The authors have no acknowledgements.

1. Bacteria Suspension Preparation:
<ol>
	<li>From a streaked culture plate, aseptically inoculate a single colony into a 250 ml&#160;sterile bottle containing 100 ml&#160;of the appropriate growth media (Table 1) by way of sterile inoculating loop.</li>
	<li>Place the bottle into an orbital shaking incubator at the appropriate conditions (Table 1) for 1-2 days, checking for growth periodically. Set the shaker speed to 140 RPM.</li>
	<li>Aseptically transfer the bacterial suspension into two sterile 50 ml&#160;conical tubes and centrifuge the tubes at 3,000 x g for 20 min at 23 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend each bacterial pellet with 15 ml&#160;of sterile peptone water. Vortex as necessary. Combine the suspended bacteria culture into one tube/container.</li>
	<li>Titer the stock culture in triplicate per section 4 to determine the stock culture titer.</li>
	<li>Refrigerate for up to 2 weeks at 4 &#177; 2 &#176;C until needed. Re-titer the culture at the time of use, see step 4.8.</li>
</ol>
2. Platelet Product Preparation:
<ol>
	<li>Collect a standard apheresis human platelet product at an accredited blood bank. The platelet product must meet the following collection specifications: 90&#8211;360 ml&#160;volume and 0.8 &#8211;3.8 x 106 platelets/&#181;l&#160;concentration.
	<ol>
		<li>Ensure that the platelet product has rested for a minimum of 2 hr&#160;prior to processing for pathogen reduction. Store the products at 22 &#177; 2 &#176;C in a platelet incubator without agitation for these 2 hr. After two hours ensure the platelet product is agitated at 22 &#177; 2 &#176;C in a platelet incubator. Use the product for up to 22 hr&#160;post collection.</li>
	</ol>
	</li>
	<li>Using a 3 ml&#160;syringe remove a 2 ml&#160;sample to measure the pH22C of the platelet product. Use a blood gas analyzer to ensure the product has a pH &#62;6.6. Use the same sample to measure the platelet concentration of the platelet product using a hematological analyzer. Ensure the product meets the 0.80&#8211;3.8 x 106 platelets/&#181;l&#160;collection concentration requirement.</li>
	<li>Visually evaluate the swirl of the platelet product. If the product has a &#8220;0&#8221; swirl the product will be discarded.</li>
	<li>Hold the platelet bag up under a direct white-light source and squeeze the platelet bag briefly. Visually observe the swirling pattern of the platelet sample. Score the product using the following scale. 
	NOTE: 2 &#8211; Positive Swirl: Swirling inhomogeneity visible throughout the entire bag with strong contrast. 1 &#8211; Intermediate Swirl: Some inhomogeneity visible, but only in a few places and with poor contrast. 0 &#8211; Negative Swirl: Turbid homogeneity remaining before and after squeezing the platelet bag</li>
</ol>
3. Riboflavin and UV Light Process:
<ol>
	<li>Transfer the platelet product to a riboflavin and UV light illumination and storage bag using a sterile tubing docking device. After transfer remove the empty platelet unit collection bag using a tubing sealer and discard the empty bag as biohazardous waste.</li>
	<li>Remove a 35 ml&#160;pouch of 500 &#956;M riboflavin stock solution from its light protective outer wrap. Sterile dock the riboflavin kit onto the inlet line of the treatment and storage bag. Allow the contents to drain into the platelet unit.</li>
	<li>Hang the riboflavin pouch / treatment and storage bag set from the riboflavin pouch and gently express residual air out of the treatment and storage bag and into the riboflavin bag. Ensure a small amount of platelet product is also expressed into the riboflavin pouch to rinse any residual riboflavin into the platelet product. Allow the riboflavin and platelet solution drain back into the treatment and storage bag and then clamp the inlet line. Remove the empty riboflavin bag using a tubing sealer and discard the empty bag as biohazardous waste.</li>
	<li>Sterile dock a 6&#8221; tubing line with female syringe connector onto the inlet line of the illumination and storage bag. Attach a 10 ml&#160;syringe barrel (remove the plunger) onto the female syringe connector.</li>
	<li>Aseptically add 5 ml&#160;of stock bacteria into the syringe barrel via a pipette. Open the clamp on the inlet line and allow the bacteria to drain into platelet product. Rinse the syringe barrel by allowing some platelet product to flow back into the 10 ml&#160;syringe barrel. Remove the 10 ml&#160;syringe barrel.</li>
