WO1996037177A2

WO1996037177A2 - Apparatus and method for detecting a contaminant in a fluid

Info

Publication number: WO1996037177A2
Application number: PCT/GB1996/001217
Authority: WO
Inventors: Janet Rosemary Rider; Robert Blanchard Chaney
Original assignee: The National Blood Authority
Priority date: 1995-05-22
Filing date: 1996-05-21
Publication date: 1996-11-28
Also published as: EP0843544A2; WO1996037177A3; GB9510262D0; AU5773196A

Abstract

The invention provides an apparatus (1; 20) for the detection of a contaminant in a fluid, and also a method of detecting a contaminant in a stored fluid using the apparatus. The apparatus comprises a primary chamber (2) for storing the fluid, and a secondary detection chamber (4; 21) which includes a test reagent or combination of reagents suitable for the detection of the contaminant in the fluid. A flow path (6; 23) from the primary chamber (2) to the secondary chamber (4; 21) is provided which is normally closed, but which is openable to enable a sample volume of fluid to pass from the primary chamber (2) to the secondary chamber (4; 21) for detection of the contaminant in the fluid.

Description

APPARATUS AND METHOD FOR DETECTING A CONTAMINANT IN A FLUID

This invention relates to an apparatus or unit for the storage of a fluid and the integrated detection of a contaminant in the fluid. More particularly, but not exclusively, the present invention relates to a closed apparatus for the integrated detection of bacterial and/or other contaminants in a stored liquid, such as a human blood product. A particular blood product which may be advantageously used with the apparatus of the invention is a blood platelet concentrate, which is a concentrated suspension (normally in blood plasma) of blood platelets.

Until recently, bacteraemia and/or septicaemia, associated with platelet transfusion, has largely gone unrecognised and dramatically under-reported. This is most probably because febrile transfusion reactions occur so frequently (up to 30%) in patients receiving platelets and because the ubiquitous bacteria involved are not always considered highly pathogenic. Nevertheless, it is very likely that transfusion-associated bacteraemia is now the most common transfusion-related infection confronting the transfusion medicine community. Because episodes of transfusion-associated infection may lead to death or other serious sequelae, there is genuine cause for concern.

Therapeutic platelet concentrates are currently derived by single-donor apheresis or pooled random-donor techniques. The incidence of bacterial infection caused by pooled random donor units is approximately twelve times that for apheresis products. This observed difference is due to a recipient's greater donor exposure from pooled products and the fact that the average storage time of apheresis platelets is often less than that for the random-donor equivalent.

SUBSTITUTESHEET(RULE25) A diverse range of bacteria, including both gram positive and gram negative species, have been isolated from platelet concentrates and are implicated in transfusion-transmitted disease. Although the highest percentage (25%) of clinical episodes involve coagulase-negative staphylococci, often Staphylococcus epider idis , many other organisms (including Staphylococcus aureus and various Bacillus, Enterobacter, Pseudomonas , Salmonella, Serratia and Streptococcus species) can also be responsible. Potential sources of contamination include the collection procedure, donor bacteraemia, and contaminated blood containers or processing equipment. Regardless of the source, most contaminants are thought to be initially below harmful levels, <10 CFU/ml. Prolonged storage, however, can permit growth to clinically significant populations.

Donor leucocyte cells are also responsible for adverse transfusion events. Such leucocyte cells are suspected of causing febrile transfusion reactions, refractoriness to transfusion, graft-versus-host disease, immunomodulation and transmission of infectious agents. In view of this, it is strongly recommended that leucodepleted products (<5 x 10⁶ leucocytes/U) are used in certain patient groups.

Production differences mean that approximately 70% of apheresis platelet concentrates are inherently leucodepleted, whereas only about 40% of pooled random donor concentrates are leucodepleted. Filtration can be used to further leucodeplete concentrates produced by either method. Irrespective of how they have been produced, it is desirable that the leucocyte load of all concentrates designated leucodepleted be determined before they are issued. The current demand for leucodepleted platelet concentrates is approximately 35% of total output. According to a first aspect of the present invention, there is provided an apparatus for the detection of a contaminant in a fluid, comprising a primary chamber for storing said fluid and a secondary detection chamber which includes a test reagent or combination of reagents suitable for the detection of said contaminant in the fluid, a flow path from the primary chamber to the secondary chamber being provided which is normally closed, but which is openable to enable a sample volume of fluid to pass from the primary chamber to the secondary chamber for detection of the contaminant in the fluid.

