FLOW CYTOMETRY EVALUATION FOR UNASSOCIATED NON-ENVELOPED VIRAL PARTICLES

Abstract

A method for flow cytometry evaluation of unassociated non-enveloped viral particles having a non-enveloped viral capsid includes preparing a fluorescently-stained fluid sample in which at least one fluorescent staining step is performed at an acidic pH in an acidic pH range and then subjecting the fluorescently-stained fluid sample to flow cytometry evaluation. A kit includes a plurality of sealed container with a first sealed container containing a fluorescent stain composition and a second sealed container including an aqueous dilution liquid at an acidic pH.

Claims

1. A method for flow cytometry evaluation of a biological material for unassociated non-enveloped viral particles having a non-enveloped viral capsid, the method comprising: preparing a fluorescently-stained fluid sample for flow cytometry evaluation, the fluorescently-stained fluid sample comprising: an aqueous liquid medium; a sample of biological material to be evaluated by flow cytometry for the unassociated non-enveloped viral particles having the non-enveloped viral capsid; at least one fluorescent stain to fluorescently stain the unassociated non-enveloped viral particles to prepare unassociated labeled particles of virus size dispersed in the aqueous liquid medium, wherein each said unassociated labeled particle comprise a said unassociated non-enveloped viral particle stained with the at least one fluorescent stain, and wherein each said fluorescent stain has a fluorescent emission response when the unassociated labeled particle is subjected to a stimulation radiation; after the preparing, subjecting the fluorescently-stained fluid sample to flow cytometry evaluation in a flow cytometer, the flow cytometry evaluation comprising flowing the fluorescently-stained fluid sample through an investigation zone of the flow cytometer and in the investigation zone subjecting the fluorescently-stained fluid sample to the stimulation radiation and detecting for the fluorescent emission response from the investigation zone and counting identified occurrences of the unassociated labeled particles; and the preparing the fluorescently-stained fluid sample comprising fluorescent staining the biological material in a fluid sample composition with a said fluorescent stain to prepare a stained fluid sample composition at an acidic pH in an acidic pH range of from pH 3.0 to pH 6.5.

2. The method of claim 1, wherein the acidic pH range is from pH 4.0 to pH 6.0.

3. The method of claim 1, wherein the at least one fluorescent stain comprises a fluorogenic dye for staining of nucleic acid content inside the viral capsid of the non-enveloped viral particles.

4. The method of claim 3, wherein the fluorogenic dye is a first fluorogenic dye and the at least one fluorescent stain comprises a second fluorogenic dye for staining of protein content of the viral capsid.

5. The method of claim 3, wherein as fed to the flow cytometer the fluorescently-stained fluid sample has a pH in the acidic pH range.

6-9. (canceled)

10. The method of claim 4, wherein the preparing the fluorescently-stained fluid sample comprises: providing a liquid dye concentrate comprising the first fluorogenic dye and the second fluorogenic dye in a stain liquid medium; and during the fluorescent staining, mixing the liquid dye concentrate with the fluid sample composition; and wherein the method comprises at least one member selected from the group consisting of: (i) the stain liquid medium comprising a mixture including water and liquid phase organic material and with the stain liquid medium comprising more than 50 percent by moles of water; (ii) the liquid dye concentrate comprising disaccharide dissolved in the stain liquid medium; (iii) the providing the liquid dye concentrate being in the absence of reconstituting the fluorogenic dyes from a dry form into the stain liquid medium; and (iv) combinations including two or more of any of (i)-(iii).

11. (canceled)

12. The method of claim 10, wherein the liquid dye concentrate comprises both (i) and (ii).

13. The method of claim 1, wherein the preparing the fluorescently-stained fluid sample comprises diluting a preliminary fluid sample with aqueous sample dilution liquid to prepare a diluted preliminary fluid sample, wherein the aqueous sample dilution liquid has a pH in the acidic pH range.

14. The method of claim 13, wherein the diluted preliminary fluid sample has a pH in the acidic pH range and the fluorescent staining comprises adding the said fluorescent stain to the diluted preliminary fluid sample as the fluid sample composition.

15. The method claim 1, wherein the at least one fluorescent stain comprises a fluorescent antibody stain for binding with an epitope of the viral capsid of the unassociated non-enveloped viral particle.

16. (canceled)

17. The method of claim 1, wherein as fed to the flow cytometer the fluorescently-stained fluid sample has a pH in the acidic pH range.

18. The method of claim 1, wherein: the at least one fluorescent stain comprises a fluorogenic dye for staining of nucleic acid content inside the viral capsid of the non-enveloped viral particles, the fluorescent staining is a first fluorescent staining the biological material and the stained fluid sample composition is a first stained fluid sample composition, and the preparing the fluorescently-stained fluid sample comprises second fluorescent staining the biological material after the first fluorescent staining; and the second fluorescent staining comprises staining the biological material with fluorescent antibody stain to prepare a second stained fluid sample composition including both the fluorescent antibody stain and the fluorogenic dye for staining of nucleic acid content, wherein the second stained fluid sample composition is at a second pH that is larger than the acidic pH by at least 0.5 pH unit, and the second pH is pH 5.5 or larger.

19-25. (canceled)

26. The method of claim 1, wherein the stained fluid sample comprises dissolved disaccharide.

27. A kit for preparing a fluorescently-stained fluid sample for flow cytometry evaluation of biological material for quantification of unassociated non-enveloped viral particles of virus size, having a non-enveloped viral capsid, the kit comprising: a plurality of sealed containers; a fluorescent stain composition for fluorescent staining the unassociated non-enveloped viral particles to prepare unassociated labeled particles of virus size, each said unassociated labeled particle of virus size comprising a said unassociated non-enveloped viral particle stained with a fluorescent stain from the fluorescent stain composition, the fluorescent stain composition comprising at least one fluorescent stain and being contained in a first said sealed container; an aqueous dilution liquid for preparing a pH-adjusted fluid sample for fluorescent staining biological material at an acidic pH of up to pH 6.5 during preparation of the unassociated labeled particles of virus size dispersed in an aqueous medium for flow cytometry evaluation of a fluorescently-stained fluid sample with the biological material, the aqueous dilution liquid having an acidic pH of no larger than pH 6.5 and being contained in a second said sealed container.

28. The kit of claim 27, wherein the first said sealed container and the second said sealed container are packaged in a common packaging enclosure.

29. The kit of claim 27, wherein the at least one fluorescent stain of the fluorescent stain composition comprises a fluorogenic dye for staining nucleic acid content inside the viral capsid of the non-enveloped viral particles.

30. The kit of claim 29, wherein; the fluorogenic dye is a first fluorogenic dye with a first fluorescent emission signature and the at least one fluorescent stain of the fluorescent stain composition comprises a second fluorogenic dye for staining of protein content of the viral capsid, the second fluorogenic dye having a second fluorescent emission signature different than the first fluorescent emission signature; and each said fluorogenic dye in the fluorescent stain composition contains at least one aromatic group susceptible to pi stacking interactions in aqueous liquids.

31-33. (canceled)

34. The kit of claim 27, wherein: the fluorescent stain composition is a first fluorescent stain composition and the kit comprises a second fluorescent stain composition comprising a fluorescent antibody stain; and the fluorescent antibody stain is either: specific for binding with an epitope of the viral capsid for direct staining of the unassociated non-enveloped viral particles; or specific for binding with a primary antibody that is specific for binding with an epitope of the viral capsid for indirect staining of the unassociated non-enveloped viral particles.

35. (canceled)

36. The kit of claim 30, wherein the fluorescent stain composition comprises a liquid dye concentrate comprising the first fluorogenic dye and the second fluorogenic dye in a stain liquid medium, and wherein the liquid dye concentrate comprises at least one member selected from the group consisting of: (i) the stain liquid medium comprising a liquid mixture including water and liquid phase organic material, with the stain liquid medium comprising more than 50 percent by moles of water; (ii) disaccharide dissolved in the stain liquid medium; and (iii) a combination including both (i) and (ii).

37-40. (canceled)

41. The kit of claim 27, wherein the acidic pH of the aqueous dilution liquid is in a range of from pH 4.0 to pH 5.6.

42-45. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0092] FIG. 1 illustrates a process diagram for an example embodiment a method of the disclosure.

[0093] FIG. 2 illustrates another process diagram for another example embodiment of a method of the disclosure.

[0094] FIG. 3 illustrates another process diagram for another example embodiment of a method of the disclosure.

[0095] FIG. 4 illustrates another process diagram for another example embodiment example implementation of a method of the disclosure.

[0096] FIG. 5 illustrates another process diagram for another example embodiment an example implementation of a method of the disclosure.

[0097] FIG. 6 illustrates an example embodiment of operation of a flow cytometer for performing a flow cytometry evaluation on a fluorescently-stained fluid sample as part of a method of this disclosure.

[0098] FIG. 7 illustrates another example embodiment of operation of a flow cytometer for performing a flow cytometry evaluation on a fluorescently-stained fluid sample as part of a method of this disclosure.

[0099] FIG. 8 illustrates an example embodiment of a kit of this disclosure for fluorescent staining unassociated non-enveloped viral particles for flow cytometry evaluation.

[0100] FIG. 9 illustrates another example embodiment of a kit of this disclosure for fluorescent staining unassociated non-enveloped viral particles for flow cytometry evaluation.

[0101] FIG. 10 illustrates another example embodiment of a kit of this disclosure for fluoresce staining unassociated non-enveloped viral particles for flow cytometry evaluation.

[0102] FIG. 11 illustrates another example embodiment of a kit of tins disclosure for fluorescent staining unassociated non-enveloped viral particles for flow cytometry evaluation.

DETAILED DESCRIPTION

[0103] Reference is made to FIG. 1 showing a generalized process block flow diagram illustrating one embodiment of a generalized method of this disclosure for flow cytometry evaluation for quantification of unassociated non-enveloped viral particles, which have a non-enveloped viral capsid. The processing illustrated in FIG. 1 includes fluid sample preparation 101 in which a sample feed 103 and a fluorescent stain feed 106 are processed to prepare a fluorescently-stained fluid sample 120, which is then subjected to flow cytometry evaluation 122. The sample feed 103 is a preliminary sample prior to staining and includes biological material to be evaluated to identify and quantify the presence of unassociated non-enveloped viral particles that are a target of the evaluation. The sample feed 103 may be supplied to the fluid sample preparation 101 in any convenient form as a starting point for processing in the fluid sample preparation 101. For example such sample feed 103 may be a crude sample harvested from a biological process in which the non-enveloped, or may be in a form that has already been subjected to some preparatory processing, for example to some purification processing, pH adjustment, dilution or other conditioning with reagents. Such purification processing could include for example centrifugation and/or filtration too remove particles larger than virus size that might interfere with or complicate the flow cytometry evaluation 122. As prepared during the fluid sample preparation 101, the fluorescently-stained fluid sample 120 includes unassociated labeled particles of virus size dispersed in aqueous liquid medium and may be at a pH in an acidic pH range, such as an acidic pH range discussed in more detail elsewhere herein, and with such unassociated labeled particles of virus size each including an unassociated non-enveloped viral particle stained with at least one fluorescent stain. Regardless of the particular state of the sample feed 103 as fed to the fluid sample preparation 101, processing during the fluid sample preparation 101 will include staining at least a portion of biological material of the sample feed 103 with at least one fluorescent stain provided in the fluorescent stain feed 106 and adjusting pH, if needed, to within the acidic pH range. Processing performed during the fluid sample preparation 101 may include any other pre-staining preparatory processing or post-staining processing to prepare the fluorescently-stained fluid sample 120 in a form and with properties, preferably including a pH in the acidic pH range, suitable for the flow cytometry evaluation 122.

[0104] During, the flow cytometry evaluation 122, the fluorescently-stained fluid sample 120 is introduced into and processed through a flow cytometer for flow cytometry evaluation for quantification of the targeted unassociated non-enveloped viral particles in the fluid sample. Quantification refers to counting particles, and preferably determining a concentration of the particles in they fluid sample and/or in a base sample stock from which the fluid sample was prepared. The flow cytometry evaluation includes flowing the fluid sample through an investigation zone of the flow cytometer and in the investigation zone subjecting the fluid sample to stimulation radiation to elicit fluorescent emission response from the fluorescent stain or stains of the unassociated labeled particles. Radiation (e.g., light) coming from the investigation zone is detected by one or more radiation detectors (e.g., light detectors) to identify the presence of such fluorescent emission response to identify occurrences of the unassociated labeled particles, which are counted, and which may be quantified in terms of particle concentration based on the volume of fluid sample processed through the investigation zone that corresponds with the number of the counted particles. When multiple fluorescent stains are used, the different fluorescent emission responses from the different fluorescent stains may be detected separately by separate detectors (e.g., photodetectors) each configured for detection of the specific fluorescent emission response of the targeted fluorescent stain. Detected fluorescent emission signatures may be processed by a data analysis system to identify detection events indicative of the unassociated labeled particles and to count identified occurrences of and determine a concentration for the unassociated labeled particles in the fluid sample. When multiple fluorescent stains are used, the excitation radiation may include a single excitation radiation source (e.g., a single laser or LED), for example when multiple fluorescent stains are suitably excited at similar wavelengths of excitation radiation. Alternatively, the excitation radiation may include multiple excitation radiation sources (e.g., multiple lasers and/or LEDs) that provide excitation radiation about different wavelengths each targeted to excitation of a different one of the fluorescent stains.

[0105] With respect to achieving the desired pH of the fluorescently-stained fluid sample 120 during the fluid sample preparation 101, pH adjustment may occur prior to and or during the fluid sample preparation 101. For example, the sample feed 103, including biological material of interest for investigation, may have been subjected to processing including pH adjustment to within the desired pH range prior to the fluid sample preparation 101, In that case, processing during the fluid sample preparation may include simply mixing one or more stain compositions, including fluorescent stain provided in the fluorescent stain feed 106, with the sample feed 103 to prepare the fluorescently-stained fluid sample at the desired pH within the acidic pH range. Alternatively, and commonly, the sample feed 103 will be in a form and with a pH that will involve more significant processing during the fluid sample preparation 101, including a step during which a pH adjustment is made into the desired acidic pH range. The fluorescent stain feed 106 may be provided in a variety of corals and may be provided in a single fluorescent stain feed composition or in multiple fluorescent stain feed compositions. Such a fluorescent stain feed may be a fluorescent stain composition as provided in a kit, for example in any kit as discussed herein, including any oldie kits illustrated in any of FIGS. 8-11. Such a fluorescent stain feed composition of the fluorescent stain feed 106, may in some implementations be used directly to stain a sample containin2, the biological material, or and in other implementations may be subjected to preparatory processing to prepare a modified fluorescent stain composition that is mixed with the sample to accomplish the fluorescent staining. When the fluorescently-stained fluid sample 120 is to be stained with multiple fluorescent stains, the fluorescent stain feed 106 will include the multiple fluorescent stains, however different ones of the fluorescent stains may be provided separately to the fluid sample preparation 101 in separate fluorescent stain teed compositions. Alternatively, multiple fluorescent stains may be provided in a single fluorescent stain feed composition.

[0106] Reference is now made to FIG. 2 illustrating one example for processing within the fluid sample preparation 101 of the general processing scheme of FIG. 1. As shown in FIG. 2, the fluid sample preparation 101 includes a step of fluorescent staining 116, in which fluorescent stain from a fluorescent stain feed 106a is used to fluorescently stain biological material provided in the sample feed 103. FIG. 2 shows some alternative processing options. In one processing alternative, in which the sample feed 103 as provided to the fluid sample preparation 101 is already in a form and at a pH in an acidic pH range generally desired for the flow cytometry evaluation 122, the sample feed 103 may the provided directly to the fluorescent staining 116, where the fluorescent stain feed 106a may be mixed into the sample feed 103 to prepare the fluorescently-stained fluid sample 120 at the desired acidic pH, in alternative processing shown in FIG. 2. When the sample feed 103 is not in a fluid form and/or not at a pH suitable for the flow cytometry evaluation 122, the sample feed 103 may be subjected to a preliminary step of sample adjustment 162 prior to processing in the fluorescent staining 116. During the sample adjustment 162, the sample feed 103 may be diluted with an aqueous sample dilution liquid 164, and the aqueous sample dilution liquid 164 may be provided at a suitable acidic pH so that when mixed with the sample feed 103, a resulting diluted sample 166 will have fluid properties and be at a pH within the acidic pH range for the flow cytometry evaluation. In some implementations, the sample adjustment may include diluting a sample feed 103 to a desired dilution factor for the flow cytometry evaluation, and with the aqueous 164 at a suitably acidic pH. The diluted sample 166 may then be subjected to the fluorescent staining 116 using fluorescent stain from the fluorescent stain feed 106a to prepare the fluorescently-stained fluid sample 120. The processing as illustrated in FIG. 2 may preferably apply in situations when the fluorescent stain feed 106a is provided in the form of a liquid stain composition with one or more fluorescent stain components dispersed, and preferably dissolved, in a compatible liquid medium for the fluorescent stain components. Such a liquid stain composition may for example include one or more fluorescent antibody stains and/or one or more fluorogenic dyes. In one example, such a liquid stain composition may include both a fluorescent antibody stain specific for binding to an epitope of the capsid of a target non-enveloped viral particle and a fluorogenic dye that is nonspecific for staining nucleic acid content within the capsid of the viral particle. In one preferred variation, the fluorescent stain feed 106a may be in the form of a liquid dye concentrate comprising a plurality of different fluorogenic dyes (e.g., the first fluorogenic dye for nonspecific nucleic acid staining and the second fluorogenic dye for nonspecific protein staining) in a liquid medium, each different said fluorogenic dye having a different fluorescent emission signature for detection when bound to a said unassociated non-enveloped viral particle, and wherein the liquid dye concentrate comprises at least one member selected from the group consisting, of: (i) the liquid medium of the liquid dye concentrate comprising a liquid mixture including water and liquid phase organic material, and preferably the liquid medium is aqueous, and more preferably the liquid medium comprises more than 50 percent by moles of water; (ii) disaccharide dissolved in the liquid medium of the liquid dye concentrate; and (iii) a combination including both (i) and (ii). An advantage of providing the fluorescent stain feed 106a in the form of a liquid dye concentrate is that complications involving reconstitution of fluorogenic dyes from a dry mixture form may be avoided. The method may include unsealing a sealed container, such as provided in a kit, containing the liquid dye concentrate and removing the liquid dye concentrator a portion thereof from the unsealed container for direct use in the fluorescent staining 116 without any need for preparatory processing other than to measure the proper quantity of the liquid dye concentrate fear use in the fluorescent staining 116. As may be appreciated, in the processing example shown in FIG. 2, the fluorescent stain feed 106a may be provided to the fluorescent staining 116 as a single fluorescent stain composition or in multiple separate stain compositions each including at least one different fluorescent stain component.

