Arrangement for detection of hemolysis

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(12) Patent Specification do) SE 536 634 C2

Sweden (21) Patent application number: 1151178-9 (45) Patent granted: 2014-04-15 (41) Application generally available: 2013-06-10 (22) Patent application submitted: 2011-12-09 (24) Maturity date: 2011- 12-09 (83) Deposit of microorganism: - (30) Priority information: - (51) International class:

G01N 33/49 (2006.01)

G01N 33/72 (2006.01) (73) Patent holder: Hemcheck Sweden AB, Hybelejens gata 2, 653 40 Karlstad SE

<td>(72) Inventor:</td><td>Mathias KARLSSON, KARLSTAD SE</td>

<td>(74) Agents:</td><td>HYNELL PATENTTJÄNST AB, Box 138, 683 23 HAGFORS SE</td>

<td>(54) Name:</td><td>Device for detecting hemolysis</td>

<td>(56) Quoted publications:</td><td>WO 9623223 Al · US 20020058342 Al</td>

(47) Summary:

The present invention relates to an apparatus for visual detection of hemolysis in a whole blood sample from a penetrable container, said apparatus comprising at least one space for visual detection and a transmission passage connected to said space for visual detection, said apparatus further comprising means for penetrating the container to the inside of said container for accessing whole blood and allowing transfer of a plasma volume from said sample to said detection space via said transfer passage, said apparatus further provided with a separation unit for separating plasma. from blood cells in said whole blood sample before said plasma reaches the detection space, wherein said apparatus is further provided with means providing capillarity for generating a capillary force which forces said plasma volume to be transmitted through the separation unit to said detection space.

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536 634

SUMMARY

The present invention relates to an apparatus for visual detection of hemolysis in a whole blood sample from a penetrable container, said apparatus comprising at least one space for visual detection and a transmission passage connected to said space for visual detection, said apparatus further comprising means for penetrating the container to the inside of said container for accessing whole blood and allowing transfer of a plasma volume from said sample to said detection space via said transfer passage, said apparatus further provided with a separation unit for separating plasma from blood cells in said whole blood sample before said plasma reaches the detection space, wherein said apparatus is further provided with means providing capillarity for generating a capillary force which forces said plasma volume to be transmitted through the separation unit to said detection space.

536 634

DEVICE FOR DETECTION OF HEMOLYSIS

TECHNICAL FIELD

The present invention relates to an apparatus for the visual detection of hemolysis in a whole blood sample from a permeable container.

BACKGROUND OF THE ART

Laboratory testing is probably the most common clinical routine performed in modem healthcare. Brain-spinal fluid and urine can be used for biochemical analysis, but blood is the body fluid most commonly used and these tests are extremely important diagnostic and prognostic tools in daily patient care. Laboratory testing can be divided into three phases.

• The preanalytic phase: all the steps before the actual analysis of a sample, including patient variables, collection, handling and processing • The analysis phase • The post-analytic phase: test reporting variables

For obvious reasons, it is of great importance that all three phases are performed correctly because errors could provide misleading information to the physicians and therefore jeopardize the well-being of individuals or patient groups. A majority of the errors noted in laboratory testing occur in the preanalytical phase, and hemolysis is one of the most significant causes of specimen rejection. By hemolysis is usually meant the release of hemoglobin and other intracellular components from erythrocytes to the surrounding plasma, as a result of damage to or rupture of the cell membrane. Hemolysis can take place either in vivo or in vitro, and is a highly undesirable condition affecting the accuracy and reliability of laboratory testing. Reasons for hemolysis interfering with several biochemical analyzes may be, for example, that hemoglobin interferes with the measurements (eg spectrophotometry methods), and also that the release of biochemical markers from the damaged red blood cells causes erroneously high values ​​of these substances.

Visible hemolysis, as a characteristic of a more generalized process of blood cell damage, usually does not appear until the separation of serum or plasma has occurred. It is usually defined as an extracellular hemoglobin concentration of 0.3 g / L (0.0186 mmol / L), resulting in a detectable pink to red hue of serum or plasma. IN

536 In general, a collected blood sample must be transported to a long-range ward where red blood cells are separated from the plasma or serum, for example by means of centrifugation, and where said color shade can be detected and reported to the patient in charge of the patient. Modern laboratories also objectively assess the degree of hemolysis in each blood sample that comes in for analysis. If hemolysis is sufficiently extensive to cause clinically relevant disruption of the assay, the result is not reported and a new sample must be collected from the patient. For obvious reasons, the above-described procedures for assessing the validity of the test are associated with a time delay that results in an undesirable situation for the patient, and which also leads to cumbersome procedures.