The apparatus of the first aspect of the present invention is suited to the detection of an infectious microorganism contaminant in a liquid, particularly bacterial contamination of blood products. One such blood product is human blood platelet concentrate, which is currently impossible to sterilize without having a detrimental effect on cell function, and which may also still contain unacceptable levels of antimicrobial drugs and/or bacterially-produced toxins. The invention is also relevant to the detection of other microorganism contaminants, such as viral and fungal contaminants in a liquid such as a blood product.

Furthermore, the apparatus of the first aspect of the present invention is suitable for detecting the presence of eukaryotic cells in a fluid. In particular, the apparatus is relevant to the detection of leucocyte cells in blood products, such as blood platelet concentrate.

The combined storage/detection apparatus of the invention may be a conventional storage bag or container for a liquid product such as a blood product, which has been adapted to incorporate a detection chamber.

SUBSTITUTE SHEET (RULE 25) The storage and detection chambers of the apparatus of the invention will normally be closed by a flexible or rigid wall, and will usually be fabricated from one or more synthetic plastic materials in a known manner. Normally, the primary chamber will include other access or delivery ports so that it can be connected to the necessary equipment for filling with the liquid product, and subsequently for connection to the delivery apparatus for delivering the product in the desired manner, for example, the delivery of a blood product to a patient.

Typically, the volume of the primary chamber will be up to about 1000 ml, and may be as low as 10 ml. The volume of the detection chamber is rather smaller, generally of the order of up to 5 ml, and normally up to about 1 ml.

It is most preferred that the primary and secondary chambers are integral, and linked by a flow path in the form of a passage which is normally closed, but which is openable to enable a quantity of fluid from the primary chamber to be delivered to the secondary chamber. By "integral" in this context, we do not mean that the primary and secondary chambers must be integrally moulded as one unit, although this could be the case, but that the primary and secondary chambers are part of a single item of equipment.

Alternatively, the apparatus of the invention may comprise a kit in which the primary chamber or reservoir includes an opening, which is normally closed, which is adapted to be connected to a suitable detection chamber which then remains united to the primary chamber throughout the test. It is important, in this respect, that the detection chamber does not become detached from the primary chamber, as one of the advantages of the apparatus of the invention is that the stored product remains united with its associated

SUBSTITUTE SHEET (RULE 25) test chamber throughout the testing procedure until a result has been obtained. It is likely that the detection chamber will remain with the storage chamber after testing until the product is used, to provide an indication to the end user that the product has been tested.

The flow path is preferably a conduit or passageway allowing communication between the primary and secondary chambers^'. The flow path should of course be sealed with respect to the outside environment to prevent contamination. The closure in the flow path may be capable of being fractured or broken in order to open a direct flow path between the primary and secondary chamber. Alternatively, the opening of the closure may be reversible, so as to close the flow path between the primary and secondary chambers after a required volume of fluid has been allowed to flow between the chambers.

It is preferred that flow between the primary and secondary chambers is unidirectional, to prevent the return of sample fluid from the detection chamber to the primary chamber, which might contaminate the product in the primary chamber with the test reagent(s) present in the detection chamber. A one-way valve may therefore be included in the passageway. It is preferred that the one way valve and closure may be combined in a one-way cannula.

The apparatus of the present invention may have a plurality of secondary detection chambers, each communicating with the primary chamber via a respective flow path. In a preferred embodiment, the apparatus is adapted to detect a plurality of contaminants in the fluid stored in the primary chamber, and comprises a corresponding plurality of secondary detection chambers, each including a test reagent or combination of test reagents suitable for the detection of a

SUBSTTTUTESHEET(RULE25) respective contaminant.

