Patent Publication Number: US-10775282-B2

Title: Automated systems and methods for preparing biological specimens for examination

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional application of and claims priority under 35 U.S.C. § 121 to U.S. patent application Ser. No. 14/833,800, filed on Aug. 24, 2015, which is a continuation application of U.S. patent application Ser. No. 13/858,531, filed on Apr. 8, 2013, now U.S. Pat. No. 9,116,087, which is a divisional application of U.S. patent application Ser. No. 13/293,050, filed on Nov. 9, 2011, now U.S. Pat. No. 8,454,908, which claims priority under 35 U.S.C. § 119(e)( 1 ) to U.S. Provisional Patent Application No. 61/510,180, filed on Jul. 21, 2011, and to U.S. Provisional Patent Application No. 61/460,775, filed on Nov. 10, 2010. The contents of each of the foregoing applications are incorporated by reference in their entirety. 
    
    
     BACKGROUND 
     For years, laboratory technologists have used dyes and stains such as those used in Romanowsky staining for preparing biological specimens to improve the contrast of a specimen during examination. Such examination typically utilizes a microscope, another device that captures images of the specimen, or, in other instances, unaided visual examination. Several different systems and methods for preparing a specimen for examination are known. For example, U.S. Pat. Nos. 6,096,271; 7,318,913; and 5,419,279, and published U.S. Patent Application Nos. 2008/0102006 and 2006/0073074 relate to machines and methods for staining a substrate during specimen processing. These publications provide various details on staining and preparing specimens for examination. 
     SUMMARY 
     The present disclosure relates to automated systems and methods for preparing biological specimens for examination. The specimens can include, for example, a blood sample containing red blood cells, white blood cells, and platelets, applied to a substrate, e.g., a microscope slide or a cover slip. Different embodiments can be used to prepare other biological specimens from biological samples including bone marrow, urine, vaginal tissue, epithelial tissue, tumors, semen, saliva, and other body fluids. Additional aspects of the disclosure include systems and methods for fixing, staining, rinsing, and agitating the specimens. In general, the systems and methods disclosed herein provide for rapid, efficient, and highly uniform specimen processing using minimal fluid quantities. The methods include one or more fixing, staining, and rinsing phases, including one or multiple agitation phases during or after one or more of the fixing, staining, and rinsing phases. The systems can be implemented as a standalone device or as a component in a larger system for preparing and examining biological specimens. 
     In general, in a first aspect, the disclosure features an apparatus for preparing a biological specimen on a substrate for examination, the apparatus including: (a) a substrate arm including a substrate gripper; (b) a first actuator connected to the substrate arm and configured to move the substrate arm between an open position and a specimen processing position; (c) a second actuator arranged and configured to agitate a substrate gripped by the substrate gripper on the substrate arm; (d) a platform having a top surface located opposite the substrate when the substrate arm is in the specimen processing position; and (e) two or more offsets arranged on the top surface of the platform such that when the substrate contacts all of the offsets in the substrate processing position, the substrate and top surface of the platform are substantially parallel and form a separation of at least about 50 microns. 
     Embodiments of the apparatus can include any one or more of the following features individually or in combination. 
     The first and second actuator can be the same actuator configured to both move the substrate arm and to agitate a substrate gripped by the substrate gripper on the substrate arm. A total surface area of the top surface of the platform can be smaller than a total surface area of the substrate. There can be at least three or more offsets arranged at outer edges of the top surface of the platform, where tips of the offsets define a plane. 
     A suction port can be located on the substrate gripper; the suction port can be connected to a suction source for providing suction to the suction port through a suction tube, to thereby hold the substrate to the substrate gripper. The apparatus can include a first stain port located on the top surface of the platform, a first stain reservoir, and a first stain conduit connected to the first stain port for providing a fluid pathway for stain to be pumped from the first stain reservoir to the first stain port and into the separation. The apparatus can include a second stain port located on the top surface of the platform at a location different from the first stain port location, a second stain reservoir, and a second stain conduit, where both the first and second stain ports are arranged on the top surface at a spacing from a specimen area on the substrate when the substrate is in the specimen processing position, and where the second stain conduit is connected to the second stain port to provide a fluid pathway for stain to be pumped from the second stain reservoir to the second stain port and into the separation. 
     The apparatus can include a first fixative port located on the top surface of the platform, a fixative reservoir, and a fixative conduit connected to the first fixative port for providing a fluid pathway for fixative to be pumped from the fixative reservoir to the first fixative port and into the separation. The apparatus can include a first rinse port located on the top surface of the platform, a rinse solution reservoir, and a rinse tube connected to the first rinse port for providing a fluid pathway for rinse fluid to be pumped from the rinse solution reservoir to the first rinse port and into the separation. 
     The apparatus can include a first vacuum port located on the top surface of the platform, a first waste container, and a first waste conduit connected to the first vacuum port for providing a pathway of negative pressure to evacuate fluid from the separation or substrate and deposit the fluid into the first waste container. The apparatus can include a second vacuum port located on the top surface of the platform and a second waste conduit connected to the second vacuum port for providing a pathway of negative pressure to evacuate fluid from the separation or substrate and deposit the fluid into the first waste container. The first and second vacuum ports can be located on opposite ends of the top surface of the platform. 
     The platform can include: a fixative port; a first stain port; a second stain port; a rinse port; a first vacuum port; and a second vacuum port. The apparatus can include a block arranged to support the platform, where the block includes: a fixative port; a first stain port; a second stain port; a rinse port; a first vacuum port; and a second vacuum port, where each port on the block is in a location corresponding to a port located in the platform. 
     The apparatus can include: a first stain reservoir; a second stain reservoir; a fixative reservoir; a rinse solution reservoir; a waste container; a pump; a plurality of fluid conduits connected to the pump and to the reservoirs and arranged for dispensing fluid from any one or more of the reservoirs; and a vacuum source for evacuating fluid from the substrate into the waste container. The apparatus can include a dryer positioned to direct a flow of air across the specimen when the substrate is located in the open position. 
     Embodiments of the apparatus can also include any of the other features, and any combinations of features, disclosed herein, as appropriate. 
     In a further aspect, the disclosure features methods of preparing a biological specimen on a substrate for examination that include: (a) positioning the substrate with respect to a surface so that the biological specimen faces the surface, and so that the substrate and the surface are substantially parallel and form a separation of at least about 100 microns; (b) sequentially dispensing (i) a first fixative solution, (ii) a first stain solution, (iii) a second stain solution, and (iv) a first rinse solution into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface; and (c) after dispensing each one of solutions (i), (ii), (iii), and (iv) in step (b), and before dispensing the next one of solutions (i), (ii), (iii), and (iv) in step (b), performing at least a first agitation cycle, where the first agitation cycle includes changing the distance between the substrate and surface while the dispensed solution contacts the specimen for the duration of the first agitation cycle, and removing the dispensed solution from the separation and from contacting the specimen. 
     Embodiments of the methods can include any one or more of the following features. 
     Each sequential dispensing step can include dispensing one of the solutions in step (b) at a flow rate of at least 70 microliters per second for no more than three seconds. The first agitation cycle can include increasing the distance between the substrate and the surface by at least ten microns, and decreasing the distance between the substrate and the surface by at least five microns. Removing the dispensed solution can include applying a pressure of at least one pound per square inch less than an atmospheric pressure to the separation for at least two seconds. 
     Embodiments of the methods can also include any of the other features disclosed herein, and any combination of features, as appropriate. 
     In another aspect, the disclosure features methods of preparing a biological specimen on a substrate for examination, where the methods include: (a) positioning the substrate with respect to a surface so that the biological specimen faces the surface, and so that the substrate and the surface are substantially parallel and form a separation of at least about 50 microns; (b) dispensing a first stain into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface; (c) performing at least a first agitation phase, wherein the first agitation phase includes changing the distance between the substrate and surface while the first stain is contacting the specimen for the duration of the first agitation phase; and (d) removing the first stain from the separation and the specimen. 
     Embodiments of the methods can include any one or more of the following features. 
     The dispensing step can include dispensing the stain at a flow rate of at least 70 microliters per second for no more than three seconds. The agitation phase can include increasing the distance between the substrate and the surface by at least ten microns, and decreasing the distance between the substrate and the surface by at least five microns. Removing the stain can include applying a vacuum force of at least one pound per square inch to the first stain in the separation for at least two seconds. 
     The methods can include: dispensing a second stain into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface; performing a second agitation phase, where the second agitation phase includes changing the distance between the substrate and surface while the second stain is contacting the specimen for the duration of the second agitation phase; and removing the second stain from the separation and the specimen. 
     Embodiments of the methods can also include any of the other features and/or steps disclosed herein, and any combinations thereof, as appropriate. 
     In a further aspect, the disclosure features methods of preparing a biological specimen on a substrate for examination, where the methods include: (a) positioning the substrate with respect to a surface so that the specimen faces the surface, and so that the substrate is positioned to form a separation between the surface and at least a portion of the substrate of at least about 50 to 250 microns, e.g., 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, or 250 microns; (b) performing a fixing phase that includes (i) dispensing a fixative into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface, (ii) performing at least a first agitation phase, where the first agitation phase includes changing the distance between the substrate and surface while the fixative is contacting the specimen for the duration of the first agitation phase, and (iii) removing the fixative from the separation and the specimen; (c) performing a first staining phase that includes (i) dispensing a first stain into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface, (ii) performing at least a second agitation phase, where the second agitation phase includes changing the distance between the substrate and surface while the first stain is contacting the specimen for the duration of the second agitation phase, and (iii) removing the first stain from the separation and the specimen; (d) performing a second staining phase that includes (i) dispensing a second stain into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface, (ii) performing at least a third agitation phase, where the third agitation phase includes changing the distance between the substrate and surface while the second stain is contacting the specimen for the duration of the third agitation phase, and (iii) removing the second stain from the separation and the specimen; and (e) performing a first rinse phase that includes (i) dispensing a first rinse into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface, (ii) performing at least a fourth agitation phase, where the fourth agitation phase includes changing the distance between the substrate and surface while the first rinse is contacting the specimen for the duration of the fourth agitation phase, and (iii) removing the first rinse from the separation and the specimen. 
     Embodiments of the methods can include any one or more of the following features. 
     The methods can include performing a second rinse phase, where the second rinse phase includes: (i) dispensing a second rinse into the separation between the substrate and the surface in an amount sufficient to contact the specimen and the surface; (ii) performing at least a fifth agitation phase, where the fifth agitation phase includes changing the distance between the substrate and surface while the second rinse is contacting the specimen for the duration of the fifth agitation phase; and (iii) removing the second rinse from the separation and the specimen. The methods can further include performing a drying cycle by directing a flow of air across the specimen. 
     The combined method steps can be performed, for example, in less than 70 seconds (e.g., in less than 60 seconds). In some embodiments, the methods can consume less than 650 microliters of fixative, first stain, second stain, and first rinse fluids. In certain embodiments, the methods can consume less than 850 microliters of fixative, first stain, second stain, first rinse, and second rinse fluids. 
     Embodiments of the method can also include any of the other features and/or steps, and any combinations thereof, disclosed herein, as appropriate. 
     In another aspect, the disclosure features automated specimen examination systems that include: an applicator station that applies a sample specimen to a substrate; any one of the biological specimen preparation apparatus disclosed herein; and an imaging station that images the biological specimen after preparation by the specimen preparation apparatus. 
     Embodiments of the automated specimen examination system can include any one or more of the features disclosed herein, as appropriate, including any one or more of the features of the biological specimen preparation apparatus&#39; disclosed herein. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
     Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. 
    
    
     
       DESCRIPTION OF DRAWINGS 
         FIG. 1  is a perspective view of an embodiment of an apparatus for preparing biological specimens for examination, with both sample grippers  20 A and  20 B in an open position. 
         FIG. 2  is another perspective view of a portion of the apparatus of  FIG. 1  (with the substrate arms and sample grippers not shown). 
         FIG. 3A  is a further perspective view of the apparatus of  FIG. 1 , with sample gripper  20 A in an open position and sample gripper  20 B in a closed (specimen processing) position. 
         FIG. 3B  is a perspective view of an indexing mechanism of the apparatus of  FIG. 1 . 
         FIG. 4  is a perspective view of the apparatus of  FIG. 1  showing connections between the apparatus and fluid reservoirs by means of multiple fluid conduits. 