Alternative detection methods have been proposed, for example, in WO96 / 23223, which describes a method and apparatus for detecting hemolysis in a blood sample which does not require laboratory environment. However, the detection method according to WO96 / 23223 requires a series of time-consuming and ineffective steps which result in a laborious process and unwanted interruptions.

OBJECTS OF THE INVENTION

It is an object of the invention to provide an improved method of establishing hemolysis in immediate connection to the collection of a blood sample, wherein said determination can be performed by a user, for example, in a treatment room without the need for a laboratory.

It is a further object of the present invention to provide a rapid method for detecting hemolysis in a whole blood sample, the determination of which can preferably be made within one minute, preferably within less than 30 seconds of the use of an apparatus according to the invention.

It is a further object of the invention to provide a method of determining hemolysis with only a very small volume of whole blood samples, preferably between 2 and 100 μΐ whole blood, preferably resulting in between 1 and 50 μΐ plasma volume for detection.

It is a further object of the present invention to provide a method of establishing hemolysis which is intuitive and easy to handle, preferably where the person collecting a blood sample can perform the steps of detecting hemolysis using only one hand.

These and still other objects of the invention will become apparent upon study of the accompanying drawings and description of the invention.

536 634

SUMMARY OF THE INVENTION

The objects of the invention are achieved by means of a device for the visual detection of hemolysis in a whole blood sample from a penetrable container, said apparatus comprising a dispenser containing a surface arranged to engage the closed container, at least one space for visual detection and a transmission passage connected to said visual detection space; said apparatus further comprising means for penetrating the sealed container to the inside of said sealed container for accessing whole blood and allowing the transfer of a plasma volume from said sample to said detection space via said transfer passage, said apparatus further provided with a separation unit. separating plasma from blood cells in said whole blood sample before said plasma reaches the detection space, wherein said apparatus is further provided with means providing capillarity for generating a capillary force which forces said plasma volume to be transmitted through the separation unit to said detection space.

The apparatus of the invention enables a quick and easy way to transfer a whole blood sample from within a container (such as a sealed collection tube, blood bag, or other container containing blood) via said transfer passage to the detection apparatus of the invention. Providing said capillary force involves the advantage of effective plasma separation through a separation element (e.g., a filter) and reliable transfer of the resulting plasma sample to the detection space, where no additional external force is required to accomplish said transfer.

It is understood that a "user" may refer to any person handling the device for detecting hemolysis and may include, for example, a practicing physician, a caregiver and / or laboratory staff or a veterinarian.

In the following description, the term "blood collection device" is to be construed to include (in a non-limiting sense) a sealed container, a collection tube, a blood collection tube, a conventional tube, a blood bag, and a capillary tube. In addition, a test tube may refer to a closed tube, a collection tube, a blood collection tube, a conventional tube, and vice versa.

A "sealed tube" means a, usually airtight, container of glass, plastic or the like, arranged to contain a volume of liquid biological sample, such as a whole blood sample. Typically, such sealed tubes are provided with an open end with a permeable plug or sealing member (of rubber or the like) placed in the open end.

536 634 end. Such construction is typical of closed test tubes which are manufactured under reduced atmospheric pressure and lose all or most of their vacuum when filled.

According to another aspect of the invention, said capillary providing means comprises a separation filter and a detection element, wherein the separation filter is arranged so as to abut an opening in the transfer passage and the detection element is arranged to abut the separation filter in such a way that the separation filter is interleaved. the opening of the transfer passage and the detection element; the detection element being visibly arranged inside the detection space (60). Thanks to the separation unit (for example, a separation filter), whole blood is effectively separated from the plasma which can then easily be analyzed as soon as it becomes visible inside the detection space.

As will be described in more detail hereinafter, the detection element may be in the form of a detection filter comprising a structure providing capillarity, or it may also be in the form of a porous structure providing capillarity. Examples of suitable materials may include fiberglass as well as any porous material giving rise to said capillary force which contributes to the transmission of the plasma. One of ordinary skill in the art will understand that "capillarity", or "capillary effect", can be interpreted as the ability of a liquid to flow towards gravity where fluid spontaneously rises in a narrow space such as in porous material such as paper or filters. Thus, said detection filter can be made of any suitable material which provides said capillarity and meets the other requirements of the present invention, such as fiberglass material, a fabric filter or a nonwoven filter, or even certain fabric materials which may prove suitable for the purpose.

