Abstract:
An apparatus and method are provided for preparing specimens of biological cells uniformly distributed over a substrate surface. The method comprises providing a suspension of biological cells in fluid, and then extracting biological target cells from the suspension to leave debris and contaminants behind. The extracted target cells are distributed over a substrate surface into a primary layer of cells coupled to the substrate surface with the remaining cells forming a secondary layer on top of the primary layer. The target cells forming the secondary layer are then removed to produce a substantially non-overlapping layer of target cells coupled to the substrate surface.

Description:
This application is a continuation of our co-pending International Patent Application No. PCT/CA98/00501 filed May 21, 1998, and claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/047,482, filed May 23, 1997. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a method for preparing a specimen from a cellular suspension of biological cells. In particular, the invention relates to a method for preparing a specimen comprising a uniform distribution of biological cells on a substrate surface. 
     BACKGROUND OF THE INVENTION 
     The collection or preservation of biological cells in fluid suspension is common in medicine and biology for the detection of disease. For example, naturally voided urine contains urothelial cells from the lining of the bladder. If the urothelial cells are separated from the urine and then placed on a substrate surface, such as a microscope slide, examination of the cells can determine the presence or absence of certain diseases. Another example is the PAP Smear Test which involves the artificial exfoliation of epithelial cells from the cervix of the uterus and the subsequent suspension of the exfoliated epithelial cells in a water/alcohol solution to preserve and protect the cells. If the epithelial cells are separated from the solution and then deposited on a microscope slide, examination of the cells can determine the presence or absence of precancerous lesions on the cervix. 
     However, current techniques for the preparation of specimens from cellular suspensions are deficient since the cellular suspensions may contain debris and contaminants which can interfere with the examination of the desired (“target”) cells. For instance, in the case of cervical epithelial specimens, the contaminants may include leukocytes, erythrocytes, bacteria and mucus. In addition, the typical specimen may contain several layers of cells and/or the cells may overlap one another, thereby rendering the detection of cell abnormalities difficult. Another reason is that, for the Pap test or indeed any other type of test requiring an exfoliation instrument, the technique of transferring the collected cells from the exfoliation instrument to the glass slide can be very inefficient. In some studies it has been shown that less than 20% of the collected sample is effectively transferred. By contrast, a liquid-based specimen allows, as a preliminary step, all of the collected cells to be rinsed or washed off of the exfoliation instrument into the collection fluid thereby improving specimen recovery and aiding in subsequent diagnostic accuracy. 
     Accordingly, there is a need for a method for preparing specimens from cellular suspensions which enhances the ease and accuracy of evaluation of biological cells for abnormalities. 
     SUMMARY OF THE INVENTION 
     According to the present invention, there is provided a method and apparatus for preparing specimens from cellular suspensions which enhances the specimen recovery as well as the ease and accuracy of evaluation of biological target cells for abnormalities. 
     The method comprises the steps of providing a suspension of biological cells in fluid wherein said cells include biological target cells; extracting a portion of the biological target cells from the suspension; distributing the extracted portion over a substrate surface into a primary layer with the remaining extracted biological target cells being located on top of said primary layer; and removing the plurality of cells located on top of the primary layer. 
     Specimens prepared according to the above method are substantially free of debris and contaminants. In addition, the cells comprising such specimens are densely packed into a single layer with little cell overlap. Therefore, the ease and accuracy of detection of cell abnormalities is greatly enhanced. 
