Abstract:
A blood test instrument using a disposable cartridge and a method of measuring a blood sample using the instrument are disclosed. The instrument includes a cell counting station for counting blood cells by electrical resistance measurement, a pressure actuating component adapted to apply a pressure alternately on two flexible receptacles of a disposable cartridge removably placed in the instrument to cause flowing of a mixture of a blood sample and a liquid agent between the two receptacles to obtain proper mixing, and a conduit adapted to deliver the mixture to the cell counting station for counting. After measuring the blood sample, the instrument withdraws a washing liquid contained in another receptacle of the disposable cartridge and uses the washing liquid to clean the instrument and to deliver the mixture back to the cartridge for disposal.

Description:
CROSS REFERENCES TO RELATED APPLICATIONS 
     This application is a division of patent application Ser. No. 12/118,603, filed May 9, 2008, now U.S. Pat. No. 7,833,746, which is a division of patent application Ser. No. 10/533,790, filed May 4, 2005 now U.S. Pat. No. 7,608,223, which is the national phase of International Patent Application No. PCT/SE03/01796, filed Nov. 19, 2003, which claims priority of Sweden Patent Application No. 0203435-3, filed Nov. 20, 2002. All prior applications are herein incorporated by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention concerns a blood testing apparatus, an instrument for its operation and a method for operating the apparatus. 
     BACKGROUND OF THE INVENTION 
     When making blood tests in the field, it is a desire to perform such tests with simple but reliable apparatus that can be handled even by relatively untrained personnel. Still, there exists the requirement that a blood sample shall be taken and handled under strict hygienic conditions, and that neither the sample itself or residues thereof, nor diluting or flushing liquids used when testing the sample shall risk to be contacted by humans. Thus, there shall be no waste matter and all contaminated material shall remain within the apparatus. 
     It is known in the state of art to count blood cells by causing a volume of diluted blood sample to pass a so-called capillary, i.e., an extremely small hole, generally in a ruby, the hole having a diameter considerably larger than the size of a blood cell, typically 80 μm. A voltage is applied over the capillary, and, when a blood cell passes through the hole, the electrical resistance changes. This is because the cells can be regarded as insulators. Each change in resistance can be detected by suitable electronic equipment, and the sum of all changes detected corresponds to the number of blood cells having passed through the capillary. In order to obtain the concentration of cells in the original sample, the concentration of cells in the diluted sample is multiplied by the dilution factor, typically 1:40000 when counting of red blood cells (RBC) is concerned. It is obvious, that measuring of sample volumes and dilution liquid volumes must be performed in an accurate and repeatable way such that not only a correct degree of dilution can always be guaranteed but also a thorough and uniform mixing of the two volumes is ensured. 
     A disposable sampling device for an apparatus for counting particles contained in a liquid, such as blood cells in a blood sample, is known from WO 99/01742. This device is capable of making one diluting step. 
     A blood testing apparatus for performing dilution of a small defined volume of blood sample contained in a capillary tube is described in U.S. Pat. No. 6,284,548. The dilution involves a pre-dilution step and a final dilution step. 
     A device for diluting and mixing a liquid sample, such as a blood sample for performing a CRP test, is described in WO 01/75416. The sample is contained in a capillary tube and is mixed in a first step with a diluting agent to provide a diluted sample. In a second step, a third medium, such as antibodies, may be mixed with the diluted sample. 
     Even if some of the prior art devices are capable of making two dilutions, none of them is capable of making two simultaneous dilutions to different dilution ratios, which is desirable in order to perform, e.g., simultaneous counting of white and red blood cells. 
     A disposable apparatus for use in blood testing, having one of the present co-inventors as single inventor, is described in SE 0103877-7 filed 21 Nov. 2001 and unpublished at the date of filing the present application. It presents one solution to the problem of providing such apparatus allowing simultaneous dilution of a blood sample to two defined dilutions ratios. It is also capable of retaining all contaminated material within itself. 