In a preferred aspect of the invention, said means for penetrating the penetrable container comprises a needle element having a first end portion for penetrating the sealing element of a penetrable container and a second end portion disposed at the housing of the apparatus and adjacent to said separation filter. It is understood that "adjacent" herein is to be interpreted in such a way that the needle element is positioned with its second end portion, and the orifice at the corresponding second end portion, positioned adjacent to the separation filter so that all whole blood passing through the needle upon exit from the needle will continue to separating filter. Preferably, there is a small distance between the needle orifice and the separation filter so that the blood volume can easily be spread on the filter as soon as it is applied to it. When a blood volume is applied to the separation filter, it will be sucked into the structure of the separation filter immediately upon exit from said transfer passage (for example, the needle), due to capillarity, whereby the plasma is separated from the red blood cells. The adjacent detection filter, in turn, is arranged to also provide capillarity, which means that the plasma volume, after passing the separation filter, will

536 634 continue to be sucked into the detection filter to the extent that it becomes visible on the opposite side of the detection filter as the plasma is transmitted therethrough. The detection filter, in turn, is preferably visibly arranged inside the detection space (60), and can thereby be readily observed by a user. Since hemolysis is visually detectable in serum or plasma, the device of the invention provides an opportunity for the person collecting a sample to visually determine if a clinically significant hemolysis is present in the sample immediately after the plasma becomes visible in the detection space. containing the sample is sent to the laboratory. Such determination of hemolysis can be done by merely observing the hue of the plasma amount absorbed by the visible detection filter inside the detection space (ie if the plasma is yellow-orange, no hemolysis has occurred, but if the plasma is light pink to red, hemolysis may be suspected and a new blood test should be taken).

In the case of discarded specimen due to hemolysis in the collected blood sample, the invention will also enable, possibly even before the test equipment is removed from the patient, the collection of a new sample more suitable for analysis. This brings many benefits. The situation for the patient is greatly improved as the risk of requiring repeated blood sampling is reduced when using the inventive detection apparatus. The time delay caused by the laboratory's hemolysis testing is eliminated, which results in faster processing of the blood test analysis, which of course means faster delivery of results / diagnosis as well as a larger proportion of successful subsequent test analyzes and reduced costs.

Thanks to the apparatus of the invention, there is provided a method of detecting hemolysis in a collected blood sample which comprises very few steps, which is simple and intuitive, which is fast, requires only a small sample volume, and which can be performed with only one hand in the immediate vicinity (e.g. the bed edge) to a patient.

Preferably, the separation filter as well as the detection element (for example, a detection filter) comprise a porous structure which produces capillarity, whereby plasma is forced through both filters. The visual control of the color shade of the plasma is performed at the time when the plasma has been sucked into the structure of the detection element to the extent that the plasma is visible through the transparent cover of the detection space. It is understood that the detection space may contain only the detection element and that the detection space is covered by a transparent cover through which the inside of the detection space can be observed. The detection space may contain the detection element in the form of a detection filter, or it may be filled with another detection element in the form of a porous material such as glass wool, which also provides the desired capillarity which

536 634 causes separated plasma to be sucked into the structure of the detection element to such an extent that the detection element is gassed by the color shade of the plasma, whereby hemolysis in the blood sample can be determined by observing the color shade of the detection element.

By providing filters (i.e., separation filters and detection filters) or porous material in the detection space, the risk of bubble formation is significantly reduced.

By means of said separation filter and detection element, a capillarity is obtained which results in efficient plasma transfer. Another advantage that is achieved by having a separation filter and a further detection filter is that the red blood cells will get stuck in the separation filter, which means that only plasma is passed through the second filter. Thus, in addition to providing extra capillary power, the second filter will also provide a shielding function by shielding any red color of the first separation filter from being detectable / visible / discernible within the detection space. This is an advantage because the detection according to the invention requires the safe and reliable determination of the color shade of the plasma, and any red color from separated blood cells could risk destroying the correct determination of hemolysis.