The test reagent or test reagents in the detection chamber should be suitable to provide an indication of the presence of the contaminant in the fluid. Where the contaminant is bacterial in nature, there are various detection methods which would be suitable. One suitable method is staining, in which the presence of bacteria in the fluid in the secondary chamber are identified by a stain specific to bacteria. A variety of reagents which may be used in such a method are available. An alternative method is to assay the fluid in the detection chamber for the presence of nucleic acid sequences specific to the microorganisms which are to be detected. The fluid may be assayed for nucleic acid sequences which are specifically found in all microorganisms but not found in humans. Where the contaminant to be detected is leucocyte cells, the fluid may be assayed for nucleic acid sequences which are specific to leucocyte cells. Each test method would require calibration to determine the response considered to be clinically unacceptable. A response at, or exceeding that level would constitute a positive result under the test and cause the unit of fluid to be rejected. Where the apparatus is used to detect the presence of leucocyte cells in stored blood products such as platelet concentrates, the response to the test may be used to determine whether the product can be considered as leucodepleted or not. Appropriate positive and negative controls may be included within each apparatus or batch of apparatuses.

Where the presence of bacteria in the fluid is to be detected by a staining process, the test reagents will include the necessary staining reagents, which will be well known in the art. It may also be necessary to incorporate in the detection chamber a

SUBSTITUTE SHEET (RULE 25) suitable lysing agent to lyse the blood cells in the fluid under test in order to give a clear solution in which the stained bacteria may be more easily visualised. Where the fluid is to be subjected to an assay for the detection of specific nucleic acid sequences, it will first be necessary to lyse the cells in which the specific nucleic acid sequences are found, whether they be prokaryotic or eukaryotic. Preferably, this is done within the secondary chamber of the apparatus.

Where the contaminant to be detected is bacterial, it is envisaged that various cell lysis techniques can be used, provided that they completely lyse a broad range of bacterial species, both gram positive and gram negative. These include one or more of sonication, extreme heating, freeze-thawing, lysozyme cell wall degradation and detergents. Lysis of bacterial cells may include submerging the secondary chamber in liquid nitrogen. If the detection of the bacterial contaminant is based on nucleic acid hybridisation, the lysis techniques must ensure that the target nucleic acid sequences are functionally intact after cell lysis and must also be compatible with the reagent(s) used in the detection method. Where the contaminant to be detected is leucocyte cells, detergents may be used for cell lysis. This has the advantage that the cytoplasmic contents of the leucocyte cells can be released without rupture of the nuclear membrane. Various methods for the detection of specific nucleic acid sequences which can be conducted in homogenous solution are available. One suitable method is that described in EP-A-0382433, the content of which is incorporated herein. In this method, fluorescence polarisation is used to detect hybridisation of a fluorescent nucleic acid probe or to detect fluorescent

SUBSTITUTE SHEET (RULE 25) pri er extension products. An alternative method is that described in the article entitled "A New Homogeneous Identification Method for DNA" by John Coates et al, J. Chem. Soc, Chem. Commun, 1994, pages 2311-2312, the content of which is incorporated herein. In this method, the nucleic acid probe is linked to a chelator to which is strongly co-ordinated a europium ion. When the probe binds to the target sequence in the presence of a secondary ligand, or sensitiser, the europium ion will luminesce. It will be appreciated that any method for detecting specific nucleic acid sequences can be used, including those based on nucleic acid hybridisation and electromagnetic signal emission. Assay methods which rely on the hybridisation of a nucleic acid probe with the target nucleic acid sequence may be increased in their sensitivity by identifying a suitable target nucleic acid sequence present in the cell type to be detected in sufficient abundance that additional amplification methods to increase the amount of target nucleic acid sequence are not necessary. The target nucleic acid may be in the genome in two or more copies, preferably ten or more copies.

The target nucleic acid sequence should be one which is specific to the microorganism(s) or eukaryotic cell(s) under test. When it is intended to screen a fluid for the presence of general bacterial contamination, a target nucleic acid sequence should be chosen which is specific to all, or the majority of, bacteria which may be expected to be present as contaminant, and which does not appear in human cells. Currently preferred target nucleic acid sequences useful in the detection of general bacterial contamination are located in bacterial ribosomal RNA (rRNA) . Bacterial rRNA is relatively abundant in all types of bacteria, constituting approximately 80% of

SUBSTITUTE SHEET (RULE 25) #RNA in bacterial cells, and has primary and secondary structures which are relatively conserved. The most preferred target nucleic acid sequences are located in homologous regions of bacterial rRNA which are not found in human rRNA. The nucleic acid probes for hybridising to these target nucleic acid sequences are preferably complementary DNA probes. Preferred DNA probes have the following sequences:

5'-GCTGCCTCCCGTAGGAGT-3' (targeting 16s RNA) [SEQ ID NO: 1]

5' -ATTACCGCGGCTGCTGGCA-3' (targeting 16s RNA)

[SEQ ID NO: 2]

5'-CCGACAAGGAATTTCGCTAC-3' (targeting 23s RNA)

[SEQ ID NO: 3] 5'-CGGTACTGGTTCACTATC-3' (targeting 23s RNA)

[SEQ ID NO: 4]

5'-CATCCCCACCTTCCTCC-3' (targeting 16s RNA)

[SEQ ID NO: 5]

It will be appreciated that nucleic acid probes having slightly different sequences to those given above will still be sufficiently complementary to the respective target sequences to hybridise with those target sequences.

When it is intended to screen a fluid for the presence of leucocyte cell contamination, a target nucleic acid sequence should be chosen which is present in adequate amounts in leucocyte cells but not in microorganisms or in the fluid stored in the primary chamber. The target nucleic acid sequence is preferably present in sufficient abundance that additional amplification methods to increase the amount of target nucleic acid sequence are not necessary. Such target nucleic acid sequences may be mRNA which is relatively abundant in leucocytes, for example the mRNA derived from the H2A.Z, H2A.X or H3.3 histone genes or the CD53 gene.

SUBSTITUTE SHEET (RULE 25) in some circumstances, where the microorganism or leucocyte cells are to be detected by an assay method in which a target nucleic acid sequence is identified, it may be necessary to use the polymerase chain reaction (PCR) to amplify the relevant target nucleic acid sequence present in the sample. However, it is preferred that such amplification is not carried out, as this would introduce an extra element of complexity to the method. According to a second aspect of the present invention, there is provided a method for the detection of a contaminant in a fluid stored in the primary chamber of an apparatus in accordance with the first aspect of the invention, said method comprising the steps of opening the flow path from the primary chamber to the or each secondary chamber and permitting a sample volume of fluid to pass from the primary chamber to the or each secondary chamber; permitting the contaminant to be assayed in the or each detection chamber; and observing the results of the or each assay. The sample volume is generally up to 5 ml, and typically up to 1 ml. Generally, the smaller the volume of sample, without compromising the validity of the assay, the more preferred. In a preferred embodiment, the method is for detecting a plurality of contaminants in the fluid, and utilises an apparatus in accordance with the first aspect of the invention which apparatus comprises a corresponding plurality of secondary chambers. The method of the second aspect of the invention is particularly suited to the analysis of a blood product for bacterial and/or leucocyte cell contamination. It is envisaged that the or each test for such contamination would be done immediately prior to issue of the blood product. Units passing the or each test would then need to be transfused within a

SUBSTITUTESHEET(RULE25) fixed time period.

In accordance with a third aspect of the present invention, there is provided a method for detecting the presence in a sample of a microorganism or of eukaryotic cells, preferably leucocyte cells, containing a target nucleic acid sequence, which method comprises the steps of: contacting the nucleic acid in the sample with a nucleotide probe capable of hybridising with the target nucleic acid sequence of the microorganism or eukaryotic cell in homogeneous solution; and detecting hybridisation of the nucleotide probe with the* target nucleic acid sequence, wherein said target nucleic acid sequence is present in the microorganism or eukaryotic cell to be detected in sufficient quantities that no additional amplification of the target nucleic acid sequence is required. The presence or absence of hybridisation may be detected by a fluorescence or luminescence technique.

In accordance with a fourth aspect of the present invention, there is provided a method for detecting the presence in a sample of a microorganism or of eukaryotic cells, preferably leucocyte cells, containing a target nucleic acid sequence, which method comprises the step of contacting the nucleic acid in the sample with a nucleotide probe capable of hybridising with the target nucleic acid sequence of the microorganism or eukaryotic cell in homogeneous solution, wherein there are at least two copies of said target nucleic acid sequence in the microorganism or eukaryotic cell to be detected. The presence or absence of hybridisation may be detected by a fluorescence or luminescence technique. The target nucleic acid sequence may be present in the genome of the microorganism. The method of the third and fourth aspects of the invention represent improvements in methods such as

