Abstract:
A biological specimen preparation system for processing and depositing a portion of a biological specimen from a specimen vial onto a slide. The biological specimen preparation specimen includes a specimen processing unit and an external actuation unit. The specimen vial is connected to an input port on the specimen processing unit and the slide is coupled to an output port. A portion of the biological specimen is injected into the specimen processing unit and prepared by sequentially passing the biological specimen under the control of the external actuation unit through a number of specimen conditioning chambers and a specimen enrichment chamber in the specimen processing unit. The specimen is then deposited on the slide through the output port. To prevent sample to sample contamination, the specimen processing unit is manufactured as a disposable component using injection moulding techniques.

Description:
FIELD OF THE INVENTION 
     The present invention relates to a system for preparing a specimen from a cellular suspension of biological cells. In particular, the invention relates to apparatus and 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 purpose of detecting 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 pre-cancerous 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 specimen samples, the contaminants may include leukocytes, erythrocytes, bacteria and mucus. In addition, the typical specimen sample 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. 
     With the exception of a membrane filter tube, all components used in the preparation of a biological specimen are reusable. This gives rise to the possibility of sample-to-sample contamination which in the context of medical applications cannot be ignored. 
     Accordingly, there remains a need for an apparatus and method for preparing specimens from cellular suspensions which enhances the ease and accuracy of evaluation of biological cells for abnormalities, and which eliminates the potential for sample-to-sample contamination. 
     SUMMARY OF THE INVENTION 
     According to the present invention, there is provided an apparatus and a method for preparing biological specimens from cellular suspensions which enhances the specimen recovery as well as the ease and accuracy of evaluation of biological target cells for abnormalities. In another aspect, the apparatus is designed to be disposable so as to eliminate the possibility of sample-to-sample contamination. 
     The biological specimen preparation system features an integrated processing unit or IPU. Advantageously, the integrated processing unit according to the present invention is inexpensive and easy to manufacture, using known injection-molded techniques. The design of the IPU also features the elimination of moving parts. The IPU is externally actuated wherein the motive force for the movement and distribution of fluids during the processing of the specimen is supplied externally. 
     In a first aspect, the present invention provides a biological specimen preparation system for depositing a portion of a biological specimen on a slide wherein the biological specimen is held in a vial, the biological specimen preparation system comprises: (a) a specimen processing unit having an input port for coupling to the vial containing the biological specimen and including a specimen conditioning chamber, and a specimen output port for depositing the conditioned specimen on the slide, an input channel connecting the input port to the specimen conditioning chamber and the specimen conditioning chamber having an output coupled to the specimen output port through an output channel, a first flow regulator for regulating the flow of the biological specimen through said input channel, and a second flow regulator for regulating the flow of the biological specimen through the output channel; (b) an actuation module for controlling the movement of the biological specimen in the specimen processing unit, the actuation module including an injector for injecting a portion of the biological specimen from said vial into the specimen conditioning chamber, a first actuator for actuating the first flow regulator and a second actuator for actuating the second flow regulator. 
     In another aspect, the present invention provides a method for preparing a biological specimen in a disposable specimen processing unit, the disposable specimen processing unit including an input port for coupling to a vial containing the biological specimen, and output port for coupling to a slide, the method comprising the steps of: (a) injecting a portion of the biological specimen from the vial to a first specimen conditioning chamber in the disposable specimen processing unit; (b) moving the biological specimen from the first specimen conditioning chamber to at least another specimen conditioning chamber in the disposable specimen processing unit for further conditioning; (c) moving the biological specimen form the last specimen conditioning chamber to a specimen enrichment chamber in the disposable specimen processing unit; (d) enriching the biological specimen in the specimen enrichment chamber by passing the biological specimen over a filter to remove contaminants from the biological specimen; (e) moving the enriched biological specimen from the specimen enrichment chamber to the output port for deposition on the slide. 
    
    
     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: 
     FIG. 1 shows in diagrammatic form a biological specimen preparation system according to the present invention; 
     FIG. 2 shows the specimen preparation system of FIG. 1 in an exploded view; 
     FIG. 3 shows in more detail the external actuation unit for the specimen preparation system of FIG. 1; and 
     FIG. 4 shows in more detail the integrated processing unit for the specimen preparation system of FIG.  1 . 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Reference is first made to FIG. 1 which shows a biological specimen preparation system according to the present invention and denoted generally by reference  10 . The specimen preparation system  10  provides an apparatus for extracting a portion of a biological sample or specimen contained in a specimen vial and depositing the biological sample on microscope slide for further analysis. 
     As shown in FIG. 1, the integrated specimen processing system  10  comprises an external actuation module (or EAS)  12 , an integrated processing unit (or IPU)  14 , a specimen vial  16  and a slide  18 . The integrated processing system  10  is shown in an exploded view in FIG.  2 . In the drawings like reference numerals indicate like elements. 