     In another aspect, the present invention provides an apparatus for preparing a biological cellular specimen on the surface of a substrate, said apparatus comprising: a filter tube including an outlet port and a porous filter medium coupled to the outlet port; a tubular member having an interior region and a pair of opposite ends, and including means for receiving said filter tube within said interior region; and means for sealingly coupling the surface of said substrate to one of the ends of said tubular member. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     Reference will now be made to the accompanying drawings, which show by way of example, a preferred embodiment of the present invention, and in which: 
     FIGS.  1 ( a ) and  1 ( b ) are schematic views of an apparatus for performing the cellular suspension method according to the present invention; 
     FIG. 2 is a schematic view of a filter tube for performing the extraction step and the distribution step; 
     FIG. 3 is a schematic view of the filter tube shown in FIG. 2, immersed in a cellular suspension; 
     FIG. 4 is a magnified schematic view of the filter tube during the extraction step, showing the biological target cells and the debris in suspension, and the membrane filter; 
     FIGS.  5 ( a ) to  5 ( c ) are magnified schematic views of the filter tube during a back-flushing stage of the extraction step, showing the biological target cells in suspension and the membrane filter; 
     FIG.  5 ( d ) is a schematic view of the filter tube being subjected to mechanical agitation during the extraction step; 
     FIG.  5 ( e ) is a schematic view of the filter tube being subjected to sonic agitation during the extraction step; 
     FIG. 6 is a magnified schematic view of the filter tube at the end of the extraction step; 
     FIGS.  7 ( a ) to  7 ( b ) are magnified schematic views of one variation of the filter tube and substrate; 
     FIG. 8 is a magnified schematic view of the filter tube and substrate during the distribution step; 
     FIG. 9 is a magnified schematic view of the filter tube and substrate at the end of the distribution step; 
     FIGS.  10 ( a ) to  10 ( b ) are magnified schematic views of a variation of the filter tube and substrate shown in FIG. 7; 
     FIG. 11 is a magnified schematic view of the biological target cells at the end of the distribution step, showing the primary and secondary layers; 
     FIG. 12 is a magnified schematic view of the biological target cells during the removal step; 
     FIG. 13 is a magnified schematic view of the biological target cells and the substrate surface at the end of the removal step; 
     FIG. 14 is a specimen of cervical epithelial cells prepared according to the invention; and 
     FIG. 15 is a flow chart showing the method steps for preparing a specimen from a cellular suspension according to the present invention. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Reference is first made to FIG.  1 ( a ) which shows a collection vessel  10  for providing a suspension of biological cells in accordance with the present invention. The collection vessel  10  is filled with a preservation fluid or medium  12 . The preservation medium  12  comprises water and alcohol, or other known anti-microbial compounds. Biological cells for the cellular suspension are introduced into the collection vessel  10  using a cell collection implement  14 . The cell collection implement  14  contains biological cells obtained through an artificial cellular exfoliation procedure, and is inserted into the preservation fluid  12  in the collection vessel  10 . For example, the collection implement  14  may contain uterine cervical epithelial cells obtained through a PAP Smear. The exfoliated cells are rinsed from the collection implement  14  into the preservation fluid  12 , which preserves the cells until a specimen is prepared as will be discussed below. 
     As shown in FIG.  1 ( a ), the collection vessel  10  includes a catch basket  16  to allow the technician to easily remove the collection implement  14  from the vessel  10 . 
     In one variation, as shown in FIG.  1 ( b ), the collection implement  14  is replaced with a cell collection vial  18 . The collection vial  18  contains naturally exfoliated cells which are obtained through a natural exfoliation process. For example, the collection vial  18  may include urothelial cells filtered from naturally voided urine. The collection vial  18  is inserted into the collection vessel  10 , and the exfoliated cells in the collection vial  18  are rinsed into the preservation fluid  12 . The preservation fluid  12  preserves the exfoliated cells until a specimen can be prepared. 
     After rinsing the exfoliated cells into the preservation fluid  12  in the collection vessel  10 , the next step involves disaggregating the cells in order to place the cells in suspension in the preservation fluid  12 . During the disaggregation step, the collection vessel  10  undergoes vortex motion in which the collection vessel  10  is repeatedly rotationally accelerated and decelerated about the longitudinal axis of the collection vessel  10  to break apart any randomly-bonded cellular groups or cell clusters. The disaggregation step continues until a desired level of cellular suspension has been obtained. 
     After the disaggregation step, a filter tube  20  as shown in FIG. 2 is used to extract the desired (“target”) biological cells from the suspension of cells. The filter tube  20  comprises a tubular member  22  having an exterior outer surface  24  (which defines an interior cylindrical volume  26 ) and a pair of open opposite ends  28 ,  30 . A disc-shaped membrane filter  32  seals one end  28  of the tubular member  22 . As shown in FIG. 2, the membrane filter  32  includes a plurality of pores. The pores are larger that the debris, mucus and contaminants which may be present in the preservation fluid  12  but smaller than the biological target cells so that the debris, mucus and contaminants are allowed to pass through the membrane filter  32 , while the biological target cells are not. 