[0107] Reference is now made to FIG. 3 illustrating another example for processing within the fluid sample preparation 101 in which a fluorescent stain feed 106b includes fluorescent stain in a dry powder stain composition. As shown in FIG. 3, the fluid sample preparation 101 includes the fluorescent staining 116 as shown in FIG. 2. However, in the processing example shown in FIG. 3, fluorescent stain is provided in the form of dry powder stain composition in the fluorescent stain feed 106b, to a step of first preparatory processing 102 to prepare a concentrated stain formulation 104, which is then processed in a step of second preparatory processing 110 to prepare an aqueous diluted stain formulation 112 that is then used in the fluorescent staining 116. During the first preparatory processing 102, the dry powder stain composition of the fluorescent stain feed 106b is combined and mixed with a reconstitution liquid medium 108 to dissolve a fluorescent stain component from the dry powder stain composition into the reconstitution liquid medium 108. Dissolving the fluorescent stain component from powder form into the reconstitution liquid medium 108 may be aided by mechanical means, such as by stirring, shaking, vortex and or centrifuging the mixture. In one preferred implementation for the processing shown in FIG. 3, the dry powder stain composition includes at least one fluorogenic dye, for example in one preferred implementation such a dry powder stain composition includes a fluorogenic dye for nonspecific nucleic acid staining within the non-enveloped viral particle capsid, and in some other preferred implementations such a dry powder stain composition includes a dry powder mixture with at least two different fluorogenic dyes, for example a first fluorogenic dye for non-specific staining of nucleic acid content inside the capsid and a second fluorogenic dye for nonspecific staining of protein content of the capsid. When the dry powder stain composition of the fluorescent stain feed 106b includes a fluorogenic dye as a fluorescent stain component, the dry powder stain composition may be referred to as a dry powder dye composition, the concentrated stain formulation 104 may be referred to as a concentrated dye formulation and the aqueous diluted stain formulation 112 may be referred to as an aqueous diluted dye composition.

[0108] With continued reference to FIG. 3, the reconstitution liquid medium 108 is a solvent for at least one, and preferably all, fluorescent stain components in dry powder form in the fluorescent stain feed 106b. The reconstitution liquid medium 108 includes an organic liquid to help facilitate effective dissolution and dispersion of fluorescent stain. Such organic liquid may include any organic liquid component or mixture of organic liquid components. One useful organic liquid component is acetonitrile, however in preferred implementations the organic liquid includes DMSO, and preferably at the largest concentration of any organic liquid components in the reconstitution liquid medium 108, for example as discussed above. The concentrated stain formulation 104 resulting from the first preparatory processing 102 includes the fluorescent stain components (e.g., fluorogenic dyes) dissolved in the reconstitution liquid medium 108, In the example processing shown in FIG. 3, the reconstitution liquid medium 108 has been pre-prepared to a desired composition, however in alternative processing when the reconstitution liquid medium 108 is to include multiple liquid components, such a reconstitution liquid medium may be prepared in situ during the first preparatory processing 102 through the mixing together of multiple such components mixing an organic liquid component with aqueous buffer solution and/or other organic solvent components) before or after mixing with the dry powder stain composition of the fluorescent stain feed 106b.

[0109] With continued reference to FIG. 3, the concentrated dye formulation 104 is processed in the second preparatory processing 110 to prepare the aqueous diluted stain formulation 112, which is an aqueous composition including the fluorescent stain components fluorogenic dyes) from the concentrated stain formulation 104 dissolved in an aqueous liquid medium. Importantly, during the second preparatory processing 110, the fluorescent stain components in the concentrated dye formulation 104 remain in solution for the duration of the second preparatory processing 110. In other words, during the second preparatory processing 110, such fluorescent stain components are not precipitated, even temporarily, during the second preparatory processing 110, and the liquid medium in which the fluorescent stain components are dissolved is converted from a first liquid medium in the concentrated stain formulation 104, and preferably an organic liquid medium, to an aqueous diluted liquid medium in the aqueous diluted stain formulation 112 while the fluorescent stain components remain in a dissolved state. As shown in FIG. 3, an aqueous stain dilution liquid 114 (e.g., aqueous buffer solution) is provided to the second preparatory processing 110 and is mixed with the concentrated stain formulation 104 to dilute the reconstitution liquid medium 108 with the aqueous stain dilution liquid 114 to an extent to which after the second preparatory processing 110 the liquid medium in which the fluorescent stain components are dissolved is assured to be aqueous in nature, in other words is comprised of at least a majority by moles of water, and preferably with a weight ratio of water to any and all organic liquid components of at least 1011 or such larger weight ratio, for example as discussed above for some implementations when the aqueous diluted dye formulation 112 is to include a relatively large weight ratio water to DMSO, and otherwise with a smaller weight ratio of water to DMSO for some other implementations when the aqueous diluted dye formulation 112 is to include a larger concentration of DMSO, as discussed above. Mixing of the aqueous stain dilution liquid 114 with the concentrated stain formulation 104 preferably follows a suitable retention, or incubation, time following mixing together the dry powder stain composition of the stain feed 106b and the reconstitution liquid medium 108. In the processing shown in FIG. 3, the aqueous stain dilution liquid 114 is shown as a single addition, however, the aqueous stain dilution liquid 114 may in alternative embodiments include multiple additions of different amounts of the aqueous it dilution liquid 114, which may or may not each have the same composition. When the aqueous diluted stain formulation 112 is to include a larger concentration of DMSO, as discussed above, DMSO in addition to that included in the first liquid medium 108 may be provided, for example, as a minor molar component in the aqueous liquid diluent 114.

[0110] With continued reference to FIG. 3, during the fluorescent staining 116, at least a portion of the aqueous diluted stain formulation 112 is mixed with and used to fluorescently stain biological material provided in the sample feed 103. Depending upon the form and properties of the sample feed 103, the sample feed 103 may be provided directly to the fluorescent staining 116, or may be subjected to a preliminary step of sample adjustment 162 using aqueous sample dilution liquid 164 to prepare the diluted sample 166 at a desired dilution factor and/or desired acidic pH in a like manner as described with respect to similar processing shown in FIG. 2, in the processing of FIG. 3, adjustment of pH may occur during the second preparatory processing 110 by providing the aqueous stain dilution liquid 114 at an appropriate acidic pH such that the aqueous diluted stain formulation 112 has an acidic pH in a desired range for the fluorescently-stain fluid sample 120. Alternatively, or additionally, pH adjustment may occur during the sample adjustment 162 using the aqueous sample dilution liquid 164 which may conveniently be provided at an appropriate acidic pH to provide the diluted sample 166 to the fluorescent staining 116 already adjusted to within an appropriate addle pH range for the fluorescently-stained fluid sample 120. In some implementations of the processing of FIG. 3 when the sample feed 103 is subjected to the sample adjustment 162, the aqueous stain dilution liquid 114 and the aqueous sample dilution liquid 164 may both be provided at the same or similar acidic pH, and in some implementations the aqueous stain dilution liquid 114 and the aqueous sample dilution liquid 164 may be the same or essentially the same aqueous composition at the same or essentially the same acidic pH for preparation of the fluorescently-stained fluid sample 120 at a pH in the desired acidic pH range.

[0111] With continued reference to FIG. 3, when the stained fluid sample 120 is prepared containing dissolved disaccharide, some or all of the disaccharide may be included in the aqueous stain dilution liquid 114 used in the second preparatory processing 110 and/or in the sample feed 103 and/or or the aqueous sample dilution liquid 164 to prepare the diluted sample 166.

[0112] Reference is now made to FIG. 4 illustrating another example for processing within the fluid sample preparation 101 of the general processing scheme of FIG. 2, and including use of both a fluorescent stain feed 106a as described with reference to FIG. 2, for example in the form of a liquid stain composition, and a fluorescent stain feed 106b as described with reference to FIG. 3, for example in the form of a dry powder stain composition. The only difference illustrated in FIG. 4 relative to the processing of FIG. 3 is that in the processing of FIG. 4, the stain feed 106a is provided to the fluorescent staining 116 to provide one or more fluorescent stain components in addition to what is provided in the aqueous diluted stain formulation 112. Such a stain feed 106b as shown in FIG. 4 may be for example as described with respect to FIG. 3 and the stain feed 106a as shown in FIG. 4 may be for example as described with respect to FIG. 2. For example, the stain feed 106b provided to the first preparatory processing 102 may include one or more fluorogenic dyes in dry powder form and the other stain feed 106a provided to the fluorescent staining 116 may include one or more fluorescent antibody stains and/or one or more fluorogenic dyes in a liquid stain composition.

[0113] Reference is now made to FIG. 5 illustrating yet another example for processing within the fluid sample preparation 101 of the general processing scheme of FIG. 2, and including both a fluorescent stain feed 106a as described with reference to FIG. 2, for example in the farm of a liquid stain composition, and a fluorescent stain feed 106b as described with reference to FIG. 3, for example in the form of a dry powder stain composition. The only difference illustrated in FIG. 5 relative to the processing of FIG. 4 is that in the processing of FIG. 3, the fluorescent staining 116 is a first fluorescent staining and the processing includes second fluorescent staining 174 after the first fluorescent staining 116, and with an intermediate step of pH adjustment 170 between the first fluorescent staining 116 and the second fluorescent staining 174, and in the processing of FIG. 5 the fluorescent stain feed 106a is introduced into the second fluorescent staining 174. In the processing of FIG. 5, the first fluorescent staining 116 is performed within an acidic pH range to prepare a first stained fluid sample composition 168 at an acidic pH within the acidic pH range. During the pH adjustment 170, the pH of the first stained fluid sample composition 168 is raised to prepare a pH adjusted stained fluid sample composition 172 at a higher pH than the acidic pH of the first stained fluid sample composition 168. As illustrated in FIG. 5, an aqueous sample dilution liquid in the form of an aqueous pH adjustment liquid 176 is fed to the pH adjustment 170. The aqueous pH adjustment liquid 176 has a higher p11 than the acidic pH of the first stained fluid sample composition 168 to raise the pH of the first stained fluid sample composition 168 to prepare the pH adjusted stained fluid sample composition 172. The aqueous pH adjustment liquid 176 may have a pH, and may be added to the first stained fluid sample composition 168 at a volume ratio, to provide a desired level of pH adjustment to prepare the pH adjusted stained fluid sample composition 172 at the desired higher pH. Such an aqueous pH adjustment liquid 176 may have a pH as described elsewhere for a second aqueous sample dilution liquid that may be provided in a kit according to a kit aspect of this disclosure. The pH adjusted stained fluid sample composition 172 is then processed in the second fluorescent staining 174 where biological material in the pH adjusted stained fluid sample composition 172 is stained with fluorescent antibody stain provided in the fluorescent stain feed 106a, to prepare a second stained fluid sample composition including both fluorescent antibody stain from the fluorescent stain feed 106a and fluorogenic dye from the fluorescent stain feed 106b. Such a second stained fluid sample composition may be used directly as the fluorescently-stained fluid sample 120 that is subjected to the flow cytometry 122.

[0114] Or the second stained fluid sample composition may be further processed to adjust properties to prepare the fluorescently-stained fluid sample 120 that is subjected to flow cytometry evaluation 122. The pH adjusted stained fluid sample composition 172 and the second stained fluid sample composition each has a higher pH than the first stained fluid sample composition 168. Each of the second stained fluid sample composition and the pH adjusted stained fluid sample composition 172 may each have a pH that is at least 0.5 pH unit, or more, larger than the acidic pH of the lust stained fluid sample composition 168. The pH of the pH adjusted stained fluid sample composition 172 and the second stained fluid sample composition resulting from the second fluorescent staining 174 may be at the same pH, or may be at a different pH, for example as a consequence of pH changes occurring, during the second fluorescent staining 174. The second stained fluid sample composition and the fluorescently-stained fluid sample 120 may be at the same pH or each may have a different pH, for example as a consequence of further processing following the second fluorescent staining 174 to prepare the fluorescently-stained fluid sample 120 that is then subjected to the flow cytometry evaluation 122. Each of the pH adjusted stained fluid sample composition 172, the second stained fluid sample composition resulting from the second fluorescent staining 174 and/or the fluorescently-stained fluid sample 120 may be at a higher pH than the acidic pH of the first stained fluid sample composition 168, and such a higher pH may be any such higher pH as described previously. For example such a higher pH may be at least 0.5 PH unit larger, at least 1.0 pH unit larger, at least 1.5 pH units larger, at least 2.0 pH units larger or even at least 2.5 pH units larger than the acidic pH of the first stained fluid sample composition 168, and such a higher pH may be pH 5.5 or larger, pH 5.8 or larger, pH 6.0 or larger, pH 6.2 or larger, or pH 6.5 or larger. As described previously, in some embodiments such a higher pH may be larger than pH 6.5, may be at least pH 6.7, may be at least pH 6.9, or may be at least pH 7.0, although such a higher pH may often be not larger than pH 8.5, not larger than pH 8.0, not larger than pH 7.5 or not larger than pH 7.2.

[0115] As alternatives to the processing shown in any of FIGS. 3-5, the fluorescent stain feed 106b may be provided in the form of a liquid dye concentrate and may be added directly to the fluorescent staining 116, and without need for performing the first preparatory processing102 or the second preparatory processing 110.

[0116] Consumable materials for use in the processing of any of FIGS. 1-5 may be provided in a kit, for example a kit as illustrated in any of FIGS. 8-11, discussed below. For example, a fluorescent stain feed 106, an aqueous sample dilution liquid 164, a reconstitution liquid medium 108, an aqueous stain dilution liquid diluent 114 and/or an aqueous pH adjustment liquid 176 may be provided in such a kit. When processing includes the use of both an aqueous stain dilution liquid 114 and an aqueous sample dilution liquid 164, the aqueous stain dilution liquid 114 and aqueous sample dilution liquid 164 may be provided in a kit in separate containers, or when the aqueous stain dilution liquid 114 and the aqueous sample dilution liquid 164 have the same composition, both may be provided in a single aqueous liquid formulation contained in a single container, and such a single aqueous liquid formulation may be used both as the aqueous stain dilution liquid 114 and the aqueous sample dilution liquid 164.

[0117] Reference is now made to FIG. 6, illustrating one example of flow cytometry processing, which may be used during the flow cytometry evaluation 122 of the fluorescently-stained fluid sample 120 according to any oldie processing examples illustrated in any of FIGS. 1-5. FIG. 6 shows partial componentry of a flow cytometer 128 including a flow cell 130 and a light detection and analysis system 138. As shown in FIG. 6, a flow of such a fluorescently-stained fluid sample 120 through and investigation zone of the flow cell 130 is subjected in the investigation zone to excitation radiation 134 from a light source (not shown) of the flow cytometer 128. Such a light source may be, for example, a laser, LED or other light source. As illustrated in FIG. 6, the fluorescently-stained fluid sample 120 includes virus-size particles 124 (e.g., unassociated non-enveloped viral particles), and the fluorescently-stained fluid sample 120 is shown as flowing through the flow cell 130 of the flow cytometer 128 in the direction of the flow arrow 135. In the example illustrated in FIG. 6, the fluorescently-stained fluid sample 120 has been hydrodynamically focused with a sheath fluid 136 prior to introduction into the flow cell 130. As illustrated in FIG. 6, the sheath fluid 136 is shown as a distinct fluid relative to the stained fluid sample 120. As may be appreciated, differentiation between such a sheath fluid 136 and fluorescently-stained fluid sample 120 may not be as distinct as illustrated in FIG. 6. The light detector analysis system 138 in FIG. 2 is illustrated to include two photodetectors for separately detecting radiation wavelength ranges associated with different fluorescent emission signatures of two different fluorescent stains that may be used to stain different features of target unassociated non-enveloped viral particles. The illustrated example of FIG. 6 shows a first time series plot 142 with an example output (C1) of a first photodetector detecting for fluorescent emission response from presence of a first fluorescent stain on an unassociated virus-size particle 124 and a second time series plot 144 with an example output (C2) of a second photodetector detecting for a different fluorescent emission response from presence of a second fluorescent stain on such a virus -size particle 124, The time series plots 142 and 144 are illustrated as plots of voltage from the respective photodetector versus time, and with voltage peaks at t1, t2 and t3 indicating detection of such an unassociated virus-size particle 124 stained with a fluorescent. stain. In this example a first fluorescent stain may be a fluorogenic dye for nonspecific nucleic acid staining and a second fluorescent stain may be a fluorogenic dye for nonspecific protein staining or alternatively the second fluorescent stain may be a fluorescent antibody stain for binding to an epitope of a viral capsid. In FIG. 6, the second time series plot 144 is illustrated with three voltage peaks at times t1, t2 and t3 indicative of passage through the flow cell of three virus-size particles 124 each labeled for example with such a second fluorescent stain. The first time series plot 142 is illustrated in FIG. 6 with a voltage peak at time t2 indicative of passage through the flow cell 130 of a vines-size particle 124 labeled for example with the first fluorescent stain.

[0118] In one example of the processing of FIG. 6 where the first fluorescent stain is a first fluorogenic dye for nonspecific staining of nucleic acid content and the second fluorescent stain is a second fluorogenic dye for nonspecific staining of protein content, the coincidence at time t2 of voltage peaks on both the first time series plot 142 and the second time series plot 144 indicates passage through the flow cell 130 of an unassociated virus-size particle 124 labeled with both the first fluorescent stain and the second fluorescent stain, such as in the case of an intact virus of a non-enveloped virus containing a non-enveloped viral capsid fluorescently labeled by the nonspecific protein stain and genome inside the capsid fluorescently labeled by the nonspecific nucleic acid stain. Conversely, in this example the occurrence of the voltage peaks at t1 and t3 on the second time series plot 144 with no corresponding coincidental occurrences of voltage peaks on the first time series plot 142 indicates passage through the flow cell 130 of virus-size particles 124 including fluorescently labeled, protein content and not including fluorescently labeled nucleic acid content, such as may be the case for a virus-size particle having a non-enveloped viral capsid but not containing genome inside the capsid, such as may be the case for virus-like particles or other particles containing protein content and not containing genome.

[0119] In another example where the first fluorescent stain is a fluorogenic dye for nonspecific staining of nucleic acid content and the second fluorescent stain is a fluorescent an stain for binding to epitope of the viral capsid, the coincidence at time t2 of a voltage peaks on both the first time series plot 142 and the second time series plot 144 indicates passage through the flow cell 130 of a virus-size particle 124 labeled with both the first fluorescent stain in the second fluorescent stain such as in the case of an intact virus of a non-enveloped virus containing a non-enveloped viral capsid with the epitope indicated by labeling with the fluorescent antibody stain and genome inside the capsid indicated by labeling with the nonspecific nucleic acid stain. Conversely the occurrence of the voltage peaks at t1 and t3 on the second time series plot 144 with no corresponding coincidental occurrences of voltage peaks on the first time series plot 142 indicates passage through the flow cell 130 of virus -size particles 124 including a non-enveloped viral capsid with the epitope but not containing genome inside the capsid, such as may be the Case for virus-like particles corresponding to a virus with which the epitope is associated.

[0120] Reference is now made to FIG. 7 illustrating another example of flow cytometry processing which may be used during the flow cytometry evaluation 122 of the fluorescently-stained fluid sample 122 according to any of the processing embodiments illustrated in any of FIGS. 1-5, and using the same flow cytometer 128 illustrated in FIG. 6. In the example illustrated in FIG. 7, only the time series plot 144 is shown for the output C2 of the second photodetector and no time series plot is shown for the first photodetector of the light detection and analysis system 138, The example illustrated in FIG. 7 may be the case for example when the fluorescently-stained fluid sample 120 includes only a single fluorescent stain, the fluorescent emission signature of which is detected by the second photodetector. Such a single fluorescent stain may, for example, be a fluorescent antibody stain for binding with an epitope of a targeted non-enveloped viral capsid, in which case the voltage peaks at t1, t2 and t3 indicate passage through the flow cell of virus size particles 124 including such a non-enveloped viral capsid with the epitope, and without distinguishing whether or not any of the virus-size particles 124 includes genome inside the capsid. In the example illustrated in FIG. 7. the virus-size particles 124 associated with the voltage peaks at t2 and t3 may be associated with passage through the flow cell of intact virions or virus-like particles.