SUBSTITUTE SHEET (RULE 25) those described in EP-A-0382433 and "A New Homogeneous Identification Method for DNA" by John Coates et al, J. Chem. Soc, Chem. Commun, 1994, pages 2311-2312. In accordance with these aspects of the invention, rather than amplify the target nucleic acid sequence using methods such as the polymerase chain reaction, a target nucleic acid sequence is identified which is present in sufficient quantities in the microorganism or leucocyte cell to be detected. In the fourth aspect, a nucleic acid sequence is identified which is present in at least two copies, and preferably at least 10 copies in the microorganism or leucocyte cell to be detected.

In accordance with a fifth aspect of the present invention, there is provided a method for detecting the presence of bacterial contamination in a sample containing a non-bacterial cellular component, comprising the step of contacting said sample with a lysing agent capable of lysing the non-bacterial cellular component, and carrying out a staining procedure to identify the presence of bacteria in the sample. Whilst staining of bacterial cells is known per se, it is not known to conduct a lysing step in order to enhance the visualisation of the stained microorganism. Preferably, the sample is a sample of a human blood product, such as a blood platelet concentrate.

For a better understanding of the present invention and to show how it may be carried into effect, reference will now be made, by way of example only, to the accompanying drawings in which:

Figure 1 is a schematic illustration of one apparatus in accordance with the first aspect of the present invention;

Figure 2 is a schematic illustration of another apparatus in accordance with the first aspect of the present invention; and

SUBSTITUTE SHEET (RULE 25) Figures 3-7 are copies of northern blots showing selective hybridisation of nucleic acid probes to target nucleic acid sequences in bacteria.

Figure 1 illustrates an apparatus 1 for the detection of a bacterial contaminant in a blood product, such as a blood platelet concentrate. The apparatus comprises a primary closed chamber 2 defined by an outer wall 3 for storage of the blood product, and a secondary detection chamber 4 defined by an outer wall 5. The detection chamber 4 includes a test reagent or combination of reagents suitable for the detection of bacterial contamination in the blood product. A flow path 6 from the primary chamber 2 to the secondary chamber 4 is provided in the form of a passageway 7 which is sealed with resect to the outside environment. The passageway 7 is normally closed. A breakable one-way cannula 8 is provided in the passageway 7 to allow the flow of fluid into the detection chamber 4 from the primary storage chamber 2, but prevent the return of fluid from the detection chamber 4 to the primary storage chamber 2.

The primary chamber 2 includes suitable access ports 9 and a transfer line 10 which is normally closed. In use, a blood product is stored in the storage chamber 2. When the blood product is to be issued, it is tested for bacterial contamination in the following manner. First, the breakable element 8 is broken to enable a sample volume of fluid, approximately 1 ml, to pass from the primary chamber 2 to the secondary chamber 4. The fluid is then allowed to react with the test reagents in the detection chamber 4, and the response noted. The response may be detected manually or by a suitable machine such as a spectrophotometer. If the response is indicative of bacterial contamination to a clinically significant degree, the

SUBSTITUTE SHEET (RULE 25) unit is rejected. Otherwise, the unit is identified as having passed the test and issued for use within a predetermined period.

It will be appreciated that apparatus 1 can be used for the detection of contaminants other than bacterial contaminants by using test reagents appropriate to the contaminant to be detected. In particular, apparatus 1 can be used for the detection of leucocyte cells in a blood product, such as a blood platelet concentrate.

Referring to Figure 2, a preferred apparatus 20 is illustrated. This differs from the apparatus of Figure 1 only in that an additional secondary chamber 21 is provided. Secondary chamber 21 is defined by an outer wall 22 and includes a test reagent or combination of reagents suitable for the detection of a contaminant the same as or different to the test reagent or combination of reagents in secondary chamber 4. A flow path 23 from the primary chamber 2 to the secondary chamber 21 is provided in the same manner as that from the primary chamber 2 to the secondary chamber 4. In this regard, there is provided a passageway 24 which is sealed with resect to the outside environment. The passageway 24 is normally closed. A breakable one-way cannula 25 is provided in the passageway 24 to allow the flow of fluid into the detection chamber 21 from the primary storage chamber 2, but prevent the return of fluid from the detection chamber 21 to the primary storage chamber 2. Apparatus 20 enables the blood product stored in the storage chamber 2 to be tested for two contaminants, e.g. leucocyte contamination as well as bacterial contamination. Alternatively, the same contaminant can be tested for twice, so as to provide a confirmatory result.