	<li>Aseptically attach a 30 ml&#160;syringe and flush the inlet port a minimum of two times to ensure the stock bacteria is mixed into the platelet product. After the product is well mixed, use a clean 5-10 ml&#160;syringe and remove a 2 ml&#160;pre-treatment sample. Using a new syringe ensure any air introduced from the inoculation step or sampling step is removed.
	<ol>
		<li>Titer the pre-treatment sample per section 4.</li>
	</ol>
	</li>
	<li>Remove the inlet line from the treatment and storage bag and weigh the product.</li>
	<li>Place the product into the illuminator and follow the onscreen commands to start a standard platelet treatment.
	<ol>
		<li>Enter in Operator ID.</li>
		<li>Secure and mount the bag to the Illuminator platen.</li>
		<li>Enter the Sample ID into the Illuminator using either the barcode reader or the manual entry keypad.</li>
		<li>Scan product type.</li>
		<li>Close the quartz clamp.</li>
		<li>The illumination device will automatically deliver 6.24 J/ml&#160;of energy. The typical treatment time takes 5-10 min.</li>
	</ol>
	</li>
	<li>Following treatment sterile dock a 6&#8221; tubing line with female syringe connector onto the sample bulb line of treatment and storage bag. Ensure the product is well mixed and then attach a 5 ml syringe and remove a 2 ml&#160;post-treatment sample.
	<ol>
		<li>Titer the post-treatment sample per section 4.</li>
	</ol>
	</li>
</ol>
4. Bacterial Titer
<ol>
	<li>Estimate the titer of the pre-treatment and post-treatment samples and prepare the appropriate number of 5 ml&#160;dilution tubes needed to perform an end point serial dilution of each sample. Aseptically fill the required tubes with 1.8 ml&#160;of peptone water.</li>
	<li>Titer each sample using a 10-fold serial dilution scheme. Aseptically transfer 0.2 ml&#160;from the undiluted sample to the first dilution tube (10-1). Vortex this dilution tube and transfer 0.2 ml&#160;to the next dilution tube. Continue the serial dilution through the required number of dilutions.
	<ol>
		<li>Aseptically transfer 100 &#181;l of each dilution to be plated to an appropriate agar plate (Table 1).</li>
	</ol>
	</li>
	<li>Using a sterile cell spreader, evenly distribute the sample onto each agar plate.</li>
	<li>Invert and incubate the plates at the appropriate conditions (Table 1) for 1-2 days checking for growth periodically.</li>
	<li>Count plates with 30&#8211;300 colonies. Plates containing greater than 300 colonies are considered too numerous to count (TNTC). Plates containing less than 30 colonies are considered to be below the limit of quantification (LOQ).</li>
	<li>Calculate the titer of each test sample and record the results as CFU/ml.
	<ol>
		<li>Use the following equation to calculate CFU/ml: 
		<img alt="Equation 1" fo:content-width="2.5in" src="/files/ftp_upload/52820/52820eq1.jpg" width="250" /></li>
		<li>If there are no colonies on the lowest dilution plate, use the following equation to calculate the Limit of Detection (LOD): 
		<img alt="Equation 2" fo:content-width="1in" src="/files/ftp_upload/52820/52820eq2.jpg" width="100" /> 
		and 
		<img alt="Equation 3" fo:content-width="1in" src="/files/ftp_upload/52820/52820eq3.jpg" width="100" /> 
		Where: 
		N =lowest number of particles (CFU) in product that can be detected with confidence. 
		P = probability that a microorganism will be undetected; for a 95% confidence of detecting a microorganism, P = .05. 
		V = total product volume in ml 
		v =volume plated (ml) x dilution level of plate</li>
	</ol>
	</li>
	<li>For Negative Control, plate 0.5 ml&#160;of the peptone water that is used for the serial dilutions onto each of two agar plates. Use the same plate type as the samples to be tested. Invert and incubate the plate at the appropriate conditions for 1-2 days checking for growth periodically.
	<ol>
		<li>If growth is observed on either agar plate, the bottle of peptone water used must be discarded; the results of the study are also invalidated.</li>
	</ol>
	</li>
	<li>For Positive Control, re-titer the stock culture of bacteria that was used. Invert and incubate the plates at the appropriate conditions for 1-2 days checking for growth periodically. The titer shall be &#177; 1.0 log CFU/ml&#160;of the recorded stock titer (See section 1.5) for the study to be considered valid.</li>
</ol>