[0121] As illustrated in FIGS. 6 and 7, the light detector and analysis system 138 of the example flow cytometer 128 includes photodetectors for detecting fluorescent emission signatures from two fluorescent stains. However, as may be appreciated, in alternative embodiments the light detector analysis system 138 may include photodetectors for detecting fluorescent emission signatures for more than two different fluorescent stains. Also, although the flow cytometer 128 is illustrated in FIGS. 6 and 7 as including a single light source providing a single excitation radiation 134 for stimulation of multiple different fluorescent emission responses from different fluorescent stains, such a flow cytometer 128 may include multiple different light sources providing different portions of excitation radiation in different wavelength ranges where the fluorescent emission responses from different fluorescent stains are stimulated by different excitation radiation wavelength ranges. Moreover, although processing in FIGS. 6 and 7 is shown using a hydrodynamically focused fluorescently-stained fluid sample 120, such a fluorescently-stained fluid sample 120 may alternatively be processed through a flow cytometer in which samples are not hydrodynamically focused, although hydrodynamically focused systems are preferred with the present disclosure. In a preferred flow cytometry evaluation, a flow rate of the hydrodynamically focused fluorescently-stained fluid sample 120 through the flow cell 130 may be very low, preferably in a range having a lower limit of 300 nanoliters per minute, 600 nanoliters per minute or 800 nanoliters per minute and an upper limit of 6000 nanoliters per minute, 3000 nanoliters per minute or 2000 nanoliters per minute, with one preferred range being from 600 nanoliters per minute to 3000 nanoliters per minute and with one even more preferred range being from 600 nanoliters per minute to 2000 nanoliters per minute,

[0122] Reference is now made to FIGS. 8-11 illustrating some example embodiments of kits of the disclosure, With reference first to FIG. 8, an example kit 200 is illustrated including a plurality of first sealed containers in the form of dry powder stain composition containers 202 each containing a dry powder stain composition with a dry powder including, at least one, and in some preferred implementations including a mixture of a plurality of different fluorogenic dyes each having a different fluorescent emission signature. Each such fluorogenic dye may contain a least one aromatic group susceptible to pi stacking interactions in aqueous liquid media, Each dry powder stain composition container 202 may include a quantity of the dry powder stain composition to prepare an aqueous diluted dye formulation to be processed contemporaneously for flow cytometry evaluation, such as in the example processing illustrated in FIGS. 3-5. The dry powder stain composition and fluorogenic dyes in the dry powder stain composition may have any or any combination of features as described elsewhere herein. As shown in FIG. 8, the plurality of dry powder stain composition containers 202 within the kit 200 are for convenience of manufacture and packaging contained, within a bag 204, which is preferably opaque. The kit 200 includes a second sealed container in the form of a dilution liquid container 208 including aqueous dilution liquid at a pH within an acidic pH range, such as described elsewhere herein, for use to adjust pH to prepare a fluorescently-stained fluid sample at a suitable acidic pH within an acidic pH range, for example as disclosed elsewhere herein. Such aqueous dilution liquid may be used, for example, as the aqueous sample dilution liquid 164 in the processing illustrated in any of FIGS. 2-5 and/or the aqueous stain dilution liquid 114 in the processing illustrated in any of FIGS. 2-5. The example kit 200 illustrated in FIG. 8 also includes a third sealed container in the form of a reconstitution liquid container 206 containing a reconstitution liquid medium, for example for use as the reconstitution liquid medium 108 in the second preparatory processing 110 in the processing illustrated in FIGS. 3-5.

[0123] Reference is now made to FIG. 9. which illustrates another example kit 220 including a first sealed container in the form of a liquid stain composition container 218 containing a liquid stain composition. In one variation, the liquid stain composition may include at least one, and optionally a plurality of, fluorescent antibody stains-As another option, the liquid stain composition in the container 218 may include a fluorescent antibody stain and a fluorogenic dye, such as for nonspecific staining of nucleic acid content. As yet another option, the liquid stain composition in the container 218 may be a liquid dye concentrate comprising a plurality of different fluorogenic dyes (e.g., the first fluorogenic dye for non-specific nucleic acid staining and the second fluorogenic dye for non-specific protein stain) in a liquid medium, with each of the different fluorogenic dyes having a different fluorescent emission signature for detection when bound to an unassociated non-enveloped viral particle, and wherein the liquid dye concentrate comprises at least one member selected from the group consisting of (i) the liquid medium of the liquid dye concentrate comprising a liquid mixture including water and liquid phase organic material, and preferably the liquid medium is aqueous, and more preferably the liquid medium comprises more than 50 percent by moles of water; iii) disaccharide dissolved in the liquid medium of the liquid dye concentrate; and (iii) a combination including both (i) and (ii). The kit 220 includes a second sealed container in the form of the dilution liquid container 208 including aqueous dilution liquid at a pH within an acidic pH range, such as described with respect to FIG. 8, for use to adjust pH to prepare a fluorescently-stained fluid sample at a suitable acidic pH within an acidic pH range for flow cytometry evaluation. Such an aqueous dilution liquid may be used, for example, as the aqueous sample dilution liquid 164 in the processing illustrated in any of FIGS. 2-5, and/or as the aqueous stain dilution liquid 114 in the processing illustrated in any of FIGS. 3-5. As shown in FIG. 9, the kit 220 optionally includes another sealed container 209, illustrated in dashed lines indicating an optional feature. The other sealed container 209 may include a second aqueous dilution liquid at a different pH than the acidic range of the aqueous dilution liquid in the dilution liquid container 208, and preferably the second aqueous dilution liquid is at a higher pH than the acidic pH range. The second aqueous dilution liquid may, for example, be used as an aqueous pH adjustment liquid to dilute and increase the pH of a preliminary stained fluid sample including biological material following a first fluorescent staining, step at an acidic pH and prior to a second fluorescent staining step of a higher pH. Alternatively, the second aqueous dilution liquid may be an aqueous sample dilution liquid initially used to prepare diluted fluid sample of a higher pH, for example in a dilution series, which may be adjusted to an acidic pH for fluorescent staining at an acidic pH using the aqueous dilution liquid in the dilution liquid container 208. Reference is now made to FIG. 10, which illustrates another example kit 240 including both the dry powder stain composition containers 202 each containing a dry powder stain composition, such as described with respect to FIG. 8, and the liquid stain composition container 218 including a liquid stain composition, such as described with respect to FIG. 9. The kit 240 also includes the reconstitution liquid container 206 containing a reconstitution liquid medium, such as described with respect to FIG. 8. The kit 240 further includes a second sealed container in the form of the aqueous dilution liquid container 208 that contains aqueous dilution liquid at a pH within an acidic pH range, such as described with respect to FIGS. 8 and 9, such as may be used as an aqueous sample dilution liquid and/or an aqueous stain dilution liquid, for example as the aqueous sample dilution liquid 164 in the processing illustrated in any of FIGS. 2-5 and/or as the aqueous stain dilution liquid 114 in the processing illustrated in any of FIGS. 3-5. As an alternative kit embodiment to any of example kits 200, 220, or 240 shown in FIGS. 8-10, any such kit may include multiple ones of the dilution liquid container 208 with one such dilution liquid container 20$ containing an aqueous stain dilution liquid and another such dilution liquid container 208 containing an aqueous sample dilution liquid, such as when aqueous dilution liquid for use to dilute a dry powder stain composition is of a different composition than aqueous dilution liquid for use to dilute a sample directly without also fluorescent staining the sample. In such an alternative kit embodiment, such an aqueous stain dilution liquid may or may not have a pH in an acidic pH range.

[0124] Reference is now made to FIG. 11, which illustrates another example kit 260 including all of the components of the kit 240 of FIG. 10, and also including another sealed container to the form of a second aqueous dilution liquid container 262 that contains a second aqueous dilution liquid that has a higher pH than an acidic pH of the aqueous sample dilution liquid contained in the aqueous dilution liquid container 208. The second aqueous sample dilution liquid may, for example, be used as an aqueous pH adjustment liquid to dilute and increase the pH of a preliminary stained fluid sample including biological material following a first fluorescent staining step and prior to a second fluorescent staining step. The second aqueous dilution liquid may for example be used as the aqueous pH adjustment liquid 176 in the processing illustrated in FIG. 5 and/or as the aqueous stain dilution liquid 114 in the processing illustrated in any of FIGS. 2-5.

[0125] The kits 200, 220, 240 and 260 illustrated in FIGS. 8-11 each includes the components of the kit within a common packaging enclosure 210, for example a common packaging box or bag. Tn alternative implementations, the any such kit may include within the coalition packaging enclosure one or more components in addition to those illustrated in FIGS. 8-11. For example, when the stained fluid sample to be prepared by any such kit 200, 220, 240 or 260 is to include dissolved disaccharide, some or all of such disaccharide may be included in the aqueous dilution liquid in a dilution liquid container 208 and/or a in a second aqueous dilution liquid container 262 and/or in the liquid stain composition in the container 118 and/or in the second aqueous liquid in the other sealed container 209.

EXAMPLES

[0126] Equipment and Reagents; The following representative equipment and reagents were used in the examples presented below:

[0127] VC3100: Virus Counter® 3100 flow cytometer (Sartorius Stedim), operated generally in accordance with the manufacturer's Operation Manual.

[0128] Combo Dyek® concentrate: A dry powder dye composition (Sartorius Stedim product no. V1R-92101), with a dry powder mixture of a first fluorogenic dye of POPO™-3 iodide (Thermo Fischer Scientific) for nucleic acid stamina and a second fluorogenic dye of SYPRO™ Red (Thermo Fischer Scientific) for protein staining.

[0129] Organic liquid medium: Acetonitrile or DMSO, for use in preparing stain formulations from Combo Dye® Concentrate.

[0130] POPO™-3 formulation: A liquid formulation product with fluorogenic dye POPO™-3 iodide at 1 mM concentration in dimethyl formamide (DMF) (Thermo Fischer Scientific Catalog NO. P3584).

[0131] Sample dilution buffer (SBD): Buffer solution used to prepare diluted fluid samples prior to staining. Representative Sample Dilution Buffer compositions used in the examples are summarized below: [0132] PBS-7.4: A representative phosphate-based buffer solution (generally corresponding to Sartorius Stedim product no. VIR-92298), with 100 mM sodium chloride and either 0.05 or 0.09% (w/v) sodium azide, at approximately pH 7.4, was generally prepared as follows: Start with 18 mΩ water at a volume of 90% of final desired volume, add calculated amounts of sodium phosphate dibasic, sodium phosphate monobasic dihydrate, and sodium chloride and by stiffing, then add 5% sodium azide solution to provide the final desired concentration of sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume, Phosphates are added in an amount to provide 20 mM concentration relative to final composition volume. [0133] PBS-8.2: A representative phosphate-based buffer solution with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 8.2, was generally prepared as follows: Start with 18 MΩ water at a volume, of 90% of final desired volume, add 1.0 calculated amounts of sodium phosphate dibasic, sodium phosphate monobasic dihydrate, and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. Phosphates are added in an amount to provide 20 mM concentration relative to final composition volume. An alternative representative phosphate-based buffer solution with 100 mM sodium chloride and 0.05 (w/v) sodium azide, at approximately pH 8.2, was generally prepared as follows: Start with 18 MΩ water at a volume of 70% of final desired volume, add calculated amounts of 0.2 M sodium monophosphate and 0.2 sodium diphosphate to a final concentration of 20 mM total phosphate; add 1 M sodium chloride solution to provide 100 mM NaCl final concentration; then add 5% sodium azide solution to provide 0.05% (w/v) sodium azide in the final composition; then add hydroxide as needed, and then add additional 18 MΩ water to bring to final volume. [0134] PBS-7.8: A representative phosphate-based buffer solution with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately 7.8, was generally prepared as follows: Start with 1.8 MD water at a volume of 90% of final desired volume, add calculated amounts of sodium phosphate dibasic, sodium phosphate monobasic dihydrate, and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium nude in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. Phosphates are added in an amount to provide 20 mM concentration relative to final composition volume. [0135] PBS-7.0: A representative phosphate-based buffer solution with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 7.0, was generally prepared as follows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of sodium phosphate dibasic, sodium phosphate monobasic dihydrate, and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring, then add additional 18 MΩ water to bring to final volume. Phosphates are added in an amount to provide 20 mM concentration relative to final composition volume. An alternative representative phosphate-based butler solution with 100 mM sodium chloride and 0.05 (w/v) sodium azide, at approximately pH 8.2, was generally prepared as follows: Start with 18 MΩ water at a volume of 70% of final desired volume, add calculated amounts of 0.2 M sodium monophosphate and 0.2 sodium diphosphate to a final concentration of 20 mM total phosphate; add 1 M sodium chloride solution to provide 100 mM NaCl final concentration; then add 5% sodium azide solution to provide 0.05% (w/v) sodium azide in the final composition; then add hydroxide as needed; and then add additional 18 MΩ water to bring o final volume. [0136] PBS-6.6: A representative phosphate-based buffer solution with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 6.6, was generally prepared as follows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of sodium phosphate dibasic, sodium phosphate monobasic dihydrate, and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. Phosphates are added in an amount to provide 20 mM concentration relative to final composition volume. An alternative representative phosphate-based buffer solution with 100 mM sodium chloride and 0.05% (w/v) sodium azide, at approximately pH 8.2, was generally prepared as follows: Start with 18 MΩ water at a volume of 70% of final desired volume, add calculated amounts o102 M sodium monophosphate and 0.2 sodium diphosphate to a final concentration of 20 mM total phosphate; add 1 M sodium chloride solution to provide 100 mM NaCl final concentration; then add 5% sodium azide solution to provide 0.05% (w/v) sodium azide in the final composition; then add hydroxide as needed; then add additional 18 MΩ water to bring to final volume. [0137] PBS-6.2: A representative phosphate-based buffer solution, with sodium phosphate dibasic and sodium phosphate monobasic dihydrate, at approximately pH 6.2, was generally prepared as follows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of sodium phosphate dibasic, sodium phosphate monobasic dihydrate, and sodium chloride and dissolve b stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. Phosphates are added in an amount to provide 20 mM concentration relative to final composition volume. An alternative representative phosphate-based buffer solution with 100 mM sodium chloride and 0.05% (w/v) sodium azide, at approximately pH 8.2, was generally prepared as follows: Start with 18 MΩ water at a volume of 70% of final desired volume, add calculated amounts of 0.2 M sodium monophosphate and 0.2 sodium diphosphate to a final concentration of 20 mM total phosphate; add 1 M sodium chloride solution to provide 100 mM NaCl final concentration; then add 5% sodium azide solution to provide 0.05% (w/v) sodium azide in the final composition; then add hydroxide as needed; and then add additional 18 MΩ water to bring to final volume. [0138] PBS-6.0: A representative phosphate-based buffer solution with 100 mM sodium chloride and 0.05% (w/v) sodium azide, at approximately pH 8.2, was generally prepared as follows: Start with 18 MΩ water at a volume of 70% of final desired volume, add calculated amounts of 0.2 M sodium monophosphate and 0.2 sodium diphosphate to a final concentration of 20 mM total phosphate. Add 1 M sodium chloride solution to provide 100 mM NaCl final concentration, Then add 5% sodium azide solution to provide 0.05% (w/v) sodium azide in the final composition, then add hydroxide as needed, and then add additional 1.8 MΩ water to bring to final volume. [0139] PBS-5.8, A representative phosphate-based buffer solution with 100 mM sodium chloride and 0.05 (w/v) sodium azide, at approximately pH 8.2. was generally prepared as follows: Start with 18 MΩ water at a volume of 70% of final desired volume, add calculated amounts of 0.2 M. sodium monophosphate and 0.2 sodium diphosphate to a final concentration of 20 mM total phosphate. Add 1 M sodium chloride solution to provide 100 mM NaCl final concentration. Then add 5% sodium azide solution to provide 0.05% (w/v) sodium azide in the final composition, then add hydroxide as needed, and then add additional 18 MΩ water to bring to final volume. [0140] EPPS-8.0: A representative buffer solution based on 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) buffering system, with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 8.0, was generally prepared as follows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated as counts of EPPS and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. EPPS is added in an amount to provide 20 mM concentration relative to final composition volume. [0141] HEPES-7.5: A representative butter solution based on [N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)] (HEPES) buffering system, with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 7.5, was generally prepared as follows: Start with 18 MNΩ water at a volume of 90% of final desired volume, add calculated amounts of HEPES and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. HEPES is added in an amount to provide 20 mM concentration relative to final composition volume. [0142] MOPS-7.0: A representative MOPS (3-(N-morpholino)propanesulfonic acid)-based buffer solution, with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 7.0, was generally prepared as hallows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of MOPS and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. MOPS is added in an amount to provide 20 mM concentration relative to final composition volume. [0143] ACES-6.5: A representative buffer solution based on N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffering system, with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately PH 6.5, was generally prepared as follows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of ACES and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric, acid or sodium hydroxide as needed, then add additional 18 MΩ water to bring to final volume. ACES is added in an amount to provide 20 mM concentration relative to final composition volume. [0144] MES-6.0: A representative buffer solution based on 2-(N-morpholino)ethanesulfonic acid (MES) buffering system, with 100 MΩ sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 6.0, was generally prepared as fellows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of MES and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. MES is added in an amount to provide 50 mM relative to final composition volume. [0145] MES-5.5: A representative buffer solution based on 2-(N-morpholino)ethanesulfonic acid (MES) buffering system, with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 5.5, was generally prepared as follows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of MES and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using, hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ. water to bring to final volume. MES is added in an amount to provide 50 mM relative to final composition volume. Alternative representative MES-based buffer solutions at about pH 5.5 were also prepared and tested using different concentrations of sodium chloride or different concentration of MES to test for salt and buffering agent concentration sensitivity, as noted in some of the examples below. [0146] ACT-5: A representative acetate-based buffer solution, with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 5.0, was generally prepared as follows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of acetic acid and sodium acetate and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. Acetate is added in an amount to provide 50 mM relative to final composition volume. [0147] ACT-4: A representative acetate-based buffer solution, with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 4.0, was generally prepared as follows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of acetic acid and sodium acetate and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide an the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, add additional 18 MΩ water to bring to final volume. Acetate is added in an amount to provide 50 mM relative to final composition volume. [0148] CIT-3: A representative citrate buffer solution, with 100 mM sodium chloride and 0.09% (w/v) sodium azide, at approximately pH 3.0, was generally prepared as follows: Start with 18 MΩ water at a volume of 90% of final desired volume, add calculated amounts of citric acid, sodium citrate, and sodium chloride and dissolve by stirring, then add 5% sodium azide solution to provide 0.09% (w/v) sodium azide in the final composition, then correct pH using hydrochloric acid or sodium hydroxide as needed, then bring then add additional 18 MΩ water to bring to final volume. Acetate is added in an amount to provide 50 mM relative to final composition volume. [0149] Combo Dye® buffer: Generally, a sodium phosphate buffer solution (Sartorius Stedim product no. VIR-92305), with 100 mM sodium chloride, 0.05% (w/v) sodium azide and Tris-EDTA, at approximately pH 7.4, used in preparing stain formulations from Combo Dye® concentrate; and generally prepared as follows: Combine PBS-7.4 and 100× TE buffer (Sigma T9285) at a volume ratio of 100:1 of PBS-7.4 to 100× TE.