     As will be described in more detail below, the integrated processing unit or IPU  14  comprises air and fluid couplings which provide both motive force for moving the biological specimen and the capability for diluting the sample before placement on the microscope slide  18 . A portion of the biological specimen is extracted from the specimen vial  16  and moved through a series of specimen conditioning chambers and a specimen enrichment chamber before being deposited on the microscope slide  18 . As will also be described, the integrated processing unit (or IPU)  14  includes a series of pinch valves which control the fluid distribution within the IPU  14 . The pinch valves are activated by the external actuation system  12 . 
     Referring to FIG. 1, the specimen vial  16  and the microscope slide  18  are coupled to the integrated processing unit or IPU  14 . The IPU  14  is then mated to the external actuation module  12  to form the biological specimen preparation system  10 . 
     Reference is made to FIG. 3 which shows the external actuation system module (or EAS)  12 . The external actuation system module  12  is designed to be a reusable component in the biological specimen preparation system  10  according to the present invention. The external actuation system module  12  comprises a support member  20 , fluid couplers  22 , push rods  24 , and a measurement opening  26 . The external actuation module  12  is coupled to the integrated processing unit (or IPU)  14 . The fluid couplers  22 , shown individually as  22   a  and  22   b , supply differential air pressure streams which provide a motive force for moving a portion of the biological specimen from the specimen vial  16  into the integrated processing unit  16 . The push rods  24 , shown individually as  24   a ,  24   b ,  24   c , control the passage of the biological specimen through the specimen conditioning chambers  38 , the specimen enrichment chamber  40 , and the specimen settling chamber  36  as will be described in more detail below with reference to FIG.  4 . The measurement opening  26  provides a window for monitoring the flow of the fluid containing the specimen during the preparation process in the IPU  14 . The measurement opening  26  is also used for measuring the density of the specimen. 
     Reference is next made to FIG. 4 which shows the integrated processing unit  14  in more detail. As shown, the integrated processing unit  14  comprises a top member  30 , a bottom member  31 , and a flexible sheet member  32 . 
     The flexible sheet member  32  is sandwiched between the top  30  and bottom  31  members. The bottom member  31  includes a specimen vial port  34  for coupling the specimen vial  16 . The bottom member  31  also includes a settling chamber  36  for depositing the processed biological specimen on the microscope slide  18 . As shown in FIG. 4, the integrated processing unit  14  includes a series of specimen conditioning chambers  38 , shown individually as  38   a ,  38   b  and  38   c , and a specimen enrichment chamber  40 . The specimen conditioning chambers  38  provide a volumetric area for agitating and settling the biological specimen and also for diluting the specimen. The first specimen conditioning chamber  38   a  receives the initial portion of the biological specimen which is injected from the specimen vial  16 . The first specimen conditioning chamber  38   a  is coupled to the specimen vial port  34  through a channel  37   a . The biological specimen moves between the first specimen conditioning chamber  38   a  and the second specimen conditioning chamber  38   b  via a channel  37   b . Similarly, the processed biological specimen moves between the second specimen conditioning chamber  38   b  and the third specimen conditioning chamber  38   c  via channel  37   c . The output of the third specimen conditioning chamber  38   c  is coupled to the specimen enrichment chamber  40  via channel  39 . The output of the specimen enrichment chamber  40  is coupled to the settling chamber  36  by a channel  41 . Preferably, the specimen conditioning chambers  38 , the specimen enrichment chamber  40 , the specimen settling chamber  36 , and the respective channels  37 ,  39 ,  41  are formed into the bottom member  31  of the integrated processing unit  14 . 
     The loading of a portion of the biological sample from the specimen vial  16  into the first specimen conditioning chamber  38   a  and the subsequent movement of the biological specimen between the conditioning chambers  38  and the enrichment chamber  40  and the settling chamber  36  is achieved by a sequential actuation of the fluid couplers  22  and the push rods  24  to deflect the flexible sheet member  32 . Preferably, the sequential activation is performed under the control of a programmable microprocessor-based interface. 
     As shown in FIG. 4, the top member  30  includes openings  50 , shown individually as  50   a  and  50   b , for the respective fluid couplers  22   a  and  22   b  (FIG. 3) on the external actuation module  12 . The flexible sheet member  32  also includes respective openings  60 , shown individually as  60   a  and  60   b , which register with the openings  50   a  and  50   b  and allow the fluid couplers  22   a  and  22   b  to communicate with the specimen vial port  34  on the bottom member  31  of the integrated processing unit  14 . The top member  30  also includes openings  52 , shown individually as  52   a ,  52   b  and  52   c , for the respective push rods  24   a ,  24   b  and  24   c  (FIG. 3) on the external actuation module  12 . The openings  52  allow the push rods  52  to move up and down against the flexible sheet member  32  and deflect regions of the sheet member  32 . The opening  52   a  is associated with the channel  37   a  which couples the specimen vial port  34  to the input of the first specimen conditioning chamber  38   a  and deflection of the flexible sheet member over the channel  37   a  by the push rod  24   a  controls the injection of the biological specimen from the specimen vial  16  into the first specimen conditioning chamber  38   a . The second opening  52   b  is associated with the channel  39  which couples the input of the specimen enrichment chamber  40  to the output of the last specimen conditioning chamber  38   c . Deflection of flexible sheet member  31  against the channel  39  by the push rod  24   b  controls the passage of the biological specimen from the conditioning chamber  38   c  to the enrichment chamber  40 . The third opening  52   c  is associated with the channel  41  which connects the output of the enrichment chamber  40  to the input of the specimen settling chamber  36 . Deflection of the flexible sheet member  31  by movement of the push rod  24   c  through the opening  52   c  controls the passage of the biological specimen from the specimen enrichment chamber  40  into the specimen settling chamber  36  and onto the microscope slide  18 . The push rods  24  are sequentially actuated to move the biological specimen between the specimen conditioning chambers  38 , the enrichment chamber  40  and the settling chamber  36 . 