<ol>
	<li>Brecher, M. E., Hay, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+contamination+of+blood+components.">Bacterial contamination of blood components.</a> Clin. Microbiol. Rev. 18 (1), 195-204 (2005).</li><li>Brecher, M. E., Holland, P. V., Pineda, A. A., Tegtmeier, G. E., Yomtovian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+bacteria+in+inoculated+platelets:+implications+for+bacteria+detection+and+the+extension+of+platelet+storage.">Growth of bacteria in inoculated platelets: implications for bacteria detection and the extension of platelet storage.</a> Transfusion. 40 (11), 1308-1312 (2000).</li><li>Cardo, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+inactivation+of+Leishmania+donovani+infantum+in+plasma+and+platelet+concentrates+using+riboflavin+and+ultraviolet+light.">Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light.</a> Vox Sang. 90 (2), 85-91 (2006).</li><li>Cardo, L. J., Salata, J., Mendez, J., Reddy, H., Goodrich, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+inactivation+of+Trypanosoma+cruzi+in+plasma+and+platelet+concentrates+using+riboflavin+and+ultraviolet+light.">Pathogen inactivation of Trypanosoma cruzi in plasma and platelet concentrates using riboflavin and ultraviolet light.</a> Transfus. Apher. Sci. 37 (2), 131-137 (2007).</li><li>Dodd, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+viral+risks+of+blood+and+blood+products.">Current viral risks of blood and blood products.</a> Ann. Med. 32 (7), 469-474 (2000).</li><li>Dodd, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+contamination+and+transfusion+safety:+experience+in+the+United+States.">Bacterial contamination and transfusion safety: experience in the United States.</a> Transfus. Clin. Biol. 10 (1), 6-9 (2003).</li><li>Dumont, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+single-donor+apheresis+platelets+for+bacterial+contamination:+the+PASSPORT+study+results.">Screening of single-donor apheresis platelets for bacterial contamination: the PASSPORT study results.</a> Transfusion. 50 (3), 589-599 (2010).</li><li>Goodrich, R. P., Gilmour, D., Hovenga, N., Keil, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+laboratory+comparison+of+pathogen+reduction+technology+treatment+and+culture+of+platelet+products+for+addressing+bacterial+contamination+concerns.">A laboratory comparison of pathogen reduction technology treatment and culture of platelet products for addressing bacterial contamination concerns.</a> Transfusion. 49 (6), 1205-1216 (2009).</li><li>Greco-Stewart, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serratia+marcescens+strains+implicated+in+adverse+transfusion+reactions+form+biofilms+in+platelet+concentrates+and+demonstrate+reduced+detection+by+automated+culture.">Serratia marcescens strains implicated in adverse transfusion reactions form biofilms in platelet concentrates and demonstrate reduced detection by automated culture.</a> Vox Sang. 102 (3), 212-220 (2012).</li><li>Keil, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Plasmodium+spp.+in+plasma+and+platelet+concentrates+using+riboflavin+and+ultraviolet+light.">Inactivation of Plasmodium spp. in plasma and platelet concentrates using riboflavin and ultraviolet light.</a> Transfusion. 53 (10), 2278-2286 (2013).</li><li>Kleinman, S., Chan, P., Robillard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risks+associated+with+transfusion+of+cellular+blood+components+in+Canada.">Risks associated with transfusion of cellular blood components in Canada.</a> Transfus. Med Rev. 17 (2), 120-162 (2003).</li><li>Larsen, C. P., Ezligini, F., Hermansen, N. O., Kjeldsen-Kragh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+years'+experience+of+using+the+BacT/ALERT+system+to+screen+all+platelet+concentrates,+and+additional+testing+of+outdated+platelet+concentrates+to+estimate+the+frequency+of+false-negative+results.">Six years' experience of using the BacT/ALERT system to screen all platelet concentrates, and additional testing of outdated platelet concentrates to estimate the frequency of false-negative results.</a> Vox Sang. 88 (2), 93-97 (2005).</li><li>Mundt, J. M., Rouse, L., Van den Bossche, J., Goodrich, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+Biological+Mechanisms+of+Pathogen+Reduction+Technologies.">Chemical and Biological Mechanisms of Pathogen Reduction Technologies.</a> Photochem. Photobiol. , (2014).</li><li>Murphy, W. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+platelet+concentrates+for+bacterial+contamination:+low+numbers+of+bacteria+and+slow+growth+in+contaminated+units+mandate+an+alternative+approach+to+product+safety.">Screening platelet concentrates for bacterial contamination: low numbers of bacteria and slow growth in contaminated units mandate an alternative approach to product safety.</a> Vox Sang. 95 (1), 13-19 (2008).</li><li>Brien, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+incidence+and+residual+risk+of+HIV,+HBV+and+HCV+at+Canadian+Blood+Services.">Current incidence and residual risk of HIV, HBV and HCV at Canadian Blood Services.</a> Vox Sang. 103 (1), 83-86 (2012).</li><li>Pearce, S., Rowe, G. P., Field, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+platelets+for+bacterial+contamination+at+the+Welsh+Blood+Service.">Screening of platelets for bacterial contamination at the Welsh Blood Service.</a> Transfus. Med. 21 (1), 25-32 (2011).</li><li>Ramirez-Arcos, S., Kou, Y., Perkins, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+universal+point-of-issue+assay+for+bacterial+detection+in+buffy+coat+platelet+components.">Evaluation of a universal point-of-issue assay for bacterial detection in buffy coat platelet components.</a> Vox Sang. 107 (2), 192-195 (2014).</li><li>Reddy, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+testing+of+a+novel+riboflavin-based+technology+for+pathogen+reduction+and+white+blood+cell+inactivation.">Toxicity testing of a novel riboflavin-based technology for pathogen reduction and white blood cell inactivation.</a> Transfus. Med. Rev. 22 (2), 133-153 (2008).</li><li>Ruane, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemical+inactivation+of+selected+viruses+and+bacteria+in+platelet+concentrates+using+riboflavin+and+light.">Photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light.</a> Transfusion. 44 (6), 877-885 (2004).</li><li>Tonnetti, L., Proctor, M. C., Reddy, H. L., Goodrich, R. P., Leiby, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Mirasol+pathogen+reduction+technology+system+against+Babesia+microti+in+apheresis+platelets+and+plasma.">Evaluation of the Mirasol pathogen reduction technology system against Babesia microti in apheresis platelets and plasma.</a> Transfusion. 50 (5), 1019-1027 (2010).</li><li>Vanlandingham, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemical+inactivation+of+chikungunya+virus+in+plasma+and+platelets+using+the+Mirasol+pathogen+reduction+technology+system.">Photochemical inactivation of chikungunya virus in plasma and platelets using the Mirasol pathogen reduction technology system.</a> Transfusion. 53 (2), 284-290 (2013).</li><li>Walther-Wenke, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+bacterial+contamination+of+blood+components+in+Germany:+effect+of+contamination+reduction+measures.">Monitoring bacterial contamination of blood components in Germany: effect of contamination reduction measures.</a> Vox Sang. 100 (4), 359-366 (2011).</li></ol>