[0150] Antibody stain reagent: Fluorescent antibody stain concentrate formulation having an antibody for binding with epitope of a targeted non-enveloped viral capsid and including a fluorescent label for operation with the Virus Counter® 3100 Flow Cytometer. The ViroTag™ affinity reagents are formulated to be added in 5 microliter aliquots to unstained fluid samples of 195 microliters to prepare stained fluid samples of 200 microliters for flow cytometry evaluation. The following ViroTag™ Affinity Reagents are used in various examples presented below: [0151] ADVX: For evaluation of Adenovirus serotypes 2 through ti, and including 3H8 monoclonal antibody (Hytest) labeled with fluorophore CF532 (Biotium), formulated to provide a total concentration of antibody stain in the stained fluid sample of about micrograms per milliliter, [0152] EV 71: For evaluation of Enterovirus 71, and including 11GH5H monoclonal antibody (Quidel) labeled with fluorophore CF532 (Biotium), formulated to provide a total concentration of antibody stain in the stained fluid sample of about 2.5 micrograms per milliliter. [0153] AAV2/3: For evaluation of Adeno-associated virus serotypes 2 and 3, and including, A20 monoclonal antibody (Progen) labeled with fluorophore CF532 (Biotium), formulated to provide a total concentration of antibody stain in the stained fluid sample of about 0.5 micrograms per milliliter. [0154] AAV8/9: For evaluation of Adeno associated virus serotypes 8 and 9, and including Adk8/9 monoclonal antibody (Progen) labeled with fluorophore CF532 (Biotium), formulated to provide a total concentration of antibody stain in the stained fluid sample of about 0.5 micrograms per milliliter. [0155] Test Procedures:

[0156] For tests using fluorescent antibody stains, a representative test protocol was generally as follows. Unstained fluid samples of 195 microliters in volume for one dilution factor or a series with multiple dilution factors were prepared containing virions non-enveloped virus for evaluation and at dilution factors and desired pit for testing using the sample dilution buffers. To the unstained fluid sample was added 5 microliters of a ViroTag™ affinity reagent for fluorescent staining the viral capsid of the non-enveloped virions and to prepare a stained fluid sample of 200 microliters in volume, which was then subjected to flow cytometry evaluation using the VC3100 flow cytometer. After staining, samples were allowed to sit for an incubation time of about 30 minutes while protected from light before being subjected to flow cytometry.

[0157] For tests using nonspecific fluorogenic dyes for nucleic acid and protein content using Combo Dye® concentrate, a representative test protocol was generally as follows. Unstained fluid samples of 100 microliters in volume were prepared containing virions of non-enveloped virus for evaluation and at dilution factors and desired pH for testing using the sample dilution buffers. To the unstained fluid sample was added 50 microliters of pre-prepared aqueous dye formulation including the fluorogenic dyes was added for fluorescent staining and to prepare a stained fluid sample of 150 microliter in volume, which was then subjected to flow cytometry evaluation using the VC3100 flow cytometer. A representative procedure for preparing the aqueous dye formulation was generally as follows: To a vial containing 2.8 micrograms of the dry powder Combo Dye® concentrate was added 5 microliters of the organic liquid medium. The mixture in the vial was then spun down briefly to assist dissolution of the dry powder mixture of the fluorogenic dyes into the first liquid medium. The organic liquid was acetonitrile except as specifically stated otherwise in the examples below. The mixture was allowed to sit for an incubation time of either 5 minutes in the case of acetonitrile or 10 minutes in the case of DMSO, following which the mixture was diluted through addition of 500 microliters of Combo Dye® buffer and that diluted mixture was briefly vortexed to prepare the aqueous dye formulation used to stain the fluid samples for flow cytometry evaluation. As will be appreciated, each such batch of prepared aqueous dye formulation is sufficient to stain 10 fluid samples of 100 microliters in volume for flow cytometry evaluation. Fluid samples were generally stained within 4hours of preparing the aqueous dye formulation. After staining, samples were allowed to sit for a time of about 30 minutes while protected from light before being subjected to flow cytometry.

[0158] For tests using POPO™-3 alone as a fluorogenic dye for staining nucleic acid content, a representative test protocol was generally as follows. Three microliters of stock POPO-3 were diluted in 22 microliters of acetonitrile. Five microliters of this solution were diluted in 45 microliters of Combo Dye® buffer. Samples were stained with either 3 microliters or 6 microliters of the diluted POPO-3 stain formulation in 195 microliters of samples and were incubated for at least 30 minutes at room temperature, protected from light before analysis.

[0159] For each virus evaluation test, a stock solution of the sample containing virions of the virus to be evaluated was prepared and from that stock solution unstained fluid samples were prepared at the desired pH for testing at different dilution factors, and each fluid sample in a dilution series is stained as summarized above, so that all stained fluid samples include the same quantity of fluorescent stain, regardless of the concentration of the virions. The pH of the stock solutions is not measured, but are often expected to be around physiological pH or somewhat higher. When diluted with much larger proportions of the aqueous dilution liquid to prepare diluted fluid samples prior to staining, the diluted fluid samples are assumed to be at approximately the PH of the aqueous dilution liquid. For each dilution series, a blank sample was prepared at the same pH and including the fluorescent stain at the same concentration hut not including virions. Each stained fluid sample and blank sample were subjected to flow cytometry evaluation. Raw flow cytometry results of virus particle concentration for the virus-containing stained fluid samples were corrected by subtraction of particle concentration results of blank samples. For some tests, multiple replicate samples (typically 3) were prepared at each dilution factor and for the blank sample, in which case raw flow cytometry particle concentration results were averaged for the multiple replicate samples to obtain an average raw virus particle concentration, which was corrected by the average particle concentration results of the blank sample replicates. References in the examples below to a titer value are to a concentration of the non-enveloped virions in the original, undiluted stock solution from which the diluted fluid samples were prepared, rather than to the concentration as directly measured by flow cytometry on the diluted fluid samples. As may be appreciated, an estimated titer value may be calculated from the determined virion particle concentration on a diluted fluid sample by multiplying the concentration for the diluted fluid sample by the corresponding dilution factor for that diluted fluid sample. Ideally, a log-log plot of particle concentration results vs. dilution factor for a dilution series should be linear, although in actual practice results are not ideal, and in particular at very large viral particle concentrations (e.g., above 1×10.sup.9 particles/mL) and at very low particle concentrations (e.g., near or below 5×10.sup.5 particles/mL) results tend to suffer and greater departure of plotted results from linearity tends to occur. An entire dilution series may be plotted as the logarithm value of determined particle concentrations vs. logarithm value of the dilution factor and evaluated by linear regression analysis to determine a linear fit of the data across the dilution series. All virus particle concentration data presented for the examples below are for corrected concentrations unless otherwise noted. Identified anomalous concentration results and concentration results below the instrument quantification limit of about 5×10.sup.5 particles per milliliter were generally excluded from analysis. All titer values shown for the examples below for a dilution series test is an average of titer values for all reported corrected virus particle concentrations for all dilution factors for the dilution series test.

Example 1

Adenovirus 5 Stained with Fluorogenic Dyes for Nucleic Acid and Protein at Various Acidic pHs vs Physiological pH

[0160] Fluid sample dilution series and corresponding blank samples with Adenovirus 5 were prepared and stained with fluorogenic dyes from Combo Dye® concentrate at different pHs, as summarized in Table 1, and were subjected to flow cytometry valuation for quantification of Adenovirus 5 particles.

TABLE-US-00001 TABLE 1 Example 1 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Butler System Approximate pH Prepared & Tested 1-1 Adenovirus 5 Fluorogenic Dyes PBS 7.4 50/150/450/1350 (Combo Dye ®) 1-2 Adenovirus 5 Fluorogenic Dyes ACES 6.5 50/150/450/1350 (Combo Dye ®) 1-3 Adenovirus 5 Fluorogenic Dyes MBS 6.0 50/150/450/1350 (Combo Dye ®) 1-4 Adenovirus 5 Fluorogenic Dyes MES 5.5 50/150/450/1350 (Combo Dye ®)

[0161] Results are summarized in Table 2. As shown in Table 2, average titer concentrations generally increase with decreasing pH of sample staining, and with most effective staining with the fluorogenic dyes indicated for fluid samples stained at pH 5.5

TABLE-US-00002 TABLE 2 Example 1 - Corrected Flow Cytometry Results Adenovirus 5 Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series 50 150 450 1350 particles/mL) 1-1 3.19 × 10.sup.7 1.50 × 10.sup.7 6.14 × 10.sup.6 2.26 × 10.sup.6 2.42 × 10.sup.9 (pH 7.4) 1-2 5.03 × 10.sup.7 1.79 × 10.sup.7 7.32 × 10.sup.6 2.29 × 10.sup.6 2.90 × 10.sup.9 (pH 6.5) 1-3 6.30 × 10.sup.7 2.49 × 10.sup.7 8.91 × 10.sup.6 2.43 × 10.sup.5 3.44 × 10.sup.9 (pH 6.0) 1-4 7.13 × 10.sup.7 2.68 × 10.sup.7 8.30 × 10.sup.6 1.98 × 10.sup.6 3.50 × 10.sup.9 (pH 5.5)

Example 2

Adenovirus 5 Stained with Antibody Stain at Various Acidic pHs vs Physiological pH

[0162] Fluid sample dilution series and corresponding blank samples with Adenovirus 5 were prepared and stained with fluorescent antibody stain at different pHs, as summarized in Table 3, and were subjected to flow cytometry evaluation for quantification of Adenovirus 5 particles.

TABLE-US-00003 TABLE 3 Example 2 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 2-1 Adenovirus 5 Fluorescent PBS 7.4 50/150/450/1350 Antibody Slain (ADVX) 2-2 Adenovirus 5 Fluorescent ACES 6.5 50/150/450/1350 Antibody Stain (ADVX) 2-3 Adenovirus 5 Fluorescent MBS 6.0 50/150/450/1350 Antibody Slain (ADVX) 2-4 Adenovirus 5 Fluorescent MBS 5.5 50/150/450/1350 Antibody Stain (ADVX)

[0163] Results are summarized in Table 4. Similar to the results for tests using fluorogenic dyes, shown in Example 1, average titer concentrations shown in Table 4 indicate staining with the fluorescent antibody stain was generally more effective as the pH of staining was reduced, with most effective staining indicated for fluid samples stained at pH 5.5.

TABLE-US-00004 TABLE 4 Example 2 - Corrected Flow Cytometry Results Adenovirus 5 Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series 50 150 450 1350 particles/mL) 2-1 6.07 × 10.sup.6 3.97 × 10.sup.6 1.20 × 10.sup.6 N/A* 4.79 × 10.sup.8 (pH 7.4) 2-2 1.17 × 10.sup.7 4.32 × 10.sup.6 1.31 × 10.sup.6 N/A* 6.03 × 10.sup.8 (pH 6.5) 2-3 1.56 × 10.sup.7 6.15 × 10.sup.6 1.81 × 10.sup.6 N/A* 8.41 × 10.sup.8 (pH 6.0) 2-4 3.85 × 10.sup.7 2.61 × 10.sup.7 1.01 × 10.sup.7 2.20 × 10.sup.6 3.34 × 10.sup.9 (pH 5.5) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL.

Example 3

Adenovirus 2 (Ad2) and Adenovirus 6 (AD6) Stained with Antibody Stain at Acidic pH vs Physiological pH

[0164] Fluid sample dilution series and corresponding blank samples with either Adenovirus 2 or Adenovirus 6 were prepared at different pHs, as summarized in Table 5, and were subjected to flow cytometry evaluation,

TABLE-US-00005 TABLE 5 Example 3 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 3-1 Adenovirus 2 Fluorescent PBS 7.4 10/100/1000/10,000 Antibody Stain (ADVX) 3-2 Adenovirus 2 Fluorescent WES 5.5 10/100/1000/10,000 Antibody Stain (ADVX) 3-3 Adenovirus 6 Fluorescent PBS 7.4 10/100/1000/10,000 Antibody Stain (ADVX) 3-4 Adenovirus 6 Fluorescent MES 5.5 10/100/1000/10,000 Antibody Slain (ADVX)

[0165] Results are summarized n Table 6, both of Adenovirus 2 and Adenovirus 6 tests, flow cytometry results summarized in Table 6 indicate more effective staining with the fluorescent antibody staining conducted at pH 5.5 compared to staining conducted at pH 7.4.

TABLE-US-00006 TABLE 6 Example 3 - Corrected Flow Cytometry Results Adenovirus 2 or 6 Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series 10 100 1000 10,000 particles/mL) 3-1 7.52 × 10.sup.5 N/A* N/A* NA* 7.52 × 10.sup.6 (Ad2, pH 7.4) 3-2 2.04 × 10.sup.8 4.42 × 10.sup.7 NA* N/A* 3.23 × 10.sup.9 (Ad2 pH 5.5) 3-3 5.31 × 10.sup.6 7.93 × 10.sup.5 N/A* N/A* 6.62 × 10.sup.7 (Ad6, pH 7.4) 3-4 1.25 × 10.sup.8 4.83 × 10.sup.7 5.27 × 10.sup.6 N/A* 3.79 × 10.sup.9 (Ad6, pH 5.5) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL.

Example 4

Enterovirus 71 (EV-71) Stained with Fluorogenic Dyes for Nucleic Add and Protein from Combo Dye® Concentrate at Various Acidic pHs vs Physiological pH

[0166] Fluid sample dilution series and corresponding blank samples with Enterovirus 71 were prepared and stained with fluorogenic dyes from Combo Dye® concentrate the different pHs, as summarized in Table 7, and were subjected to flow cytometry evaluation for quantification of Enterovirus 71 particles.

TABLE-US-00007 TABLE 7 Example 4 - Stained Fluic Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 4-1 Enterovirus 71 Fluorogenic Dyes PBS 7.4 100/1000/10,000 (Combo Dye ®) 4-2 Enterovirus 71 Fluorogenic Dyes PBS 6.2 100/1000/10,000 (Combo Dye ®) 4-3 Enterovirus 71 Fluorogenic Dyes MES 5.5 100/1000/10,000 (Combo Dye ®)

[0167] Results are summarized in Table 8, As shown in Table 8, flow cytometry results indicate that staining with the fluorogenic dyes at the different pHs did not result in materially different flow cytometry performance.

TABLE-US-00008 TABLE 8 Example 4 - Corrected Flow Cytometry Results Enterovirus 71 Concentration (virus particles/mL) Dilution Dilution Dilution Test Factor Factor Factor Titer (virus Series 100 1000 10,000 particles/mL) 4-1 8.22 × 10.sup.7 1.81 × 10.sup.7 1.25 × 10.sup.6 1.30 × 10.sup.10 (pH 7.4) 4-2 9.92 × 10.sup.7 1.39 × 10.sup.7 1.16 × 10.sup.6 1.18 × 10.sup.10 (pH 6.2) 4-3 1.25 × 10.sup.8 1.32 × 10.sup.7 9.83 × 10.sup.5 1.19 × 10.sup.10 (pH 5.5)

Example 5

Enterovirus 71 (EV-71) Stained with Fluorogenic Dyes for Nucleic Acid and Protein at Various Acidic pHs vs Physiological pH

[0168] Fluid sample dilution series and corresponding blank samples with Enterovirus 71 were prepared and stained with fluorogenic dyes from Combo Dye® concentrate at different pHs, as summarized in Table 9, and were subjected to low cytometry evaluation for quantification of Enterovirus 71 particles.

TABLE-US-00009 TABLE 9 Example 5 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 5-1 Enterovirus 71 Fluorogenic Dyes PBS 7.4 10/100/1000/10,000 (Combo Dye ®) 5-2 Enterovirus 71 Fluorogenic Dyes MES 5.5 10/100/1000/10,000 (Combo Dye ®) 5-3 Enterovirus 71 Fluorogenic Dyes ACT 4.0 10/100/1000/10,000 (Combo Dye ®) 5-4 Enterovirus 71 Fluorogenic Dyes CIT 3.0 10/100/1000/10,000 (Combo Dye ®)

[0169] Results are summarized in Table 10. Flow cytometry results shown in Table 10 indicate similar staining efficacy at pH 7A, pH 5.5 and pH 4.0, with efficacy of staining at the acidic pH of pH 5.5 being somewhat better than staining at pH 7.4 or pH 4.0. Flow cytometry results for staining at pH 3.0 indicate reduced staining efficacy relative to staining at: the higher acidic pHs of either pH 5.5 or pH 4.0.

TABLE-US-00010 TABLE 10 Example 5 - Corrected Flow Cytometry Results Enterovirus 71 Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series 10 100 1000 10,000 particles/mL) 5-1 8.27 × 10.sup.7 1.60 × 10.sup.7 1.58 × 10.sup.6 N/A* 1.33 × 10.sup.9 (pH 7.4) 5-2 1.04 × 10.sup.8 2.23 × 10.sup.7 1.54 × 10.sup.6 N/A* 1.60 × 10.sup.9 (pH 5.5) 5-3 8.82 × 10.sup.7 1.66 × 10.sup.7 1.10 × 10.sup.6 N/A* 1.22 × 10.sup.9 (pH 4.0) 5-4 2.80 × 10.sup.7 2.00 × 10.sup.6 N/A* N/A* 2.40 × 10.sup.8 (pH 3.0) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL.

Example 6

Enterovirus 71 (EV-71) Stained with Antibody Stain at Acidic pH vs Physiological PH

[0170] Fluid sample dilution series and corresponding blank samples with Enterovirus 71 were prepared and stained with fluorescent antibody stain at different pHs, as summarized in Table 11 and were subjected to flow cytometry evaluation for quantification of Enterovirus particles.

TABLE-US-00011 TABLE 11 Example 6 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 6-1 Enterovirus 71 Fluorescent PBS 7.4 10/100/1000/10,000 Antibody Stain (EV71) 6-2 Enterovirus 71 Fluorescent MES 5.5 10/100/1000/10,000 Antibody Stain (EV71)

[0171] Results are summarized in Table 12. The flow cytometry results summarized in Table 12 indicate effective staining of fluid sample at the acidic pH of pH 5.5, whereas staining at about physiological pH at pH 7.4 was ineffective.

TABLE-US-00012 TABLE 12 Example 6 - Raw** (Uncorrected) Flow Cytometry Results Enterovirus 71 Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Series 10 100 1000 10,000 6-1 N/A* N/A* N/A* N/A* (pH 7.4) 6-2 2.08 × 10.sup.7 4.35 × 10.sup.6 6.48 × 10.sup.5 9.11 × 10.sup.5 (pH 5.5) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL. **Blank sample results anomalous for test series 6-2 and results are presented are raw (uncorrected) flow cytometry results.

Example 7

Enterovirus 71 (EV-71) Stained with as Stain at Various Acidic pHs vs Physiological pH

[0172] Fluid samples at a single dilution factor of 130 and corresponding blank samples with Enterovirus 71 were prepared and stained with fluorescent antibody stain at different pHs, as summarized in Table 13, and were subjected to flow cytometry evaluation for quantification of Enterovirus 71 particles.