         FIG. 5  is a perspective view of a specimen examination system that includes an automated substrate mover and an embodiment of a specimen preparation apparatus as described herein. 
         FIG. 6A  is an expanded perspective view of a portion of the apparatus of  FIG. 1  showing specimen gripper  20 B, platform  60 B, and block  80 B in detail. 
         FIG. 6B  is a perspective view of a ball joint mechanism of the apparatus of  FIG. 1 . 
         FIG. 6C  is a cross-sectional view of the ball joint mechanism of  FIG. 6B . 
         FIG. 7A  is a flow chart showing a series of steps for moving substrate arms from an open position to closed (specimen processing) position. 
         FIG. 7B  is a schematic diagram of an embodiment of a specimen preparation apparatus as described herein. 
         FIG. 8A  is a flow chart showing an alternate series of steps for moving substrate arms from an open position to a specimen processing position. 
         FIG. 8B  is a schematic diagram of an apparatus for preparing biological specimens for examination that includes two actuators. 
         FIG. 9  is a flow chart showing a series of steps for applying fixative to a specimen. 
         FIG. 10  is a flow chart showing a series of steps for applying stain to a specimen. 
         FIG. 11A  is a flow chart showing a series of steps for removing excess fluid from a substrate. 
         FIG. 11B  is a flow chart showing an alternate series of steps for removing excess fluid from a substrate. 
         FIG. 12  is a flow chart showing a series of steps for rinsing a specimen. 
         FIG. 13  is a flow chart showing a series of steps for agitating a specimen. 
         FIG. 14  is a flow chart showing a series of steps for drying a specimen. 
         FIG. 15  is a perspective view of a specimen preparation apparatus as used in a larger specimen examination system. 
         FIG. 16  is a flow chart showing a series of steps for processing a specimen mounted on a substrate. 
         FIG. 17  is a graph showing volume of fluid consumed as a function of time in the flow chart of  FIG. 16 . 
         FIGS. 18A and 18B  are perspective views of the apparatus of  FIG. 1  that show placement of a substrate onto a substrate arm by an automated substrate mover. 
     
    
    
     Like reference symbols in the various drawings indicate like elements. 
     DETAILED DESCRIPTION 
     Disclosed herein are methods and systems for automated biological specimen processing. The automated specimen processing methods and systems described herein provide advantages over manual and other automated processing methods, including enhanced processing speed while using minimal reagent volumes and concurrently producing a highly uniform sample preparation that significantly reduces the variability associated with the application of stains, fixatives, and other reagents as compared to specimens processed by hand or by other systems. 
     Conventional automated processing methods typically have relatively high processing throughput while at the same time consuming large volumes of processing fluids, or have relatively low processing throughput while consuming reduced volumes of fluids. For many applications, however, both high throughput operation and low fluid consumption are desirable. 
     By maintaining high throughput, specimens can be efficiently processed for subsequent examination. By keeping fluid consumption low, the amount of processing waste is reduced along with the required volume of processing reagents, keeping operating costs low. The systems and methods disclosed herein permit rapid automated processing of specimens (e.g., more than 100 specimens per hour by a single machine) using low volumes of processing fluids (e.g., less than 1 mL of fluids per specimen), while producing highly uniform and repeatable results. 
     Biological Specimen Preparation Systems and Methods 
     Before specimens are examined, they are prepared in a series of steps to enhance the visual appearance of certain features in the specimens.  FIG. 1  illustrates an embodiment of an apparatus or machine  1  for preparing a biological specimen for examination or imaging on a substrate  2  such as a microscope slide, cover slip, or other transparent surface. Machine  1  can be incorporated into an overall system for preparing and analyzing specimens comprising body fluids or other biological samples containing cells, such as system  2000  shown in  FIG. 15  and described below. Machine  1  can generally include, or form a portion of, a system that features a first station that obtains a specimen, a second station that applies the specimen to a substrate, third and fourth stations for fixing and staining the specimen, respectively, a fifth station that dries the specimen, a sixth station that images the specimen, and a seventh station for analyzing the images and data obtained from the specimen. Certain embodiments of machine  1  are compatible with system  2000 ; some embodiments of machine  1  can be used in other specimen preparation systems, and/or as stand-alone devices. 
     Machine  1  can include or connect to a control system  5  as shown in  FIG. 4 , which provides another perspective view of machine  1 . Control system  5  can include one or more computers each containing a central processing unit capable of executing software instructions stored on computer readable media such as a hard drive, optical drive, or memory. Additionally, control system  5  can include electrical circuitry for executing the software instructions. Control system  5  can include a user interface for receiving user commands to control the operation of machine  1 . Software stored on or provided to the computer can include programs that control the operation of components of machine  1  during specimen processing, such as fluid pumps and vacuums. For example, the software can include instructions for directing the machine  1  to apply various fixatives, stains, and rinses to the specimen, and to perform several agitation steps during specimen processing. 
     In addition, the software can include default settings, and the user interface may contain customization features for providing the user with the ability to change these defaults settings. For example, the user interface can contain customization features for allowing a user to customize the speed, frequency, or order of fixing, staining, and rinsing phases, as well as agitation parameters (further described below). Control system  5  can also communicate via a network protocol (such as Appletalk®, IPX, or TCP/IP). For example, the network protocol may use cables (such as twisted pair cables) and/or a wireless connection such as WiFi. The control system may be connected to a laboratory information system using the network protocol. The laboratory information system can contain a server and/or database for storing information relating to specimens processed on machine  1 . For example, the database may contain a table that provides information about the person or source of the specimen (e.g., name, date of birth (DOB), address, time specimen was taken, gender, etc.), information relating to processing of specimen (processed on date ##/##/####, specimen number #, etc.), a copy of any images acquired of the specimen, and copies of any results obtained by analyzing the images. 
     Referring to  FIG. 1 , machine  1  can include supports  110 A and  110 B to secure the device to a location within a system or a laboratory workstation. Machine  1  also includes one or more substrate arms  10 A and  10 B, each connected at their base to an actuator  30 A and  30 B. The opposite ends of the substrate arms  10 A and  10 B include substrate grippers  20 A and  20 B for receiving and holding substrates during specimen processing. Each substrate gripper  20 A and  20 B receives and holds a substrate  2  while machine  1  completes all specimen processing steps (described below). The substrate may be or include a microscope slide, a cover slip, or other transparent material suitable for holding a specimen during specimen processing and microscopic examination after specimen processing. The embodiment of  FIG. 1  depicts a glass microscope slide, substrate  2 , which includes a biological specimen  3 . Using suction ports, substrate grippers  20 A,  20 B can hold the substrate  2  to substrate arms  10 A,  10 B during specimen processing. A suction tube  23  provides suction to the substrate grippers  20 A and  20 B through suction ports  21 A and  21 B, and  22 A and  22 B (note that ports  21 A and  22 A are positioned behind the slide  2  in  FIG. 1 , and are shown in dashed lines). 
     The machine  1  embodiment shown in  FIGS. 1-3  is a dual substrate machine, capable of holding and processing a substrate on each of substrate arms  10 A and  10 B. Other embodiments provide for processing a single substrate or three or more substrates, sequentially or simultaneously. Further, while the embodiments depicted in  FIGS. 1-6  use suction to attach the substrates  2  to the substrate arms  10 A and  10 B, alternative embodiments can use various types of clamps, fingers, or magnets (if the substrate is magnetized) to attach a substrate  2  to a substrate arm  10 A during specimen processing. 
     In the embodiments shown in  FIGS. 5 and 18A -B, machine  1  receives a substrate  2  carrying a specimen  3  from an automated substrate mover  120  or manually from an individual. As an example, the substrate mover  120  can be a device that transports a substrate between stations (e.g., station  121  to station  122  to station  123 , to station  124 , and to station  125 ).  FIG. 5  shows a system having a first label reader station  121 , an applicator station  122 , a staining station  123  that includes machine  1 , a camera or imaging station  124 , and a second label reader station  125 . The first label reader station  121  is configured to read information from substrate  2  such as a bar code and/or “fingerprint” information that is used to identify the particular substrate  2  and specimen  3  thereon. The second label reader station  125  functions in the same manner, and the information it reads is used to verify that the specimen  3  that is imaged at station  124  is the same as the substrate that was processed. 
     Substrate mover  120  can include a gripper  127  for holding the substrate  2 , and registration circuitry or software to enable the mover  120  to determine whether the substrate  2  is mounted in the mover  120 . In one embodiment, substrate mover  120  can include a hydraulic cylinder for moving substrate  2  from a first station  121  to a second station  122 . After specimen processing, the substrate mover  120  may remove the processed substrate from staining station  123  and transport the substrate  2  to another station for substrate examination, such as a microscope or station  124 . Alternatively, an individual may manually remove a substrate from machine  1  after specimen processing. 
     The substrate arms  10 A and  10 B can rotate about an axis to enable the substrate to move from an open position for loading, to a specimen processing position, and back to the open position for unloading after specimen processing.  FIG. 7A  shows a flow chart  500  that includes a series of steps for moving substrate arms from an open position to a processing position. Flow chart  500  is further described below with reference to  FIG. 7B , which shows a schematic diagram of machine  1 . 
     Note that machine  1  in  FIG. 1  is configured to accept and examine two substrates. In the following discussion and figures, reference may be made to only one set of components in machine  1  (e.g., substrate gripper  20 A, actuator  30 A, substrate arm  10 A, etc.). However, it is to be understood that the same steps, features, and attributes that are disclosed in connection with one set of components can also apply to the other set of components in machine  1  (e.g., substrate gripper  20 B, actuator  30 B, substrate arm  10 B, etc.). Thus, while the discussion herein focuses only on one set of components for clarity and brevity, it is understood that machines for specimen examination such as machine  1  can include two or more than two sets of components, each set having some or all of the features discussed herein. 
     Returning to  FIGS. 7A and 7B , in a first step  502  of flow chart  500 , substrate mover  120  places a substrate  2  in contact with a substrate gripper  20 A. In step  504 , substrate  2  is positioned on the substrate gripper in a “specimen up” or “open” position. Next, in step  506 , actuator  30 A rotates substrate arm  10 A by approximately 180° (see  FIG. 7B ) to position substrate  2  in a “specimen down” or “specimen processing” or “closed” position (step  508 ), directly above platform  60 A, so that substrate  2  is in a processing position in step  510 . 
     Then, in step  512 , machine  1  stains specimen  3  positioned on substrate  2  by directing suitable fluids including stains, wash fluids, and fixatives to be pumped from reservoirs  210 A,  211 A,  212 A, and  213 A into contact with specimen  3  through ports  42 A,  43 A,  44 A, and  45 A. Excess fluids are removed from specimen  3  by vacuum pumping through ports  40 A and  41 A, and are collected in waste collectors  230  and  231 . 
     In step  514 , following staining of specimen  3 , actuator  30 A rotates substrate arm  10  by approximately 180° (reversing the rotation of step  506 ) to return the substrate to the “specimen up” position. Finally, in step  516 , substrate mover  120  removes the processed substrate from substrate gripper  20 A. Other open or “specimen up” positions can also be used, provided that an operator or automated substrate mover can load and unload substrates from machine  1 . For example, the specimen up position can be rotated 100° or more (e.g., 120° or more, 130° or more, 140° or more) from the specimen processing position. In some embodiments, the specimen up position can be rotated less than 100° (e.g., less than 90°, less than 80°, less than 70°) from the specimen processing position, provided that an operator or substrate mover can load and unload substrates from machine  1 . 
     Actuators  30 A and/or  30 B may include an electric motor, pneumatics, magnetic systems, or other hardware (e.g., a worm gear) to move arm  10 A and/or  10 B. When substrate arms  10 A and  10 B are in an open position as depicted in  FIG. 1 , grippers  20 A and  20 B can each receive a substrate  2 . Once loaded onto a substrate gripper  20 A or  20 B, actuators  30 A and/or  30 B then rotate arms  10 A and/or  10 B, and thus substrate  2 , from the open (“specimen up”) position to a processing position (“specimen down,” as shown for arm  10 B in  FIG. 3A ) for application of fixative, stain, and rinse solutions, including agitation steps, and back to an open position for unloading after processing. 