Preferably, said detection filter has a color which allows for easy assessment of the color hue of the plasma, for example a white filter color, which means that the detection filter facilitates detection of a color change indicating the occurrence of hemolysis. This means that the detection filter, in addition to the above advantages, also facilitates the actual detection since it provides a detection surface which makes it easier to detect hemolysis. The color of the detection filter may be any color other than white to further facilitate the proper detection of the color hue of the plasma. For example, said detection filter may have a light blue color to enhance color differences and facilitate correct detection: a yellow-orange plasma on light blue filter would result in a final greenish detection color while pink-colored plasma on light blue filter would give a purple detection color.

According to yet another aspect of the invention, said capillary providing means comprises a separation filter, a detection filter and a separating distribution surface, wherein the separation filter is arranged to abut an opening in the transfer passage, the separating distribution surface is interleaved between the separation filter and the detection filter, further defines the bottom portion of said detection space. The separating distribution surface has the advantage that it further ensures that any red color on the separation filter as a consequence of the separated red blood cells will not interfere with the visual assessment of the color of the plasma within the detection space. Furthermore, it has separating

536 634 distribution area the function of distributing the plasma from the separation filter before coming into contact with the detection filter so that the plasma is more evenly distributed in the detection space.

According to another aspect of the invention, said detection space and said separation unit are disposed within a housing, and the means for penetrating the closed container comprises a needle element having a first end portion for penetrating a sealed element sealing member and a second end portion disposed within said housing. separating filter.

According to another aspect of the invention, the filter cross-sectional area of ​​said separation filter is substantially larger than the cross-sectional surface of said transfer passage to eliminate the risk of clogging of the filter.

According to yet another aspect of the invention, said separation unit is a separation filter (or separation membrane) arranged to separate plasma from the cell components in whole blood samples without lysis. It is to be understood that the filter may be any known conventional filter or membrane which meets the separation requirements of the present equipment, including membranes made of synthetic as well as natural polymers, preferably but not necessarily a hydrophilic membrane. In one embodiment, the separation filter is asymmetrical, which means that the filter pores have varying sizes. The filter may have any suitable geometry or shape, for example, substantially flat or three-dimensional, for example cylinder size. The size and / or volume of the filter depends on the type of filter and the specific plasma volume to be separated thereby.

According to yet another aspect of the invention, said at least one detection space may be provided with chemical means for direct visual detection of hemoglobin. The chemical agents for visual detection can cause a color change if hemolysis has taken place, allowing for safer and more reliable test results and easier assessment, especially if there is only a slight shade of pink where accurate assessment by considering only the color of the plasma could prove difficult . The chemical agents may be dry chemical agents and may, for example, be dried in the structure of the detection element (for example in the filter).

In yet another aspect, the detection element is a fiberglass filter having a thickness between 0.5 and 1 mm. According to yet another aspect, the detection element is a filter made of a porous material having a thickness between 0.5 and 5 mm.

536 634

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in more detail with reference to the accompanying drawings, in which:

Fig. 1A is a perspective view schematically showing a preferred embodiment of the apparatus according to the invention;

Fig. 1B is a cross-sectional view of the apparatus of Fig. 1A.

Figures 2A-C schematically illustrate the use of an apparatus according to the invention, and

Fig. 3 schematically illustrates the upper portion of a conventional collection tube.

DETAILED DESCRIPTION OF THE INVENTION

The above aspects and many of the ensuing advantages of this invention will be more readily appreciated when better understood with reference to the following detailed description taken in conjunction with the accompanying figures. Further, the description, and the examples contained therein, are provided solely for the purpose of describing and illustrating certain embodiments of the invention, and are not intended in any way to limit the scope of the invention.

Figs. 1a, b show a preferred embodiment of the invention. Here, Fig. 1a, b schematically illustrates a detection apparatus 3 arranged to visually indicate hemolysis in a blood sample 12, wherein Fig. 1a shows a perspective view of the assembled apparatus 3, and Fig. 1b shows a simplified and schematic cross-section of a detection apparatus 3 according to Figs. the exemplary embodiment of the invention in Fig. 1a.

Figs. 2a-c further illustrate the principle of performing a rapid instantaneous test of hemolysis of a blood sample 12 in a collection tube 2, using a detection apparatus 3 of the present invention.