TABLE-US-00013 TABLE 13 Example 7 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilation Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 7-1 Enterovirus 71 Fluorescent PBS 8.2 130 Antibody Stain (EV71) 7-2 Enterovirus 71 Fluorescent PBS 7.4 130 Antibody Stain (EV71) 7-3 Enterovirus 71 Fluorescent PBS 7.0 130 Antibody Stain (EV71) 7-4 Enterovirus 71 Fluorescent PBS 6.6 130 Antibody Stain (EV71) 7-5 Enterovirus 71 Fluorescent PBS 6.2 130 Antibody Stain (EV71) 7-6 Enterovirus 71 Fluorescent PBS 6.0 130 Antibody Slain (EV71) 7-7 Enterovirus 71 Fluorescent PBS 5.8 130 Antibody Stain (EV71) 7-8 Enterovirus 71 Fluorescent MES 5.5 130 Antibody Stain (EV71)

[0173] Results are summarized in Table 14. As shown in Table 14, Enterovirus 71 concentration determined by flow cytometry evaluation generally increases with decreasing pH for acidic pHs of 6.6, 6.2, 6.0, and 5.8, and with best results obtained when staining was performed at pH 5.8. Although the results at pH 5.5 indicate less effective staining at that pH, based on results for other tests on this virus, the result appears to be anomalous and a problem with the lest is suspected.

TABLE-US-00014 TABLE 14 Example 7 - Corrected Flow Cytometry Results Enterovirus 71 Concentration (virus particles/mL) Test Series Dilution Factor 130 7-1 N/A* (pH 8.2) 7-2 9.76 × 10.sup.5 (pH 7.4) 7-3 6,65 × 10.sup.5 (pH 7.0) 7-4 2.53 × 10.sup.6 (pH 6.6) 7-5 2.72 × 10.sup.6 (pH 6.2) 7-6 4,51 × 10.sup.6 (pH 6.0) 7-7 4,87 × 10.sup.6 (pH 5.8) 7-8 6.87 × 10.sup.5 (pH 5.5) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL.

Example 8

Adeno-Associated Virus 8 (AAV8) and Adeno-Associated Virus 9 (AAV9) Stained with Antibody Stain at Acidic pH vs Physiological pH

[0174] Fluid sample dilution series and corresponding blank samples with either Adeno-associated virus serotype 8 or 9 were prepared and stained with fluorescent antibody stain (AAV819) at both near physiological pH (pH 7.4) and at an acidic pH (pH 5.5), as summarized in Table 15, and were subjected to flow cytometry evaluation for quantification of the respective virus particles. Fluid sample dilution series were also prepared with fluid samples including Adeno-associated virus 2 (AAV2) particles as a negative control for testing specificity of the AAV8/9 fluorescent antibody stain at different staining pH conditions.

TABLE-US-00015 TABLE 15 Example 8 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 8-1 AAV2 Fluorescent PBS 7.4 10/100/1000/10,000/100,000 Antibody Stain (AAV8/9) 8-2 AAV2 Fluorescent MES 5.5 10/100/1000/10,000/100,000 Antibody Stain (AAV8/9) 8-3 AAV8 Fluorescent PBS 7.4 10/100/1000/10,000/100,000 Antibody Stain (AAV8/9) 8-4 AAV8 Fluorescent MES 5.5 100/1000/10,000/100,000 Antibody Stain (AAV8/9) 8-5 AAV9 Fluorescent PBS 7.4 10/100/1000/10,000/100,000 Antibody Stain (AAV8/9) 8-6 AAV9 Fluorescent MES 5.5 10/100/1000/10,000/100,000 Antibody Stain (AAV8/9)

[0175] Results are summarized in Table 16, For Adeno-associated virus 2, usable flow cytometry results were not obtained for staining at either pH, indicating that binding specificity of the fluorescent antibody stain AAV 9 does not appear to be affected by the shin in staining pH. For both Adeno-associated virus 8 and Adeno-associated virus 9, results indicate significantly enhanced staining at an acidic pH of pH 5.5 relative to staining at about physiological pH of pH 74. Notably, for the acidic pH tests flow cytometry particle counts were above the instrument quantification limit and therefore usable across a greater range of dilution factors than for the pH 7.4 tests.

TABLE-US-00016 TABLE 16 Example 8 - Corrected Flow Cytometry Results Adeno-Associated Virus 2, 8 or 9 Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Factor Titer (virus Series 10 100 1000 10,000 100,000 particles/mL) 8-1 N/A* N/A* N/A* N/A* N/A* — (AAV2 pH 7.4) 8-2 N/A* NA* N/A* N/A* N/A* — (AAV2 pH 5.5) 8-3 9.57 × 10.sup.6 1.47 × 10.sup.6 N/A* N/A* N/A* 1.21 × 10.sup.8 (AAV8 pH 7.4) 8-4 — 1.08 × 10.sup.8 4.15 × 10.sup.7 4.17 × 10.sup.6 8.68 × 10.sup.5 .sup. 4.52 × 10.sup.10 (AAV8 pH 5.5) 8-5 9.88 × 10.sup.5 N/A* N/A* N/A* N/A* 9.88 × 10.sup.6 (AAV9 pH 7.4) 8-6 3.76 × 10.sup.7 5.55 × 10.sup.6 5.09 × 10.sup.5 N/A* N/A* 4.80 × 10.sup.8 (AAV9 pH 5.5 ) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL.

Example 9

Adeno-Associated Virus 8 (AAV8) Stained with Antibody Stain at Various Acidic pHs

[0176] Fluid samples at a single dilution factor and corresponding blank samples with Adeno-associated virus 8 were prepared and stained with fluorescent antibody stain at a variety of acidic pHs, as summarized in Table 17, and were subjected to flow cytometry evaluation for quantification of Adeno-associated virus 8 particles.

TABLE-US-00017 TABLE 17 Example 9 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilation Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 9-1 AAV8 Fluorescent PBS 6.6 1000 Antibody Stain (AAV8/9) 9-2 AAV8 Fluorescent PBS 6.2 1000 Antibody Stain (AAV8/9) 9-3 AAV8 Fluorescent PBS 6.0 1000 Antibody Stain (AAV8/9) 9-4 AAV8 Fluorescent PBS 5.8 1000 Antibody Stain (AAV8/9) 9-5 AAV8 Fluorescent MES 5.5 1000 Antibody Stain (AAV8/9)

[0177] Results are summarized in Table 18. As shown in Table 18, flow cytometry results indicate more effective staining of fluid samples as the staining pH decreases, with best results obtained for staining at pH 5.5. Results at the higher pHs (pH 6.6 and pH 6.2) did not provide useful results.

TABLE-US-00018 TABLE 18 Example 9 - Corrected Flow Cytometry Results Adeno-Associated Virus 8 Concentration (virus particles/mL) Test Series Dilution Factor 1000 9-1 N/A* (pH 6.6) 9-2 N/A* (pH 6.2) 9-3 3.57 × 10.sup.6 (pH 6.0) 9-4 5.23 × 10.sup.6 (pH 5.8) 9-5 7.02 × 10.sup.6 (pH 5.5) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL.

Example 10

Feline Panleukopenia Virus (FPV) Stained with Fluorogenic Dyes for Nucleic Acid and Protein at Various Acidic pHs vs Physiological pH and Basic pH

[0178] Fluid sample dilution series and corresponding blank samples with Feline panleukopenia virus were prepared and stained with fluorogenic dyes from Combo Dye® concentrate at different pHs ranging from pH 8.0 to 5.5, as summarized in Table 19, and were subjected to flow cytometry evaluation for quantification of Feline panleukopenia virus particles.

TABLE-US-00019 TABLE 19 Example 10 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 10-1 FPV Fluorogenic Dyes EPPS 8.0 10/100/1000/10,000 (Combo Dye ®) 10-2 FPV Fluorogenic Dyes PSB 7.4 10/100/1000/10,000 (Combo Dye ®) 10-3 FPV Fluorogenic Dyes ACES 6.5 10/100/1000/10,000 (Combo Dye ®) 10-4 FPV Fluorogenic Dyes MES 5.5 10/100/1000/10,000 (Combo Dye ®)

[0179] Results are summarized in Table 20, which indicate more effective staining at the acidic pHs of pH 6.5 and pH 5.5 compared to staining at pH 8.0 or pH 7.4, with most effective staining, indicated for the tests with staining at pH 6.5

TABLE-US-00020 TABLE 20 Example 10 - Corrected Flow Cytometry Results Feline Panleukopenia virus Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series W 300 1000 10,000 particles/mL) 10-1 1.99 × 10.sup.7 7.14 × 10.sup.6 9.20 × 10.sup.5 N/A* 6.11 × 10.sup.8 (pH 8.0) 10-2 1.63 × 10.sup.7 4.72 × 10.sup.6 9.03 × 10.sup.5 N/A* 5.43 × 10.sup.8 (pH 7.4) 10-3 3.67 × 10.sup.7 7.21 × 10.sup.6 2.06.× 10.sup.6 6.25 × 10.sup.5 2.35 × 10.sup.9 (pH 6.5) 10-4 6.95 × 10.sup.7 9.68 × 10.sup.6 2.04 × 10.sup.6 N/A* 1.23 × 10.sup.9 (pH 5.5) *Results excluded as anomalous, outside of identified optimal performance range or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL.

Example 11

Feline Panleukopenia Virus (FPV) Stained with Fluorogenic Dyes for Nucleic Acid and Protein at various Acidic pHs vs physiological pH

[0180] Fluid sample dilution series and corresponding blank samples with Feline panleukopenia virus were prepared and stained with fluorogenic dyes from Combo Dye® concentrate at different pHs ranging from pH 7.4 to pH 3.0, as summarized in Table 21, and subjected to flow cytometry evaluation for quantification of Feline panleukopenia virus particles.

TABLE-US-00021 TABLE 21 Example 11 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 11-1 FPV Fluorogenic Dyes PSB 7.4 10/100/1000/10,000 (Combo Dye ®) 11-2 FPV Fluorogenic Dyes MES 5.5 10/100/1000/10,000 (Combo Dye ®) 11-3 FPV Fluorogenic Dyes ACT 4.0 10/100/1000/10,000 (Combo Dye ®) 11-4 FPV Fluorogenic Dyes CIT 3.0 10/100/1000 (Combo Dye ®)

[0181] Results are summarized in Table 22. As shown in Table 22, staining appears to be more effective at both pH 5.5 and pH 4.0 than staining at physiological pH of about pH 7.4. However, results for staining at pH 3M show degradation of flow cytometry results, indicating that staining at such a low pH may be outside an optimal acidic pH range for effective staining of this virus,

TABLE-US-00022 TABLE 22 Example 11 - Corrected Flow Cytometry Results Feline Panleukopenia virus Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series 10 100 1000 10,000 particles/mL) 11-1 1.93 × 10.sup.7 —** 3.94 × 10.sup.6 N/A* 2.07 × 10.sup.9 (pH 7.4) 11-2 4.07 × 10.sup.7 2.58 × 10.sup.7 5.81 × 10.sup.6 —** 2.93 × 10.sup.9 (pH 5.5) 11-3 7.21 × 10.sup.7 3.24 × 10.sup.7 3.91 × 10.sup.6 4.29 × 10.sup.5 3.04 × 10.sup.9 (pH 4.0) 11-4 N/A* 1.38 × 10.sup.6 N/A* —  1.38 × 10.sup.8 (pH 3.0) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL. **Sample vial broke and sample lost

Example 12

Rotavirus Stained with Fluorogenic Dyes for Nucleic Acid and Protein at Various Acidic pHs vs Physiological pH and Basic pH

[0182] Fluid sample dilution series and corresponding blank samples with a rotavirus, human Rotavirus A, strain Wa (tissue culture adapted), were prepared and sunned with fluorogenic dyes from Combo Dye® concentrate at different pHs ranging from pH 8.02 pH 5.5, as summarized in Table 23, and subjected to flow cytometry evaluation for quantification of rotavirus particles.

TABLE-US-00023 TABLE 23 Example 12 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 12-1 Rotavirus Fluorogenic Dyes EPPS 8.0 10/100/1000/10,000 (Combo Dye ®) 12-2 Rotavirus Fluorogenic Dyes PBS 7.4 10/100/1000/10,000 (Combo Dye ®) 12-3 Rotavirus Fluorogenic Dyes ACES 6.5 10/100/1000/10,000 (Combo Dye ®) 12-4 Rotavirus Fluorogenic Dyes MBS 5 5 10/100/1000/10,000 (Combo Dye ®)

[0183] Results are summarized in Table 24, which indicate more effective staining at the acidic pHs of pH 6.5 and 5.5, with best results obtained with staining at the most acidic pH tested, pH 5.5.

TABLE-US-00024 TABLE 24 Example 12- Corrected Flow Cytometry Results Rotavirus Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series 10 100 1000 10,000 particles/mL) 12-1 3.52 × 10.sup.7 6.06 × 10.sup.6 N/A* N/A* 4.79 × 10.sup.8 (pH 8.0) 12-2 3.95 × 10.sup.7 7.74 × 10.sup.6 6.48 × 10.sup.5 N/A* 6.06 × 10.sup.8 (pH 7.4) 12-3 3.61 × 10.sup.7 8.00 × 10.sup.6 7.17 × 10.sup.5 N/A* 6.26 × 10.sup.8 (pH 6.5) 12-4 2.19 × 10.sup.7 8.28 × 10.sup.6 1.07 × 10.sup.6 N/A* 7.06 × 10.sup.8 (pH 5.5) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL

Example 13

Adeno-Associated Virus 2 (AAV2) with Fluorogenic Dyes for Nucleic Acid and Protein at Various Acidic pHs vs Physiological pH and Basic pH

[0184] Fluid sample dilution series and corresponding blank samples with adeno-associated virus 2 were prepared and stained with fluorogenic dyes from Combo Dye® concentrate at different pHs ranging from pH 8.0 to pH 5.5, as summarized in Table 25, and subjected to flow cytometry evaluation t′or quantification of Adeno-associated virus 2 particles

TABLE-US-00025 TABLE 25 Example 13 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 13-1 AAV2 Fluorogenic Dyes EPPS 8.0 0/100/1000/10,000 (Combo Dye ®) 13-2 AAV2 Flurogenic Dyes PBS 7.4 0/100/1000/10,000 (Combo Dye ®) 13-3 AAV2 Fluorogenic Dyes ACES 6.5 0/100/1000/10,000 (Combo Dye ®) 13-4 AAV2 Fluorogenic Dyes MES 5.5 0/100/1000/10,000 (Combo Dye ®

[0185] Results are summarized in Table 26, which indicate more effective staining as the pH is reduced from pH 8.0 to pH 5.5, with most effective staining indicated for tests with staining at pH 5.5.

TABLE-US-00026 TABLE 26 Example 13- Corrected Flow Cytometry Results Adeno-associated Virus 2 Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series 10 100 1000 10,000 particles/mL) 13-1 6.11 × 10.sup.7 9.95 × 10.sup.6 9.05 × 10.sup.5 N/A* 8.37 × 10.sup.8 (pH 8.0) 13-2 5.89 × 10.sup.7 1.01 × 10.sup.7 1.13 × 10.sup.6 N/A* 9.12 × 10.sup.8 (pH 7.4) 13-3 7.03 × 10.sup.7 1.13 × 10.sup.7 1.53 × 10.sup.6 N/A* 1.12 × 10.sup.9 (pH 6.5) 13-4 1.75 × 10.sup.8 1.77 × 10.sup.7 2.15 × 10.sup.6 N/A* 1.89 × 10.sup.9 (pH 5.5) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL.

Example 14

Adeno-Associated Virus 2 (AAV2) with Fluorogenic Dyes for Nucleic Acid and Protein at Various Acidic PHs vs Physiological pH, using DMSO as Organic Liquid to Dissolve the Dry Powder Dye Mixture to Prepare the Aqueous Dye Formulation

[0186] Fluid sample dilution series and corresponding blank samples with adeno-associated virus 2 were prepared and stained with fluorogenic dyes from Combo Dye at different pHs ranging from pH 7.4 to pH 4.0, as summarized in Table 27, and subjected to flow cytometry evaluation for quantification of Adeno-associated virus 2 particles. In this example, the aqueous dye formulation was prepared using DMSO is the organic liquid.

TABLE-US-00027 TABLE 27 Example 14 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample Dilution Factors Series Particles Stain Buffer System Approximate pH Prepared & Tested 14-1 AAV2 Fluorogenic Dyes PSB 7.4 100/1000/10,000 (Combo Dye ®) 14-2 AAV2 Fluorogenic Dyes MES 5.5 10/100/1000/10,000 (Combo Dye ®) 14-3 AAV2 Fluorogenic Dyes ACT 5.0 10/100/1000/10,000 (Combo Dye ®) 14-4 AAV2 Fluorogenic Dyes ACT 4.0 10/100/1000/10,000 (Combo Dye ®)

[0187] Results are summarized in Table 28. As shown in Table 28, staining appears to be somewhat more effective a pH 74 than pH 5.5, but results tend to indicate similar staining performance at all of pH 7.4, pH 5.5 and pH 5.0.

TABLE-US-00028 TABLE 28 Example 14- Corrected Flow Cytometry Results Adeno-associated Virus 2 Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series 10 100 1000 10,000 particles/mL) 14-1 — 5.24 × 10.sup.7 6.27 × 10.sup.6 N/A* 5.76 × 10.sup.9 (pH 7.4) 14-2 2.87 × 10.sup.8 3.57 × 10.sup.7 5.11 × 10.sup.6 N/A* 3.85 × 10.sup.9 (pH 5.5) 14-3 2.66 × 10.sup.8 2.98 × 10.sup.7 3.27 × 10.sup.6 N/A* 2.92 × 10.sup.9 (pH 5.0) 14-4 6.78 × 10.sup.7 2.47 × 10.sup.7 8.65 × 10.sup.5 N/A* 1.34 × 10.sup.9 (pH 4.0) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL.

Example 15

Adenovirus 5 (Ad5) with Antibody Stain at Acidic pH with Varying Concentrations of Buffering Agent in Sample Dilution Buffer

[0188] Fluid sample dilution series and corresponding blank samples with Adenovirus 5 were prepared and stained with fluorescent antibody stain at an acidic elf of pH 5.5 using MES-based sample dilution buffer at about pH 5.5, but prepared with varying concentrations of MES, as summarized in Table 29. The stained fluid sample and blank samples were subjected to flow cytometry evaluation for quantification of Adenovirus 5 particles.

TABLE-US-00029 TABLE 29 Example 15 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample MES Concentration Dilution Factors Series Particles Stain Buffer System Approximate pH (mM) Prepared & Tested 15-1 Ad5 Fluorescent MES 5.5 20 100/200/400 Antibody Stain (ADVX) 15-2 Ad5 Fluorescent MES 5.5 50 200/400 Antibody Stain (ADVX) 15-3 Ad5 Fluorescent MES 5.5 100 100/200/400 Antibody Stain (ADVX) 15-4 Ad5 Fluorescent MES 5.5 200 100/200/400 Antibody Stain (ADVX)

[0189] Results are summarized in Table 30, indicating enhanced performance with MES concentrations of 50 mM and 100 mM relate to the lowest tested MES concentration of 20 mM and the highest tested MES concentration of 200 mM.

