RIBOFLAVIN PHOTOCHEMICAL TREATMENT (RPT)-BASED INACTIVATION METHOD OF PATHOGENS IN BIOLOGICAL LIQUID SAMPLE

Abstract

The present disclosure relates to a riboflavin photochemical treatment (RPT)-based inactivation method of pathogens in a biological liquid sample. Aiming at the problems existing in the current riboflavin-based pathogen inactivation methods, a technical solution of the present disclosure is to provide an RPT-based inactivation method of pathogens in a biological liquid sample, including the following steps: adding riboflavin to a biological liquid sample to be treated, and conducting irradiation on the biological liquid sample with light; where the light is narrow-spectrum ultraviolet (UV) light with a wavelength of 360 nm to 370 nm and/or 390 nm to 400 nm. In the present disclosure, parameters such as an irradiation time, an irradiation intensity, and a riboflavin concentration are further optimized. The inactivation method can achieve an excellent pathogen inactivation effect, and has little damage to other components in the biological liquid sample.

Claims

1. A riboflavin photochemical treatment (RPT)-based inactivation method of pathogens in a biological liquid sample, comprising the following steps: adding riboflavin to a biological liquid sample to be treated, and conducting irradiation on the biological liquid sample with light; wherein the light is narrow-spectrum ultraviolet (UV) light with a wavelength of 390 nm to 400 nm; the irradiation is conducted on the biological liquid sample with the light for 10 min to 30 min at a light energy range of 0.2 J/ml to 5 J/ml and an ambient temperature of 20° C. to 24° C.; and 40 μM to 60 μM of the riboflavin is added to the biological liquid sample; and the biological liquid sample is a blood product.

2.-9. (canceled)

10. The RPT-based inactivation method of pathogens in a biological liquid sample according to claim 1, wherein the light is UV light with a peak at 395 nm.

11. The RPT-based inactivation method of pathogens in a biological liquid sample according to claim 1, wherein the blood product is selected from the group consisting of whole blood, leukoreduced whole blood, packed red blood cells, manual platelets, apheresis platelets, plasma, and cryoprecipitate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows inactivation effects of narrow-band UV light in different wavelength ranges on Escherichia coli in the RPT-based inactivation method of pathogens in blood products; and

[0021] FIG. 2 shows inactivation effects of narrow-band UV light with a wavelength of 395 nm±5 nm and narrow-band UV light with a wavelength range of 309 nm to 313 nm on Staphylococcus aureus in platelets.

DETAILED DESCRIPTION

[0022] The technical solutions of the present disclosure are further described below with reference to specific examples.

[0023] In the present disclosure, specific operations and devices of the RPT-based inactivation method of pathogens in a biological liquid sample can be conducted with reference to contents disclosed in the prior art. In the following examples, the specific operations and devices used were consistent with the methods and devices disclosed in the Chinese patent “CN201910975223.2, Equipment and method for inactivating pathogens in blood components by RPT”. The difference was that the wavelength range of light irradiation, irradiation time, irradiation energy, and riboflavin concentration had been changed.

Example 1

[0024] In this example, the plasma of healthy blood donors containing 50 μM of riboflavin was irradiated with LED lamp beads in a series of wavelength ranges for 15 min at an irradiation intensity of 1 W. The growth of E. coli in the blood products was then detected by a Reed-Muench method.

[0025] The results were shown in FIG. 1. Under the same irradiation time and irradiation intensity, in a series of narrow-band UV light, LED lamp beads with a wavelength of 365 nm f 5 nm and a wavelength of 395 nm±5 nm had a desirable pathogen inactivation effect on E. coli in the RPT-based inactivation system.

Example 2

[0026] In this example, the apheresis platelets containing 50 μM of riboflavin were separately irradiated with LED lamp beads with a wavelength of 395 nm±5 nm and fluorescent tubes with a wavelength of 309 nm to 313 nm for 30 min at irradiation intensities of 1 W (395 nm f 5 nm) and 9 W (309 nm to 313 nm), respectively. The growth of Staphylococcus aureus in the blood products was then detected by a Reed-Muench method.

[0027] The results were shown in FIG. 2, and the data of a control experimental group without RPT were also shown in this figure. It was seen from the figure that both the 395 nm±5 nm LED lamp beads and the fluorescent tubes with a wavelength of 309 nm to 313 nm could inactivate the S. aureus. However, under the same irradiation time and irradiation intensity, the inactivation effect of 395 nm±5 nm LED lamp beads was better than that of fluorescent tubes with a wavelength of 309 nm to 313 nm.