     With reference to  FIG. 3A , actuator  30 B has rotated substrate arm  10 B from the open position depicted in  FIG. 1  to a “closed” or processing position.  FIG. 3A  shows that the substrate  2  on substrate arm  10 B has been flipped over and rotated approximately 180° from its loading position shown in  FIG. 1  to a downward-facing position where specimen  3  on substrate  2  is substantially parallel to the surface of platform  60 B. As discussed in connection with  FIG. 7A  above, while substrate  2  is positioned proximal to platform  60 B in the specimen processing position shown, machine  1  applies various fixatives, stains, and rinses to specimen  3  on substrate  2  through several processing phases, which will be described in greater detail below. To remove substrate  2  from the processing position, actuator  30 B rotates substrate arm  10 B back to the open position shown in  FIG. 1  (both arms) and  FIG. 3A  (where only arm  10 A is in the open position). 
     In certain embodiments, control system  5  can detect the position of the arms utilizing one or more sensors  105 A and  105 B to detect indicator arms  101 A and  101 B (as shown in  FIGS. 1 and 3 ). Sensors  105 A and  105 B can be proximity sensors, e.g., photoelectric sensors, utilizing, e.g., infrared light or various other technologies (lasers, motion detectors, etc.) to detect the presence or absence of the arms. For example, proximity sensors  105 A or  105 B can have a detection field, and the sensors can determine whether or not a substrate arm (e.g., arm  10 A and/or  10 B) or a substrate gripper (e.g., gripper  20 A and/or  20 B) is within the detection field. Control system  5  can receive information from the sensors to determine the positions of substrate arms  10 . For example, when substrate arm  10 B (not shown in  FIG. 3A ) is rotated to a processing position, proximity sensor  105 B on the proximal end of indicator arm  101 B senses target substrate gripper  20 B, and notifies control system  5  that substrate arm  10 B is rotated to a specimen processing position. In this position, proximity sensor  105 B on the distal end of indicator arm  101 B will not send a signal to control system  5 , because the sensor does not detect any target (e.g., a substrate arm or substrate gripper). 
     When substrate arm  10 B rotates to an open position (as shown in  FIG. 1 ), proximity sensor  105 B on the distal end of indicator arm  101 B senses target substrate gripper  20 B, and notifies control system  5  that substrate arm  10 B is rotated to an open position. Stated differently, when substrate arm  10 B has rotated away from the sensor  105 B, the sensors send a “not present” signal to the control system  5 . When arm  10 B is rotated into the open position, arm  10 B is closer to the sensor  105 B, and the sensor can send a “present” signal to the control system  5 . In alternate configurations, the sensor can be mounted on substrate  10 B and can detect the presence of the indicator arm  101 B. In some embodiments, control system  5  can be used to calibrate the position of actuators  30 A and  30 B to known open and specimen processing positions, and/or to actively monitor the movement and position of substrate arms  10 A and  10 B based on control signals and/or feedback received from actuators  30 A and  30 B. 
     The structure and axis of rotation for substrate arms  10 A and  10 B in  FIG. 1  may be varied in other embodiments of the invention.  FIG. 8A  shows a flow chart  600  that includes an alternate series of steps for moving substrate arms from an open position to a processing position. Flow chart  600  is further described below with reference to  FIG. 8B , which shows a schematic diagram of machine  1 . 
     In step  602  of flow chart  600 , substrate mover  120  places substrate  2  on substrate gripper  20 A in a “specimen up” orientation. Then, in step  604 , a first actuator  30 A rotates substrate  2  by approximately 180° in a plane perpendicular to the plane of  FIG. 8B , so that substrate  2  remains oriented in a “specimen up” position above platform  60 A. In step  606 , a second actuator  35 A receives substrate  2  oriented in the “specimen up” position. Then, in step  608 , second actuator  35 A (e.g., positioned between substrate arm  10 A and substrate gripper  20 A) rotates the substrate  2  into a “specimen down” orientation. Second actuator  35 A can also move substrate  2  downward toward platform  60 A so that substrate  2  contacts offsets  70 A and  70 B. 
     Next, with substrate  2  in the processing position in step  610 , machine  1  stains specimen  3  on substrate  2  by applying stains, fixatives, and wash solutions as discussed above in connection with step  512  of flow chart  500 . After staining is complete, second actuator  35 A rotates substrate  2  from a “specimen down” orientation to a “specimen up” orientation (step  614 ), and then first actuator  30 A rotates substrate  2  by approximately 180° (e.g., in a plane perpendicular to the plane of  FIG. 8B , reversing the rotation applied in step  606 ) so that the substrate remains oriented in a “specimen up” position. Finally, in step  618 , substrate mover  120  removes the processed substrate from substrate gripper  20 A. 
     In general, machine  1  may include one or more (e.g., two, three, four, five, or more than five) platforms  60 A and  60 B as shown in  FIGS. 1-3  for specimen processing. As shown in  FIG. 2 , platform  60 A can include lateral sides for supporting a top side of the platform. A shield  100 , shown in  FIGS. 1 and 3 , can be positioned between the platforms  60 A and  60 B to prevent fluids from splattering between the platforms  60 . In some embodiments, shield  100  can be formed from a transparent material that blocks fluids from one of platforms  60 A and  60 B from contaminating the other platform. In certain embodiments, shield  100  can be formed from a material that is translucent or opaque. In  FIGS. 1 and 3 , shield  100  is depicted as being formed from a transparent material to allow other components positioned behind shield  100  to be shown in the same figure. Shield  100  could also have been shown as being formed from an opaque material, in which case portions of some components such as platform  60 A and block  80 A would have been obscured. 
       FIG. 3B  shows an indexing mechanism  50 A that can be used to translate the machine  1  to provide substrates  2  from each of the substrate grippers  20 A,  20 B to a position for specimen processing. The indexing mechanism  50 A can be in many forms, such as electromechanical devices (e.g., a rack and pinion gear set powered by an electric motor), linear actuators (e.g., pneumatic actuators, hydraulic actuators, or electromagnetic actuators). Although, in the illustrated embodiment, the indexing mechanism  50 A translates the machine  1  linearly between two positions, other translation paths are possible based on the number of platforms included on the machine  1 , and their configuration and layout, such as circular or semi-circular (e.g., an indexing table that can move in an arcuate path). As shown, the indexing mechanism  50 A can include a gear rack  50 B attached to a base  50 C of the machine  1  and a pinion gear  50 D attached to an electric motor  50 E that is fixed to the base  50 C. The machine  1  can be attached to the base  50 C using one or more sliding devices  50 F so that the machine  1  can move smoothly when translated by the indexing mechanism  50 A. During use, the indexing mechanism  50 A can move the machine  1  so that the multiple substrate grippers  20 A and/or  20 B of the machine  1  to receive a substrate  2  from a substrate mover  120  (shown in  FIG. 5 ) so that a sample disposed on the substrate  2  can be prepared by the machine  1 , and also so that, once prepared, the substrate gripper  20 A and/or  20 B can provide the substrate  2  having a prepared sample can be provided to the substrate mover  120  for sample processing. 
     For machines having two platforms  60 A and  60 B, as in the illustrated embodiment, substrates  2  are typically provided to, and from, the substrate mover  120  in an alternating manner. In some embodiments, a first substrate  2  is provided from the substrate mover  120  to a first substrate gripper  20 A, to be processed at a first platform  60 A, while the machine  1  is in a first position. While the first substrate  2  is processed at the first platform  60 A, the indexing mechanism  50 A can translate the machine  1  to a second position so that a second substrate gripper  20 B can receive a second substrate, to be processed at the second platform  60 B, from the substrate mover  120 . While the second substrate is processed at the second platform  60 B, the indexing mechanism  50 A can translate the machine  1  back to the first position so that the substrate mover  120  can remove the first substrate  2  from the first substrate gripper  20 A. Once the substrate  2  is removed from the first gripping platform  20 A, a next substrate can be provided to the first gripping platform  20 A. This method for providing substrates to alternating gripping platforms can be implemented for more than two (e.g., three, four, five, or more than five) platforms thereby increasing throughput of specimens prepared for further evaluation. 
     Platforms  60 A and  60 B are typically formed from one or more materials that are relatively chemically inert with respect to the fluids used during specimen processing and provide a suitable surface tension. Exemplary materials that can be used to form platforms  60 A and  60 B include engineering thermoplastics, such as polyoxymethylene (e.g., Delrin® manufactured by DuPont), high molecular weight fluorocarbons, such as polytetrafluoroethylene (PTFE) (e.g., Teflon® manufactured by DuPont), and metals such as aluminum, steel, and titanium, provided they are manufactured and/or treated to provide a suitable surface tension that acts to assist in evenly distributing and confining the processing fluids to the space between substrate  2  and the platforms, and allowing suitable evacuation of the processing fluids as well. By selection of suitable materials, the platforms can also advantageously reduce or minimize the formation of bubbles or spaces within the fluids as they are distributed, and at the same time maintain a sufficient surface tension such that fluid leakage out of the separation between the platforms and substrate  2  is reduced or eliminated. 
     In general, the surface area of platforms  60 A and  60 B can be selected as desired for purposes of substrate handling and fluid delivery. Factors such as the surface area of platforms  60 A and  60 B can also influence the selected surface area of substrate  2 . For example, in some embodiments, the surface area of platform  60 A (e.g., the area of the surface of platform  60 A that faces substrate  2 ) is slightly smaller than the area of the surface of substrate  2  that faces platform  60 A. By maintaining such a relationship between the areas of the facing surfaces of platform  60 A and substrate  2 , fluid leakage from the region between the surfaces can be reduced or eliminated. Typically, for example, the area of the surface of substrate  60 A that faces substrate  2  is smaller than the area of the surface of substrate  2  by 2% or more (e.g., 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more). 
     Platforms  60 A and  60 B can be attached to blocks  80 A and  80 B, respectively. Block  80 A includes lateral sides  81 A- 84 A supporting a top side  85 A as shown in  FIG. 2 . Blocks  80 A and  80 B can be made of the same or similar materials to those used for the platforms, including metals, ceramics, and/or plastics. Thus, materials such as Delrin® can be used to form blocks  80 A and  80 B, particularly in embodiments that implement Romanowsky staining of specimens. Other materials that can be used in embodiments include metals, and Teflon® brand polytetrafluoroethylene-coated aluminum, steel, or titanium. 
     In some embodiments, platforms  60 A and/or  60 B can be raised as shown in  FIGS. 1-3 . Alternatively, in certain embodiments, platforms  60 A and/or  60 B can be flush with the upper surface of blocks  80 A and  80 B, respectively. In either case, certain features of machine  1  as well as surface tension of fluids and surface energy of the platform or block prevent excess fluids from flowing past the edges of platforms  60 A/ 60 B and/or blocks  80 A/ 80 B. 
     As shown in  FIGS. 1 and 2 , platform  60 A can include offsets  70 A- 70 D to provide a separation between the surface of platform  60 A and substrate  2 , and prevent substrate  2  from contacting platform  60 A. Platform  60 B can include a corresponding set of offsets  71 A- 71 D. Offsets can include standoffs, pins, pegs, rods, beads, walls, or other structures that provide separation between the surface of platform  60 A and/or  60 B and substrate  2 . Offsets  70 A- 70 D and  71 A- 71 D ensure that the surfaces of platforms  60 A and  60 B and substrate  2  remain substantially parallel when substrate  2  contacts the offsets. The benefit of maintaining these two surfaces in parallel is that the volume enclosed between these two surfaces is thus defined and can be precisely controlled. If the two surfaces are not substantially parallel, and the angle between them changes, then the volume between them also changes and is not fixed and precisely controlled. In addition, the fluids may not apply uniformly to the specimen if such two surfaces are not substantially parallel. 
     As used herein, the phrase “substantially parallel” means that two surfaces are exactly parallel or nearly parallel, so that imperfections in the surface flatness of substrate  2  are reduced or eliminated when substrate  2  contacts the offsets. For example, although great care is taken in the production of substrates, certain substrates may have imperfections such as twist and/or non-coplanar corners. In the systems and methods disclosed herein, the use of offsets assists in correcting these imperfections by improving the surface flatness of substrate  2  where needed, orienting substrate  2  in a substantially parallel relationship to platforms  60 A and  60 B in the process. The phrase “substantially parallel” covers situations in which the two surfaces are not perfectly flat, but the offsets are all the same size or height, so that at least the contact points of a surface of the substrate with the offsets are in the same plane. 