     The other end  30  of the tubular member  22  includes a disc-shaped lid  34  which seals the end of the member  22 . The lid  34  includes outlet ports  36 ,  38  which communicate with the interior of the tubular member  22 . The outlet port  36  is coupled to a suction tube  40  through a valve  44 . Similarly, the other outlet port  38  is coupled to a drain/fill tube  42  through another valve  46 . The valves  44 ,  46  allow the respective tubes  40 ,  42  to be selectively opened and closed. 
     During the disaggregation step, the tubular member  22  is inserted into the collection vessel  10  with the end  28  containing the membrane filter  32  immersed in the preservation fluid  12 , as shown in FIG.  3 . The suction tube  40  is coupled to a vacuum/pressure source (not shown) and the valves  44 ,  46  are closed. At this point, the vacuum/pressure source is activated and the valve  44  to the suction tube  40  opened so as to create a vacuum inside the interior of the tubular member  22  and a pressure gradient across the membrane filter  32 . This causes the preservation fluid  12  to be drawn through the membrane filter  32  and up into the interior of the tubular member  22 . As depicted in FIGS. 3 and 4, the membrane filter  32  comprises a series of pores  33  which are smaller than the biological target cells (indicated by reference  100 ), but larger than the debris, mucus and other contaminants (indicated by reference  102 ) which may be present and suspended in the preservation fluid  12 . As a result, the membrane filter  32  allows the debris, mucus and other contaminants to pass as indicated by arrow A (FIG. 4) while trapping the biological target cells  100 . 
     The next step involves extracting the biological target cells  100 . The density and number of biological target cells  100  trapped on the membrane filter  32  is controlled by varying the vacuum inside the tubular member  22 . Preferably, the vacuum inside the tubular member  22  is controlled so that the biological target cells  100  are distributed uniformly over the surface of the membrane filter  32 . However, debris, mucus and other contaminants  102  contained in the preservation fluid  12  may also become trapped, or “caked” on the membrane filter  32  during the extraction step, together with the biological target cells  100 . To minimize the collection of debris  102  on the membrane filter  32 , the flow rate of preservation fluid  12  across the membrane filter  32  is kept relatively low by maintaining a low vacuum inside the tubular member  22 , to produce a flow rate in the range of 5 to 500 microliters per second. 
     Despite the maintenance of a low flow rate of preservation fluid  12  through the membrane  32 , it is still possible that some debris  102  will become caked on the membrane filter  32 . According to one variation of the extraction step, the tubular member  22  undergoes mechanical or sonic agitation while the preservation fluid  12  is slowly drawn across the membrane filter  32 . The mechanical or sonic agitation loosens the trapped debris  102  from the membrane filter  32 , thereby allowing the debris  102  to more readily pass through the membrane filter  32 . Advantageously, the mechanical or sonic agitation also allows the biological target cells  100  to be more uniformly distributed over the surface of the membrane filter  32 . The application of mechanical agitation to the filter tube  20  is depicted in FIG.  5 ( d ), while the application of sonic agitation to the filter tube  20  is depicted in FIG.  5 ( e ). 
     Although the low flow rate of preservation fluid  12  allows an automated control system to control the density and number of biological target cells  100  accumulated on the membrane filter  32  while minimizing the collection of debris  102  on the membrane filter  32 , the low flow rate also increases the duration of the extraction step. In another variation of the extraction step, the flow rate of preservation fluid  12  across the membrane filter  32  is kept relatively high by maintaining a high vacuum inside the tubular member  22  (depicted in FIG.  5 ( a )). While the high flow rate shortens the duration of the extraction step, it also increases the amount of caking on the membrane filter  32 . To reduce the accumulation of debris  102  on the membrane filter  32 , the rapid draw of preservation fluid  12  across the membrane filter  32  is momentarily interrupted with a back flushing step (depicted in FIG.  5 ( b )). 
     During the back flushing step, the valve  44  to the suction tube  40  is momentarily closed and the valve  46  to the drain/fill tube  42  is momentarily opened. This equalizes the pressure in the tubular member  22  with the atmospheric pressure and allows the preservation fluid  12  in the tubular member  22  to drain out through the membrane filter  32 , as indicated by arrows B in FIG.  5 ( b ). Since the direction of flow of the preservation fluid  12  across the membrane filter  32  during the back flushing step is directly opposite to the direction of flow of preservation fluid  12  prior to the back flushing step, the debris  102  and biological target cells  100  trapped on the membrane filter  32  are released from the membrane filter  32  and become substantially uniformly distributed in the preservation fluid  12 . 
