80,000,¹ far greater than the risk of 1:2,000,000 for viral transmission of HIV in blood transfusions. Technologies for rapid detection of bacterial contamination at point of issue (POI), which offer promise of reduction of this transfusion risk, have received FDA clearance, yet there is reluctance to accept the need to transition to this new methodology and the added cost of testing.²

The existing testing protocol for bacterial contamination of platelets represents the compromise accepted nearly a decade ago based on the technology available at that time. Bacterial contamination in platelets for transfusion is a dynamic system. If present in platelets, bacteria may dramatically increase in hours to days. Platelets, on the other hand, are perishable; viability decreases daily. A test for bacterial contamination needs to be sufficiently sensitive and robust to detect the widest range of potential bacterial contaminants, yet sufficiently rapid to achieve results within the reasonable use life of the platelets.

The FDA and industry struggled with the compromises required with this dynamic system. In the United States, platelet shelf life is currently five days at room temperature. In 1984 it had been extended to seven days, but in 1986 it was reduced to five days due to the increased risk of septic reaction in recipients. In 2005 another attempt was made to clear seven-day platelet shelf life under restrictions of early (24-hour) culture testing.  This trial was discontinued within two years as further testing validated that the risk of significant contamination of extended, six- and seven-day platelets was not reduced by early 24-hour culture. Bacteria, for reasons of dormancy, slow growth, or overcoming the bacteriostatic capabilities of the platelet milieu, could be undetectable at 24 hours but reach measureable levels later in the platelet life.³

Until recently there have not been tests available which are universal bacterial detectors, capable of detecting bacterial loads in platelets within a time frame that does not compromise the limited shelf life of the platelets.

To maintain viability, platelets for transfusion are stored at room temperature in oxygen-permeable bags, and under these conditions bacteria can proliferate. This requires a different testing model than for viral contaminants, which are static on collection. Initial contamination of bacteria may be less than 1 cfu/ml, yet with reasonable lag phase and doubling time this can grow to 10⁵ cfu/ml in days.^4,5 Work by Yomtovian and Jacobs identified that clinically the 10⁵ cfu/ml load in platelets increased the risk of any reaction by a factor of 4.0 and a severe septic reaction by a factor of 3.4.⁶

 Work by Wagner and Eder at American Red Cross⁷ has shown that the sample volume is quite critical for detection of low contamination levels. By proposing that the sample volume increase from 4 ml to 8 ml, they calculate that an increase in the probability of detecting very low levels of contamination for a transfusable unit of platelets increases from about 15% to 40%. This had motivated the industry to double the sample volume to 8 ml for culture. This has improved the current culture protocol, but has not reduced the risk of transfusion of contaminated platelets to an industry-wide acceptable level.

Culture methodologies for the detection of bacteria

There are currently two culture methods that are FDA-cleared and in routine use for the detection of bacteria in platelets. The FDA clearance for these tests is for QC of platelets.

One is an automated device which continuously monitors culture bottles for bacterial growth. The culture bottle is inoculated with the sample and when placed in the instrument rack is monitored for color change in a disc in the base of the bottle. Active bacterial metabolism generates CO2, which will change the pH of the disc, which changes the color of the pH indicator in the disc. The photometric monitoring will indicate the color change and identify a contaminated sample. The sensitivity of the bottles is approximately 10⁷ cfu/ml.  The average time for an inoculated bottle to turn positive is 12 hours to 22 hours.⁸

The second culture-based method is a single-use test. Sample is inoculated into a nutrient-rich bag and incubated for 24 hours. The oxygen in the head space of the bag is then sampled, and a decrease in oxygen is indicative of bacterial growth.

In the U.S. approximately 95% of platelets are tested by culture for bacterial contamination, with at least 95% of these being performed with the first device. The general protocol in the U.S. for this device requires a 24-hour waiting time between platelet collection and subsequent sampling of 8 ml of platelet solution for culturing. If the sample has not turned positive after an additional 24 hours, the platelets are released as “No growth to date.” The culture bottle remains in the instrument for ongoing monitoring. This process consumes the first two days of the five-day shelf life.