Referring to Figs. 1a, b, said detection apparatus 3 comprises at its first end a means 1 for penetrating a closed container 2 in the form of a needle element 1 which, when not in the operating position, is preferably provided with a protective cover (not shown) e.g. made of rubber. It is understood that the apparatus 3 shown for example in FIG. 1a, b should not be considered as limited to the specific dimensions shown there, and that, for example, the needle element 1 may in fact be longer than in the drawings. The needle 1 has a first end portion 1A which creates a tip for penetrating the sealing member of a sealed container 2 to the inside of said sealed container 2 to access whole blood 12 therein. The needle 1 has

536 634, further a second end disposed within a housing 30 of the apparatus 3 and abutting a separation unit, preferably a separation filter 40. A transfer channel 7 is defined by the path / passage between the needle tip 1A and the needle end at the housing 30, permitting passage of blood 12 from the container 2 to a visual detection space 60, preferably located at the other end of said detection apparatus 3, said second end being opposite said first end. The needle 1 and the transfer channel 7 together form a transfer passage 1, 7 for the sample. After the needle has pierced a closure element of a container 2 and come into contact with the sample 12 therein, said transfer passage 1, 7 is provided to enable transfer of a plasma volume from said container 2 to said detection space 60 via separation filter 40 (which will be described in greater detail). be). Thus, the separation filter 40 is arranged to contact a blood sample 12 and allow passage of free hemoglobin and stop passage of red blood cells, thus allowing passage of blood plasma 14. The plasma is sucked through the separation filter 40 by means of capillarity which is generated as the plasma enters. contact with the filter body, due to the porous material constituting the filter 40. Hereby, a blood volume is sucked into the structure of the separation filter 40 immediately upon exit from said transfer passage 1, 7 (for example, a needle), thanks to capillarity, whereby the plasma is separated from the red blood cells. As can be seen in Fig. 1b (showing an embodiment of the invention), a detection device in the form of a filter 41 abuts the separation filter 40. The abutting detection filter 41 is arranged to also provide capillarity, which means that after passing the separation filter 40, the plasma volume will continue to be sucked into the detection filter 41 as well. The plasma will thereby be transferred to the detection filter 41 to the extent that the plasma is moistened. the entire thickness of the filter structure 41 and becomes visible from the other side of the filter 41. Since the detection filter 41 also constitutes the lower portion of the visual detection space 60, the plasma can be readily observed by a user.

It is to be understood that the separation filter 40 and the detection element 41 (for example, the filter 41) may be positioned in relation to each other other than shown in Fig. 1b. As previously described, the separation filter 40 and the detection filter 41 in Fig. 1b are placed vertically adjacent to one another, and abutting one another, so that plasma will be transmitted along a path in line with the longitudinal axis of the needle. In another embodiment, it is possible to place the separation filter 40 and the detection filter 41 substantially side by side (along a horizontal line) within said housing 30. This means that, as in the previously described embodiment, blood is first applied to the separation filter 40 from the needle, which results in separation of red blood cells from the plasma. On the opposite side of the separation filter, a channel may be provided which leads to the separated plasma

536 634 detection filter 41. (Alternatively, the separation filter 40 and the detection filter 41 may partially overlap, said detection filter 41 being placed below the separation filter 40.) Next, when plasma has been transferred through the separation filter 40, it can reach the detection filter 41 via said channel. As soon as the plasma comes into contact with the detection filter body 41, it will be sucked into the detection filter structure due to. said capillarity.

Preferably, the surface of the filters 40, 41, which are arranged to be in contact with the blood sample, is substantially much larger (e.g., at least ten times) than the cross-sectional area of ​​the needle 1, to eliminate the risk of clogging of the filters 40, 41. It is understood that the separation filter 40 and the detection filter 41 may have different dimensions (e.g., diameters); for example, the detection filter 41 may have a smaller diameter than the separation filter 40.

Between the separation filter 40 and the detection filter 41, a distribution surface (not shown) may be provided. The distribution surface is preferably provided with passages (e.g. channels, apertures, pores, slits or any other suitable passage type) to allow passage of plasma from the filter 4 and at the same time cause a plasma distribution over the adjacent detection filter 41 so that the plasma to be examined is evenly distributed over said filter 41. An even plasma distribution will lead to more secure determination of hemolysis. The distribution surface is preferably arranged so that it does not allow any passage of color (i.e., red color from filtered blood cells), and may for this reason, for example, be formed of a non-translucent material which blocks any light from passing / shining through the body.