All authors of this paper are currently employed by Terumo BCT, developer and manufacturer of the Mirasol PRT system.&#160; Publication fees for this video-article have been paid by Terumo BCT.

Bacterial screening has become common practice in blood banking institutions and for many locations it serves as the primary method to prevent transfusion of bacterially contaminated platelets. Although bacterial screening typically interdicts the majority of fast growing Gram negative bacteria like S. marcescens, E. coli and E. cloacae, it struggles to detect slow growing Gram positive bacteria like S. aureus, Streptococcus spp. and S. epidermidis8. Unfortunately, these three Gram positive bacteria are implicated in upwards of 50% of all contaminated platelet units and transfusion of platelet products containing these three organisms has lead to patient mortality1.
Bacterial screening relies on the bacterial titer in a platelet product to be sufficiently high enough so that at least one organism is contained within the typical 4-8 ml screening sample. If the bacterial titer is not sufficiently high for a bacterium to be captured in the 4-8 ml sample volume, the unit is incorrectly flagged as negative and it is released for transfusion. An alternative approach to bacterial screening could be to employ a proactive process that can be performed on all platelet units and that could render the contaminating organisms as non-functional.
In this study, high titers of bacteria were purposely added to platelet units to test the total reduction capacity of the riboflavin and UV light pathogen reduction process. Although the bacterial loads tested in this study far exceed those normally found in blood products at the time of donation, this study helps to demonstrate that the riboflavin and UV light process is able to significantly reduce the titer of viable bacteria in a contaminated platelet unit by 2.6 to 5.4 log (99.7-99.9996% reduction of contaminating bacteria) (Figure 2). To obtain an accurate log reduction value, it is critical that the operator have good aseptic technique along with precise pipetting skills when performing the bacterial titer steps. Imprecise pipetting when performing serial dilutions can lead to an accumulation of errors, thus an inaccurate titer measurement.
The data collected in this study supports previous work with riboflavin and UV light, which challenged the system against low level, clinically relevant bacteremia (&#60;20 CFU/unit). The previous study was designed to mimic an actual clinical contamination event and the results showed riboflavin and UV light was effective in preventing bacterial growth over the 7-day platelet storage period. Modeling based on the data from the previous study suggests that the current risk estimates of bacterial contamination of platelet products could be reduced by as much as 98% from current levels8. The riboflavin and UV light process compares favorably to bacterial screening, which only demonstrated a 66% effectiveness to detect bacterial contaminants in platelet units when challenged with a clinically relevant bacterial load8.
As with all technologies, PRT treatment of platelet products to prevent complications associated with bacterially contaminated platelet units has its limitations. PRT systems are not effective against bacterial spores and do not inactivate endotoxin, thus even if a PRT system can inactivate high titers of Gram negative bacteria the remaining endotoxin can still cause septic shock in the recipient. PRT is best performed early during storage before the bacteria can propagate to high titers.
In conclusion, as demonstrated by the combination of these two studies, the riboflavin and UV light process is effective at reducing the bacterial loads of both Gram negative and Gram positive organisms and may offer a viable alternative to bacterial screening. Routinely treating platelet products with riboflavin and UV light also has the added benefit of reducing the potential transmission of other blood borne agents like viruses and parasites.

Transfusion of platelets, plasma, packed RBCs and whole blood products play a major role in modern medicine and can be used to treat various medical conditions, replace vital fluids and ultimately save lives. Platelets are a cellular product that are either isolated from whole blood and pooled into a transfusable dose or are collected through the process of platelet apheresis. The main role of platelets in the body is to stop bleeding at wound sites and to help maintain hemostasis. Patients suffering from a low platelet cell count (thrombocytopenia) are susceptible to spontaneous bleeding events and are transfused with platelets to bring their platelet cell count back into a normal range. Collected platelets are stored for a maximum of 5-7 days and are stored at 22 &#177; 2 &#176;C while under constant agitation.