TABLE-US-00030 TABLE 30 Example 15- Corrected Flow Cytometry Results Adenovirus 5 Concentration (virus particles/mL) Dilution Dilution Dilution Test Factor Factor Factor Titer (virus Series 100 200 400 particles/mL) 15-1 7.65 × 10.sup.7 3.44 × 10.sup.7 1.31 × 10.sup.7 6.58 × 10.sup.9  (20 mM MES) 15-2 — 9.77 × 10.sup.7 4.98 × 10.sup.7 1.97 × 10.sup.10 (50 mM MES) 15-3 9.03 × 10.sup.7 6.45 × 10.sup.7 4.03 × 10.sup.7 1.27 × 10.sup.10 (100 mM MES) 15-4 5.27 × 10.sup.7 3.37 × 10.sup.7 2.01 × 10.sup.7 6.68 × 10.sup.9  (200 mM MES)

Example 16

Adenovirus 5 (Ad5) with Antibody Stain at Acidic pH with Varying Salt Concentrations in Sample Dilution Buffer

[0190] Fluid sample dilution series and corresponding blank samples with Adenovirus 5 were prepared and stained with fluorescent antibody stain at an acidic pH of 5.5 using MES-based sample dilution buffer at a pH of about 5.5, but prepared with varying concentrations of sodium chloride, as summarized in Table 31. The stained fluid samples and blank samples were subjected to flow cytometry evaluation for quantification of Adenovirus 5 particles.

TABLE-US-00031 TABLE 31 Example 16 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample NaCl Concentration Dilution Factors Series Particles Stain Buffer System Approximate pH (mM) Prepared & Tested 16-1 Ad5 Fluorescent MES 5.5 50 100/300/900 Antibody Stain (ADVX) 16-2 Ad5 Fluorescent MES 5.5 100 100/300/900 Antibody Stain (ADVX) 16-3 Ad5 Fluorescent MES 5.5 300 100/300/900 Antibody Stain (ADVX) 16-4 Ad5 Fluorescent MES 5.5 500 100/300/900 Antibody Stain (ADVX)

[0191] Results are summarized in Table 32, indicating a generally declining performance with increasing concentration of sodium chloride for the concentrations tested.

TABLE-US-00032 TABLE 32 Example 16- Corrected Flow Cytometry Results Adenovirus 5 Concentration (virus particles/mL) Dilution Dilution Dilution Test Factor Factor Factor Titer (virus Series 100 300 900 particles/mL) 16-1 1.83 × 10.sup.8 1.13 × 10.sup.8 4.32 × 10.sup.7 3.04 × 10.sup.9 (50 mM NaCl) 16-2 1.18 × 10.sup.8 5.35 × 10.sup.7 9.37 × 10.sup.6 1.21 × 10.sup.9 (100 mM NaCl) 16-3 1.37 × 10.sup.7 3.40 × 10.sup.6 8.42 × 10.sup.5 1.05 × 10.sup.9 (300 mM NaCl) 16-4 1.02 × 10.sup.7 3.41 × 10.sup.6 8.18 × 10.sup.5 9.26 × 10.sup.8 (500 mM NaCl)

Example 17

Adenovirus 5 (Ad5) and Adenovirus 4 (Ad4) with Antibody Stain at Acidic pH with Varying Salt Concentrations in Sample Dilution Buffer

[0192] Fluid sample dilution series and corresponding blank samples with either Adenovirus 5 Adenovirus 4 were prepared and stained with fluorescent antibody stain at an acidic pH of 5.5 using MES-based sample dilation buffer at a pH of about 5.5, but prepared with varying concentrations of sodium chloride, as summarized in Table 33. The stained fluid samples and blank samples were subjected to flow cytometry evaluation for quantification of Adenovirus 5 or Adenovirus 4 particles.

TABLE-US-00033 TABLE 33 Example 17 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample NaCl Concentration Dilution Factors Series Particles Stain Buffer System Approximate pH (mM) Prepared & Tested 17-1 Ad5 Fluorescent MES 5.5 50 100/300/900 Antibody Stain (ADVX) 17-2 Ad5 Fluorescent MES 5.5 100 100/300/900 Antibody Stain (ADVX) 1.7-3 Ad5 Fluorescent MES 5.5 300 100/300/900 Antibody Stain (ADVX) 17-4 Ad4 Fluorescent MES 5.5 50 100/300/900 Antibody Stain (ADVX) 17-5 Ad4 Fluorescent MES 5.5 100 100/300/900 Antibody Stain (ADVX) 17-6 Ad.4 Fluorescent MES 5.5 300 100/300/900 Antibody Stain (ADVX)

[0193] Results are summarized in Table 34, indicating generally acceptable results for both virus serotypes at sodium chloride concentrations of 50 mM and 100 mM and generally unacceptable results at the sodium chloride concentration of 300 mM.

Example 18

Blank Particle Counts

[0194] Concentrated dye formulations with both POPO™-3 iodide stain: and SYPRO™ Red stain were prepared in 5 microliters of DMSO from 2.8 microliters of dry powder dye composition and then diluted 500:1 by volume with aa regular aqueous stain dilution buffer prepare an aqueous diluted dye formulation with both fluorogenic dyes. Three replicate blank fluid samples containing no particles for evaluation were then prepared by adding, 50 microliters of the diluted aqueous dye formulation to 100 microliters of a regular sample dilution buffer at about physiological pH, to prepare blank fluid sample of 150 microliters volume each at about physiological pH. The blank fluid samples were subjected to flow cytometry evaluation and particle emus were separately evaluated on detection channel 1 (detecting, for the emission of signature from POPO™-3 iodide stain) and detection channel 2 (detecting for the emission signature from SYPRO™ Red stain) of the Virus Counter® 3100 flow cytometer to determine a background blank particle count separately for each fluorogenic dye. Average blank particle counts on detection channel 1 (POPO™-3 iodide stain) were relatively low, on the order of 10.sup.5 blank particle counts per milliliter, whereas average blank particle counts on detection channel 2 (SYPRO™ Red stain were on the order of 10.sup.7 blank particle counts per milliliter.

[0195] Additional tests were performed in which such a concentrated dye formulation with both fluorogenic dyes is diluted with modified aqueous stain dilution buffer to prepare aqueous diluted dye formulations, which is used to prepare modified 150 microliter blank fluid samples at approximately physiological pH. The modified aqueous stain dilution buffer is prepared with the same composition as the noted regular aqueous stain dilution buffer, except also including 15% w/v percent dissolved sugar, which when diluted at a 1:2 volume dilution ratio with sample dilution buffer to prepare the blank fluid sample results in the blank fluid sample containing 5% w/v of the dissolved sugar as fed to the flow cytometer. Sugars tested include a monosaccharide (galactose), disaccharides (sucrose, lactose and trehalose) and trisaccharides (maltotriose and raffinose). The monosaccharide galactose was found to detrimentally increase blank counts on detection Channel 1. The trisaccharides maltotriose and raffinose did not improve blank particle count performance, and with maltotriose increasing blank counts on detection channel 2. All of the tested disaccharides sucrose, lactose and trehalose significantly reduced blank particle counts on detection channel 2, with trehalose exhibiting the greatest reduction. These tests demonstrate beneficial effect of adding disaccharide, even though the tests were not performed at an acidic pH.

TABLE-US-00034 TABLE 34 Example 17- Corrected Flow Cytometry Results Adenovirus 5 and Adenovirus 4 Concentration (virus particles/mL) Dilution Dilution Dilution Test Factor Factor Factor Titer (virus Series 100 300 900 particles/mL) 17-1 1.89 × 10.sup.7 3.41 × 10.sup.6 N/A* 1.46 × 10.sup.9 (Ad5, 50 mM NaCl) 17-2 5.59 × 10.sup.6 6.37 × 10.sup.5 N/A* 3.75 × 10.sup.8 (Ad5, 100 mM NaCl) 17-3 N/A* N/A* N/A* — (Ad5, 300 mM NaCl) 17-4 4.66 × 10.sup.7 1.68 × 10.sup.7 4.04 × 10.sup.6 4.44 × 10.sup.9 (Ad4, 50 mM NaCl) 17-5 2.66 × 10.sup.7 6.90 × 10.sup.6 1.19 × 10.sup.6 1.93 × 10.sup.9 (Ad4, 100 mM NaCl) 17-6 N/A* N/A* N/A* — (Ad4, 300 mM NaCl) *Results excluded as anomalous or because uncorrected counts below instrument quantification limit of 5 × 10.sup.5 particles/mL

Example 18

Adeno-Associated Virus 2 (AAV2) Stained with Fluorogenic Dye for Nucleic Acid at Acidic pH vs Physiological pH and Basic pH

[0196] Fluid sample dilution series and corresponding, blank samples with Adeno-associated virus 2 were prepared and stained with fluorogenic stain from nucleic acid content from POPO™-3 formulation at different pHs ranging from pH 8.0 to pH 5.5 and with sample dilution buffers including either 50 or 100 mM sodium chloride concentration, as summarized in Table 35. and were subjected to flow cytometry evaluation for quantification of Adeno-associated virus 2 particles. For these tests, the fluorogenic staining composition was prepared using 3 of the diluted POPO™-3 stain formulation.

TABLE-US-00035 TABLE 35 Example 18 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample NaCl Concentration Dilution Factors Series Particles Stain Buffer System Approximate pH (mM) Prepared & Tested 18-1 AAV2 Fluorogenic TRI 8.0 50 100 nucleic acid stain (POPO ™-3 formulation) 18-2 AAV2 Fluorogenic TRI 8.0 100 100 nucleic acid stain (POPO ™-3 formulation) 18-3 AAV2 Fluorogenic PBS 7.4 50 100 nucleic acid stain (POPO ™-3 formulation) 18-4 AAV2 Fluorogenic PBS 7.4 100 100 nucleic acid stain (POPO ™-3 formulation) 18-3 AAV2 Fluorogenic MES 5.5 50 100 nucleic acid stain (POPO ™-3 formulation) 18-6 AAV2 Fluorogenic MES 5.5 100 100 nucleic acid stain (POPO ™-3 formulation)

[0197] Results are summarized in Table 36, indicating much more effective staining of AAV2 at pH 5.5 compared to staining at either pH 8.0 or pH 7.4 at both sodium chloride concentrations tested.

TABLE-US-00036 TABLE 36 Example 18- Corrected Flow Cytometry Results Adeno-associated virus 2 Concentration (virus particles/mL) Test Series Dilution Factor 100 18-1 1.59 × 10.sup.6 (pH 8.0, 50 mM NaCl) 18-2 1.64 × 10.sup.6 (pH 8.0, 100 mM NaCl) 18-3 5.71 × 10.sup.5 (pH 7.4, 50 mM NaCl) 18-4 1.21 × 10.sup.6 (pH 7.4, 100 mM NaCl) 18-5 1.20 × 10.sup.8 (pH 5.5, 50 mM NaCl) 18-6 9.15 × 10.sup.7 (pH 5.5, 100 mM NaCl)

Example 9

Adeno-Associated Virus 2 (AAV2) Stained with Fluorogenic Dye for Nucleic Acid at Acidic pH vs Physiological pH and Basic pH

[0198] Fluid sample dilation series and corresponding blank samples with Adeno-associated virus 2, were prepared and stained with fluorogenic stain from nucleic acid content from POPO™-3 formulation at different pHs ranging from pH 8.0 to pH 5.5 and with sample dilution butlers including either 50 or 100 mM sodium chloride concentration as summarized in Table 37 and subjected to flow cytometry evaluation for quantification of Adeno-associated virus 2 particles. For these tests, the fluorogenic staining composition was prepared using 6 μL of the diluted POPO™-3 stain formulation,

TABLE-US-00037 TABLE 37 Example 19 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample NaCl Concentration Dilution Factors Series Particles Stain Buffer System Approximate pH (mM) Prepared & Tested 19-1 AAV2 Fluorogenic TRI 8.0 50 100 nucleic acid stain (POPO ™-3 formulation) 19-2 AAV2 Fluorogenic TRI 8.0 100 100 nucleic acid stain (POPO ™-3 formulation) 19-3 AAV2 Fluorogenic PBS 7.4 50 100 nucleic acid stain (POPO ™-3 formulation) 19-4 AAV2 Fluorogenic PBS 7.4 100 100 nucleic acid stain (POPO ™-3 formulation) 19-5 AAV2 Fluorogenic MES 5.5 50 100 nucleic acid stain (POPO ™-3 formulation) 19-6 AAV2 Fluorogenic MES 5.5 100 100 nucleic acid stain (POPO ™-3 formulation)

[0199] Results are summarized in Table 38, indicating much more effective staining of AAV2 at pH 5.5 compared to staining at either pH 8.0 or pH 7.4. Compared to Example 18, titer concentrations are somewhat higher due to the larger amount of POPO™-3 formulation used to prepare fluorogenic staining compositions,

TABLE-US-00038 TABLE 38 Example 19- Corrected Flow Cytometry Results Adeno-associated virus 2 Concentration (virus particles/mL) Test Series Dilution Factor 100 19-1 2.82 × 10.sup.6 (pH 8.0, 50 mM NaCl) 19-2 3.52 × 10.sup.6 (pH 8.0, 100 mM NaCl) 19-3 1.11 × 10.sup.6 (pH 7.4, 50 mM NaCl) 19-4 3.69 × 10.sup.6 (pH 7.4, 100 mM NaCl) 19-5 1.27 × 10.sup.8 (pH 5.5, 50 mM NaCl) 19-6 1.24 × 10.sup.8 (pH 5.5, 100 mM NaCl)

Example 20

Adeno-Associated Virus 2 (AAV2) Stained with Fluorogenic Dye for Nucleic Acid and with Antibody Stain at Acidic pH vs Physiological pH

[0200] Fluid sample dilution series and corresponding blank samples with Adeno-associated virus 2 were prepared and stained at various pHs ranging from pH 8.0 to pH 5.5 with either fluorogenic stain for nucleic acid content using POPO™-3 formulation or using AAV2/3 fluorescent antibody stain for staining the non-enveloped viral capsid, as summarized in Table 39. Sodium Chloride concentrations in the sample dilution buffer for each test series is shown in Table 39. For the test using POPO™-3, the fluorogenic staining composition was prepared using 6 μL of the diluted POPO™-3 stain formulation. Stained fluid samples and blank samples were subjected to flow cytometry evaluation for quantification of Adeno-associated virus 2 particle concentration.

TABLE-US-00039 TABLE 39 Example 20 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample NaCl Concentration Dilution Factors Series Particles Stain Buffer System Approximate pH (mM) Prepared & Tested 20-1 AAV2 Fluorogenic PBS 7.4 100 100 nucleic acid stain (POPO ™-3 formulation) 20-2 AAV2 Fluorescent PBS 7.4 100 100 Antibody Stain (AAV2/3) 20-3 AAV2 Fluorogenic MES 5.5 100 100 nucleic acid stain (POPO ™-3 formulation) 20-4 AAV2 Fluorescent MES 5.5 100 100 Antibody Stain (AAV2/3) 20-5 AAV2 Fluorogenic MES 5.5 50 100 nucleic acid stain (POPO ™-3 formulation) 20-6 AAV2 Fluorescent MES 5.5 50 100 Antibody Stain (AAV2/3)

[0201] Results are summarized in Table 40, indicating much more effective staining AAV2 at pH 5.5 compared to staining at pH 7.4 both for fluorogenic staining using POPO™-3 and for fluorescent antibody staining using AAV2/3. It is noted that determined concentrations based staining with POPO™-3 were generally higher than concentration based on staining with AAV2/3, Which may be an indication of some viral particles having empty non-enveloped capsids, that is not containing stainable nucleic acid content.

TABLE-US-00040 TABLE 40 Example 20- Corrected Flow Cytometry Results Adeno-associated virus 2 Concentration (virus particles/mL) Test Series Dilution Factor 100 20-1 1.92 × 10.sup.6 (pH 7.4, POPO ™-3, 100 mM NaCl) 20-2 7.94 × 10.sup.7 (pH 7.4, AAV2/3, 100 mM NaCl) 20-3 1.51 × 10.sup.7 (pH 5.5, POPO ™-3, 100 mM NaCl) 20-4 3.39 × 10.sup.8 (pH 5.5, AAV2/3, 100 mM NaCl) 20-5 1.72 × 10.sup.8 (pH 5.5, POPO ™-3, 50 mM NaCl) 20-6 3.18 × 10.sup.8 (pH 5.5, AAV2/3, 50 mM NaCl)

Example 21

Adeno-Associated Virus 2 (AAV2) Stained with Fluorogenic Dyes for Nucleic Acid and Protein Content at Acidic pH vs Physiological pH

[0202] Fluid sample dilution series and corresponding blank samples with Adeno-associated virus 2 were prepared and stained with fluorogenic dyes from Combo Dye® concentrate at pH 7.4 or pH 5.5, as summarized in Table 41. Sodium Chloride concentrations in the sample dilution buffer for each test series is shown in Table 41. Stained fluid samples and blank samples were subjected to flow cytometry evaluation for quantification of Adeno-associated virus 2 particle concentration.

TABLE-US-00041 TABLE 41 Example 21 - Stained Fluid Samples Prepared and Tested Test Virus Fluorescent Sample Dilution Stained Fluid Sample NaCl Concentration Dilution Factors Series Particles Stain Buffer System Approximate pH (mM) Prepared & Tested 21-1 AAV2 Fluorogenic Dyes PBS 7.4 100 10/100/1000/10,000 (Combo Dye ®) 20-2 AAV2 Fluorogenic Dyes MES 5.5 50 100/1000/10,000 (Combo Dye ®)

[0203] Results are summarized in Table 42, indicating much more effective staining of AAV2 at pH 5.5 compared to staining at pH 7.4. Notably, counts for nucleic acid and protein stains both increase for fluid samples stained at pH 5.5 relative to staining at pH 7.4, but increase in particle counts is more pronounced for nucleic acid stain than for protein stain, indicating enhanced accessibility of nucleic acid content in the capsid for staining by the fluorogenic nucleic acid stain.