       FIG. 6A  shows substrate  2  with specimen  3  (specimen not shown), substrate gripper  20 B, blocks  80 A,  80 B, platforms  60 A,  60 B, offsets  70 A- 70 D and  71 A- 71 D, and separation  92  between substrate  2  and platform  60 B. Separation  92  allows fluids to travel between the surface of platform  60 B containing ports  40 B- 45 B and substrate  2  containing specimen  3 . The separation distance required for optimal specimen fixing, staining, and rinsing will vary depending on the flow rate of fluids dispensed from ports  40 B- 45 B (and/or ports  40 A- 45 A), port diameter, the viscosity of the fluids applied during processing, and the amount of suction available for removing fluids from the substrate, separation, and platform. 
     In some embodiments, for example, offsets providing a separation  92  of about 100-200 microns between the surface of platform  60 B and substrate  2  enable fixing, staining, and rinsing for specimens comprising blood cells in embodiments capable of dispensing fluids at flow rates ranging from 70 to 140 microliters per second (e.g., 90, 115, or 125 microliters per second) from ports  40 B- 45 B having a diameter ranging from 500 to 1,500 microns. In general, the size or height of separation  92  can vary from about 50 microns to 1,000 microns for certain embodiments (e.g., from about 50 to 500 microns, from about 75 to 250 microns, from about 100 to 200 microns), provided such embodiments are capable of overcoming surface tension from fluids in the separation while dispensing and removing fluid during specimen processing. In addition, in certain embodiments, the diameters of ports located on platform  60 A and/or  60 B can vary from about 125 microns to 5,000 microns. 
       FIGS. 6B and 6C  show a ball joint mechanism  25  that can be used to align a substrate gripper  20 A to be parallel with a platform  60 A. The ball joint mechanism  25  can include a ball member  25 A that is rigidly fixed to the substrate gripper  20 A, a deflection element  25 B (e.g., a spring), a lower socket  25 C that is rigidly connected to the substrate arm  10 A, an upper socket  25 D, a cap  25 E that is fixed to the lower socket  25 C (e.g., using fasteners), and a set screw  25 F. In some embodiments, during manufacturing and/or set up of the machine  1  and substrate grippers  20 A and/or  20 B, the ball joint mechanism  25  can be adjusted to compensate for any misalignment that may be present due to tolerance stack-up or fabrication problems. To adjust the ball joint mechanism  25 , in some embodiments, the set screw  25 F is loosened and the substrate arm  10 A is moved to the closed position. Since the set screw  25 F is loosened, the substrate gripper  20 A, while gripping a substrate  2 , is able to lay substantially parallel to the platform  60 A while the substrate  2  positioned along the contact offsets  70 . Alternatively, in some embodiments, the number of offsets on platform  60  can be reduced or eliminated completely; a shim with a thickness corresponding to the desired separation distance can be used temporarily during set up or calibration of machine  1  in conjunction with ball joint mechanism  25  to set separation  92  at a desired distance for specimen processing. Although the ball joint mechanism  25  is loosened, the deflection element  25 B applies a force to keep the substrate gripper  20 A semi-fixed to the substrate arm  10 A so that it is able to move independently, but it is not so loose and not free to move so much as to interfere with, or cause damage to, other components of the machine  1 . Once the substrate  2  is pressed firm in a closed position so the substrate  2  is substantially parallel to the platform  60 A, the set screw  25 F can be tightened to secure the ball joint mechanism  25 . As shown, when tightened, the set screw  25 F applies a downward force on the upper socket  25 D and thus applies a frictional force to the top of the ball member  25 A via the upper socket  25 D. Since the lower socket  25 C is fixed to the cap  25 E, the force created by the set screw  25 F also lifts the lower socket  25 C such that the lower socket  25 C applies a frictional force to the bottom side of the ball member  25 A to constrain the ball member  25 A within the upper and lower sockets  25 C,  25 D. Once constrained to the ball member  25 A, the substrate gripper  20 A becomes fixed to the substrate arm  10 A. 
     Typically, once the substrate gripper  20 A is positioned and constrained with the set screw  25 F, the ball joint mechanism  25  need not be adjusted again during normal use. However, if the substrate gripper  20 A becomes misaligned and therefore the ball joint mechanism  25  requires adjustment (e.g., due to damage, machine repair, poor performance, or other reasons), the set screw  25 F can be loosened, the substrate gripper  20 A can be moved to a closed position to position so that a substrate gripped by the substrate gripper  20 A is substantially parallel to the platform  60 A, and then set screw  25 F can be tightened to secure the ball joint mechanism  25 . 
     In general, actuators  30 A and/or  30 B can be configured to adjust the position of substrate arms  10 A and/or  10 B to vary the extent of separation between the surface of platforms  60 A and/or  60 B and substrate  2 . Varying this separation provides greater flexibility in embodiments that allow for adjusting the fluids assigned to each port, flow rates, fluid viscosities, and evacuation forces from platforms  60 A and/or  60 B. For example, a 100 micron separation  92  can provide sufficient specimen fixing, staining, and rinsing when fluids applied from platform  60 A are dispensed at a flow rate of 70 microliters per second from ports  40 A- 45 A having port diameters ranging from 500 microns to 1,500 microns. Alternatively, with a separation  92  distance between the surface of platform  60 A and substrate  2  of approximately 200 microns, a higher flow rate for fluids dispensed from ports  40 A- 45 A, such as 115-140 microliters per second, can be used for specimen processing. 
     As disclosed above, machine  1  may contain a series of ports and tubes for dispersing and removing fluids applied during specimen processing. The following discussion describes various ports, tubes, and other components associated with platform  60 A, but similar considerations apply to platform  60 B and its associated components.  FIG. 2  shows a close up view of the apparatus shown in  FIG. 1 , and shows in detail ports  40 A- 45 A on platform  60 A and tubes  50 A- 55 A connected to block  80 A. Tubes  52 A- 55 A distribute certain fluids including one or more fixatives, stains, and rinse solutions across the platform, into the separation, and onto the substrate. 
     Referring to  FIG. 2 , the top side of platform  60 A includes six ports  40 A- 45 A that are connected to tubes  50 A- 55 A. Fluids are driven by one or more pumps through the tubes and ports onto substrate  2 . One or more fluid reservoirs  210 A- 213 A (such as a first stain reservoir  211 A, a second stain reservoir  212 A, a fixative reservoir  210 A, and a rinse solution reservoir  213 A), e.g., as shown in  FIG. 4 , can direct fluid onto platform  60 A and substrate  2 . The diameters of ports  40 A- 45 A shown in  FIGS. 1-3  range from approximately 500 microns to 1,500 microns, although the diameters can also be smaller or larger in certain embodiments. In some embodiments, the diameters of the vacuum ports  40 A and  41 A are more than twice the diameters of fluid ports  42 A- 45 A. 
     Each of ports  40 A- 45 A is typically dedicated to a particular fluid or vacuum source. Alternatively, more than one port may be used for each fluid or vacuum source, or multiple tubes from various fluid and vacuum sources may connect to a single port located on platform  60 A. For example, in some embodiments, only one port on platform  60 A may be used for waste removal, but when using more viscous fluids, the single port may not provide sufficient suction to evacuate residual fluid from the platform. Thus, it may be desirable in certain embodiments to provide two suction ports at different positions on the platform (e.g., one suction port at each end of the platform) for removing excess stain, fixative, and rinse fluids as shown with ports  40 A and  41 A in  FIG. 2 . Further highlighting the variability of fluid-to-port configurations, in certain embodiments, a single port on platform  60 A may be dedicated for a particular stain, while in other embodiments multiple ports are used for applying stains during specimen processing. Indeed, various combinations relating to the number of ports, port locations, and fluids assigned to each port and fluid tube may be used in different embodiments of the invention. 
     Ports  40 A- 45 A can generally be positioned as desired on platform  60 A to provide for fluid delivery to, and fluid removal from, substrate  2 . Typically, each of the fluid ports is positioned on platform  60 A such that the port&#39;s aperture is not positioned directly adjacent or beneath specimen  3  on substrate  2  when the specimen is undergoing processing. With certain combinations of specimens and stains, for example, if stains are dispensed from a port located directly adjacent or beneath a portion of specimen  3 , a larger quantity of stain may be applied to cells in that portion (in the vicinity of the port) than to cells in other portions of the specimen. As a result, cells receiving the larger quantity of stain may appear darker in specimen images, and this non-uniform staining of specimen cells can complicate manual and automated evaluation of the specimen and introduce errors into diagnostic measurements and analytical outcomes based on the images. Thus, fluid ports that deliver stain to specimen  3  can be spaced a certain distance from the specimen-containing area of a slide to improve staining results. 
     In addition, the use of pairs of ports, e.g., multiple pairs of ports, located opposite each other, can also improve staining uniformity. For example, in some embodiments, two ports are used to deliver stain to specimen  3 . The two ports can be located on platform  60 A at positions spaced a certain distance (e.g., are offset) from the edges of specimen  3 , and located opposite each other in a direction parallel to the short edges of platform  60 A. When stain is dispensed from the two spaced ports, a relatively uniform quantity of stain is deposited on the cells in different regions of specimen  3 , and improved staining homogeneity is observed in specimen images. 
     Similarly, while ports  40 A- 45 A can generally be positioned as desired to remove excess fluids from the surface of substrate  2  using one or more vacuum sources, in some embodiments ports that are used for fluid removal are spaced at a distance from positions on platform  60 A that are directly beneath cells within specimen  3  on substrate  2 . Positioning waste removal ports in this manner (i.e., not directly opposing a portion of specimen  3 ) reduces the chances that when such ports are actuated to evacuate fluids from substrate  2 , cells from specimen  3  are inadvertently damaged or drawn into the fluid removal ports. In certain embodiments, due to the difference in lengths of the long and short sides of platform  60 A, the waste removal ports are spaced apart from the edge of the specimen area and arranged opposite each other along a direction parallel to the long edges of platform  60 A. 
     Fixative Phases 
     Fluid tubes  52 A- 55 A and  52 B- 55 B can be positioned to deliver fixative to platforms  60 A and  60 B, separation  92 , substrate  2 , and specimen  3  during specimen processing. Fixatives that can be used include chemicals used for protecting biological samples from decay, and such fixatives can impede biochemical reactions occurring in the specimen and increase the mechanical strength and stability of the specimen. Various fixatives can be used including, but not limited to, methanol, ethanol, isopropanol, acetone, formaldehyde, glutaraldehyde, EDTA, surfactants, metal salts, metal ions, urea, and amino compounds. 
     Referring to  FIG. 4 , one or more fluid tubes  52 - 55 A can be connected to a port inside platform  60 A and a respective fixative reservoir  210 A. The fluid tubes may also include a connection to a pump  200 A and/or a valve capable of directing fixatives from the reservoir through the tube and a port located on the platform, and onto a substrate and specimen. As an example, pump  200 A can direct fixative from reservoir  210 A through tube  54 A, through block  80 A, out from port  44 A, onto platform  60 A, into the separation  92  between the platform  60 A and substrate  2 , and onto substrate  2  containing specimen  3 . After applying a specific quantity of fixative to substrate  2 , a vacuum or other suction source  220 A and/or  221 A can evacuate residual fixative from platform  60 A, the separation  92 , and substrate  2  into waste container  230 A and/or  231 A via one or more of ports  40 A and/or  41 A through waste tubes  50 A and  51 A. 
       FIG. 9  shows a flow chart  700  that includes a series of steps for applying fixative to a specimen. In step  702 , a pump (e.g., pump  200 A) directs fixative (e.g., methanol) from a reservoir (e.g., reservoir  210 A) into a fixative tube (e.g., tube  54 A). In step  704 , the fixative is directed into port  44 A attached to block  80 A. Then, in step  706 , the fixative is directed out of port  44 A in platform  60 A. In step  708 , the fixative is directed out through port  44 A and into separation  92  between substrate  2  and platform  60 A. Finally, in step  710 , specimen  3  on substrate  2  is fixed by the fixative solution. 
     In some embodiments, pump  200 A directs methanol through tube  54 A and port  44 A, onto platform  60 A and into the separation  92  at a flow rate of 70 microliters per second for a period of four seconds. A vacuum or other suction source  220 A and/or  221 A then removes residual methanol present in separation  92  and/or on the platform  60 A and substrate  2  using ports  40 A and/or  41 A and waste tubes  50 A and/or  51 A (further described below). Next, the pump  200 A can again direct methanol through tube  54 A and port  44 A, and onto platform  60 A at a flow rate of 70 microliters per second for a period of four seconds, followed by a second fluid evacuation process. This process of fixing and evacuating can be repeated again, using the same or a different fixative, depending on the type of biological specimen requiring fixation. Further, machine  1  is capable of varying the frequency and flow rates for each fixing phase. Other flow rates sufficient to overcome any surface tension in the fluid located in separation  92  and fix specimen  3  for further processing and evaluation can also be used. By adjusting the frequency and/or flow rate of the fixing phases, machine  1  can achieve optimal fixation for various specimens using several different fixatives. Machine instructions for different types of specimens can be hardwired or preprogrammed in control unit  5  and selected by a system operator as needed. 