[0028] The properties and component contents of the platelet samples in the three groups of experiments were compared, and the results were as follows:

TABLE-US-00001 TABLE 1 Quality of platelet preservation Control Test item 395 nm ± 5 nm 309 nm to 313 nm (no irradiation) PH 7.45 ± 0.01 7.26 7.53 Na.sup.+ mmol/L 153.6 ± 0.49  151 152 K.sup.+ mmol/L 2.7 ± 0.sup.  3 2.6 Glu mmol/L 26.3 ± 0.09 23.6 26 Lac mmol/L 9.24 ± 0.08 10.6 8.1 HCO.sub.3-mmol/L 8.54 ± 0.22 8.5 10 HCO.sub.3 std 15.26 ± 0.15  12.2 17.2 TCO.sub.2 8.94 ± 0.22 9.1 10.4 PLT 714.2 ± 6.49  297 717 PDW 10.84 ± 0.16  17.3 10.9 MPV 9.5 ± 0.sup.  12.1 9.4 P-LCR 21.2 ± 0.2  36.7 20.7 PCT 0.68 ± 0.01 0.36 0.68

[0029] From the data in Table 1, it was seen that after inactivating pathogens on platelets by RPT, the parameters of various properties and component contents deviated from those of the control group without light. However, an irradiation effect of the 395 nm±5 nm LED lamp beads on the various properties and component contents was significantly smaller than an irradiation effect of the 309 nm to 313 nm fluorescent tubes on the various properties and component contents. This showed that the light irradiation of 395 nm±5 nm had significantly less damage to the platelet samples.

Example 3

[0030] In this example, the light dose of 309 nm to 313 nm narrow-band UV light and the light dose of 395 nm±5 nm narrow-band UV light were compared under a same inactivation effect.

[0031] The specific implementation steps were as follows:

[0032] 1. 10 μL of a S. aureus culture solution was added to 150 ml of a bag of plasma from a healthy blood donor (supported by the local ethics committee), to obtain a bacterial plasma suspension of about 4 log to 5 log.

[0033] 2. 9 mL of the bacterial plasma suspension was mixed with 1 mL of physiological saline as a control, and placed in a refrigerator at 4° C.

[0034] 3. A 500 μmon riboflavin-containing physiological saline (CAS: 83-88-5; purchased from Sigma-Aldrich, St. Louis, Missouri, USA) was added to the bacterial plasma suspension, such that a final riboflavin concentration was 50 μmol/L.

[0035] 4. 300 μL of a riboflavin-added bacterial plasma suspension was transferred to a sterile 24-well plate (with a well diameter of 1.5 cm), and then separately exposed to 9 W 309 nm to 313 nm fluorescent tubes (UVB narrow-band PL-L/PL-S, Philips, Amsterdam, The Netherlands) and 1 W 395 nm±5 nm LED lamp beads for irradiation under a temperature-controlled environment (20° C. to 24° C.).

[0036] The light dose of the 309 nm to 313 nm fluorescent tubes was 9.76 J/mL, and an irradiation time was 30 min. The light dose of the 395 nm±5 nm LED lamp beads was 1.25 J/mL, and an irradiation time was 10 min. The two experimental samples were conducted in parallel for 6 groups.

[0037] 5. After the irradiation was over, the experimental sample and the control sample were serially diluted by 101 to 106.

[0038] 6. 100 μL of each diluted sample was added to a center of a sterile plate, the inoculation of each diluted sample was repeated 8 times, and the bacterial growth was determined.

[0039] 7. After culturing in a 37° C. biochemical incubator (SHP-080, Jinghong, China, Shanghai) for 24 h to 48 h, the growth of bacteria in each well was observed and recorded, and a bacterial titer was calculated using a Reed-Muench method.

[0040] The experimental results showed that after irradiating with 309 nm to 313 nm UV light at a higher light dose and using 395 nm±5 nm UV light at a lower light dose, the bacteria growth at 309 nm to 313 nm and 395 nm±5 nm were 2.01 log±1.99 log and 2.22 log±1.80 log, respectively. There was no statistical difference between the two (P=0.568), that is, the inactivation effect on pathogens was equivalent.

[0041] It was seen from the above examples that the narrow-band UV light in the preferred wavelength range of the present disclosure had a better inactivation effect on pathogens and less damages to other components in biological liquid samples (such as blood products). In addition, using the narrow-band UV light in the preferred wavelength range of the present disclosure could select shorter irradiation time and lower irradiation energy, thereby further reducing the damages of light irradiation to other components in biological liquid samples (such as blood products).