The detection space 60 is visibly arranged in a lower portion of the main structure 30. of the detection apparatus 3. and whether the visible hue of such plasma indicates that hemolysis has occurred. In one embodiment, the side of the detection filter 41 which is intended to face the transparent cover and which will correspond to the white surface of the detection space 60 has white color in order to facilitate the determination of the color of the plasma (e.g. yellow orange or pink). In one embodiment of the invention, said detection space consists merely of said detection filter, covered / protected by a transparent cover plate on the underside of housing 30.

In one embodiment of the invention, it is conceivable that in the lower portion of the apparatus 3 (either inside the detection space 60 or beside the visual detection well on the outside of the main structure 30 of the detection apparatus) a color reference scale for

536 634 comparison with the color shade of the plasma. Such a reference scale could further simplify a correct determination of hemolysis.

Preferably, the detection filter 41 has a thickness between 0.5 and 5 mm. The diameter of the detection space 60 is preferably adapted for convenient visual detection, ie. adapted so that a user can easily observe the inside of such a detection space. Preferably, the detection apparatus 3 is arranged to filter a volume of between 2 and 100 μΐ of whole blood, resulting in approximately 1-50 μΐ of plasma for visual observation. In one embodiment, said detection filter 41 is provided with chemical agents for direct visual detection, which means that one or more reagents may be deposited on and dried into detection filter 41 and react with hemoglobin to provide a color to indicate whether hemolysis has occurred.

The apparatus 3 may further comprise a transparent hydrophilic strip to facilitate transfer of plasma to the detection space 60. For the same reason (i.e. facilitating plasma transfer), the surface of said detection filter 41 may comprise a hydrophilic surface treatment such as coating, surfactant or plasma surface treatment for enhancement. .

Figures 2a-c illustrate an exemplary use of a detection apparatus 3 according to an embodiment of the invention. Typically, a collection tube 2 referred to in this preparation is constructed of glass material or injection molded plastic such as polypropylene, polystyrene, polyethylene terephthalate or any other suitable polymer. Preferably, the collection tube 2 has an elongated shape with a circular wall, with a closed end and an open end defining a chamber therein to receive samples of a collected fluid (e.g., blood 12) from a patient. The open end is tightly sealed with an elastic sealing element (see Fig. 3). The sealing member 10 (for example, a sealing plug) may be of rubber or other suitable resilient material, and is provided at the open end of the tube 2 to close the chamber and hermetically seal the inside of the tube. Further, but need not, the open end of tube 2 may be protected by a protective cap 9 fixed to the tube 2 and over the sealing member 10. If covered by a cap, said cap 9 includes a central aperture 90 intended to pass through a needle 1 arranged to penetrate the sealing member 10. It is understood that the tube 2 illustrated in Fig. 3 is to be viewed schematically only and that the specific the dimensions, for example of the central opening 90, should only be regarded as explanatory and for the reader's better understanding. Thus, the dimensions of the pipe in FIG. 3 is seen as limiting the function and / or use of the present invention. Similarly, the tubes of Figures 2a-c are shown without the said lid 9, but one skilled in the art will understand that both types (i.e. with or without lid 9) are conceivable for the function of the present invention.

536 634

The discharge of a blood volume 12 from inside the collection tube 2 (the sealed container 2) to the detection apparatus 3 is accomplished by a method described in US5344666, which is hereby incorporated by reference. The principle of dispensing a blood volume is as follows. As can be seen in Fig. 2a, the apparatus 3 is mounted against the sealing element 10 by pressing the tip of the needle 1A through the center of the sealing element 10, for example, but not necessarily when the container is in an upright position. It is also conceivable to press the sealing member 10 against the tip 1A of the needle if the apparatus is positioned with the needle pointing vertically upwards, said apparatus 3 resting on a surface. Optionally, said apparatus 3 can then be placed inside any supporting holder structure (not shown) which ensures that it is held in the correct position and does not fall or move. The latter option allows a user to use only one hand in carrying out the method of the invention. However, if the container is in (or placed in) upright position when the appliance 3 is connected to it, any pressure difference that could exist between the container 2 inside and the atmosphere is neutralized by air being able to pass through the needle 1. The needle 1 is held in a heel such as the annular abutment member 31 (also called a dispenser 31) containing a surface arranged to engage the sealed container 2. The dispenser 31 limits the length of the needle 1 which can penetrate the rubber sealing member 10. This length is sufficient to penetrate the sealing member 10 and penetrates an additional distance in the space of the well immediately adjacent the inner surface of the sealing member to contact the liquid sample (whole blood) located therein.