     In general, a wide variety of fixatives can be applied to specimens during fixative phases. For example, 85% methanol can be used as the fixative. For some stains, an ethyl alcohol or formaldehyde based fixative can be used. Additional fixative formulations that can be used to prepare the specimen are disclosed, for example, in U.S. Provisional Patent Application No. 61/505,011, the entire contents of which are incorporated by reference herein. 
     Staining Phases 
     Machine  1  also includes tubes and ports configured to apply one or more dyes or stains to a specimen fixed to a substrate in one or more staining phases. Staining a specimen increases the contrast of the specimen when it is viewed or imaged under a microscope or other imaging device. Romanowsky stains and/or other dyes or stains can be used, including hematoxylin and eosin, fluorescein, thiazin stains using antibodies, nucleic acid probes, and/or metal salts and ions. Additional stain formulations that can be used to prepare the specimen are disclosed, for example, in U.S. Provisional Patent Application No. 61/505,011. 
       FIG. 10  is a flow chart  800  that includes a series of steps for applying stain to a specimen. In step  802 , a pump (e.g., pump  201 A) directs dye or stain from a reservoir (e.g., reservoir  211 A) into a stain tube (e.g., tube  52 A). In step  804 , the stain is directed into a port (e.g., port  42 A) attached to block  80 A. Next, in step  806 , the stain flows out of port  42 A in platform  60 A. In step  808 , the stain flows into separation  92  between substrate  2  and platform  60 A and thereafter, in step  810 , stains specimen  3  on substrate  2 . 
     In some embodiments, multiple tubes and ports can be used to apply stain to specimen  3 . For example, a second pump (e.g., pump  202 A) can direct stain (e.g., the same stain or a different stain from that dispensed from reservoir  211 A) from reservoir  212 A through tube  53 A and port  43 A and onto platform  60 A. In certain embodiments, two or more fluid tubes may connect to a shared stain reservoir or pump and/or valve used to direct stain through the ports and onto the platform. Referring back to  FIG. 2 , tube  52 A may deliver red stain, such as a fluorescein dye, to the platform, substrate  3 , and specimen  2 . Tube  53 A may deliver blue stain, such as a thiazin dye. In  FIGS. 1-6 , the numbers, locations, and sizes of the ports on platform  60 A are selected to optimize the application of stain to a specimen fixed to the substrate. If other stains are selected, a different number, locations, and sizes of ports may be preferable depending on the viscosity of the stain. 
     Each of ports  40 A- 45 A (and  40 B- 45 B) can include both an input channel for receiving fluid and an output channel for outputting fluid. In some embodiments, the output channels of the rinse  45 A, fixative  44 A, and staining ports  42 A- 43 A are on the upper surface of platform  60 A, and the input channels of vacuum ports  40 A and  41 A may be on opposite ends of the upper surface of platform  60 A. The input channels of the rinse  45 A, fixative  44 A, and staining ports  42 A- 43 A may be situated on the same lateral side of block  80 A, and the output channels of the vacuum ports  40 A and  41 A can be positioned on opposite lateral sides of block  80 A. 
     By way of example and with reference to  FIGS. 2 and 10 , control system  5  instructs a pump (e.g., pump  201 A) in step  802  to direct a stain (e.g., a stain comprising fluorescein dye) from a stain reservoir into fluid tube  52 A. In step  804 , the stain enters port  42 A from the fluid tube. Then, in step  806 , the stain leaves port  42 A at a flow rate of 140 microliters per second, for a five second period, and in step  808 , the stain is deposited into separation  92  between platform  60 A and substrate  2  containing specimen  3 . In step  810 , specimen  3  on substrate  2  is stained. Following staining, a vacuum or other suction source (e.g., pumps  220  and/or  221 ) may then evacuate residual stain present in separation  92 , on platform  60 A, and on substrate  3  using ports  40 A- 41 A and waste tubes  50 A- 51 A. 
     Machine  1  can be programmed to repeat these staining and evacuation phases after a delay (e.g., a delay of between 3 seconds and 10 seconds, such as a five second delay), following the first staining phase. A second pump  202 A can be instructed by control system  5  to direct thiazin dye from a stain reservoir through fluid tube  53 A, out port  43 A at a flow rate of 140 microliters per second, and onto platform  60 A for a period of time, e.g., three seconds. A vacuum or other suction source (e.g., pump  220 A and/or  221 ) may then evacuate residual thiazin dye present in separation  92  and/or on platform  60 A and/or on substrate  2  using ports  40 A- 41 A and waste tubes  50 A- 51 A. As with the fixing phases, machine  1  is capable of varying the frequency, delay times, and flow rates for each staining phase. The flow rate may range, e.g., from 70 to 140 microliters per second, or may be smaller or greater than the outer limits of this range (e.g., 10 to 500 microliters per second) provided the flow rate is sufficient to overcome any surface tension present in the fluid located in separation  92  and desirably stain the specimen for the intended evaluation. 
     Exemplary stains that can be applied to specimens include, but are not limited to: Wright-Giemsa stain, Giemsa stains, and Romanowsky stains. Other agents such immunocytochemical reagents or other markers of specific cell components can also be applied to specimens. 
     Waste Fluid Removal 
     As referenced above, a vacuum or other suction source  220  and/or  221  can evacuate residual fluid from substrate  2 , separation  92 , and platform  60 A during or between fixing and staining phases. Referring to  FIG. 1 , one or more waste tubes can be connected to sides  82 A and  84 A of block  80 A. Waste or vacuum tubes  50 A and  51 A are used to withdraw fluid and small particulate matter from platform  60 A, separation  92 , and substrate  2  into a waste container or other location separate from machine  1 . With reference to  FIG. 2 , waste tubes  51 A and  51 B may be connected to separate vacuum sources  220  and  221 , and waste containers  230  and  231 , at the distal ends of the waste tubes. Alternatively, two or more waste tubes can be connected to a single vacuum source, and the same waste container, as shown in  FIG. 4 . Waste tubes  50 A and  50 B may extend through pinch valves  90 A and  90 B, respectively. 
     A vacuum or other source (e.g., vacuum pump  220  and/or  221 ) for applying suction may be connected to one or more of waste tubes  50 A,  50 B,  51 A, and  51 B to draw fluid from the platforms  60 A and/or  60 B, separation  92 , and substrate  2  into waste containers  230  and  231 . The vacuum force applied within the waste tubes may be equivalent to negative one to negative ten pounds per square inch (“psi”) to provide sufficient suction for removing fluids when the separation between the substrate  2  and the platform is between 100 to 200 microns. In general, as used herein, “negative” pressure refers to a pressure less than the ambient pressure within machine  1  or the environment surrounding machine  1 . For example, in some embodiments, the environment surrounding machine  1  has an ambient air pressure of approximately one atmosphere. “Negative” pressures refer to pressures that are less than this ambient air pressure (e.g., a pressure of negative one psi applied to a fluid is a pressure of one psi less than the ambient air pressure exerted on the fluid). Other vacuums ranging from negative 0.1 psi to negative 14 psi (e.g., negative six psi), or greater, can be used provided such vacuums are sufficient to overcome any surface tension in the fluid present in the separation and remove all residual fluid in the separation and on the substrate and specimen. In addition, immediately prior to applying vacuum to evacuate fluids from the separation, actuator  30 A can raise the proximate edge of substrate  2  a distance of 15-35 microns from the specimen processing position. This increased separation between substrate  2  and platform  60  can improve evacuation of any residual fluids in separation  92  during a vacuum phase. 
     In some embodiments, control system  5  is configured to vary the frequency and vacuum applied for fluid removal during specimen processing.  FIG. 11A  includes a flow chart  900  that features a series of steps for removing excess fluid from a substrate. Following a fixing phase, for example, control system  5  can open pinch valves  90 A and/or  90 C in step  902  and apply a vacuum of negative 5 psi in the waste tubes (e.g., waste tubes  50 A and  51 A) for a five second period. During this period, fixative is removed (step  904 ) the separation, substrate, and platform through ports  40 A and  41 A. The fluid travels through the waste tubes in step  906 , and is deposited in into one or more waste containers (e.g., containers  230  and/or  231 ) in step  908 . Once the evacuation period expires, control system  5  can instruct one or more of the pinch valves  90 A,  90 C to close off the waste tubes  50 A and/or  51 A in step  910 , thereby preventing further evacuation by the vacuum  220 - 221 . Control system  5  may direct machine  1  to repeat this fluid removal step after each fixing phase. 
       FIG. 11B  includes a flow chart  1000  that features an alternate series of steps for removing excess fluid from a substrate. The method in flow chart  1000  does not use pinch valves to seal waste tubes. Instead, after a fluid application phase, suction source  220  and/or  221  are initialized in step  1002  and enter an active state in step  1004 . The suction source applies a vacuum of negative 3 psi in waste tubes  50 A and/or  51 A for a four second period to remove fluid from separation  92 , substrate  2 , and platform  60 A through ports  40 A and  41 A in step  1006 . The evacuated fluid travels through waste tubes  50 A and/or  51 A in step  1008 , and is deposited in one or more waste containers  230 ,  231  in step  1010 . Machine  1  may repeat this fluid removal step after each fluid application phase. By varying the frequency and pressure applied during fluid removal steps, machine  1  may achieve optimal fixing, staining, and rinsing of biological specimens. 
     Pinch values  90 A,  90 B,  90 C, and  90 D close off waste tubes  50 A,  50 B,  51 A, and  51 B, as shown in  FIG. 1 . The pinch valves  90 A- 90 D may be mechanically, electrically, hydraulically, or pneumatically actuated through actuators contained within or external to the valves. Pinch valves  90 A- 90 D operate to prohibit fluid flow through waste tubes  50 A,  50 B,  51 A, and  51 B. For example, when changing or emptying a full waste container  230  from machine  1 , it may be desirable to close the pinch valves ( 90 A- 90 D) to prevent leakage of residual fluids present in the waste tubes. Different valve types or other mechanisms such as clamps or stoppers may be used with embodiments of machine  1  to close the waste tubes  50 A,  50 B,  51 A, and  51 B. 
     Rinsing Phases 
     Rinse solutions can be applied during specimen processing with machine  1  in one or more rinse phases. For example, it may be desirable to remove residual and/or excess fluids from specimen  3  on substrate  2 , separation  92 , and platforms  60 A and/or  60 B between fixing phases, between staining phases, and/or between fixing and staining phases. Rinse solutions compatible with the present systems and methods include distilled water; buffered, aqueous solutions; organic solvents; and mixtures of aqueous and organic solvents, with or without buffering. Additional formulations for rinse solutions that can be used to prepare the specimen are disclosed, for example, in U.S. Provisional Patent Application No. 61/505,011. 
       FIG. 12  includes a flow chart  1100  featuring a series of steps for rinsing a specimen. In step  1102 , a pump (e.g., pump  203 A) directs rinse solution (e.g., comprising distilled water) from a reservoir (e.g., reservoir  213 A) into a rinse tube (e.g., rinse tube  55 A). In step  1104 , the rinse solution enters port  45 A connected to block  80 A. In step  1106 , the rinse solution flows onto platform  60 A through the output channel of port  45 A, and in step  1108 , the rinse solution enters separation  92  between substrate  2  and platform  60 A. In step  1110 , rinsing of specimen  3  is performed. Finally, in step  1112 , a vacuum source  220 ,  221  applies suction to one or more of waste tubes  50 A and  51 A to remove rinse solution from separation  92  and substrate  2 ; the rinse solution is transported to waste container  230  and/or  231 . 
     In some embodiments, control system  5  may direct pump  203 A to apply the rinse solution at a flow rate of, e.g., 70 microliters per second for a period of, e.g., five seconds. As with fixing phases, control system  5  may vary the duration and flow rate of each rinse phase and the number of rinse phases. In addition, control system  5  may adjust the placement of one or more rinse phases during specimen processing. Control system  5  may, for example, direct that a rinse phase occur once, after completion of all fixing phases, and that a second rinse phase occur once, after completion of all staining phases. Alternatively, rinse phases may be interspersed between two or more fixing phases or between two or more staining phases. 
