Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

The overall goal of this procedure is to evaluate the reduction of common bacterial contaminants in platelet products using a riboflavin and UV based pathogen reduction process. This is accomplished by first preparing a stock of high titer bacteria. The second step is to obtain a human platelet product and evaluate the unit cell quality.

Next, the platelet unit is dosed with riboflavin and inoculated with a known concentration of bacteria. The final step is treating the unit with UV light. Ultimately, bacterial reduction is measured by comparing pre-treatment and post-treatment samples for viable bacteria as measured by an endpoint dilution plating assay.

The main advantage of this technique over existing methods like bacterial screening, is that riboflavin and UV light can inactivate other pathogens like H-I-V-H-C-V, malaria and Babesia. The implications for this technology extend beyond bloodborne pathogens because the technology is also capable of inactivating white blood cells and donated blood products. Those have been associated with adverse transfusion events such as transfusion associated graft versus host disease.

I first came up with the thought of using this method to inactivate pathogens in blood through studies I was doing with the interaction of compounds, photochemical compounds with nucleic acids. The technique utilizes the fact that pathogens, unlike red blood cells, platelets and plasma proteins contain genomic nucleic acids that are essential for the replication and for transmission of disease. So by targeting nucleic acid chemistry in the milieu of blood products that are present, we can more selectively inactivate the pathogens while preserving the quality of red cell platelet and plasma protein constituents Demonstrating the procedure.

Today will be Nick HOA, laboratory scientist and Denise Gilmore, laboratory technician. First, prepare a sterile 250 milliliter bottle containing 100 milliliters of growth medium specific for the bacterium of interest. Using a sterile inoculating loop inoculate a single bacterial colony from a streak culture plate into the medium.

Next, place the bottle into an orbital shaking incubator. Set the shaker speed to 140 RPM and incubate the culture for one to two days. Periodically check the culture for bacterial growth.

After incubation, transfer the bacterial suspension into two sterile 15 milliliter conical tubes. Centrifuge the tubes at 3000 times G for 20 minutes at 23 degrees Celsius. Next, aspirate the supernatant and resuspend the bacterial pellet with 15 milliliters of sterile peptide water.

Vortex the tubes to aid in suspension, and then pool the resuspended bacterial cultures to tighter the stock culture. Fill five milliliter dilution tubes each with 1.8 milliliters of peptide water. Transfer 200 microliters of undiluted sample into the first tube, vortex the tube, and then transfer 200 microliters to the next tube.

Repeat until the appropriate dilution is reached. Transfer 100 microliters of each dilution to an appropriate agri plate Using a sterile cell spreader, even lead. Distribute the sample across the AER for a negative control plate.

500 microliters of the peptide water onto two agar plates. Using a sterile cell spreader evenly, distribute the sample across the aer, invert all the plates and incubate them at the appropriate temperature for one to two days after incubation. If growth is observed on either of the negative control plates, the peptide water is contaminated and the titer is invalid.

If the negative control plates are negative, count the colonies on all plates with 30 to 300 colonies. Then calculate the titer of each test sample, and record the results to see a few per milliliter. Refrigerate the bacterial cultures for up to two weeks at four degrees Celsius.

For accuracy, re titer the culture before use. Obtain a standard apheresis human platelet product of 90 to 360 milliliters with a concentration of 0.80 to 3.8 million platelets per microliter. Allow the bag to rest for at least two hours in a platelet incubator at 22 degrees Celsius, and then gently agitate at 60 RPM until use.

Remove a two milliliter sample of the platelet product and use a blood gas analyzer at 22 degrees Celsius to ensure the pH is above 6.6. Using the same sample, measure the platelet concentration with a hematological analyzer to ensure it falls in the desired range. Next, visually inspect the platelet bag by holding it under a direct white light source.

Squeeze the bag briefly and observe the swirling pattern of the sample. If a turid homogeneity remains before and after squeezing the bag, do not use the platelets with a tubing docking device. Transfer the platelets to a riboflavin and UV light illumination and storage bag.

Remove the empty platelet bag using a tubing sealer and discard it as biohazardous waste. Next, remove the light protective outer wrap of the 500 micromolar riboflavin stock solution sterile. Dock the riboflavin pouch onto the inlet line of the treatment and storage bag, and drain the contents into the platelet unit.

Then hang the pouch and treatment bag set from the riboflavin pouch. Gently press residual air out of the treatment bag and into the riboflavin bag. Rinse any residual riboflavin into the platelet pouch with a small amount of platelet product.

Allow the riboflavin and platelet solution to drain back into the treatment bag. Then clamp the inlet line. Remove the empty riboflavin bag with the tubing sealer and discard the empty bag appropriately.Sterile.

Dock a six inch tubing line with a female syringe connector onto the inlet line of the illumination and storage bag. Attach a 10 milliliter syringe barrel to the tubing line. Next, add five milliliters of the bacterial stalk suspension into the syringe barrel.

Then open the inlet line clamp and allow the bacteria to drain into the platelet product. Rinse the syringe barrel by allowing some platelet product to flow back. After rinsing, remove the syringe barrel.

Attach a 30 milliliter syringe and flush the inlet port at least twice after the product is well mixed. Use a clean five milliliter syringe and draw a two milliliter pretreatment sample. Use a new syringe to remove any air introduced to the bag.

Then remove the inlet line from the treatment bag and weigh the platelet solution. Begin platelet treatment by first entering the operator ID and then mount the bag on the illuminator pla. Next, enter the sample ID using a barcode reader.

Enter the product type and enter its weight into the software. Next, close the quartz clamp to automatically deliver 6.24 joules per milliliter of ultraviolet energy. Typical treatment lasts five to 10 minutes.

In the meantime, titer the pre-treatment sample after UV treatment, titer a post-treatment sample to assay bacterial survival, and then titer the stock bacterial culture as a positive control. The stock culture titer should be within one log unit CFU per milliliter of its recorded titer. The reduction of 11 different bacterial species was evaluated using riboflavin and a UV light-based pathogen reduction technology process.

At least six platelet products were evaluated per organism and each was inoculated. With enough bacteria teel, a final titer greater than 10, 000 CFU per milliliter samples were taken prior to treatment, as well as immediately following treatment with riboflavin and UV when compared to pre-treatment solutions. All bacterial loads were reduced by at least 2.6 log units were approximately 400 fold by treatment with riboflavin and UV light.

Following these studies with high titer bacteria, additional studies can be done using more clinically relevant spike titers. These actually represent the levels of bacteria that might be present in products at the time of donation. The goal of those studies with low titer spiking is to evaluate the ability, the method to prevent the growth of bacteria and to allow them to be maintained culture negative during the period of storage, extended periods of storage at room temperature.

Following the introduction of this technology, it has allowed the use by researchers and medical professionals to evaluate the ability to inactivate a broad spectrum of pathogens and to use this technology to prevent the spread of transfusion transmitted diseases. After watching this video, you should have a good understanding of how to measure log reduction of bacteria in a highly contaminated platelet product treated with the with riboflavin and UV light.