     This prior apparatus comprises a block-shaped housing having a first and a second receptacle; a first and a second cylinder, each having a piston moveable therein and each containing a defined volume of a diluent; a valve including a valve body having three valve body channels extending therethrough and being positionable in three distinct positions. In one position the receptacles are put in simultaneous communication with one each of the cylinders through pairs of the channels. 
     One of the receptacles, as a first means for receiving a blood sample, is adapted to receive a blood sampling capillary tube. 
     Although fulfilling the objectives stated, this apparatus presents some inconveniences. One relates to the manufacture of the block-shaped housing, which is expensive and complicated due to its various cylinders, and makes it unsuited for injection moulding. Another relates to the use of cylinders as means for containing the diluent, and pistons movable within the cylinders to displace the diluent. It manifests itself particularly during air transportation when the pistons tend to move uncontrolled due to a varying surrounding air pressure. 
     SUMMARY OF THE INVENTION 
     The present invention has as its object to present an alternative solution to problem of providing a disposable apparatus for use in blood testing allowing simultaneous dilution of a blood sample to two defined dilutions ratios and being capable of retaining all contaminated material within itself. 
     To fulfill this object, the present invention proposes the disposable apparatus having the characterizing features, an instrument for controlling the apparatus and a method of controlling the apparatus, as defined in the appended claims. 
     According to the present invention there is provided a block-shaped housing or cartridge having depressions in at least one of its sides. The depressions form and define main portions of receptacles and channels in the housing open towards the side of the housing in which they are formed. A diaphragm is positioned over at least portions of that side to seal the respective receptacles and channels and to define one Side thereof. Portions of the diaphragm positioned over receptacles are moveable relative to the plane of the side of the housing so as to cause a variation in the pressure within the receptacle, or, in response to a pressure variation, or, a volume variation therein. The channels interconnect the various receptacles and a valve provided within the housing to control flow between the receptacles. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
       An embodiment of the invention will now be described, reference being made to the accompanying drawings, 
         FIG. 1  is a perspective view of a cartridge having receptacles and channels in one of its sides, an associated sampling tube, and a photometer arrangement; 
         FIG. 2  is a perspective longitudinal central section through the cartridge of  FIG. 1 ; 
         FIG. 2   a  is a perspective view of the capillary holder; 
         FIG. 3  is a plan view showing the cartridge in its transport position; 
         FIG. 4  is a plan view showing the cartridge in its sampling position and also showing the sampling device and a portion of a finger tip; 
         FIG. 5  is a plan view showing the cartridge in its first dilution or mixing position; 
         FIG. 6  is a plan view showing the cartridge in its second dilution or mixing position; 
         FIG. 7  is a longitudinal central section through the cartridge taken along line VII-VII in  FIG. 8 ; 
         FIG. 8  is a plan view of one half of the cartridge showing portions of the diaphragm cut away; 
         FIG. 9  is a perspective view of the valve slide; 
         FIG. 10  is a section through the cartridge taken along line X-X in  FIG. 8  showing more in detail the operation of the device; and 
         FIG. 11  is a schematic view showing the measuring step and the subsequent cleaning step. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A cartridge  20  according to the present invention is shown in perspective in  FIG. 1 . It comprises a block-shaped, preferably molded housing  21  made from a preferably translucent material. It has a generally paralellepipedic shape including opposed longer side walls  22 ,  23 , opposed shorter side walls  24 ,  25 , a bottom side  26  and a top side  27 . In the generally flat top side  27  is formed a plurality of depressions, in this embodiment six relatively large depressions defining receptacles  28 - 33 , and five relatively narrow depressions defining channels  34 - 39 . 
     A diaphragm  40  ( FIGS. 2 ,  7 ,  8  and  10 ) is sealingly attached to the top side  27  of the housing  21  so as to cover all depressions forming the receptacles and channels and to seal them relative to the environment. 
     A hole  41  extends centrally from the shorter side wall  24  towards the middle of the housing (see particularly  FIGS. 2 and 7 ). It includes an outer, relatively wide, cylindrical portion  41   a  merging by an intermediate, frustoconical portion  41   b  with an inner, relatively narrow cylindrical portion  41   c.    
