a riboflavin and uv light treatment of platelet products to eliminate bacteria

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

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 2-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 turbid 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 6-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 5 milliliters of the bacterial stock 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 5-milliliter syringe, and draw a 2-milliliter pre-treatment 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 platen. 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 5 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.

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1. 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 volume and 0.8 &#8211;3.8 x 106 platelets/&#181;l concentration.
	<ol>
		<li>Ensure that the platelet product has rested for a minimum of 2 hr 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 hours post-collection.</li>
	</ol>
	</li>
	<li>Using a 3 ml syringe, remove a 2 ml 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 collection concentration requirement.</li>
	<li>Visually evaluate the swirl of the platelet product. If the product has a &#34;0&#34; 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>
2. 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 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 to 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&#34; tubing line with a female syringe connector onto the inlet line of the illumination and storage bag. Attach a 10 ml syringe barrel (remove the plunger) onto the female syringe connector.</li>
	<li>Add 5 ml of stock bacteria into the syringe barrel via a pipette. Open the clamp on the inlet line and allow the bacteria to drain into the platelet product. Rinse the syringe barrel by allowing some platelet product to flow back into the 10 ml syringe barrel. Remove the 10 ml syringe barrel.</li>
	<li>Aseptically attach a 30 ml syringe and flush the inlet port at least 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 syringe and remove a 2 ml 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 3.</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 of energy. The typical treatment time takes 5-10 min.</li>
	</ol>
	</li>
	<li>Following treatment, sterile dock a 6&#34; tubing line with a 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 post-treatment sample.
	<ol>
		<li>Titer the post-treatment sample per section 3.</li>
	</ol>
	</li>
</ol>
3. Bacterial Titer
<ol>
	<li>Estimate the titer of the pre-treatment and post-treatment samples and prepare the appropriate number of 5 ml dilution tubes needed to perform an end-point serial dilution of each sample. Aseptically fill the required tubes with 1.8 ml of peptone water.</li>
	<li>Titer each sample using a 10-fold serial dilution scheme. Aseptically transfer 0.2 ml from the undiluted sample to the first dilution tube (10-1). Vortex this dilution tube and transfer 0.2 ml 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="Figure 1" src="/files/ftp_upload/21977/21977_Equation1.jpg" /></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="Figure 2" src="/files/ftp_upload/21977/21977_Equation2.jpg" /> 
		and 
		<img alt="Figure 3" src="/files/ftp_upload/21977/21977_Equation3.jpg" />3 
		Where: 
		N =lowest number of particles (CFU) in a 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 of the peptone water that is used for the serial dilutions onto each of the 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 of the recorded stock titer (See section 1.5) for the study to be considered valid.</li>
</ol>
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 4" src="/files/ftp_upload/21977/21977_Table1.jpg" />

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8226;T diff</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Gas Analyzer</td>
			<td>Siemens</td>
			<td>RapidLab 1265</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>25 ml Disposable Serological Pipette</td>
			<td>Costar</td>
			<td>4489</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Keil, S. D., et al. <a href="https://app.jove.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 ...

Take a platelet product into a riboflavin and UV illumination storage bag.
Introduce riboflavin molecules into the platelet product.
Inoculate the platelet product with clinically relevant bacteria, ensuring thorough mixing.
Retrieve a small volume of the sample and perform a serial dilution.
Spread each appropriate dilution on a suitable agar medium, and incubate it to measure the initial bacterial load.
Expose the platelet product to UV&#160; light.
Riboflavin is absorbed into the bacterial cell, where it interacts with the bacterial nucleic acid.
The UV light exposure leads to photodegradation of the riboflavin-nucleic acid complex, resulting in strand breakage. This irreversible damage inactivates the bacteria.
Platelets remain unaffected by the treatment due to the lack of nucleic acid.
Post-treatment, retrieve the platelet sample.
Repeat serial dilution and spread plate procedure to quantify the post-treatment bacterial load.
Compare initial and post-treatment bacterial loads to gauge the efficacy of riboflavin and UV light treatment.

32 조회수. 박테리아를 제거하기 위한 혈소판 제품의 리보플라빈과 자외선 처리

비디오: 박테리아를 제거하기 위한 혈소판 제품의 리보플라빈과 자외선 처리 - 실험

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

자외선 VI와 라만 분광학 면역 플랫폼 제조

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

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Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

공막 래빗 모델에 축 방향 근시의 예방 리보플라빈 및 자외선-A 방사선을 사용하여 교차 결합

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

의학

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.

리보플라빈-5'-인산염의 가시광선 광분해를 통한 병원체 불활성화

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

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

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