     Agitation Phases 
     Specimen processing in certain embodiments may include one or more agitation phases to disperse fixative, stain, and/or rinse fluids throughout separation  92 , substrate  2  containing specimen  3 , and platforms  60 A and/or  60 B during the fixing, staining, and/or rinsing phases.  FIG. 13  includes flow chart  1200  that features a series of steps for agitating a specimen. Actuator  30 A and/or  30 B, shown in  FIG. 3A , can provide fine movement adjustment for changing the position of substrate  2  relative to platform  60 A and/or  60 B. 
     Control system  5  can include software and/or hardware for instructing the actuator  30 A and/or  30 B to initiate an agitation phase. Actuator  30 A and/or  30 B can be configured to move substrate arm  20 A and/or  20 B up and down upon an agitation initiation command from the control system. The agitation phase may repeat for a predetermined number of agitation cycles. The term “agitation cycle,” as used herein, refers to motion from a starting position in an upward direction, followed by movement in a downward direction opposite to the upward direction. In some embodiments, one or more agitation cycles return substrate  2  to the starting position at the conclusion of each cycle, or at least at the conclusion of some cycles. In certain embodiments, substrate  2  does not return to the starting position at the conclusion of some or all of the agitation cycles, but each cycle still includes an upward motion followed by a downward motion. Actuator  30 A and/or  30 B typically continues moving substrate  2  in one or more agitation cycles until a stop command is sent to the actuator from the control system  5 . An agitation phase may temporarily increase the separation size (separation distance) between substrate  2  and the surface of platform  60 A and/or  60 B, and then return the substrate to the specimen processing position. In addition, an agitation phase may include a series of movements that shift substrate  2  between an angular position relative to the surface of platform  60 A and/or  60 B and the specimen processing position. Surface tension in the fluids dispensed into the separation between the platform and substrate  2  causes a redistribution of fluid molecules on the substrate when the substrate moves from the specimen processing position during the agitation phase and can advantageously improve fluid distribution across the specimen. 
     Other methods can also be used to move substrate  2  relative to the platforms during agitation phases. For example, in some embodiments, the positions of one or more of offsets  70 A-D and/or  71 A-D (e.g., the amount by which the offsets extend above the surfaces of platforms  60 A and/or  60 B) can be rapidly adjusted to agitate specimen  3 . In certain embodiments, the positions of platforms  60 A and/or  60 B can be adjusted to cause agitation of specimen  3 . For example, platforms  60 A and/or  60 B can be moved alternately up and down (e.g., corresponding to the direction of movement of substrate  2  described above) to cause agitation of specimen  3 . 
     In some embodiments, agitation of specimen  3  can be effected by varying the extent to which actuator  30 A and/or  30 B drives substrate  2  towards offsets  70 A-D and/or  71 A-D when the substrate arms are made of a material that flexes, as discussed below. Strain gauges can be used to measure and adjust the frequency of the agitation applied to substrate  2  by detecting the variation in strain in the substrate arms as a function of time. 
     Referring to  FIG. 13 , in a first step  1202 , an agitation phase is initiated. In step  1204 , control system  5  instructs actuator  30 A to begin an agitation cycle. In response to this instruction, actuator  30 A rotates substrate  2  upward in step  1206 , increasing the distance between substrate  2  and platform  60 A. Then, in step  1208 , actuator  30 A rotates substrate  2  downward toward platform  60 A, reducing the distance between the substrate and platform  60 A. In decision step  1210 , if the agitation phase is to continue, control returns to step  1204  and the rotation of substrate  2  by actuator  30 A occurs again in another agitation cycle. If the agitation phase is to terminate, then control passes from step  1210  to step  1212 , where substrate  2  is returned to its initial position with agitation complete. 
     The agitation phase can include one or more agitation cycles applied through actuator  30 A and/or  30 B. Further, agitation phases can occur once or multiple times during each of the fixative, stain, and/or rinse phases and in varying frequencies between each of the fixing, staining, and/or rinsing phases. For example, and referring to  FIG. 3A , actuator  30 A and/or  30 B may raise the proximate edge of substrate  2  vertically a distance of 35 microns from the specimen processing position and subsequently return substrate  2  to the specimen processing position three times, once after each fixing, staining, and rinse phase. Actuator  30 A and/or  30 B may complete each agitation cycle in two seconds (e.g., one second to raise the proximate edge of substrate  2  vertically a distance of 35 microns from the specimen processing position and one second to return the substrate to the specimen processing position). Machine  1  is capable of carrying out instructions to vary the agitation frequency and distance for each agitation cycle and/or phase. For example, an agitation phase may include actuator  30 A and/or  30 B raising the proximate edge of substrate  2  vertically a distance of 5 microns from the specimen processing position and then returning the substrate to the specimen processing position, 10 to 20 times per second. 
     Alternative combinations of agitation distances and frequencies can also be used. For example, in some embodiments, the agitation distance is 5 microns or more (e.g., 15 microns or more, 25 microns or more, 50 microns or more, 100 microns or more, 150 microns or more, 200 microns or more, 250 microns or more, 300 microns or more, 500 microns or more, 700 microns or more, 1 mm or more. For example, in certain embodiments, the agitation distance is between 35 microns and 350 microns. 
     In some embodiments, the agitation cycle frequency is one cycle per second or more (e.g., two cycles per second or more, three cycles per second or more, four cycles per second or more, five cycles per second or more, seven cycles per second or more, ten cycles per second or more). 
     Additional agitation techniques can also be used. For example, in some embodiments, substrate gripper  20 A and/or  20 B may include an actuator that rotates the substrate about an axis perpendicular to the rotational axis of actuator  30 A and/or  30 B depicted in  FIGS. 1 and 3 . 
     Alternatively, platform  60 A and/or  60 B may be equipped with an offset adjuster for raising or lowering the one or more offsets  70 A-D and/or  71 A-D during fixing, staining, and rinsing phases. To implement the offset adjuster, platform  60 A and/or  60 B can include offsets that are attached to an internal plate in the platform. The height of the plate may be varied using an internal actuator, thus varying the height of the offsets. Alternatively, the position of the offsets  70 A-D and  71 A-D relative to substrate  2  can be changed by instructing the actuator to move platform  60 A and/or  60 B, or block  80 A and/or  80 B, thereby changing the separation distance during the agitation phase. Control system  5  can adjust the frequency of fluid cycles, flow rate, offset height, separation distance, and agitation parameters and frequency to process specimens more efficiently, using significantly less fluid volumes during the specimen preparation process as compared to conventional staining and preparing techniques. 
     In some embodiments, substrate arms may be made of a material that flexes such that if a substrate in the specimen processing position rests against only two offsets extending from the platform, an actuator or other motive force element may rotate the slide further towards the platform surface until the slide rests against all four offsets. Varying the position of the substrate between these two positions may accomplish sufficient agitation during specimen processing. Substrate arms may include strain gauges to monitor the strain in the substrate arm, and may be used to inform control system  5  of the position of the substrate relative to the platform offsets. In addition, the control system may include information corresponding to the thickness imperfections of the substrate, which the control systems may account for when placing the substrate in the specimen processing position or during agitation phases. 
     Drying Phases 
     In certain embodiments, the control system  5  can dry the specimen using a dryer  4  attached to machine  1 .  FIG. 14  includes a flow chart  1300  that features a series of steps for drying a specimen. Following the initial step  1302  in which the completion of the staining and other phases (e.g., one or more rinsing phases) is verified, in step  1304  the dryer  4  directs a flow of air across the specimen. The drying process continues in step  1306 , until a signal is received from the control unit to stop the drying. When the signal is received, the dryer stops the flow of air across the specimen and the drying phase terminates at step  1308 . 
     In general, machine  1  can be controlled to vary the temperature of the air, the flow rate, the duration of the applied air flow, and the phase(s) during specimen processing for drying the specimen  3 . For example, after completing a staining phase, dryer  4  can direct a flow of air at approximately 120° F. at a rate of 10 liters per minute for a period of 7 seconds across the specimen. Other air temperatures (e.g., ambient temperature up to 300° F.), air flow rates (e.g., one liter per minute to 100 liters per minute), and air flow periods (e.g., from a few seconds to several minutes) can also be used. 
     Specimen Examination Systems 
     The automated specimen preparation machines and apparatus disclosed herein, including machine  1 , can generally be used with, and/or incorporated into, larger specimen examination systems, such as those described in U.S. Patent Application Publication No. 2009/0269799, the entire contents of which are incorporated herein by reference. For example,  FIG. 15  shows a schematic diagram that illustrates one possible embodiment of a specimen examination system  2000 . System  2000  includes a platform  2100 , a light receiving device  2200 , a computer  2300 , an applicator  2400 , a gas circulation device  2500 , a light source  2600 , a dispenser  2800 , a discharge device  2900 , a slide labeler  3000 , and slide label reader  3100 . An advancer  2110  may be configured to receive one or more slides or other substrates  2700 . The advancer  2110  may be attached to a surface, such as the top surface  2101 , of the platform. The advancer  2110  may take the form of a belt, and the system may use a mechanical arm, gravity, magnetism, hydraulics, gears, or other locomotion techniques to move substrate-mounted specimens along the surface  2101  of the platform. 
     The platform  2100  may also include a feeder  2102  and a collector  2106  for respectively feeding and collecting substrates  2700  (e.g., slides) from or to a stack or rack. Feeder  2102  may be equipped with a feeder propulsion mechanism  2103  (such as rubberized wheels) for pushing the specimens onto advancer  2110 . Alternatively, a mechanical arm could be used to grab substrates  2700  and place the substrates on the advancer directly. Alternate mechanisms to propel the substrates out of feeder  2102  may be used such as magnets or hydraulics. The feeder may include a sensor for determining how many slides are present. The sensor could measure the weight of substrates  2700  for example to determine how many substrates are present. Collector  2106  can also include a sensor for determining how many substrates are present. The sensor can be configured to inform the computer  2300  when a preset number of specimens have been analyzed, and/or can inform the computer of the receipt of a specimen mounted on a substrate on an ongoing basis. 
     Light receiving device  2200  can be a microscope (such as brightfield microscope), a video camera, a still camera, or other optical device that receives light. Embodiments that include a standard brightfield microscope can also include an automated stage (e.g., a substrate mover  2201 ) and an automated focus. In some embodiments, a microscope can be attached to a motorized stage and a focus motor attachment. The microscope can have a motorized nosepiece for allowing different magnification lenses to be selected under the control of computer  2300 . A filter wheel can be used to enable the computer  2300  to automatically select narrow band color filters in the light path. LED illumination can be substituted for the filters, and the use of LEDs can reduce the image acquisition time as compared to the time required for filter wheel rotation. For example, a 1600×1200 pixel FireWire® (IEEE1394 High Performance Serial Bus) camera can be used to acquire the narrow band images. 
     In some embodiments, light receiving device  2200  receives light reflected from substrate  2700  and stores one or more images formed by the reflected light. Alternatively, or in addition, in some embodiments, fluorescent emission from the specimen on the substrate can be detected by light receiving device  2200 . 
     In certain embodiments, light receiving device  2200  is configured to obtain transmission images of specimens on substrates. For example, light emission source  2600  can be positioned below the platform and may direct light so that it passes through platform  2100  and substrate  2700  into light receiving device  2200 . 
     Light receiving device  2200  and any of the other components shown in  FIG. 15  can be interfaced with the computer  2300  through links ( 2011 - 2014 ), which can provide energy to the component, provide instructions from computer  2300  to the component, and/or allow the component to send information to computer  2300 . Links  2011 - 2014  can be wired links or wireless links. 
     Light receiving device  2200  may be capable of X, Y, and Z axial movement (in other embodiments, a motorized stage or substrate mover  2201  may provide X, Y, and Z movement). Light receiving device  2200  can include pan, tilt, and/or locomotive actuators to enable computer  2300  to position light receiving device  2200  in an appropriate position. Light receiving device  2200  can include a lens  2210  that focuses incoming light. 
     Light receiving device  2200  can be selected to capture black and white and/or color images. In some embodiments, two or more light receiving devices can be used to divide the processing time associated with capturing the images. For example, a low magnification imaging station can be followed by a high magnification imaging station. Similarly, in some embodiments, system  2000 , platform  2100 , computer  2300 , and/or light receiving device  2200  can direct substrate mover  2201  to move substrate  2700  to ensure the capture and storage of one or more images of all, or most, of the cells on the substrate or on a specific portion of the substrate. 