     As shown in FIG. 4, the top member  30  of the integrated processing unit  14  also includes a specimen enrichment chamber  54  having a membrane filter. The specimen enrichment chamber  54  is coupled to the specimen enrichment port  40  and provides a mechanism for enriching the biological specimen. The specimen enrichment chamber  54  includes a disc-shaped membrane filter  56  (shown in broken outline inside the chamber  54 ). The membrane filter  56  registers with the specimen enrichment port  40  and includes a plurality of ports. The ports are larger than the debris, mucus and other contaminants which may be present in the fluid sample containing the biological specimen, but smaller than the biological target cells, so that the debris, mucus and contaminants are allowed to pass through the membrane filter  56 , while the biological target cells are retained in specimen enrichment port  40 . The biological specimen is enriched by increasing the number of biological target cells through the removal of the debris, mucus and contaminants. In one embodiment, the specimen enrichment chamber  54  is made from a transparent material so that a specimen density measurement can be made by the EAS  12 . In another embodiment, the specimen density is pre-determined through an optical turbidity measurement which is taken with the biological specimen in the specimen vial  16 . Once sufficient specimen density is reached, the push rod  24   c  is released to open the output channel  41  and allow the specimen to move from the specimen enrichment chamber  54  to the settling chamber  36  and onto the slide  18 . 
     The biological specimen contained in the specimen vial  16  comprises a preservation fluid (e.g. water and alcohol, or other known anti-microbial compounds) and biological cells obtained through an artificial cellular exfoliation procedure. For example, the biological cells may comprise uterine cervical epithelial cells obtained through the well-known PAP smear. The preservation fluid preserves the exfoliated cells until a specimen can be processed and deposited on the microscope slide  18 . 
     The biological target cells moved to the specimen settling chamber  36  from the specimen enrichment chamber  40  via the channel  41  are allowed to settle onto the upper substrate surface of the microscope slide  18  under the influence of gravity. Since the biological target cells are substantially uniformly distributed over the surface of the membrane filter  56 , the biological target cells will also be substantially uniformly distributed over the upper substrate of the microscope slide  18 . Preferably, the upper substrate of the slide  18  is provided with a polymer layer, such as poly-L-lysine, which improves the strength of attachment between the upper substrate surface of the slide  18  and the biological target cells received from the specimen enrichment chamber  40 . Advantageously, the finished microscope slide  18  comprises a single layer of biological target cells which do not overlap and are ready for subsequent preparation steps, such as staining according to the well-known Papanicolaou test, cover-slipping, etc. 
     In operation, the external activation module  14  provides a differential air pressure through the fluid couplers  22   a ,  22   b  to place the biological specimen in the specimen vial  16  into suspension and to drive it into the first specimen conditioning chamber  38   a  via the input channel  37   a . The push rod  24   a  controls injection of the biological specimen into the first specimen conditioning chamber  38   a  by pinching and opening the input channel  37   a . The biological specimen is then directed through the other specimen conditioning chambers  38   b  and  38   c . Each specimen chamber  38  provides a volumetric area for controlling the flow of the specimen through the operation of the fluid couplers  22   a ,  22   b  and the push rod  24   a . After the third specimen conditioning chamber  38   c , the biological specimen is directed to the specimen enrichment chamber  54  through the channel  39  which is pinched and closed by the operation of the push rod  24   b . At the specimen enrichment chamber  54 , the specimen is enriched through a succession of fluid motions (under the control of the fluid couplers  22   a ,  22   b ) which drive the specimen across the membrane filter  56 . Since the openings in the membrane filter  56  are larger that the debris and mucus present in the fluid sample containing the specimen, the debris and mucus pass through the filter  56 , whereas the larger biological target cells are retained in the specimen enrichment port  40 . The biological specimen is enriched by increasing the number of fluid motions to increase the debris, mucus and other contaminants from the fluid sample. Before the specimen is passed to the settling chamber  36  for deposition on the slide  18 , the specimen density needs to be determined to ensure the density is sufficient. The density of specimen may be measured by the external actuation system module  12  through the specimen enrichment chamber  54 . In another embodiment, the specimen density is determined by making an optical turbidity measurement of the biological specimen in the specimen vial  16 . The optical turbidity of the specimen is related to the density of cells in the specimen. By knowing the optical turbidity, a specified volume of the specimen is measured out from the specimen vial  16  to provide a specimen with the required cell density. Once the required cell density for the specimen is attained, the push rod  24   c  is actuated to open the channel  41  and allow the specimen to flow into the settling chamber  36  and onto the surface of the slide  18 . 
     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.