     Relatively close to its exterior end, a hole  42  connects the hole portion  41   a  to one end of the channel  34 , whose opposite end opens in the receptacle  28 . 
     At the inner end of the hole  41 , a hole  43  connects its more narrow portion  41   c  to a first end of the channel  37 , whose second, opposite end connects to a hole  44   a  opening into an aperture  45  extending crosswise through the housing  21  between its longer side walls  21  and  22 . 
     The opening  41  serves for receiving a holder  46  for a capillary tube  47  in the housing  21 . The capillary holder is particularly shown in  FIGS. 1 and 2   a , and is shown sectioned in its entirely introduced state in  FIG. 7 . It comprises a body  48  fitting into the wider portion  41   a  of the hole  41  and having a frustoconical portion  48   a  matching the frustoconical portion  41   b  of the hole  41 . A cap portion  48   b  in the rear end of the body  48  abuts a recessed portion  24 ′ of the side wall  24  around the mouth of the hole  41  so as to limit the extent of introduction of the capillary holder into the hole  41  ( FIG. 7 ). A central hole  49  extending from the forward end of the capillary holder  46  receives a major portion of the capillary tube  47 , the forward, free end of which opens close to where the hole  43  opens into the narrow portion  41   c  of the hole  41 . The capillary tube  47  has a smaller outer diameter than the inner diameter of the hole portion  41   c , thus leaving an annular space  50  between the capillary tube and the wall of the hole portion  41   c . An annular groove  51  is formed in the body  48  to be located opposite to the hole  42 , and a cross-hole  52  extends diametrically therethrough to open at opposed locations in the groove  51 . The hole  52  intersects the hole  49  so as to establish communication between the annular groove  51  and the capillary tube  47  via the cross-hole  52 , and, thereby, from the receptacle  28 , through the channel  34 , the hole  42 , the groove  51 , the cross-hole  52 , the capillary tube  47 , and the hole  43  to the channel  37 . 
     As partly seen in  FIG. 1  and more clearly in  FIG. 7 , the body  48  of the capillary holder  46  has a slot  53  extending from its foremost end to the annular groove  51 , thus establishing communication between the annular space  50  and the annular groove  51 . The slot  53  also makes the capillary holder flexible so as to facilitate introduction of the capillary tube  47  therein. 
     In the aperture  45  open one respective end of the channels  35 ,  36 ,  37 ,  38  and  39 . The respective opposite end of these channels open in the receptacles  29 ,  30 ,  32  and  33 . 
     Channels  35  and  36  are aligned, as are channels  37  and  39 . 
     Channels  35  and  37  are mutually parallel, as are channels  36 ,  38  and  39 . The lateral spacing between channels  35  and  37  is equal to that between channels  36  and  39  as well as between channels  39  and  38 . 
     The aperture  45  serves to slidingly receive a valve slide  54  shown in  FIGS. 2 and 7 , and separately shown in  FIG. 9 . It comprises a parallelepipedic body having opposed parallel surfaces  54   a ,  54   b . In the surface  54   a  is formed a straight channel  55  extending in the crosswise direction of the aperture  45  and having a well-defined volume, typically 10 μl, and an L-shaped channel  56  having a first leg  56   a  extending in the crosswise direction of the aperture  45 , i.e., parallel to the straight channel  55 , and a second leg  56   b  extending perpendicularly thereto. The spacing between the channel  55  and the first leg  56   a  is equal to the lateral spacing between the channels  35 / 37  and  36 / 39 / 38 , and the length of the second leg  56   b  is equal to the spacing between the channels  36  and  38 . 