Despite the life saving properties of platelet transfusions there remains a slight risk to transfusion recipients due to contamination of these products by parasites, bacteria and viruses11. The implementation of viral nucleic acid testing (NAT) has significantly decreased the risk of viral transmission of major blood borne agents, such as hepatitis C virus (HCV), hepatitis B virus (HBV) and human immuno-deficiency virus (HIV)5. A recent publication from the Canadian Blood Services estimates the residual risk for these agents to be 1 per 8 million donations for HIV, 1 per 6.7 million donations for HCV and 1 per 1.7 million donations for HBV15.
Although bacteria typically garner less attention in the general public, the frequency of bacterial contamination in platelet products has been estimated to be as high as 1:1,0007 and because millions of platelet products are transfused each year many recipients are exposed to potentially life threatening complications like sepsis6. Research suggests that the bacterial load at the time of contamination is low, &#60;100 colony forming units (CFU)/product2,16, however the nutrient rich environment and room temperature storage allows contaminating bacteria to proliferate to dangerously high titers prior to transfusion. Currently, the only approved methods available to prevent bacterially contaminated products from reaching a platelet recipient is through the use of culture based systems and rapid point of care testing. Briefly, for culture based systems platelet products are stored on an agitator at 22 &#176;C for 12-24 hr to allow bacteria to proliferate in the product, upon which a 4-8 ml sample is removed from the platelet product and inoculated into a bottle containing nutrient media. The bottle is placed into an instrument which monitors the bottle for bacterial growth. If the instrument detects bacterial growth in the bottle it is flagged and the corresponding platelet unit is discarded. While this process is reasonably successful at detecting fast growing organisms, many of the slow growing species do not grow to a high enough titer to be detected, thus creating the potential for false-negative units to be released for transfusion7,12,14,16,22. Unlike culture based detection systems, rapid point of care testing is typically performed later in the platelet storage period when the bacterial load has increased significantly. The higher titers are required since point of care tests are less sensitive than the culture based systems, and only reliably detect bacteria once they reach a titer &#8805;1 x 103 CFU/ml17. However such tests can provide results within 1 hour of sampling. Variability in the performance of these tests have led to the release of a false negative product, causing a fatal septic reaction in the recipient9.
An alternative way to combat the issue of bacterial contamination of platelet products is through the routine use of a pathogen reduction process that can inactivate contaminating bacteria instead of try to detect them. Using riboflavin as a photosensitizer, in combination with UV light, has been shown to reduce the infectivity of a broad range of pathogenic blood-borne contaminants, including bacteria3,4,8,10,19-21.The use of riboflavin and UV light for pathogen reduction is non-toxic and non-mutagenic, and riboflavin and UV light-treated components have been shown to be safe for transfusion recipients as well as for those handling blood products18. Briefly, riboflavin molecules can associate with the nucleic acids (DNA and RNA) of bacteria, parasites, viruses and any nucleated cell (e.g. white blood cells). Exposure to UV light activates riboflavin, causing a chemical alteration to functional groups of the nucleic acids (primarily guanine bases), thus preventing replication and/or transcription of the nucleic acids and leaving the organism inactivated13. Anucleated cells like platelets and red blood cells are not affected by the riboflavin chemistry due to the lack of nucleic acid.
Previous work with riboflavin and UV light technology evaluated a select group of bacteria using an experimental design intended to mimic a platelet product contaminated with a clinically relevant bacterial load (&#60;20 CFU/product)8. The goal of this study was to evaluate the riboflavin and UV light process against high titer bacterial contamination (&#62;1.0 x 105 CFU/ml) in platelet products treated in plasma in order measure the total bacterial reduction capacity of the system. Based on data collected from hemovigilance studies1, a panel of commonly occurring gram negative and gram positive organisms was selected for evaluation in this study and included the following species: Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus mitis, Streptococcus pyogenes, Serratia marcescens, Yersinia enterocolitica, Brucella neotomae, Bacillus cereus (vegetative form), Esherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. All organisms, except B. cereus, were obtained from ATCC and a priority was placed on obtaining bacterial strains that had been identified as being isolated from blood components. The B. cereus tested in this study is a clinical strain isolated internally from a contaminated platelet product.