Preferably, apparatus 20 is used to test the blood

SUBSTITUTESHEET(RULE25) product stored in the storage chamber 2 for leucocyte contamination as well as bacterial contamination. The test for bacterial contamination is carried out in a manner identical to that for apparatus 1. However, additionally leucocyte contamination can be tested by breaking breakable element 25 to enable a sample volume of fluid, approximately 1 ml, to pass from the primary chamber 2 to secondary chamber 21. The fluid is then allowed to react with the test reagents in the detection chamber 21, and the response noted. If the response is indicative of a leucocyte load below a predetermined level, the unit will be designated "leucodepleted". Otherwise, it will be designated "standard" . Where nucleic acid hybridisation is to be used to detect general bacterial contamination of the blood product contained in primary chamber 2, DNA probes having the following sequences can be used for selective hybridisation to target sequences in bacterial rRNA:

Probe 1: 5'-GCTGCCTCCCGTAGGAGT-3' (targeting 16s RNA)

Probe 2: 5' -ATTACCGCGGCTGCTGGCA-3' (targeting 16s RNA)

Probe 3: 5' -CCGACAAGGAATTTCGCTAC-3' (targeting 23s RNA) Probe 4: 5'-CGGTACTGGTTCACTATC-3' (targeting 23s RNA)

Probe 5: 5'-CATCCCCACCTTCCTCC-3' (targeting 16s RNA)

In order to test that the probes are sufficiently specific for bacterial rRNA, the probes were labelled with digoxygenin at the 3' end (Boehringer Mannheim

Inc.) and were used to detect bacterial rRNA and human rRNA using conventional northern blotting techniques. The detection system employed in these studies comprised a proprietary chemiluminescent substrate as the vehicle to measure probe:target interactions. The detection materials and protocol were provided for in a kit by (Boehringer Mannheim Inc.) .

The results of the detection of bacterial rRNA and human rRNA are illustrated in Figures 3-7 which are copies of the northern blots obtained for probes 1-5 respectively. In Figures 3-7, A = E. coli standard, B = E. coli , C = Pseudomonas aeruginosa, D = Serratia marcescens, E = Salmonella typhimurium, F = Enterobacter cloacae, G = Staphylococcus aureus, H = Staphylococcus epidermis, I = Streptococcus pyogenes, J = Bacillus cereus, K = Calf rRNA and L = Human leucocyte rRNA. It can be seen that, with the exception of probe 2 (Fig. 4) , all probes hybridised specifically to bacterial targets and not to human rRNA.

SUBSTITUTE SHEET (RULE 25) SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: The National Blood Authority

(B) STREET: Oak House, Reeds Crescent

(C) CITY: Watford

(D) STATE: Hertfordshire

(E) COUNTRY: United Kingdom

(F) POSTAL CODE (ZIP) : WD1 1QM

(A) NAME: Janet Rosemary Rider

(B) STREET: 29 Chapel Lane, Old Sodbury

(C) CITY: Bristol

(E) COUNTRY: United Kingdom

(F) POSTAL CODE (ZIP) : BS17 6NQ

(ϋ) TITLE OF INVENTION: DETECTION METHOD (iii) NUMBER OF SEQUENCES: 5

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1

GCTGCCTCCC GTAGGAGT 18

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATTACCGCGG CTGCTGGCA 19

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3

CCGACAAGGA ATTTCGCTAC 20

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY" linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4

CGGTACTGGT TCACTATC 18

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5

CATCCCCACC TTCCTCC 17

Claims

CLAIMS :

1. Apparatus (1;20) for the detection of a contaminant in a fluid, comprising a primary chamber (2) for storing said fluid, and a secondary detection chamber (4;21) which includes a test reagent or combination of reagents suitable for the detection of said contaminant in the fluid, a flow path (6;23) from the primary chamber (2) to the secondary chamber (4;21) being provided which is normally closed, but which is openable to enable a sample volume of fluid to pass from the primary chamber (2) to the secondary chamber (4;21) for detection of the contaminant in the fluid.