     The cross-sectional shape of the aperture  45  appears best from  FIG. 10 . It includes a central portion having parallel sides  45   a ,  45   b  for slidingly supporting the valve slide surfaces  54   a ,  54   b , respectively. The valve slide  54  is laterally guided by parallel ribs  57  protruding from the surface  45   b . In order not to make the material between the side  45   b  of the aperture and the bottom surface  26  of the body  21  flex due to introduction of the valve slide into the aperture  45 , the aperture is widened beyond the ribs  57 , and the material thickness is reduced along parallel, part-cylindrical recesses  45   c ,  45   d , thereby imparting the remaining material spring properties urging the valve slide  54  towards the surface  45   a  of the aperture  45 . Alternatively, the slide may be made with a certain degree of resilience to ensure proper sealing. 
     In the opposed longer side walls  22  and  23  of the housing are provided openings  58 ,  59 ,  60  and  61  providing access to the receptacles  31 ,  32 ,  30  and  33 , respectively. After filling of these receptacles (see below), the openings are closed by plugs that are pierceable by an injection needle or the like in order to inject a fluid into or retract a fluid from the respective receptacles of the plugs, only plugs  62  and  63  sealing receptacles  31  and  32 , respectively, are seen in  FIG. 1 . 
     The sequence of operation of the cartridge  20  will now be described with reference to  FIGS. 3-6  showing four subsequent steps. 
     In  FIG. 3  is shown the preparatory or transport position of the cartridge  20 . In advance, a well-defined volume, typically 2 ml, of a liquid diluting agent D 1  has been filled into the receptacle  33  having a capacity of typically 3 ml. Likewise, a well-defined volume, typically 2 ml, of a liquid diluting agent D 2  has been filled into the receptacle  30  also having a capacity of typically 3 ml. The diluting agents are typically an isotonic sodium chloride solution. Furthermore, a well-defined volume, typically 2 ml of a liquid haemolysis agent H has been filled into the receptacle  32 , typically having a capacity of 2 ml. (It should be noted here, that a dry haemolysis agent may be used as an alternative.) Finally, the receptacle  31 , typically having a capacity of 3 ml, is filled with a washing liquid, typically isotonic sodium chloride solution. The receptacles  29  and  28 , each typically having a capacity of 1 ml, are empty. 
     In the transport position, the capillary holder  46  is not completely introduced into the hole  41 . The valve slide  54  is in a position where the mouths of the channels  35 ,  36 ,  37 ,  38  and  39  are covered by smooth, unrecessed portions of the valve slide surface  54   a . Consequently, all these channels are closed in relation to the valve slide. 
       FIG. 4  shows the sampling position. The capillary holder has been pulled out of the hole  41 , and a blood sample S is taken with the capillary tube as illustrated in  FIG. 4 . The tube is approached to a drop of blood B formed on a punctured finger tip F, and the drop is sucked up by capillary action to completely fill the capillary tube  46  with a defined volume of sample S, typically 10 μl. After the sample is taken, the capillary tube is re-inserted into the hole  41  and pushed into its fully inserted position shown in  FIG. 7 . In that position, the rear portion of the capillary holder body  48  having a non-shown O-ring effectively seals the body  48  in the hole  41 . The valve slide is still in its original position. 
       FIG. 5  shows the first diluting or mixing step. The valve slide  54  is displaced such that one end of its cross-channel  55  communicates with the channel  37  and the other end thereof communicates with the channel  39 . Since the annular groove  52  is located opposite to the hole  42  in the fully introduced position, communication is now established between the receptacle  28  and the receptacle  33 . 
     Now, diluting agent D 1  is caused to flow from the receptacle  33  through the channel  39 , the valve channel  55 , and the channel  37  into the relatively narrow portion  41   c  of the hole  41 . There, a part of the flow is directed through the capillary tube  47 , thus displacing the blood sample S contained therein into the cross-channel  52  and the annular groove  51 . Another part of the flow is directed along the exterior of the protruding end of the capillary tube into the slot  53  and from there into the cross-channel  52  and the annular groove  51  where it meets the blood sample and mixes therewith and dilutes it. Together the two flows will end up in the receptacle  28 . The mixture is then caused to flow back along the same two paths into the receptacle  33 , where it mixes with the remainder of the diluting agent D 1  still contained therein. 