     Computer  2300  can be a laptop, a server, a workstation, or any other type of computing device. The computer can include a processor, a display  2320 , an interface  2310 , and internal memory and/or a disk drive. Computer  2300  can also include software stored in the memory or on computer readable, tangible media such as an optical drive. The software may include instructions for causing the computer to operate light receiving device  2200 , applicator  2400 , gas circulation device  2500 , platform  2100 , advancer  2110 , light source  2600 , dispensers  2450  and/or  2800 , specimen preparation machine  1 , or any component within or connected to one of these components. Similarly, the computer is arranged to receive information from any of these components. 
     For example, the software may control the rate of dispersal of substrates from the feeder  2102 , and feeder  2102  may inform the computer about the number of substrates present. In addition, computer  2300  can also be responsible for performing the analysis of the images captured by light receiving device  2200 . Through the analysis process, the computer can be arranged and controlled to calculate the number of a specific type of cell in a particular volume of blood, for example for blood, red cell, white cell, and platelet counts and other measured and derived components of the complete blood count such as: hemoglobin content, red blood cell morphology, or white blood cell count differential could be calculated. The image analysis software can analyze each individual field and sum the total red and white cell counts. To calculate the total counts per microliter in a patient blood sample, the number counted on the slide can be multiplied by the dilution ratio and volume of the sub-sample. Results of the counts, morphologic measurements, and images of red blood cells and white blood cells from the slide may be shown on the display  2320 . 
     In some embodiments, computer  2300  is configured to display numerical data, cell population histograms, scatter plots, and direct assessments of cellular morphology using images of blood cells displayed on the monitor. The ability to display cellular morphology provides users of system  2000  the ability to quickly establish the presence or absence of abnormalities in cell morphology that may warrant preparing an additional slide for manual review by an experienced technician or other professional. The software can also provide the computer with instructions to display images  2331  received from the light receiving device or may cause display  2330  to show the results  2332  (in perhaps a chart or graph, for example) of an analysis of the images. Similarly, computer  2300  can be controlled to enumerate the number of cells of a specific type in a particular blood volume or enumerate the number of damaged cells, cancerous cells, or lysed cells in a particular volume of blood. The software enables the computer to perform the analysis process. The computer can use one or more magnifications during the analysis. 
     Although shown as one component, computer  2300  can include multiple computers; a first computer can be used for controlling the components of system  2000 , and a second computer can be used for processing the images from light receiving device  2200 . The various computers can be linked together to allow the computers to share information. Computer  2300  can also be connected to a network or laboratory information system to allow the computer to send and receive information to other computers. 
     In certain embodiments, applicator  2400  can include a syringe, a manual or motor driven pipettor, or a motor-controlled pump attached through a tube to a pipette tip. Applicator  2400  applies a specimen to substrate  2700  in controlled fashion. Exemplary features, attributes, and methods of using applicator  2400  are disclosed, for example, in U.S. Patent Application Publication No. US 2009/0269799. The specimen can include one or more blood components, cells, tissue, or other biological components. 
     Once the specimen has been applied to substrate  2700 , the applied specimen is processed using machine  1 . Machine  1  functions as described herein to apply one or more stains, fixatives, and/or other solutions to the specimen on the substrate. 
     In some embodiments, system  2000  can be configured to achieve minimal overlapping between cells deposited on substrate  2700  by laying down non-touching rows of cells from the tip of applicator  2400 . Increasing viscosity of the diluted fluid or the type or amount of diluent may affect the width of the final settlement positions of specimen flows from the applicator. By selecting a distance between rows to allow for the typical variation in blood samples, all cells can be counted in all samples. 
     Gas movement device  2500 , which can be a separate device as shown in  FIG. 15 , or can be incorporated into machine  1  as discussed previously, can include a fan and/or may include other gas movement devices such as a compressor or a bellows for example. Gas movement device  2500  may be connected directly to the computer  2300  or may be connected through another component such as platform  2100  or applicator  2400 . The gas movement device pushes gas (in some cases atmospheric air) across the substrate to control the rate at which substances on the substrate dry. Moving too much air too quickly (i.e., too high of a fan speed) across the substrate can cause cells in the specimen to burst due to rapid drying, and too little air too slowly (i.e., too low of a fan speed) across the substrate can cause the cells to dry too slowly and appear to shrink. 
     Computer  2300  can select and control the amount of air that moves across the substrate in a period of time (i.e., the cubic feet or cubic centimeters of air per second) based upon the distance the gas movement device is from the substrate, the type of fluid being analyzed, the width of the flows, the temperature of the gas (e.g., air), and the average thickness of the flows. Gas movement device  2500  can be positioned so that the device directs gas so that the gas strikes the substrate at an angle of 30°-60° (e.g., 45°) for a period of about 15 to 20 seconds. In some embodiments, computer  2300  can control humidity and temperature settings in the vicinity of the system to allow the drying process to occur without the use of a gas movement device  2500 . 
     Light emission device  2600 , and the various components thereof, are described by way of example in U.S. Patent Application Publication No. US 2009/0269799. Various wavelengths of light can be generated by light emission device  2600  and detected by light receiving device  2200 . For example, wavelengths such as 415 nm are useful for obtaining a hemoglobin-only image for assessing RBC morphology and hemoglobin content. Light emitted at 600 nm may be useful to provide high contrast images for platelets and nuclei. Other wavelengths may be chosen in order to best discriminate the colors of basophils, monocytes, lymphocytes (all shades of blue), eosinophils (red), and neutrophils (neutral color). 
     EXAMPLES 
     The disclosure is further described by the following examples, which are not intended to limit the scope of the invention recited in the claims. 
     Example 1 
       FIG. 16  is a flow chart  1400  showing a series of exemplary steps for processing a specimen mounted on a substrate. The steps in flow chart  1400  can be used to prepare a biological specimen for examination. Although the description of this process at times refers to specific steps having specific ranges, and/or discloses steps occurring in a specific sequence, this description is intended solely as a non-limiting example. With reference to  FIG. 16 , machine  1  is connected to a control system  5  for commanding the operation of various machine components during the processing steps. In a specimen initiation step, a biological specimen  3  that includes red blood cells, white blood cells, and platelets from an aliquot of blood is applied to a substrate  2  consisting of a glass microscope slide. This can be performed using a different station such as one or more of the stations described in co-pending U.S. Patent Application Publication No. 2008/0102006. In a positioning step  1402 , substrate  2  containing specimen  3  is loaded onto substrate gripper  20 A of substrate arm  10 A as shown in  FIG. 1 . Control system  5  instructs suction source  222  (step  1404 ) to evacuate air from the substrate gripper  20 A. Suction applied through suction ports  21  and  22  (step  1406 ) adheres the substrate  2  to the substrate gripper  20 A during specimen processing. Control system  5  instructs (step  1408 ) the actuator  30 A to rotate the substrate  3  from an open position shown in  FIG. 1  to a specimen processing position shown in  FIG. 3A . In the specimen processing position, specimen  3  faces the surface of platform  60 A while substrate  2  rests against offsets  70 A-D shown in  FIG. 2 . The offsets prevent the substrate  2  from making contact with the surface of platform  60 A. In this example process, the separation  92  between the specimen-containing surface of substrate  2  and the surface of platform  60 A is approximately 100 microns. 
     During the fixation phase (step  1412 , see also  FIG. 10 ), a pump applies fixative to the specimen  3  in step  1414 . Pump  200 A connected to fluid tube  54 A shown in  FIG. 2  propels fixative comprising methanol from a fixative reservoir  210  through tube  54 A, out port  44 A, onto platform  60 A, onto substrate  2  containing specimen  3 , and into the separation  92  between platform  60 A and substrate  2 . Pump  200 A propels methanol from port  44 A at a flow rate of 70 microliters per second for a two second period T 1 , thereby directing a total of 140 microliters of methanol, V 1 , onto substrate  2  containing specimen  3 . 
     Next, in a first agitation step  1416 , control system  5  agitates the substrate by directing actuator  30 A (step  1418 ) to raise the proximate edge of substrate  2  vertically a distance of 35 microns from the specimen processing position and returning the substrate to its specimen processing position. Machine  1  repeats this agitation step four more times. The machine  1  completes the five agitation movements in approximately ten seconds, T 2 , as shown in  FIG. 17 . After agitation, the control system initiates a vacuum or evacuation step  1420 . A vacuum force of negative five psi is applied for one and a half seconds, T 3 , evacuating any residual methanol (step  1422 ) present in the separation, on the platform, or on the substrate via ports  40 A and  41 A, and waste tubes  50 A and  51 A. The evacuated methanol is collected in a waste container  230  and/or  231 . 
     Following the fixing phase, control system  5  initiates (step  1424 ) a first staining phase. In doing so, control system  5  directs the machine  1  to stain the specimen (step  1426 ). Referring to  FIG. 2  and the flowchart of  FIG. 11 , pump  201  connected to fluid tube  52 A propels fluorescein dye from a stain reservoir  211 A out port  42 A, onto platform  60 A, onto substrate  2  containing specimen  3 , and into the separation  92  between the platform  60 A and substrate  2 . Pump  201  dispenses fluorescein dye through port  42 A at a flow rate of 70 microliters per second for a two second period, T 4 , thereby directing 140 microliters of dye, V 2 , onto the substrate. 
     After applying fluorescein dye to specimen  3 , machine  1  performs a second agitation step  1428  by directing actuator  30 A to raise, in step  1430 , the proximate edge of substrate  2  vertically a distance of 35 microns from the specimen processing position and then return the substrate to its specimen processing position. Control system  5  causes the machine  1  to repeat this agitation step two more times and complete the three agitations over a period of approximately six seconds, T 5 , as shown in  FIG. 17 . 
     Next a second vacuum or evacuation phase is initiated in step  1432 . A vacuum of negative five psi applied for three seconds, T 6 , in step  1434  to evacuate any residual fluorescein dye present in the separation  92  or on the platform and substrate via ports  40 A and/or  41 A, and waste tubes  50 A and  51 A. The evacuated fluorescein dye is collected in a waste container  230 A and/or  231  A. 
     After staining the specimen with fluorescein dye, machine  1  initiates a second staining phase in step  1436  using thiazin dye. Pump  202  connected to fluid tube  53 A propels thiazin dye from a stain reservoir through port  43 A, onto platform  60 A, onto substrate  2 , and into the separation  92  between platform  60 A and substrate  2  (step  1438 ). Machine  1  dispenses thiazin dye through port  43 A at a flow rate of 70 microliters per second for a two second period, T 7 , thereby directing a total of 140 microliters of thiazin dye, V 3 , onto the substrate. 
     After applying stain to specimen  3 , machine  1  initiates a third agitation phase in step  1440  by directing actuator  30 A to raise the proximate edge of substrate  2  (step  1442 ) a distance of 35 microns from the specimen processing position and then return the substrate containing specimen  3  to its specimen processing position. Machine  1  repeats this agitation step three more times. The machine completes the four agitation movements over a period of approximately eight seconds, T 8 . 
     A third vacuum or evacuation step  1444  is then initiated. A vacuum of negative five psi is applied for two seconds, T 9 , to evacuate residual thiazin dye in step  1446  present in the separation or on the platform  60 A and substrate  2  via ports  40 A and/or  41 A, and waste tubes  50 A and/or  51 A, after agitation. The evacuated thiazin dye is collected in a waste container  230 A and/or  231 A. 
     Machine  1  then performs two rinse-agitation-vacuum phase sequences. The first sequence of phases is initiated at step  1448  when control system  5  instructs machine  1  to initiate a first rinse phase. A reservoir  213 A containing rinse solution of distilled water is connected to a pump  203  and fluid tube  55 A. Pump  203  directs distilled water through wash tube  55 A that feeds into port  45 A, into the separation  92 , and onto platform  60 A and substrate  2  to rinse specimen  3  in step  1450 . Alternatively, in some embodiments, wash fluid is directed through two or more of fluid ports  42 A to  45 A. Pump  203  directs distilled water out of ports  45 A at a flow rate of 70 microliters per second for two seconds, T 10 , thereby directing a total of 140 microliters, V 4 , of water onto the substrate containing the specimen. 