TABLE-US-00042 TABLE 42 Example 21- Corrected Flow Cytometry Results Adeno-associated virus 2 Concentration (virus particles/mL) Dilution Dilution Dilution Dilution Test Factor Factor Factor Factor Titer (virus Series 10 100 1000 10,000 particles/mL) 21-1 N/A* 5.57 × 10.sup.5 4.75 × 10.sup.6 N/A* 2.40 × 10.sup.9  (pH 7.4, Combo Dye ®) 21-2 — 2.77 × 10.sup.7 9.58 × 10.sup.7 6.35 × 10.sup.6 5.42 × 10.sup.10 (pH 5.5, Combo Dye ®) *Results excluded as anomalous or because uncorrected counts below instrument

EXEMPLARY IMPLEMENTATION COMBINATIONS

[0204] Some other contemplated embodiments of implementation combinations for various aspects of this disclosure, with or without additional features as disclosed above or elsewhere herein, are summarized in exemplary numbered combinations presented below, and in the appended claims:

[0205] 1. A method for flow cytometry evaluation of a biological material for unassociated non-enveloped viral particles having a non-enveloped viral capsid, the method comprising: [0206] preparing; a fluorescently-stained fluid sample lot flow cytometry evaluation, the fluorescently-stained fluid sample comprising: [0207] an aqueous liquid medium; [0208] a sample of biological material to be evaluated by flow cytometry for the unassociated non-enveloped viral particles having the non-enveloped viral capsid; [0209] at least one fluorescent stain to fluorescently stain the unassociated non-enveloped viral particles to prepare unassociated labeled particles of virus size dispersed in the aqueous liquid medium, wherein each said unassociated labeled particle comprise a said unassociated non-enveloped viral particle stained with the at least one fluorescent stain, and wherein each said fluorescent stain has a fluorescent emission response when the unassociated labeled particle is subjected to a stimulation radiation; [0210] after the preparing, subjecting the fluorescently-stained fluid sample to flow cytometry evaluation in a flow cytometer, the flow cytometry evaluation comprising flowing the fluorescently-stained fluid sample through an investigation zone of the flow cytometer and in the investigation zone subjecting the fluorescently-stained fluid sample to the stimulation radiation and detecting for the fluorescent emission response from the investigation zone and counting identified occurrences of the unassociated labeled particles; and [0211] the preparing the fluorescently-stained fluid sample comprising fluorescent staining the biological material in a fluid sample composition with a said fluorescent stain to prepare a stained fluid sample composition at an acidic pH in an acidic pH range of from pH 3.0 to pH 6.5. [0212] 2. The method of combination 1, wherein the acidic pH of stained fluid sample composition is not larger than pH 6.0. [0213] 3. The method of either one of combination 1 or combination 2, wherein the acidic pH of the stained fluid sample composition is not smaller than pH 4.5. [0214] 4. The method o. any one of combinations 1-3, wherein the acidic pH range is from pH 4.0 to pH 6:0. [0215] 5. The method of any one of combinations 1-4, wherein the at least one fluorescent stain comprises a fluorogenic dye for staining of nucleic acid content inside the viral capsid of the non-enveloped viral particles. [0216] 6. The method of combination 5, wherein the fluorogenic dye is a first fluorogenic dye and the at least one fluorescent stain comprises a second fluorogenic dye for staining of protein content of the viral capsid. [0217] 7. The method of either one of combination 5 or combination 6. wherein the fluorescently-stained fluid sample as prepared during the preparing and as fed to the flow cytometer comprises an organic liquid component as a minor component, on a molar basis, of the aqueous liquid medium, optionally with the fluorescently-stained fluid sample comprising at 0.25 weight percent or at least 0.5 weight percent of the organic liquid component or another minimum concentration as described elsewhere herein, and with a separate or further option being that the fluorescently-stained fluid sample comprises up to 30 weight percent of the organic liquid component or up to another maximum concentration as described elsewhere herein. [0218] 8. The method of combination 7, wherein the organic liquid component is dimethyl sulfoxide (DMSO), [0219] 9. The method of combination 7, wherein the organic liquid component is acetonitrile. [0220] 10. The method of one of combinations 7-9, wherein the aqueous liquid medium comprises a weight ratio of water to the organic liquid component in a range of from 30:1 to 3000:1. [0221] 11. The method daily one of combinations 7-10, wherein the preparing the fluorescently-stained fluid sample comprises: [0222] first preparatory processing to prepare a concentrated stain formulation with the fluorogenic dye containing at least one aromatic group susceptible to pi stacking interactions in aqueous liquids, the first preparatory processing comprising dissolving the fluorogenic dye into a first liquid medium from a dry powder dye composition with the fluorogenic dye, wherein the first liquid medium comprises the organic liquid component; [0223] after the first preparatory processing, second preparatory processing while the fluorogenic dye remains in solution to prepare an aqueous diluted stain formulation comprising the fluorogenic dye dissolved in a diluted aqueous liquid comprising the organic liquid component, the second preparatory processing comprising diluting the first liquid medium with aqueous stain dilution liquid at a volume ratio of the aqueous stain dilution liquid to the first liquid medium of at least 10:1 to prepare the diluted aqueous liquid with a weight ratio of water to the organic liquid component of at least 10:1; and [0224] during the fluorescent staining, mixing at least a portion of the aqueous diluted stain formulation with the fluid sample composition, and optionally with the fluid sample composition already at a pH within the acidic pH range prior to the mixing. [0225] 12. The method of combination 11, wherein the first preparatory processing comprises mixing the dry powder dye composition with the first liquid medium and after the mixing permitting the resulting mixture to sit for a residence time of at least 3 minutes prior to diluting the first liquid medium with the aqueous stain dilution liquid during the second preparatory processing; and preferably for a residence time of at least 7 minutes when the organic liquid component is DMSO. [0226] 13. The method according to either one of combination 11 or combination 12, wherein, [0227] the fluorogenic dye is a first fluorogenic dye with a first fluorescent emission signature and the at least one stain comprises a second fluorogenic dye for staining of protein content of the viral capsid, the second fluorogenic dye containing at least one aromatic group susceptible to pi stacking interactions in aqueous liquids and having a second fluorescent emission signature that is different than the first fluorescent emission signature; [0228] the dry powder dye composition comprises a dry powder mixture including the first fluorogenic dye and the second fluorogenic dye; [0229] the first preparatory processing comprises dissolving the second fluorogenic dye into the first liquid medium from the dry powder dye composition to prepare the concentrated stain formulation including the second fluorogenic dye, wherein the stained fluid sample composition includes both the first fluorogenic dye and the second fluorogenic dye; [0230] the second preparatory processing comprises, while the second fluorogenic dye remains in solution, preparing the aqueous diluted stain formulation comprising the first fluorogenic dye and the second fluorogenic dye dissolved in the diluted aqueous liquid; and [0231] the flow cytometry evaluation comprises detecting and counting occurrences of the unassociated labeled particles stained with both the first fluorogenic dye and the second fluorogenic dye, [0232] 14. The method of any one of combinations 11-13, wherein the aqueous stain dilution liquid has a pH in the acidic pH range. [0233] 15. The method of any one of combinations 11-14, wherein the diluted aqueous liquid of the aqueous diluted stain formulation has a pH in the acidic pH range. 16. The method of any one of combinations 6-10, wherein the preparing the fluorescently-stained fluid sample comprises: [0234] providing a liquid dye concentrate comprising the first fluorogenic dye and the second fluorogenic dye in a stain liquid medium; and [0235] during the fluorescent staining, mixing the liquid dye concentrate with the fluid sample composition; [0236] and wherein the method comprises at least one member selected from the group consisting of: [0237] (i) the stain liquid medium comprising a mixture including water and liquid phase organic material, and preferably the stain liquid medium aqueous, and more preferably the stain liquid medium of the liquid dye concentrate comprising more than 50 percent by moles of water; [0238] (ii) the liquid dye concentrate comprising disaccharide dissolved in the stain liquid medium; [0239] (iii) the providing the liquid dye concentrate being in the absence of reconstituting the fluorogenic dyes from a dry form into the stain liquid medium; and [0240] (iv) combinations including two or more of any of (i)-(iii). [0241] 17. The method of claim 16, wherein the fluorescent staining comprises combining the fluid sample composition and the liquid dye concentrate at a volume ratio of volume of the liquid dye concentrate to volume of the fluid sample composition of at least 1:125. [0242] 18. The method of either one of combination 16 or combination 17, wherein the fluorescent staining comprises combining the fluid sample composition with a volume of the liquid dye concentrate in a volume ratio of the volume of the liquid dye concentrate to a volume of the fluid sample composition of no greater than 1:24. [0243] 19. The method of any one of combinations 16-18, comprising the providing the liquid dye concentrate being in the absence of reconstituting the first and second fluorogenic dyes from a dry form into the stain liquid medium. [0244] 20. The method of any one of combinations 16-19, wherein the providing the liquid dye concentrate comprises unsealing a sealed container containing the aqueous liquid dye concentrate and removing from the unsealed container a quantity of the liquid dye concentrate for combining with the fluid sample composition during the staining. [0245] 21. The method of combination 20, wherein the sealed container containing the liquid dye concentrate is provided in a kit also comprising a different sealed container containing an aqueous sample dilution liquid, optionally with the sealed containers packaged in a common packaging enclosure, and optionally with the aqueous sample dilution liquid at an acidic pH of up to pH 6.5, and preferably not smaller than pH 3.0, and optionally the pH of the aqueous sample dilution liquid is in the acidic pH range. [0246] 22, The method of combination 21, comprising: [0247] providing a plurality of said fluid sample compositions each with a portion of a biological material from a stock source and each diluted to different dilution ratios with the aqueous sample dilution liquid; [0248] and separately subjecting each said fluorescently-stained fluid sample to the flow cytometry. [0249] 23. The method of any one of combinations 16-22, wherein the stain liquid medium comprises a liquid mixture including water and liquid phase organic material, [0250] 24. The method of combination 23, wherein the fluorescently-stained fluid sample comprises a concentration of the liquid phase organic material of at least 0.25 weight percent, optionally at least 0.35 weight percent or optionally at least 0.5 weight percent. [0251] 25. The method of either one of combination 23 or combination 24, wherein the fluorescently-stained fluid sample comprises a concentration of the liquid phase organic material of no larger than 2 weight percent, optionally no larger than 1.25 weight percent or optionally no larger than 0.75 weight percent, and with one preferred range for the concentration of the liquid phase organic material being from 0.25 weight percent to 1.25 weight percent and a more preferred range being from 0.5 weight percent lo 1.25 weight percent. [0252] 26. The method of any one of combinations 1-25, wherein the fluorescently-stained fluid sample comprises a disaccharide dissolved in the stain liquid medium. [0253] 27. The method of combination 26, wherein the fluorescently-stained fluid sample comprises a concentration of the disaccharide of at least 0.1 weight percent, optionally at least 0.2 weight percent or optionally at least 0.3 weight percent. [0254] 28. The method of either one of combination 26 or combination 27, wherein the fluorescently-stained fluid sample comprises a concentration of the disaccharide of no larger than 3 weight percent, optionally no larger than 2 weight percent, optionally no larger than 1 weight percent or optionally no larger than 0.75 weight percent; and with one preferred range for the concentration for the concentration of the disaccharide being from 0.1 weight percent to 2 weight percent and a more preferred range being from 0.2 weight percent to 1 weight percent. [0255] 29., The method of any one of combinations 1-28, wherein the preparing the fluorescently-stained fluid sample comprises diluting a preliminary fluid sample with aqueous sample dilution liquid to prepare a diluted preliminary fluid sample, wherein the aqueous sample dilution liquid has a pH in the acidic pH range. [0256] 30. The method of combination 29, wherein the diluted preliminary fluid sample has a pH in the acidic pH range. [0257] 31. The method of either one of combination 29 or combination 30, wherein the fluorescent staining comprises adding the said fluorescent stain to the diluted preliminary fluid sample as the fluid sample composition. [0258] 32. The method of any one of combinations 29-31 wherein the diluting comprises adding to the preliminary fluid sample a quantity of the aqueous sample dilution liquid to prepare the diluted preliminary fluid sample at a dilution factor of at least 5 relative to the preliminary fluid sample. [0259] 33. The method of any one of combinations 1-32, wherein the at least one fluorescent stain comprises a fluorescent antibody stain for binding with an epitope of the viral capsid of the unassociated non-enveloped viral particle. [0260] 34. The method of combination 33, wherein the preparing the fluorescent-stained fluid sample comprises: [0261] mixing the fluorescent antibody stain with the biological material and [0262] after the mixing not separating an unbound portion of the fluorescent antibody stain from the biological material prior to the flow cytometry evaluation, wherein the fluorescently-stained fluid sample as subjected to flow cytometry comprises the unbound portion of the fluorescent stain. [0263] 35. The method of either one of combination 33 or combination 34, wherein: [0264] the fluorescently-stained fluid sample as fed to the flow cytometer includes the unbound portion of the fluorescent antibody stain at a concentration within a range having a lower limit and an upper limit; [0265] the lower limit is 0.25 microgram per milliliter, optionally 0.35 microgram per milliliter, as another option 0.5 microgram per milliliter, as another option 0.75 microgram per milliliter, as another option 1 microgram per milliliter and as yet another option 1.5 micrograms per milliliter; and [0266] the upper limit is 10 micrograms per milliliter, optionally 8 micrograms per milliliter, as another option 6 micrograms per milliliter, as another option 5 micrograms per milliliter, as another option 4 micrograms per milliliter and as yet another option 3 micrograms per milliliter. [0267] 36. The method of any one of combinations 33-35 wherein the fluorescently-stained fluid sample as fed to the flow cytometer includes a primary antibody for binding with the epitope, and wherein the fluorescent antibody stain comprises a secondary antibody with fluorophore to bind with the primary antibody to indirectly fluorescently stain the unassociated non-enveloped viral particles. [0268] 37. The method of any one of combinations 33-36, wherein the epitope is a conformational epitope of the viral capsid. [0269] 38. The method of any one of combinations 33-36, wherein the epitope is a sequential epitope of the viral capsid. [0270] 39. The method of any one of combinations 33-38, wherein the fluorescent antibody stain comprises a said fluorescent stain in the fluorescent staining and the stained fluid sample composition at the acidic pH includes the fluorescent antibody stain. [0271] 40. The method of combination 39, wherein: [0272] the at least one fluorescent stain comprises a fluorogenic dye for staining of nucleic acid content inside the viral capsid of the non-enveloped viral particles; and [0273] the fluorogenic dye for staining of nucleic acid comprises a said fluorescent stain in the fluorescent staining and the stained fluid sample composition at the acidic pH includes the fluorogenic dye. [0274] 41. The method of any one of combinations 1-40, wherein as fed to the flow cytometer the fluorescently-stained fluid sample has a p11 in the acidic pH range. [0275] 42. The method of combination 41, wherein as fed to the flow cytometer the fluorescently-stained fluid sample is the stained fluid sample composition from the fluorescent staining. [0276] 43. The method of any one of combinations 33-3920-25, wherein the at least one fluorescent stain comprises a fluorogenic dye for staining of nucleic acid content inside the viral capsid of the non-enveloped viral particles. [0277] 44. The method of combination 43, wherein the fluorogenic dye for staining of nucleic acid content comprises a said fluorescent stain in the fluorescent staining and the stained fluid sample composition includes the fluorogenic dye for staining of nucleic acid content. [0278] 45. The method of combination 44, wherein as fed to the flow cytometer the fluorescently-stained fluid sample has a pH in the acidic pH range. [0279] 46. The method of combination 45, wherein as fed to the flow cytometer the fluorescently-stained fluid sample is the stained fluid sample composition of the fluorescent staining. [0280] 47. The method of combination 43, wherein: [0281] the fluorescent staining, is a first fluorescent staining the biological material and the stained fluid sample composition is a first stained fluid sample composition, and the preparing the fluorescently-stained fluid sample comprises second fluorescent staining the biological material after the first fluorescent staining: and [0282] the second fluorescent staining comprises staining the biological material with the fluorescent antibody stain to prepare a second stained fluid sample composition including both the fluorescent antibody stain and the fluorogenic dye for staining of nucleic acid content, wherein the second stained fluid sample composition is at a second pH that is larger than the acidic pH. [0283] 48. The method of combination 47, wherein t e second pH is at least 0.5 pH unit larger than the acidic pH. [0284] 49. The method of either one of combination 47 or combination 48, wherein the second pH is pH 5.5 or larger. [0285] 50. The method of combination 49, wherein the second pH is pH 6.0 or larger. [0286] 51. The method of combination 49, wherein the second pH is larger than pH 6.5. [0287] 52. The method of any one of combinations 47-51 wherein the preparing the fluorescently-stained fluid sample comprises after the first fluorescent staining and prior to the second fluorescent staining; [0288] adjusting pH, comprising raising the pH of the first stained fluid sample composition to prepare a pH adjusted stained fluid sample composition at a higher pH than the acidic pH, wherein the higher pH is at least 0.5 pH units larger than the acidic pH. [0289] 53. The method of combination 52, wherein the second fluorescent staining comprises adding the fluorescent antibody stain to the pH adjusted fluid sample. [0290] 54. The method of any one of combinations 1-53, wherein the aqueous liquid medium comprises aqueous buffer solution, optionally including buffering agent based on a buffering system selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES), citric acid, acetic acid, cacodylic acid, phosphoric acid, a salt of any of the foregoing and combinations thereof. [0291] 55. The method of any one of combinations 1-54, wherein the flow cytometry evaluation comprises flowing the fluorescently-stained fluid sample through the investigation zone at a flow rate in a range of from 300 nanoliters per minute to 6000 nanoliters per minute while subjecting the fluorescently-stained fluid sample to the stimulation radiation, and preferably the flow rate is in a range of float 600 nanoliters per minute to 3000 nanoliters per minute. [0292] 56. The method of any one of combinations 1-55, wherein the flow cytometry evaluation comprises evaluating only fluorescent emission response from the investigation zone to identify occurrences of the unassociated labeled particles, and not evaluating light scatter from the investigation zone. [0293] 57, The method of any one of combinations 1-56, wherein the at least one fluorescent stain includes a plurality of fluorescent stains and the flow cytometry evaluation comprises detecting for a separate fluorescent emission response from each of the said plurality of fluorescent stains. [0294] 58. The method of combination 57, wherein the flow cytometry evaluation comprises identifying coincidences of at least two different fluorescent emission responses from the plurality of fluorescent stains indicative of passage through the investigation zone of a said unassociated labeled particle including the at least two different fluorescent stains. [0295] 59. The method of combination 58, wherein the flow cytometry evaluation comprises counting as occurrences of said unassociated non-enveloped viral particles identified coincidences of the at least two different fluorescent emission responses. [0296] 60. The method of any one of combinations 1-59. wherein the unassociated non-enveloped viral particles are virions. [0297] 61. The method of any one of combinations 1-59, wherein the unassociated non-enveloped viral particles are virus-like particles. [0298] 62. The method of any one of combinations 1-61, wherein the unassociated non-enveloped viral particles are genetically modified. [0299] 63. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Adenoviridae, and optionally of an adenovirus. [0300] 64. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Parvoviridae, and optionally of an adeno adeno-associated virus, and as another option of a Minute virus of mice. [0301] 65. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Parvoviridae, and optionally of an enterovirus. [0302] 66. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Reoviridae, and optionally of a rotavirus. [0303] 67, The method of any one of combinations 1-62, wherein the viral capsid is of virus family Papillomaviridae. [0304] 68. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Polyomaviridae. [0305] 69. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Bidnaviridae. [0306] 70. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Circoviridae. [0307] 71. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Astroviridae. [0308] 72. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Calciriviridae. [0309] 73. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Hepeviridae. [0310] 74. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Potyviridae. [0311] 75. The method of any one of combinations 1-62, wherein the viral capsid is of virus family Papovaviridae. [0312] 76. The method of any one of combinations 1-62, wherein the viral capsid is of a virus family selected from the group consisting of Myoviridae; Podoviridae; Siphoviridae; Rudiviridae; Clavaviridae; Corticoviridae; Marseilleviridae; hycodnaviridae; Sphaerolipoviridae; Tectiviridae; Turriviridae; Lavidaviridue; Spiraviridae; Anelloviridae; Geminiviridae; Genomoviridae; Inoviridae; Microviridae; Nanoviridae; Spiraviridae; Amalgaviridae; Birnaviridae; Chrysovirus; Endornaviridae; Hypoviridae; Megabirnaviridae; Partitiviridae; Totiviridae; Quadriviridae; Dicistroviridae; flaviridae; Marnaviridae; Secoviridae; Alphaflexiviridae; Betaflexiviridae; Gammaflexiviridae; Tymoviridae; Barnaviridae; Benyviridae; Bromoviridae; Carmotetraviridae; Closteroviridae; Leviviridae; Luteoviridae; Nodaviridae; Permutotetraviridae; Tombusviridae; Virgaviridae and Aspiviridae. [0313] 77. The method of any one of combinations 1-62, wherein the viral capsid is of a bacteriophage. [0314] 78. The method of any one of combinations 1-77, wherein the unassociated non-enveloped viral particles have a particle size of 2 microns or smaller, optionally 1 micron or smaller, as another option 600 nanometers or smaller, as another option 300 nanometers or smaller, as another option 200 nanometers or smaller and as yet a further option 100 nanometers or smaller. [0315] 79. The method of any one of combinations 1-78, wherein the unassociated non-enveloped viral particles have a particle size of at least 10 nanometers, optionally at least 20 nanometers, as another option at least 30 nanometers, and as yet another option at least 40 nanometers. [0316] 80. The method of any one of combinations 1-79, wherein the unassociated labeled particles have a particle size of 2.1 microns or smaller, optionally 1.1 microns or smaller, as another option 625 nanometers or smaller, as another option 425 nanometers or smaller, as another option 325 nanometers or smaller, as another option 225 nanometers or smaller and as yet another option 125 nanometers or smaller. [0317] 81. A kit for preparing a fluorescently-stained fluid sample for flow cytometry evaluation of biological material for quantification of unassociated non-enveloped viral particles of virus size, having a non-enveloped viral capsid, the kit comprising: [0318] a plurality of sealed containers; [0319] a fluorescent stain composition for fluorescent staining the unassociated non-enveloped viral particles to prepare unassociated labeled particles of virus size, each said unassociated labeled particle of virus size comprising a said unassociated non-enveloped viral particle stained with a fluorescent stain from the fluorescent stain composition, the fluorescent stain composition comprising at least one fluorescent stain and being contained in a first said sealed container; [0320] an aqueous dilution liquid for preparing a pH-adjusted fluid sample for fluorescent staining biological material at in an acidic pH of up to pH 6.5 during preparation of the unassociated labeled particles of virus size dispersed in an aqueous medium for flow cytometry evaluation of a fluorescently-stained fluid sample with the biological material, the aqueous dilution liquid having an acidic pH of no larger than pH 6.5 and being contained in a second said sealed container. [0321] 82. The kit of combination 81, wherein the first said sealed container and the second said sealed container are packaged in a common packaging enclosure. [0322] 83. The kit of combination $2, wherein the common packaging enclosure is a hermetically sealed enclosure. [0323] 84. The kit of any one of combinations 81-83, wherein the at least one fluorescent stain of the fluorescent stain composition comprises a fluorogenic dye for nonspecific staining nucleic acid content inside the viral capsid of the non-enveloped viral particles. [0324] 85. The kit of combination 84, wherein the fluorogenic dye is a first fluorogenic dye with a first fluorescent emission signature and the at least one fluorescent stain of the fluorescent stain composition comprises a second fluorogenic dye for nonspecific staining of protein content of the viral capsid, the second fluorogenic dye having a second fluorescent emission signature different than the first fluorescent emission signature. [0325] 86. The kit of combination 85, wherein each said fluorogenic dye in the fluorescent stain composition contains at least one aromatic group susceptible to pi stacking interactions in aqueous liquids. [0326] 87. The kit of any one of combinations 84-86, comprising a reconstitution liquid medium, the reconstitution liquid medium being a solvent for each said fluorogenic dye to provide each said fluorogenic dye in solution in the reconstitution liquid medium prior to mixing with the aqueous dilution liquid to prepare the pH-adjusted fluid sample, the reconstitution liquid medium comprising an organic liquid component; [0327] and optionally, the reconstitution liquid medium is contained in the common packaging enclosure of either one of combination 82 or combination 83. [0328] 88. The kit of combination 874, wherein the organic liquid component is dimethyl sulfoxide (DMSO), and the reconstitution liquid medium comprises at least 34 percent by moles of DMSO. [0329] 8976. The kit of combination 87, wherein the organic liquid component is acetonitrile, and the reconstitution liquid medium comprises at least 34 percent by moles of the acetonitrile. [0330] 90. The kit of any one of combinations 87-89, wherein the reconstitution liquid medium is contained in a said sealed container, other than the first sealed container or the second sealed container, and optionally the other sealed container with the reconstitution liquid medium is contained in the common packaging enclosure of either one of combination 82 or combination [0331] 91. The kit of any one of combinations 87-90, wherein the fluorescent stain composition comprises a dry powder dye composition including each said fluorogenic dye. [0332] 92. The kit of any one of combinations 87-89, wherein the fluorescent stain composition comprises the reconstitution liquid medium, [0333] 93. The kit of combination 92, wherein the fluorescent stain composition comprises each said fluorogenic dye dissolved in the reconstitution liquid medium. [0334] 94. The kit of any one of combinations 84-86, wherein the fluorescent stain composition comprises a dry powder stain composition including each said fluorogenic dye. [0335] 95. The kit of any one of combinations 84-94, wherein: [0336] the fluorescent stain composition is a first fluorescent stain composition and the kit comprises a second fluorescent stain composition comprising a fluorescent antibody stain; and [0337] the fluorescent antibody stain is either: [0338] specific for binding with an epitope of die viral capsid for direct staining of the unassociated non-enveloped viral particles: or [0339] specific for binding with a primary antibody that is specific for binding with all epitope of the viral capsid for indirect staining of the unassociated non-enveloped viral particles. [0340] 96. The kit of combination 95, wherein the kit comprises another said sealed container, other than the first sealed container or the second sealed container, containing the second fluorescent stain composition, and optionally the other sealed container with the second fluorescent stain composition is contained in the common packaging enclosure of either one of combination 69 or combination 70. [0341] 97. The kit of either one of combination 85 or combination 86, wherein the fluorescent stain composition comprises a liquid dye concentrate comprising the first fluorogenic dye and the second fluorogenic dye in a stain liquid medium, and wherein the liquid dye concentrate comprises at least one member selected from the group consisting of: [0342] (i) the stain liquid medium comprising a liquid mixture including water and liquid phase organic material, and preferably the stain liquid medium is aqueous, and more preferably the stain liquid medium comprises more than 50 percent by moles of water: [0343] (ii) disaccharide dissolved in the stain liquid medium: and [0344] (iii) a combination including both (i) and (ii). [0345] 98. The kit of combination 97, wherein the stain liquid medium comprises the liquid mixture including water and liquid organic phase material. [0346] 99. The kit of combination 98, wherein the stain liquid medium is an aqueous liquid medium comprising a major proportion on a molar basis of water and a minor proportion on a molar basis of the liquid phase organic material in the liquid mixture. [0347] 100. The kit of either one of combination 98 or combination 99, wherein the liquid mixture is a single liquid phase comprised of mutually soluble liquid components. [0348] 101. The kit of any one of combinations 98-100, wherein the liquid phase organic material comprises dimethyl sulfoxide (DMSO). [0349] 102. The kit of combination 101, wherein the liquid phase organic material consists essentially of only DMSO, [0350] 103. The kit of any one of combinations 98-100, wherein the liquid phase organic material comprises acetonitrile. [0351] 104. The kit of any one of combinations 98-103, wherein the stain liquid medium comprises at least 10 weight percent of the liquid phase organic material, optionally at least 15 weight percent of the liquid phase organic material or optionally at least 20 weight percent of the liquid phase organic material. [0352] 105. The kit of any one of combinations 98-104, wherein the stain liquid medium comprises no greater than 50 weight percent of the liquid phase organic material, optionally no greater than 35 weight percent of the liquid phase organic material or optionally no greater than 30 weight percent of the liquid phase organic material. [0353] 106. The kit of any one of combinations 98-105, wherein the stain liquid medium comprises at least 50 weight percent water, optionally at least 60 weight percent water, optionally at least 65 weight percent water or optionally at least 70 weight percent water. [0354] 107. The kit of any one of combinations 98-106, wherein the stain liquid medium comprises no greater than 90 weight percent water. [0355] 108. The kit of any one of combinations 97-107, wherein the liquid dye concentrate comprises the disaccharide dissolved in the stain liquid medium, [0356] 109. The kit of combinations 108, wherein a concentration of the disaccharide in the liquid dye concentrate is at least 1 weight percent, optionally at least 2 weight percent, further optionally at least 3 weight percent, further optionally a least 4 weight percent, further optionally at least 6 weight percent, further optionally at least 9 weight percent or further optionally at least 12 weight percent. [0357] 110. The kit of either one of combination 108 Or combination 109, wherein the concentration of the disaccharide in the liquid dye concentrate is no greater than 45 weight percent, optionally no greater than 36 weight percent, further optionally no greater than 30 weight percent, further optionally no greater than 24 weight percent, further optionally no greater than 15 weight percent, further optionally no greater than 10 weight percent or further optionally no greater than 8 weight percent. [0358] 111. The kit of any one of combinations 81-83, wherein the at least one fluorescent stain comprises a fluorescent antibody stain, and the fluorescent antibody stain is either: [0359] specific for binding with an epitope of the viral capsid for direct staining of the unassociated non-enveloped viral particles; or [0360] specific for binding with a primary antibody that is specific for binding with an epitope of the viral capsid for indirect staining of the unassociated non-enveloped viral particles. [0361] 112. The kit of any one of combinations 95, 96 and 111, wherein the fluorescent antibody stain is specific for binding with an epitope of the viral capsid, and optionally the viral capsid is of a virus family of any one of combinations 63-76, and as another option the viral capsid is of a bacteriophage. [0362] 113. The kit of combination 112, wherein the fluorescent antibody stain is specific for binding with a conformational epitope of the viral capsid. [0363] 114. The kit of combination 113, wherein the fluorescent antibody stain is specific for binding with a sequential epitope of the viral capsid. [0364] 115. The kit of any one of combinations 95, 96 and 111, wherein the fluorescent antibody stain is specific for binding with a primary antibody that is specific for binding with an epitope of the viral capsid for indirect staining of the unassociated non-enveloped viral particles. [0365] 116. The kit of combination 115, comprising the primary antibody, optionally contained in the common packaging enclosure of either one of combination 81 or combination 82. [0366] 117. The kit of combination 116, wherein the primary antibody is contained in a said sealed container separate from the fluorescent antibody stain. [0367] 118. The kit of combination 116, wherein the primary antibody is contained in a said sealed container together with the fluorescent antibody stain. [0368] 119, The kit of any one of combinations 81-118, wherein the acidic pH of the aqueous dilution liquid is not larger than pH 6.0. [0369] 120. The kit of any one of combinations 81-119, wherein the acidic pH of the aqueous dilution liquid is not larger than 5.8. [0370] 121. The kit of any one of combinations 81-120, wherein the acidic pH of the aqueous dilution liquid is not larger than 5.6. [0371] 122. The kit of any one of combinations 81-121, wherein the acidic pH of the aqueous dilution liquid is not smaller than pH 3.0. [0372] 123. The kit of any one of combinations 81-122, wherein the acidic pH of the aqueous dilution liquid is not smaller than pH 4.0. [0373] 124. The kit of any one of combinations 81-123, wherein the acidic of the aqueous dilution liquid is not smaller than pH 4.5. [0374] 125. The kit of any one of combinations 81-124, wherein the acidic pH of the aqueous dilution liquid is not smaller than pH 5.0. [0375] 126. The kit of any one of combinations 81-125, wherein the aqueous dilution liquid comprises an aqueous buffer solution, optionally including a but agent based on a buffering system selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES), citric acid, acetic acid, cacodylic acid, phosphoric acid, a salt of any of the foregoing and combinations thereof. [0376] 127. The kit of any one of combinations 81-126, comprising a quantity of the fluorescent stain composition and a quantity of the aqueous dilution liquid for preparing at least 10 of the fluorescently-stained fluid samples for the flow cytometry evaluation. [0377] 128. The kit of combination 127, comprising a plurality of the first said sealed containers each comprising a portion of the quantity of the fluorescent stain composition. [0378] 129. The kit of any one of combinations 81-128, wherein the kit is for preparing a fluorescently-stained fluid sample comprising unassociated labeled particles of virus size dispersed in an aqueous liquid medium according the method of any one of combinations 1-67. [0379] 130. The kit of any one of combinations 81-129, wherein the aqueous dilution liquid is an aqueous sample dilution liquid to dilute a preliminary fluid sample including the biological material prior to a fluorescent staining step. [0380] 131. The kit of combination 130, comprising another said sealed container, other than the first sealed container and the second sealed container, containing an aqueous stain dilution liquid to prepare an aqueous diluted stain formulation to fluorescently stain the biological material, and optionally the other sealed container with the aqueous stain dilution liquid is packaged in the common packaging enclosure of either one of combination 82 or combination 83. [0381] 132. The kit of combination 131, wherein the aqueous sample dilution liquid and the aqueous stain dilution liquid have different compositions. [0382] 133. The kit of either one of combination 131 or combination 105, Wherein the aqueous stain dilution liquid has a higher pH than the acid pH of the aqueous sample dilution liquid. [0383] 134. The kit of any one of combinations 131-133, wherein the aqueous stain dilation liquid has a pH of larger than pH 6.5, optionally at least pH 7.0, and preferably no larger than pH 8.0. [0384] 135. The kit of any one of combinations 131-133, wherein the aqueous stain dilution liquid has a pH in an acidic pH range of from pH 3.0 to 6.5. [0385] 136. The kit of any one of combinations 130-135, wherein; [0386] the aqueous sample dilution liquid is a first aqueous sample dilution liquid and the kit comprises a second aqueous sample dilution liquid to dilute a preliminary stained fluid sample including the biological material following a fluorescent staining step; [0387] the second aqueous sample dilution liquid has a pH that is higher than the acidic pH of the first aqueous sample dilution liquid; and [0388] the second aqueous sample dilution liquid is contained in another sealed container, other than the first sealed container and the second sealed container, and optionally the other sealed container with the second aqueous sample dilution liquid is packaged in the common packaging enclosure of either one of combination 82 or combination 83. [0389] 137. The kit of combination 136, wherein the second aqueous sample dilution liquid has a composition that is different than, and is contained in a said fluid container that is different than any said fluid container that contains, any aqueous stain dilution liquid in the kit. [0390] 138. The kit of either on of combination 136 or 137, wherein the second aqueous sample dilution liquid has a pH that is at least 0.5 pH units larger than the acidic pH of the first aqueous sample dilution liquid. [0391] 139. The kit of any one of combinations 136-138, wherein the second aqueous sample dilution liquid has a pH of larger than pH 6.5, optionally at least pH 7.0, and preferably no larger than pH 8.0. [0392] 140. The kit of any one of combinations 81-139, comprising a disaccharide. [0393] 141. The kit of combination 140, wherein at least a portion of the dissolved disaccharide is in the aqueous dilution liquid. [0394] 142. The kit of either one of combination 140 or combination 141, wherein at least a portion of the dissolved disaccharide is in an aqueous sample dilution liquid. [0395] 143. The kit of any one of combinations 140-142, wherein at least a portion of the dissolved disaccharide is in an aqueous stain dilution liquid. [0396] 144. Use of the kit of any one of combinations 81-143 for preparation and flow cytometry evaluation of a fluorescently-stained fluid sample comprising unassociated labeled particles of virus size dispersed in an aqueous liquid medium. [0397] 145. The use of combination 144, wherein the flow cytometry evaluation is according to the method of any one of combinations 1-80. [0398] 146. The method of any one of combinations 1-80, wherein the stained fluid sample comprises dissolved disaccharide. [0399] 147. The method of combination 146, wherein at least a portion of the dissolved disaccharide is provided in an aqueous dilution liquid. [0400] 148. The method of either one of combination 146 or combination 147, wherein at least a portion of the dissolved disaccharide is provided in an aqueous sample dilution liquid. [0401] 149. The method of any one of combinations 146-149, wherein at least a pardon of the dissolved disaccharide is in an aqueous stain dilution liquid. [0402] 150. The method or kit of any of the previous combinations that comprise a disaccharide, wherein the said disaccharide comprises one or more than one member selected from the group consisting of trehalose, sucrose, lactose, lactulose, melibiose, melibitdose, cellobiose, nigerose, isomaltose, isomaludose, maltulose, rutinose, β-laminaribiose, and maltose. [0403] 151. The method or kit of combination 150, wherein the said disaccharide comprises trehalose. [0404] 152. The method or kit of combination 151, wherein the said disaccharide consists essentially of only trehalose.