<table><tbody><tr><td>Mirasol Illuminator</td><td>Terumo BCT</td><td>N/A</td><td></td></tr><tr><td>Mirasol Treatment/Storage Bag</td><td>Terumo BCT</td><td>N/A</td><td></td></tr><tr><td>Hematology Analyzer</td><td>Beckman-Coulter</td><td>Ac&amp;bull;T diff&amp;nbsp;</td><td></td></tr><tr><td>Blood Gas Analyzer</td><td>Siemens</td><td>RapidLab 1265&amp;nbsp;</td><td></td></tr><tr><td>Sterile Docking Device</td><td>Terumo BCT</td><td>TSCD</td><td></td></tr><tr><td>Platelet Incubator</td><td>Helmer</td><td>PC2200</td><td></td></tr><tr><td>Orbital Incubator Shaker</td><td>Lab-Line</td><td>4628</td><td></td></tr><tr><td>Dry Incubator</td><td>Fisher</td><td>Iso-temp</td><td></td></tr><tr><td>Class II A/B3 Biosafety Cabinet</td><td>Thermo-forma</td><td>1286</td><td></td></tr><tr><td>Tubing Sealer</td><td>Sebra</td><td>Smart Sealer II</td><td></td></tr><tr><td>Table Top Centrifuge</td><td>IEC</td><td>Centra GP8R</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>30 ml Syringe</td><td>BD</td><td>309650</td><td></td></tr><tr><td>5 ml Dilution Tube</td><td>VWR</td><td>211-0058</td><td></td></tr><tr><td>50 ml Conical Tube</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>Micropipette</td><td>Gilson</td><td>P-200</td><td></td></tr><tr><td>Micropipette</td><td>Rainin</td><td>R-200</td><td></td></tr><tr><td>Repeat Pipette</td><td>Eppendorf</td><td>4780</td><td></td></tr><tr><td>1-200 &amp;micro;l Filter Tip</td><td>Costar</td><td>4810</td><td></td></tr><tr><td>25 ml Disposable Serrological Pipette</td><td>Costar</td><td>4489</td><td></td></tr><tr><td>5 ml Disposable Serrological Pipette</td><td>Costar</td><td>4487</td><td></td></tr><tr><td>35 ml 500 &amp;micro;M Riboflavin</td><td>Terumo BCT</td><td>35 mL 500 &amp;micro;M&amp;nbsp;</td><td></td></tr><tr><td>Brucella Agar with 5% Horse Blood</td><td>BBL</td><td>221547</td><td></td></tr><tr><td>Brain Heart Infusion</td><td>Teknova</td><td>B9993</td><td></td></tr><tr><td>Peptone Water</td><td>BD</td><td>257087</td><td></td></tr><tr><td>Trypticase Soy Broth</td><td>BD</td><td>299113</td><td></td></tr><tr><td>Trypticase Soy Agar</td><td>Remel</td><td>R01917</td><td></td></tr><tr><td>Heat Inactivated Horse Serum</td><td>Gibco</td><td>26050</td><td></td></tr></tbody></table>