     Next, control system  5  initiates a fourth agitation phase in step  1452 , directing actuator  30 A (step  1454 ) to raise the proximate edge of substrate  2  vertically a distance of 35 microns from the specimen processing position and returning the substrate to its specimen processing position. Control system  5  may direct the machine  1  to repeat this agitation phase, and complete the two agitations in approximately four seconds, T 11 . 
     Then, a vacuum or evacuation phase is initiated in step  1456 . A vacuum of five psi applied for five and a half seconds, T 12 , in step  1458 , evacuates residual distilled water present in the separation  92  or on the platform  60 A and substrate  2  via ports  40 A and/or  41 A, and waste tubes  50 A and/or  51 A after agitation. 
     Thereafter, in step  1460 , control system  5  directs machine  1  to begin the second rinse-agitation-vacuum phase sequence by initiating a second rinse phase. A second rinse phase (steps  1460 ,  1462 ), a fifth agitation phase (steps  1464 ,  1466 ), and a fifth vacuum phase (steps  1468 ,  1470 ) are performed in the same manner as disclosed above for the first rinse-agitation-vacuum phase. During the second rinse-agitation-vacuum phase, the amount of wash fluid, V 5 , and the processing times T 13 , T 14 , and T 15  are generally the same as in the first rinse-agitation-vacuum phase sequence. 
     After the specimen has been fixed, stained with fluorescein and thiazin stains, and rinsed, machine  1  initiates a drying phase in step  1472 . Dryer  4  directs an air flow of approximately 120° at a 10 liter-per-minute flow rate (step  1474 ) for an eight second period, T 16 , across the specimen. 
     Following completion of these steps, substrate  2  is returned to its original position in step  1476 . In this step, actuator  30 A rotates substrate  2  from the specimen processing position to the open position as depicted in  FIG. 1 . Substrate  2  may then be removed by a substrate mover, and a new substrate may be loaded for processing a new specimen. 
     Example 2 
     The processing steps described above for Example 1 may be adjusted in other embodiments of the invention as follows. In addition, fixative, stains, and rinse solution formulations disclosed in U.S. Provisional Patent Application No. 61/505,011 can be used in the following example processing steps. 
     During a first fixation phase (step  1412 , see also  FIG. 10 ), a pump applies a fixative solution to the specimen  3  in step  1414 . Pump  200 A connected to fluid tube  54 A shown in  FIG. 2  propels a fixative solution comprising methanol from a fixative reservoir  210  through tube  54 A, out port  44 A, onto platform  60 A, onto substrate  2 , and into the separation  92  between platform  60 A and substrate  2 . Pump  200 A propels the fixative solution from port  44 A at a flow rate of 115 microliters per second for a two second period T 1 , thereby directing a total of 230 microliters of the fixative solution, V 1 , onto substrate  2 . 
     Next, in a first agitation step  1416 , control system  5  agitates the substrate by directing actuator  30 A (step  1418 ) to raise the proximate edge of substrate  2  vertically a distance of 35 microns from the specimen processing position and returning the specimen to its specimen processing position. Machine  1  repeats this agitation step five more times. The machine  1  completes the six agitation movements in approximately 12 seconds. After agitation, the control system initiates a vacuum step  1420 . A vacuum force of negative six psi is applied for one and a half seconds, T 3 , evacuating any residual fixative solution (step  1422 ) present in the separation, on the platform, or on the substrate via ports  40 A and  41 A, and waste tubes  50 A and  51 A. The evacuated fixative solution is collected in a waste container  230  and/or  231 . 
     Thereafter, in a second fixation phase including a second agitation step, the foregoing steps of the first fixation phase and first agitation step are repeated. 
     Following the fixing phases, control system  5  initiates (step  1424 ) a first staining phase. In doing so, control system  5  directs the machine  1  to stain the specimen (step  1426 ). Referring to  FIG. 2  and the flowchart of  FIG. 11 , pump  201  connected to fluid tube  52 A propels a first stain solution comprising eosin Y from a stain reservoir  211 A out port  42 A, onto platform  60 A, onto substrate  2  including specimen  3 , and into the separation  92  between the platform  60 A and substrate  2 . Pump  201  dispenses the first stain solution through port  42 A at a flow rate of 115 microliters per second for a two second period, T 4 , thereby directing 230 microliters of the first stain solution, V 2 , onto the substrate. 
     After applying a first stain solution to specimen  3 , machine  1  performs a second agitation step  1428  by directing actuator  30 A to raise, in step  1430 , the proximate edge of substrate  2  vertically a distance of 35 microns from the specimen processing position and then return the specimen to its specimen processing position. Control system  5  causes the machine  1  to repeat this agitation step two more times and complete the three agitations over a period of approximately six seconds, T 5 , as shown in  FIG. 17 . 
     Next a second vacuum phase is initiated in step  1432 . A vacuum of negative five psi applied for three seconds, T 6 , in step  1434  to evacuate any residual first stain solution present in the separation  92  or on the platform and substrate via ports  40 A and/or  41 A, and waste tubes  50 A and  51 A. The evacuated first stain solution is collected in a waste container  230 A and/or  231  A. 
     After staining the specimen with the first stain solution including eosin Y, machine  1  initiates a second staining phase in step  1436  using a second stain solution including azure B and methylene blue. Pump  202  connected to fluid tube  53 A propels the second stain solution from a stain reservoir through port  43 A, onto platform  60 A, onto substrate  2 , and into the separation  92  between platform  60 A and substrate  2  (step  1438 ). Machine  1  dispenses the second stain solution through port  43 A at a flow rate of 115 microliters per second for a two second period, T 7 , thereby directing a total of 230 microliters of the second stain solution, V 3 , onto the substrate. 
     After applying stain to specimen  3 , machine  1  initiates a third agitation phase in step  1440  by directing actuator  30 A to raise the proximate edge of substrate  2  (step  1442 ) a distance of 35 microns from the specimen processing position and then return the specimen  3  to its specimen processing position. Machine  1  repeats this agitation step two more times. The machine completes the three agitation movements over a period of approximately six seconds, T 8 . 
     A third vacuum step  1444  is then initiated. A vacuum of negative six psi is applied for two seconds, T 9 , to evacuate residual second stain solution in step  1446  present in the separation or on the platform  60 A and substrate  2  via ports  40 A and/or  41 A, and waste tubes  50 A and/or  51 A, after agitation. The evacuated second stain solution is collected in a waste container  230 A and/or  231 A. 
     Machine  1  then performs two rinse-agitation-vacuum phase sequences. The first sequence of phases is initiated at step  1448  when control system  5  instructs machine  1  to initiate a first rinse phase. A reservoir  213 A containing a rinse solution is connected to a pump  203  and fluid tube  55 A. Pump  203  directs the rinse solution through wash tube  55 A that feeds into port  45 A, into the separation  92 , and onto platform  60 A and substrate  2  to rinse specimen  3  in step  1450 . Alternatively, in some embodiments, rinse solution is directed through two or more of fluid ports  42 A to  45 A. Pump  203  directs the rinse solution out of ports  45 A at a flow rate of 115 microliters per second for two seconds, T 10 , thereby directing a total of 230 microliters, V 4 , of water onto the substrate. 
     Next, control system  5  initiates a fourth agitation phase in step  1452 , directing actuator  30 A (step  1454 ) to raise the proximate edge of substrate  2  vertically a distance of 35 microns from the specimen processing position and returning the specimen to its specimen processing position. Control system  5  then directs the machine  1  to repeat this agitation phase three more times, and complete the four agitations in approximately eight seconds, T 11 . 
     Then, a vacuum phase is initiated in step  1456 . A vacuum of five psi applied for five and a half seconds, T 12 , in step  1458 , evacuates residual rinse solution present in the separation  92  or on the platform  60 A and substrate  2  via ports  40 A and/or  41 A, and waste tubes  50 A and/or  51 A after agitation. 
     Thereafter, in step  1460 , control system  5  directs machine  1  to begin the second rinse-agitation-vacuum phase sequence by initiating a second rinse phase. A second rinse phase (steps  1460 ,  1462 ), a fifth agitation phase comprising six agitations completed in approximately 12 seconds, and a fifth vacuum phase (steps  1468 ,  1470 ) are performed in the same manner as disclosed above for the first rinse-agitation-vacuum phase. During the second rinse-agitation-vacuum phase, the amount of rinse solution, V 5 , and the processing times T 13 , T 14 , and T 15  are generally the same as in the first rinse-agitation-vacuum phase sequence. In addition, immediately prior to the vacuum phase, actuator  30 A raises the proximate edge of substrate  2  a distance of 15-35 microns from the specimen processing position. This increased separation between substrate  2  and platform  60  improves evacuation of any residual fluids in separation  92  during the final vacuum phase. 
     After the specimen has been fixed, stained with a first stain solution containing eosin Y and a second staining solution containing azure B and methylene blue, and rinsed, machine  1  initiates a drying phase in step  1472 . Dryer  4  directs an air flow of approximately 120° at a 10 liter-per-minute flow rate (step  1474 ) for an eight second period, T 16 , across the specimen. 
     Following completion of these steps, substrate  2  is returned to its original position in step  1476 . In this step, actuator  30 A rotates substrate  2  from the specimen processing position to the open position as depicted in  FIG. 7 . Substrate  2  may then be removed by a substrate mover, and a new substrate may be loaded for processing a new specimen. 
     As illustrated in the example specimen processing steps described above, the systems and methods disclosed herein provide for more efficient specimen processing by consuming fewer reagents as compared to conventional specimen processing methods including automated and manual specimen preparation techniques. Referring to Example 2, machine  1  consumed less than one and a half milliliters of reagents for fixing, staining, and rinsing the specimen during the exemplary processing steps (e.g., 460 microliters of fixative solution+230 microliters of first stain solution+230 microliters of second stain solution+460 microliters of rinse solution=1380 microliters of reagents). In some embodiments, more or less than 1380 microliters of fluids can be used during specimen processing. For example, the amount of fluid used in processing a specimen can be approximately 1150 microliters (e.g., by eliminating one of the rinse phases) or less than 1,000 microliters (e.g., by further eliminating one of the fixative phases). With respect to  FIG. 17 , for Example 1, machine  1  consumed less than one milliliter of reagents for fixing, staining, and rinsing the specimen during the exemplary processing steps (e.g., 140 microliters of methanol fixative+140 microliters of fluorescein dye+140 microliters of thiazin dye+280 microliters of rinse solution=700 microliters of reagents). In some embodiments, more or less than 700 microliters of fluids can be used during specimen processing. For example, the amount of fluid used in processing a specimen can be approximately 560 microliters (e.g., by eliminating one of the rinse phases). 
     In general, the total volume of fluids consumed can be 500 microliters or more (e.g., 520 microliters or more, 540 microliters or more, 560 microliters or more, 580 microliters or more, 600 microliters or more, 650 microliters or more, 700 microliters or more, 750 microliters or more) and/or 2 mL or less (e.g., 1.5 mL or less, 1.4 mL or less, 1.3 mL or less, 1.2 mL or less, 1.1 mL or less, 1.0 mL or less, 900 microliters or less). 
     Referring to  FIG. 17  and Example 1, the specimen preparation process is completed in slightly more than one minute (e.g., 13.5 seconds elapsed during the fixing phase+11 seconds elapsed during the fluorescein dye phase+12 seconds elapsed during the thiazin dye phase+23 seconds elapsed during the rinse phases+8 seconds elapsed during the drying phase=67.5 seconds total elapsed time). In certain embodiments, specimen preparation can be completed in more, as in Example 2, or less than 67.5 seconds. For example, specimen processing can be completed in 180 seconds or less (e.g., 150 seconds or less, 120 seconds or less, 90 seconds or less, 80 seconds or less, 70 seconds or less, 60 seconds or less, 50 seconds or less, or 40 seconds or less). 
     Further, while the foregoing exemplary process describes processing time for a single specimen, systems and methods for processing multiple substrates (e.g., machine  1  in  FIG. 1 , configured to process two substrates, and/or systems configured to process three or more substrates) are capable of processing more than 100 specimens per hour (e.g., between 60 specimens and 120 specimens per hour). Use of the systems and methods disclosed herein in laboratory settings can result in faster throughput on a per specimen basis, while consumption of fluids (e.g., fixative, stain, and rinse fluids) is reduced compared to conventional automated systems and manual specimen preparation techniques. 
     OTHER EMBODIMENTS 
     It is to be understood that while the invention has been described in conjunction with the detailed description, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.