     After the “back flushing step”, the valve  46  to the drain/fill tube  42  is closed and the valve  44  to the suction tube  40  is re-opened to re-establish the high vacuum inside the tubular member  22  and the high flow rate of preservation fluid  12  across the membrane filter  32  (depicted in FIG. 5 c ). Since the back flushing step substantially uniformly distributes the biological target cells  100  throughout the preservation fluid  12 , the biological target cells  100  become substantially uniformly distributed over the surface of the membrane filter  32  as also depicted in FIG.  5 ( c ). 
     The draw of the preservation fluid  12  across the membrane filter  32  continues for a fixed period of time until a desired density and number of biological target cells  100  on the membrane filter  32  is reached. At this point, the valve  44  to the suction tube  40  is closed to keep the biological target cells  100  trapped on the membrane filter  32 . Alternately, since the resistance to fluid flow across the membrane filter  32  is a function of the density and number of biological target cells  100  trapped on the membrane filter  32 , the fluid flow resistance across the membrane filter  32  is continuously monitored. When the fluid flow resistance reaches a threshold level, indicative of the desired density and number of biological target cells  100 , further draw of preservation fluid  12  across the membrane filter  32  is terminated by closing the valve  44 . 
     With the biological target cells  100  substantially uniformly distributed over the surface of the membrane filter  32 , the extraction step is completed by removing the filter end  28  of the tubular member  22  from the collection vessel  10 . The biological target cells are retained on the membrane filter  32  due to the pressure gradient maintained across the membrane filter  32 . The tubular member  22  is then inverted, as shown in FIG. 6, and the valve  46  to the drain/fill tube  42  is opened to allow any preservation fluid  12  remaining in the tubular member  22  to drain out the drain/fill tube  42  in the direction of the arrow C. The valve  46  is then closed, and the tubular member  22  inverted in preparation for a biological target cell distribution step. 
     The biological target cell distribution step comprises bringing the tubular member  22  in close proximity to a substrate  48 , as shown in FIG.  7 ( a ). The substrate  48  comprises a microscope slide  50  having an upper substrate surface  52  and a settling chamber  54 . The settling chamber  54  is tubular in shape and includes an interior region  56  bounded by an inner cylindrical wall  58 , a lower end face  60 , and a first resilient O-ring  62  secured to the inner tubular wall  58 . A second resilient O-ring  64  is permanently secured to the lower end face  60  and the upper substrate surface  52  so as to provide a leak resistant seal between the lower end face  60  and the upper substrate surface  52 . 
     As shown in FIG.  7 ( b ), the settling chamber  54  receives the tubular member  22  inside the interior region  56 . As part of the biological cell distribution step, the tubular member  22  is inserted into the interior region  56  so that the first O-ring  62  seals the outer tubular surface  24  of the tubular member  22  to the settling chamber  54 . The tubular member  22  is moved towards the microscope slide  50  until the end  28  of the member  22  is proximate to the upper substrate surface  52 . 
     Next, the valve  46  to the drain/fill tube  42  is opened and fluid., such as the preservation fluid  12 , introduced into the interior  24  of the tubular member  22  through the drain/fill tube  42 , in the direction of the arrow D as depicted in FIG.  8 . The biological target cells  100  are detached, or washed, from the membrane filter  32  and placed in fluid suspension in the interior region  56  between the upper substrate surface  52  and the end  28  of the tubular member  22 . 
     Referring next to FIG. 9, the biological target cells  100  are allowed to settle onto the upper substrate surface  52  of the slide  52  under the influence of gravity. Since the biological target cells  100  are substantially uniformly distributed over the surface of the membrane filter  32  prior to the distribution step, the biological target cells  100  will also be substantially uniformly distributed over the upper substrate surface  52 . To enhance the uniform distribution of the biological target cells  100 , the settling chamber  54  is gently agitated during the distribution step in a further variation. 