[0405] The foregoing description of the present invention and various aspects thereof has been presented for purposes of illustration and description. Furthermore, the description is not intended to limit the invention to the form disclosed herein. Consequently, variations and modifications commensurate with the above teachings, and skill and knowledge of the relevant art, are within the scope of the present invention. The embodiments described hereinabove are further intended to explain known modes of practicing the invention and to enable others skilled in the art to utilize the invention in such or other embodiments and with various modifications required by the particular application(s) or use(s) of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.

[0406] The description of a feature or features in a particular combination do not exclude the inclusion of an additional feature or features in a variation of the particular combination. Processing steps and sequencing are for illustration only, and such illustrations do not exclude inclusion of other steps or other sequencing of steps to an extent not necessarily incompatible. Additional steps may be included between any illustrate processing steps or before or after any illustrated processing step to an extent not necessarily incompatible.

[0407] The terms “comprising”, “containing”, “including” and “having”, and grammatical variations of those terms, are intended to be inclusive and nonlimiting in that the use of such terms indicates the presence of a stated condition or feature, but not to the exclusion of the presence also of any other condition or feature. The use of the terms “comprising”, “containing”. “including” and “having”, and grammatical variations of those terms in referring to the presence of one or more components, subcomponents or materials, also include and is intended to disclose the more specific embodiments in which the term “comprising”, “containing”, “including” or “having”, (or the variation of such term) as the case may be, is replaced by any of the narrower terms “consisting essentially or” or “consisting of” or “consisting of only” (or any appropriate grammatical variation of such narrower terms). For example, a statement that something “comprises” a stated element or elements is also intended to include and disclose the more specific narrower embodiments of the thing “consisting essentially of” the stated element or elements, and the thing “consisting of” the stated element or elements. Examples of various features have been provided for purposes of illustration, and the terms “example”, “for example” and the like indicate illustrative examples that are not limiting and are not to be construed or interpreted as limiting a feature or features to any particular example. The term “at least” followed by a number (e.g., “at least one”) means that number or more than that number. The term at “at least a portion” means all or a portion that is less than all. The term “at least a part” means all or a part that is less than all. The term “at least a majority” means all or a majority part that is less than all. When reference is made to a “liquid medium” in which fluorogenic dyes are dissolved, such as in the case of the liquid medium of the liquid dye concentrate, it is meant the liquid medium of a liquid composition in which the fluorogenic dyes are dispersed in solution, and not including the fluorogenic dyes themselves. Such a liquid medium as used herein also does not include any suspended solids that may be carried by such a liquid composition. Such a liquid medium may include one or more than one normally-liquid components (e.g., DMSO and/or water and/or one or more other organic solvent components) and a liquid composition including the liquid medium may include one or more normally-solid materials (e.g., dissolved salts and other additives of buffer solutions) that may be in solution in the liquid medium of the liquid composition.