2. Apparatus (1;20) as claimed in claim 1, in the form of a kit, said primary chamber (2) including an opening, which is normally closed and which is, in use, connected to said detection chamber (4;21) to form said flow path.

3. Apparatus (1;20) as claimed in claim 1, wherein the primary chamber (2) and the secondary chamber (4;21) are integral, said flow path (6;23) being formed by a passage between the primary chamber (2) and the secondary chamber (4;21) .

4. Apparatus (1;20) as claimed in claim 1, 2 or 3, wherein said flow path (6;23) is unidirectional to prevent the return of fluid from the secondary chamber (4;21) to the primary chamber (2) .

5. Apparatus (1;20) as claimed in claim 4, wherein said flow path (6;23) includes a one-way valve.

6. Apparatus (1;20) as claimed in any preceding claim, wherein said flow path (6;23) includes a closure capable of being fractured or broken in order to enable said sample volume of fluid to pass from the primary chamber (2) to the secondary chamber (4;21).

7. Apparatus (1;20) as claimed in any one of claims 1 to 5, wherein said flow path (6;23) includes a closure which is reversible, so as to close the flow

SUBSTITUTESHEET(RULE25) path (6;23) after said sample volume of fluid has passed from the primary chamber (2) to the secondary chamber (4;21) .

8. Apparatus (1;20) as claimed in claim 6 or claim 7 when appended to claim 5, wherein the one-way valve and closure are combined in a one-way cannula (8;25) .

9. Apparatus (1;20) as claimed in any preceding claim, wherein said test reagent or combination of reagents is for the detection of one or more microorganisms in a blood product.

10. Apparatus (1;20) as claimed in any one of claims 1 to 8, wherein said test reagent or combination of reagents is for the detection of leucocyte cells in a blood product.

11. Apparatus (1;20) as claimed in any preceding claim, wherein said test reagent or combination of reagents comprises a stain for the detection of said contaminant in the fluid.

12. Apparatus (1;20) as claimed in claim 11, wherein said test reagent or combination of reagents comprises a stain for the detection of one or more microorganisms in a blood product.

13. Apparatus (1;20) as claimed in claim 11, wherein said test reagent or combination of reagents comprises a stain for the detection of leucocyte cells in a blood product.

14. Apparatus (1;20) as claimed in claim 12 or claim 13, wherein said test reagent or combination of reagents further comprises a lysing agent to lyse blood cells in the blood product.

15. Apparatus (1;20) as claimed in any one of claims 1 to 9, wherein said test reagent or combination of reagents comprises an assay for the presence of a target nucleic acid sequence specific to one or more microorganisms which is/are to be detected in a blood

SUBSTITUTE 5HEET (RULE 25) product .

16. Apparatus (1;20) as claimed in claim 153, wherein said test reagent or combination of reagents further comprises a lysing agent to lyse the or each microorganism in the blood product.

17. Apparatus (1;20) as claimed in claim 15 or claim 16, wherein said target nucleic acid sequence is present in the or each microorganism in a sufficient quantity that additional amplification of the target nucleic acid sequence is unnecessary.

18. Apparatus (1;20) as claimed in claim 15, 16 or 17, wherein said test reagent or combination of reagents comprises a nucleic acid probe which is for hybridising with the target nucleic acid sequence.

19. Apparatus (1;20) as claimed in any one of claims 15 to 18, wherein said target nucleic acid sequence is located in bacterial rRNA.

20. Apparatus (1;20) as claimed in claim 19, wherein said target nucleic acid sequence is located in bacterial 16s rRNA.

21. Apparatus (1;20) as claimed in claim 20, wherein said nucleic acid probe has the following sequence: 5' -GCTGCCTCCCGTAGGAGT-3' ; 5' - ATTACCGCGGCTGCTGGCA-3' ; or 5' -CATCCCCACCTTCCTCC-3' .

22. Apparatus (1;20) as claimed in claim 19, wherein said target nucleic acid sequence is located in bacterial 23s rRNA.

23. Apparatus (1;20) as claimed in claim 22, wherein said nucleic acid probe has the following sequence: 5' -CCGACAAGGAATTTCGCTAC-3' or 5' - CGGTACTGGTTCACTATC-3' .