     The flow back and forth is repeated until a proper mixing is ensured. When the first mixing step is completed, a defined volume of first step diluted sample (S+D 1 ) remains within the valve slide channel  55 . This is due to the typical volume relations between the receptacles  28  and  33 , that ensures that the receptacle  33  will never be emptied. With the typical volumes stated, the dilution ratio after the first step is 1:200. 
     The second dilution and mixing step is shown in  FIG. 6 . The valve slide  54  is further displaced such that one end of its cross-channel  55  communicates with the channel  35  and the other end thereof communicates with the channel  36 , thus establishing communication between the receptacles  29  and  30 . 
     In the displacement of the valve slide, the defined volume of first stage diluted sample previously entrapped in the slide channel  55  is brought along. Simultaneously, one end of the first leg  56   a  of the L-shaped channel  56  is brought into communication with the channel  37 , one end of the second leg  56   b  is brought into communication with the channel  38 , and the common other end of the legs  56   a  and  56   b  is brought into communication with the channel  39 . Thus, there is established simultaneous communication between the receptacles  28 ,  32  and  33 . 
     The second mixing step includes two parallel parts. 
     A first part takes place between receptacles  29  and  30 . The diluting liquid D 2  in the receptacle  30  is caused to flow through the channel  36 , into the slide channel  55  displacing the entrapped volume of first stage diluted sample, through the channel  35  and into the originally empty receptacle  29 . 
     As before, the mixture is then caused to flow back and forth along the same path until a proper mixing is ensured. This first part of the second mixing step is stopped with the two step diluted sample (S+D 1 +D 2 ) remaining in the receptacle  30 , resulting, with the typical volumes stated, in a dilution ratio of 1:40000. This dilution ratio is typical for RBC testing. 
     A second part takes place between the receptacles  28 ,  32  and  33 , the receptacles  28  and  33  both containing the first step diluted sample (S+D 1 ) and the receptacle  32  containing a haemolysis agent H. The liquids are caused to flow back and forth between the three receptacles until a proper mixing is ensured. In case the haemolysis agent is dry, it will be successively dissolved during the repeated flushing of the receptacle  32 . The second part of the second mixing step is stopped with a main portion of the mixture (S+D 1 +H) remaining within the receptacle  33 . This mixture has a dilution ratio of 1:400 with the typical volumes stated, and is for white blood cell testing. 
     The housing  21  is preferably made from a translucent synthetic resin. This enables the provision of a light path  64  through the housing. A portion of the receptacle  33  is formed with a recess  65  having an accurately defined length and parallel end walls  66 ,  67 . The recess extends diagonally across a corner of the housing  21 , and the walls  22  and  25  of the housing are formed with planar wall portions  68 ,  68 ′, parallel to the respective end wall  66 ,  67 . The light path further includes a light source  69 , preferably a light diode, and a light sensor  70 . The light path enables photometric determination of certain parameters of the liquid contained in the receptacle  33 , such as, initially, a reference value of the diluting liquid and the opposed walls of the recess  65 , and then certain values of the final mixture. 
     Other tests to be performed on the diluted samples contained within the receptacles  30  and  33 , involve withdrawal of fluid from the respective receptacles. This is performed by a measurement system schematically shown in  FIG. 11 . It includes two conduits  71 ,  72 , each starting with a needle portion  73 ,  74 , respectively. The needle portions are inserted through the respective plug sealing the openings  60 ,  61  into the receptacles  30 ,  33 , respectively. Each of the conduits  71 ,  72  is provided with a cell counting station, each comprising an orifice  75 ,  76 . As is known in the art, the orifices are small apertures allowing statistically only one blood cell to pass at a time. By means of non-shown electric wires, a voltage may be applied over the orifices, and any change in the resistance across the orifices, indicating the passage of a blood cell to be counted, may be detected by suitable electronic equipment included in the system, and the sum of all resistance changes detected corresponds to the number of blood cells having passed through the orifice. 