The diameter of the annular dispenser 31 is smaller than the average concave diameter of the concave recess of the sealing member, and the dispenser 31 is also longer than the maximum depth of the concave recess of the sealing member 10 so that the dispenser 31 can always cause a bend or deformation of the rubber plug to in the container.

As previously described, the apparatus 3 is very easily connected to a collection tube 2 by forcing the penetrating tip 1A of the needle 1 through the center portion of the sealing member 10 until the annular dispenser 31 reaches the depth of the concave depression of the sealing member 10 (see Fig. 2a), ready for use. as shown in Fig. 2b. In FIG. 2b, the sealed tube 2 with the attached device 3 is shown in the upside down operating position after it has been mounted in the manner already described. When container and apparatus 3 are pressed by manual force against a surface, the downward force is counteracted by the underlying surface (for example, a bench surface). This creates an internal compression force within the sealing element 10 which deforms the sealing element 10 thereby reducing the volume inside the container 2 and ejecting a sealing element 10.

536 634 small amount of fluid (the blood 12) through the needle 1 and further on the previously described filters 40, 41. Said small volume of whole blood 12 will thereby be sucked through the separation unit due to. capillarity, and plasma is thus transferred to the detection space as previously described, after which the color shade of the plasma can be determined (illustrated in Fig. 2c).

As shown in Fig. 2c, the user 13 can rotate the tube 2, which is still connected to the detection apparatus 3, to visually inspect the detection space 60 and the plasma therein, and thus be able to determine whether hemolysis has occurred in the blood sample 12 or not: if the plasma is yellow-orange, no hemolysis has occurred, but if the plasma is pink, hemolysis may be suspected and the blood sample 12 should be replaced with a new one. To simplify the determination of hemolysis, the apparatus may be provided with a color reference for comparison with the sample plasma, for example, showing a border color, whereby hemolysis may be suspected if the color of the plasma is darker than the reference and vice versa. Such color reference may, for example, be arranged next to the space for visual detection on the upper part of the apparatus 3.

If no hemolysis has taken place, the detection apparatus 3 is removed from the tube 2 and discarded as waste, and the tube 2 with the sample 12 can be sent for further analysis.

An application where said detection apparatus 3 is initially positioned with its needle element 1 pointing upwards may have the advantage that the transfer and separation of plasma can occur very quickly, preferably within one minute, preferably within 30 seconds, and substantially in one movement and with only one hand. Thus, the visual detection is essentially a "direct" visual detection in that it provides the result almost immediately. However, as previously described, the invention should not be limited to such use. Those skilled in the art will understand that it is also possible to apply a detection apparatus 3 to an upright collection tube, with its sealing member facing up.

Those skilled in the art will appreciate that even though a "needle element" is conventionally made of steel material, the needle element referred to in this specification should not be limited thereto. In some circumstances, it would be sufficient to have a needle in some other hard material suitable for the specific function, such as hard plastic or glass.

The detection filter 41 may be provided with chemical means for direct visual detection. Such chemical agents for visual detection can cause a color change if hemolysis has taken place, allowing for safer and more reliable test results as well as easier assessment. For example, a common method for colorimetric detection of hemoglobin is Drabkin's reagent, which consists of potassium cyanide. Other alkali cyanides and ferric cyanides

536 634 could also be used in such an analysis. Further examples of chemical agents for visual detection may include colorimetric methods utilizing the peroxidase activity of the hemoglobin, based on a chromogen such as benzidine compounds with peroxides as substrates. The chemical agents (reagents) may be deposited within the detection space 60 either in dried form or as wet reagents, or as a combination of dry and wet reagents.

The described method for detecting hemolysis with the detection apparatus 3 according to the invention can be carried out very easily, quickly and in direct connection with the taking of blood sample 12 from a patient. A user 13 needs only one hand to perform all the necessary steps to detect hemolysis; no preparation is required, and the time from the application of a test tube 2 to a detection apparatus 3 to read the result is extremely short, preferably less than 1 minute, more preferably less than 30 seconds.

Many modifications and other embodiments of the inventions presented in this preparation will become apparent to those skilled in the art to which these inventions relate using the teachings set forth in the above descriptions and the accompanying figures. Therefore, it is understood that the inventions are not to be considered limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included in the scope of the appended claims. Although specific terms are used in this preparation, they are used only in a generic and descriptive sense and not for limiting purposes.

536 634