This protocol using culture, while traditional and analytically highly sensitive, is of limited effectiveness, since it has been shown that it is only 25.9% to 40% effective, mostly due to the low bacterial count even after 24 hours.

Point-of-issue assays for the detection of bacteria in platelets

Newer technologies approach the issue as a clinical and not an analytical problem. The goal of the new technologies is to detect clinically relevant bacterial loads rapidly, in less than one hour, so that testing might be closer to time of transfusion. These methods may not be as sensitive as culture, but they are rapid and generally capable of measuring bacterial loads below 10.⁵ Currently two technologies have been cleared by the FDA for testing prior to transfusion. They are both cleared to be used in conjunction with a growth-based QC assay method.

The first of these technologies uses a novel methodology for the detection of bacteria. It incorporates a protein which detects peptidoglycan, which is the cell wall component of all clinically relevant bacteria. When peptidoglycan is present from bacteria in platelets, it triggers a reaction cascade which ends in the development of a red color. The analyzer is a dedicated photometer which monitors the color development. Software interprets the absorbance to determine whether the sample is bacterially contaminated. Since bacterial cell walls are variable within and across species, there is variation in detection limits for each species.

Some pre-analytical treatment of the sample is required to break down cellular material and expose the peptidoglycan structure, if present. This processed sample is then placed in the single-use reaction tube, and this is placed in a dedicated analyzer for monitoring and reporting. The entire assay, about 15 minutes of processing and 30 minutes of photometric monitoring, requires about 45 minutes total. The analyzer can accommodate up to eight tubes. A new random access analyzer has been unveiled recently.

Data in the package insert states lowest detection limits from 1.3 x 10³ for Staph epidermidis to highest 7.6 x 10⁴ for e. coli in leuko-reduced apheresis platelets for cultured bacterial strains from ATCC, below the target of 10⁵ cfu/ml. Publications have shown that this method of detection of bacteria is robust. Four publications studying about 20 clinical isolates have shown detection of clinical isolates also is below the 10⁵ cfu/ml threshold.

The second POI technology uses a lateral flow assay format for detection of bacteria. It incorporates a mix of antibodies for lipoteichoic acid and lipopolysaccharides, which are also cell wall components of bacteria. The assay, in a single-use cassette format, has separate windows to distinguish reactions of gram positive or gram negative bacteria as well as a control window to confirm that sample was applied to the cassette. Reading and interpretation of final result is manual.

Pre-analytical treatment of the sample is needed to expose the bacterial cell wall constituents to react with the specific antibodies in the lateral flow unit. The package insert recommends that initial readings begin within 20 minutes after addition of sample, but that readings should be repeated every 10 minutes for up to 60 minutes if a reading is not seen.

Data in the package insert states detection limits of 8.2 x 10³ for Pseudomonas aeroginosa to 8.6 x 10⁵ for Serratia marscescens for cultured ATCC strains in leuko-reduced apheresis platelets.

A third technology has been reported⁹ in the literature that uses a physical principle of changes in impedance of the sample. The method reports that by adding a stressor to the platelet concentrate the differential impedance of the stressed sample versus an unstressed sample is an indication of the presence of bacteria. It was reported that testing seventeen different bacteria by this technology could detect 10³ cfu/ml in 30 minutes.

This method, as reported, is a direct test on platelet samples and does not require the pre-analytical separation steps. As yet, there are no reports of clinical trials of this method.

Currently, POI tests for bacteria in platelets represent an added test to confirm quality of platelets. Use of a POI assay in addition to the early culture method has been shown to reduce transfusion of bacterially contaminated platelets by 75%, significantly reducing patient risk.¹⁰ At this time there is no requirement to perform POI testing, although it is expected that the FDA will create guidelines to include pre-transfusion testing for bacteria in platelets.