     Each of the conduits  71 ,  72  has a branch  77 ,  78 , including a valve  79 ,  80 , and a measuring portion  81 ,  82 , respectively. 
     The measuring portions are provided with counting start detectors  81   a ,  82   a  and counting stop detectors  81   b ,  82   b  spaced defined distances. 
     The branches  77 ,  78  are joined to a common conduit  83  having a valve  84  and a pump  85  therein. A branch  86  from the conduit  83  between the branches  77 ,  78  and the valve  84  has a valve  87  and is open to the atmosphere. 
     Beyond the respective branch  77 ,  78 , the conduits  71 ,  72  have a valve  88 ,  89 , respectively, and are joined to a common conduit  90  having in its end a needle portion  91  introduced through the non-shown plug  62  into the receptacle  31  containing washing liquid. 
     In the start position of measurement, the conduits  71 ,  72 , and possibly also the conduit  90 , are filled with a liquid, typically the same isotonic sodium chloride solution as that used in receptacles  30  and  31 . The valves  88 ,  89  and  87  are closed, whereas the valves  79 ,  80  and  84  are opened. The pump  85  is started to withdraw liquid from the receptacles  30  and  33 . When the liquid originally in the conduits  71  and  72  reach the respective counting start detector  81   a ,  82   a , counting in the orifices  75 ,  76  is started. At that time, liquid from the respective receptacle  30 ,  33  has reached the orifices. 
     Counting stops when the liquid has reached the respective counting stop detector  81   b ,  82   b . At that time, liquid from the receptacles  30 ,  33  may not have reached into the branches  77 ,  78 . 
     Subsequently, the valve  84  is closed and the valve  87  is opened, whereupon liquid is caused to return to the receptacles  30 ,  33 , thereby returning the liquid within the measuring portions  81 ,  82  at least to the counting start detectors  81   a ,  82   a . Then the valves  79 ,  80  and  87  are closed, and the valves  88  and  89  are opened to perform a cleaning step. 
     In the cleaning step, more liquid is caused to enter the receptacles  30  and  33 , but is now withdrawn from the receptacle  31  at least until fresh liquid therefrom has reached both receptacles  30 ,  33 . In this position, with all possibly contaminated objects and liquids safely kept within the cartridge, the needles  73 ,  74 ,  91  are withdrawn, and the cartridge is disposed of. 
     To perform displacement of liquids from and to the various receptacles, there are various methods available, from simply pressing a finger against a portion of the diaphragm  40  over a chosen receptacle, to applying a hydraulic or pneumatic pressure over selected portions of the diaphragm. According to the present invention it is preferred to apply a vacuum over portions of the diaphragm corresponding to a selected receptacle. To perform this operation, the cartridge is placed in an instrument indicated in  FIGS. 5 and 6 , including also the measurement system shown in  FIG. 11 . In  FIG. 10 , merely a portion of the instrument is shown. It includes a plurality of vacuum domes having outlines corresponding to those of selected receptacles. A vacuum dome  92 , as a pressure actuating component, is shown to be located over the receptacle  28 . It has a rim  93  sealing against the diaphragm  40  and a tubular stem portion  94  attachable to a non-shown source of vacuum. A vacuum applied in the dome  92  causes the diaphragm  40  to strive to adopt the upwardly convex shape illustrated at  40   a , thus sucking liquid into the receptacle  28  from, e.g., the receptacle  33  in the position according to  FIG. 5 . A vacuum applied to the corresponding vacuum dome associated with the receptacle  33  causes the liquid to be withdrawn from the receptacle  28  and the diaphragm to assume the shape indicated at  40   b . Evidently, the same shape would result from an overpressure within the vacuum dome. 
     Although a slide valve has been described herein, it is obvious that other kinds of valves may be used, such as primarily a turning valve. 
     Also, it is within the scope of the present invention to provide an apparatus having depressions defining receptacles and channels in more than one of its sides, e.g., in two opposite sides, and a valve between these sides.