24. Apparatus (1,-20) as claimed in any one of claims 1 to 8 and 10, wherein said test reagent or combination of reagents comprises an assay for the presence of a target nucleic acid sequence specific to leucocyte cells which are to be detected in a blood product .

25. Apparatus (1;20) as claimed in claim 24, wherein said test reagent or combination of reagents further comprises a lysing agent to lyse the leucocyte cells in the blood product.

26. Apparatus (1;20) as claimed in claim 24 or claim 25, wherein said target nucleic acid sequence is present in the leucocyte cells in a sufficient quantity that additional amplification of the target nucleic acid sequence is unnecessary.

27. Apparatus (1;20) as claimed in claim 24, 25 or 26, wherein said test reagent or combination of reagents comprises a nucleic acid probe which is for hybridising with the target nucleic acid sequence.

28. Apparatus (1;20) as claimed in any one of claims 24 to 27, wherein said target nucleic acid sequence is located in mRNA of leucocyte cells.

29. Apparatus (1;20) as claimed in claim 27, wherein said target nucleic acid sequence is located in mRNA for the H2A.Z, H2A.X or H3.3 histone genes or the CD53 gene of leucocyte cells.

30. Apparatus (1;20) as claimed in any one of claims 9 to 29, wherein said blood product is a human blood product.

31. Apparatus (1;20) as claimed in claim 30, wherein said human blood product is a blood platelet concentrate.

32. Apparatus (20) as claimed in any preceding claim, having a plurality of secondary detection chambers (4, 21), each communicating with the primary chamber (2) via a respective flow path (6,23).

33. Apparatus (20) as claimed in claim 32, which is adapted to detect a plurality of contaminants in the fluid stored in the primary chamber (2) , each secondary detection chamber (4,21) comprising a test reagent or combination of test reagents suitable for the detection

SUBSTITUTE SHEET (RULE 25) of a respective contaminant.

34. Apparatus (20) as claimed in claim 32, having first and second secondary detection chambers (4,21), the first secondary detection chamber (4) including a test reagent or combination of test reagents suitable for the detection of bacteria in a blood product stored in said primary chamber (2) , and the second secondary detection chamber (21) including a test reagent or combination of test reagents suitable for the detection of leucocyte cells in the blood product.

35. A method for the detection of a contaminant in a fluid stored in the primary chamber of an apparatus as claimed in any preceding claim, said method comprising the steps of opening the flow path from the primary chamber to the secondary chamber and permitting a sample volume of fluid to pass from the primary chamber to the or each secondary chamber; permitting the contaminant to be assayed in the or each secondary chamber; and observing the results of the or each assay.

36. A method for detecting the presence in a sample of a microorganism or of eukaryotic cells containing a target nucleic acid sequence, which method comprises the steps of: contacting the nucleic acid in the sample with a nucleotide probe capable of hybridising with the target nucleic acid sequence of the microorganism or eukaryotic cells in homogeneous solution; and detecting hybridisation of the nucleotide probe with the target nucleic acid sequence, wherein said target nucleic acid sequence is present in the microorganism or eukaryotic cells to be detected in sufficient quantities that no additional amplification of the target nucleic acid sequence is required.

37. A method as claimed claim 36, wherein said eukaryotic cells are leucocyte cells.

38. A method for detecting the presence of

SUBSTITUTE SHEET (RULE 25) bacterial contamination in a sample containing a non- bacterial cellular component, comprising the step of contacting said sample with a lysing agent capable of lysing the non-bacterial cellular component, and carrying out a staining procedure to identify the presence of bacteria in the sample.

39. A method as claimed in claim 36, 37 or 38, wherein the sample is a sample of a human blood product.

40. A DNA molecule having the following sequence: 5' -GCTGCCTCCCGTAGGAGT-3'

41. A DNA molecule having the following sequence: 5' -ATTACCGCGGCTGCTGGCA-3'

42. A DNA molecule having the following sequence: 5' -CCGACAAGGAATTTCGCTAC-3'

43. A DNA molecule having the following sequence: 5' -CGGTACTGGTTCACTATC-3'

44. A DNA molecule having the following sequence: 5'-CATCCCCACCTTCCTCC-3'

SUBSTITUTE SHEET (RULE 25)