     In another variation of the distribution step, a filter tube denoted by  120  in FIG.  10 ( a ) is used to extract the target biological cells  100  during the extraction step. The filter tube  120  is identical to the filter tube  20  (FIG. 7) except that the first resilient O-ring  62  is permanently secured to the outer tubular surface  26  of the tubular member  22  and the inner tubular wall  58  of the settling chamber  54  so as to provide a leak resistant seal between the tubular member  22  and the settling chamber  54 . As shown in FIG.  10 ( b ), the second resilient O-ring  64 , provided at the lower end face  60  of the settling chamber  54 , is pressed against the upper substrate surface  52  of the microscope slide  50  so as to provide a leak resistant seal between the lower end face  60  and the upper substrate surface  52 . The biological target cells  100  are then washed onto the upper substrate surface  52  as described above. 
     As shown in FIG. 11, by the end of the distribution step the biological target cells  100  will be distributed over the upper substrate surface  52  into a primary layer  66  and at least one secondary layer  68 . The primary layer  66  is located immediately adjacent the upper substrate surface  52  and the secondary layer  68  located above the primary layer  66  as shown in FIG.  11 . Due to the proximity of the primary layer  66  with the upper substrate surface  52 , the biological target cells  100  located in the primary layer  66  become attached to the upper substrate surface  52 . 
     Referring to FIG. 12, the next step comprises a cellular removal step. The cellular removal step comprises spraying the biological target cells  100  with a fluid stream  12 ′, such as the preservation fluid, as shown in FIG.  12 . Since the biological target cells  100  located in the primary layer  66  form a stronger bond with the upper substrate surface  52  than do the biological target cells  100  located in the secondary layers  68 , the biological target cells  100  in the secondary layers  68  will be washed away while the biological target cells  100  in the primary layer  66  will remain attached to the upper substrate surface  52  as shown in FIG.  13 . 
     In one variation of the above cellular removal step, the upper substrate surface  52  is provided with a polymer layer  53  (FIG.  13 ), such as poly-L-lysine, which improves the strength of attachment between the upper substrate surface  52  and the biological target cells  100  in the primary layer  62 . In another variation, the biological target cells  100  in the secondary layers  68  are removed by immersing the substrates  48  in a fluid, such as the preservation fluid  12 , and then agitating the substrate  48  in the fluid. 
     Reference is next made to FIG. 14 which shows a cervical epithelial cell specimen  111  prepared according to the described invention, using a microscope slide  50  coated with poly-L-lysine polymer. Advantageously, the specimen  111  comprises a single layer of biological target cells which do not overlap and are ready for subsequent preparation steps, such as staining, cover-slipping etc. For example, the specimen  111  depicted in FIG. 14 represents the distribution and density of cervical epithelial cells taken from the uterine cervix and subjected to the standard Papanicolaou protocol (i.e. PAP test). 
     Reference is next made to FIG. 15 which shows a flow chart summarizing the method steps for preparing a specimen from a cellular suspension according to the present invention. The first step in block  201  comprises introducing the biological cells  10  in a preservation fluid  12  (FIG.  1 ). The second step in block  202  comprises disaggregating the cells to put them into suspension in the preservation fluid  12 . The third step in block  203  involves extracting the target biological cells  100  (FIG. 5) from the suspension of cells. The extraction step  203  may include the application of mechanical or sonic agitation (block  203   a  shown in broken outline) to reduce “caking” of cells on the membrane filter  32  (FIG.  2 ). The next step in block  204  involves back flushing to reduce the accumulation of debris on the membrane filter  32 . The back flushing step  204  involves momentarily interrupting the draw of preservation fluid through the membrane filter  32 . The next step in block  205  involves continuing the draw of preservation fluid  12  through the membrane filter  32  until the desired density and number of target biological cells  100  are accumulated on the membrane filter  32 . The next step in block  206  comprises distributing the target biological cells from the membrane filter  32  to a substrate  50  (FIG.  7 ). The last principle step in block  207  comprises a cellular removal step in which the secondary layer(s)  68  (FIG. 11) of cells  100  on top of the primary layer  66  on the substrate  50  are removed. The cells  100  in the secondary layer  68  are washed away so that the target cells  100  in the primary layer  66  remain attached to the substrate  50 . 
     The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the presently discussed embodiments are considered to be illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.