treatment of platelet products with riboflavin and uv light: effectiveness against high titer bacterial contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 &#176;C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805;5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

The reduction of eleven different bacterial species was evaluated using the riboflavin and UV light based PRT process. These agents represent some of the more common contaminants of platelet products and include five Gram positive and six Gram negative organisms. At least six platelet products were evaluated per organism and each product was inoculated with enough bacteria to yield &#62;1.0 x 105 CFU/ml&#160;final titer of bacteria in the platelet product. Prior to treatment a sample was removed to determine the pre-treatment titer, after which the platelet product was promptly treated with the riboflavin and UV process. Following treatment of the platelet products the following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805; 5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%) (Figure 2). All units met the system specifications as stated above for the riboflavin and UV process. Additional reduction testing was performed with S. pyogenes using buffy coat derived platelets. No significant difference was observed in the reduction of S. pyogenes in buffy coat derived platelets versus apheresis platelets (data not shown).
<img alt="Table 1" src="/files/ftp_upload/52820/52820table1.jpg" /> 
Table 1: Growth conditions for bacterial species evaluated using riboflavin and UV light. A panel of both Gram negative and Gram positive bacteria that have been shown to contaminate platelet products were selected for this study. The growth conditions (temperature and media type) used to propagate and titer the organisms are shown.
<img alt="Figure 1" src="/files/ftp_upload/52820/52820fig1.jpg" /> 
Figure 1: Riboflavin and UV light pathogen reduction process. Riboflavin was dissolved in 0.9% saline at a nominal concentration of 500 &#181;M. The solution was sterile connected and drained into each platelet product. Bacteria was added aseptically to each platelet unit and then treated with 6.2 J/ml&#160;of UV energy (approximately 6&#8211;10 min). In this in vitro study the post-treatment platelet unit was sampled to determine the final bacterial titer.
<img alt="Figure 2" src="/files/ftp_upload/52820/52820fig2.jpg" /> 
Figure 2: Reduction of different bacterial species when treated with riboflavin and UV light.&#160;Riboflavin and UV light was used to reduce the bacterial loads of common bacterial contaminants of platelet products. The log10 reduction is the difference between the average pre-treatment titer and the average post-treatment titer. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52820/52820fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination at JoVE.com

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

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

treatment-platelet-products-with-riboflavin-uv-light-effectiveness

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.6K Views.  Scientific Affairs. The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

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

Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System

Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.
Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (&ge; 2.0 x1011 platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.
Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g. pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.

This article describes the process used by &#214;rebro University Hospital to produce double dose buffy coat platelet concentrates prepared from whole blood donations and treated with the INTERCEPT Blood System for pathogen inactivation. The in vitro quality of the final platelet units are evaluated over 7 days of storage.

Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the ...

Medicine

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Bioengineering

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

Biochemistry

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

Biology

Analyzing Platelet Phosphatidylserine and Microvesicle Release

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

A Riboflavin and UV Light Treatment of Platelet Products to Eliminate Bacteria

Source: Keil, S. D., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52820">Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination.</a> J. Vis. Exp. (2015).
This video demonstrates the riboflavin and UV light-based pathogen reduction process against high-titer bacterial contamination in human platelet products. This process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

1. Platelet Product Preparation

	Collect a standard apheresis human platelet product at an accredited blood bank. The platelet product must meet the ...

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

Summary

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