Abstract:
A method for preserving the temperature integrity of cryogenically preserved biological samples is presented, involving initiating a countdown sequence upon removal of a sample from a controlled portable or bulk storage environment, requiring operator action to terminate the countdown on restoration of the sample to a controlled environment. Audible and visual warnings are provided to an operator prior to attainment of a critical temperature, beyond which damage may accrue to the sample. A portable device facilitating execution of the method is described.

Description:
FIELD OF THE INVENTION 
     This invention relates to a method and to an associated device or apparatus for maintaining the temperature integrity of cryogenically preserved materials. More particularly, this invention relates to a method and to an associated apparatus for safeguarding the cryogenic preservation of biological specimens during transfer of the specimens to or from a long-term storage container. 
     BACKGROUND OF THE INVENTION 
     When properly treated, biological specimens including human tissue and cell lines may be viably stored almost indefinitely at temperatures approaching that of liquid nitrogen, so long as that temperature is maintained. As long as a specimen is stored in a bulk storage facility it is relatively easy to maintain that specimen at a steady liquid nitrogen temperature. However, once the temperature of a specimen is raised significantly, the integrity of the specimen suffers. More specifically, unintended (upward) temperature excursions (UTE&#39;s), even falling far short of thawing, may permit the growth of ice crystals inside stored biological samples. Ice crystals may disrupt cell membranes, destroy cellular organelles and genetic material, and render the biological samples inviable. Such damage is irreversible and, if not recorded or reported at the time, may go undetected until the samples are deliberately thawed for use, years or possibly decades later. 
     When it becomes necessary to move or transfer a specimen, the possibility for uncontrolled and unrecorded temperature excursions occurs. The problem is compounded because an operator may not be aware that a removed specimen has undergone an unacceptable temperature excursion, or, inadvertently allowing such an excursion to occur, may not wish to record such an event out of concern for his or her continued employment and the possibly accurate perception that the damage is unlikely to be discovered during his or her tenure, or possibly, lifetime. 
     For these reasons, any installation undertaking the long-term cryogenic storage of viable tissue samples and cell lines must embody quality control and quality assurance measures, both to render unintended upward temperature excursions of stored material improbable, and to assure end-users of the improbability of such excursions and of the over-all reliability of the storage facility, to which irreplaceable samples may be entrusted. There is a continuing need for improved devices which will assist in maintaining the temperature integrity of biological samples undergoing cryogenic processing, and in particular, during operations which require the temporary removal of individual specimens from a controlled bulk storage facility and exposure to ambient conditions during such transfer, transportation, or other intermediate steps prior to a final deliberate thawing and use. There is yet a further need for methods and devices which will assure the temperature integrity of a multitude of portable individual samples within a bulk storage facility without the necessity of expensive sensing devices or permanent sensor leads affixed to each cell. 
     OBJECTS OF THE INVENTION 
     An object of the present invention is to provide a method and/or an apparatus for assisting in maintaining the temperature integrity of specimens stored at or within a preselected temperature range. 
     A more particular object of the present invention is to provide such a method and/or apparatus which will facilitate the maintenance of temperature integrity of cryogenically preserved biological specimens temporarily removed from a low-temperature storage container for processing or transport. 
     Yet another object of the present invention is to provide such a method and/or apparatus which will minimize possibilities for operator error during a processing operation requiring temporary removal of a cryogenically preserved biological specimen from a storage apparatus. 
     These and other objects of the present invention will be apparent from the drawings and descriptions herein. 
     SUMMARY OF THE INVENTION 
     The present invention addresses methods and devices for overcoming these limitations of bulk cryogenic storage facilities and in providing temperature control assurance, particularly for the storage of a multitude of small sub-containers (e.g., vials or ampules) which may be intermittently moved among units of the storage facility or between the facility and portable transport containers. 
     A bulk cryogenic storage facility includes one or more bulk containers or storage units each containing a multitude of addressable storage locations or receptacles for the receipt of vials or ampules. Such a bulk storage facility is disclosed in U.S. Pat. No. 5,921,102. A transfer operation comprises removing one or more vials containing cryogenically preserved biological material from respective storage locations and subsequently inserting the vials in different storage locations in the same or different bulk container or in a portable cryogenic storage unit. A portable cryogenic storage unit is a portable device capable of maintaining the temperature of one or more vials or other sub-containers in a cryogenic temperature range during transport. Typically, a portable cryogenic storage unit will take the form of an insulated container including a reservoir for liquid nitrogen, identifiable storage locations for one or more sub-containers, and a temperature monitoring device. 
     A method in accordance with the present invention for maintaining temperature integrity of a cryogenic specimen during a transfer operation comprises triggering an automatic process tracking the temperature of an individual specimen container upon removal of that container from a storage location in a bulk or portable cryogenic storage unit. In general, the temperature of the individual specimen container will rise following removal from a bulk or portable cryogenic storage unit. Upon exceeding a critical temperature T c , irreversible damage is presumed to begin accumulating in the specimen. Therefore, prior to reaching temperature T c , action is initiated at an alarm temperature T A =T c −Δ, where Δ is a temperature increment pre-chosen to allow time for corrective action to be completed prior to incipient accumulation of irreversible specimen damage at temperature T c . 
     Action initiated automatically upon determining that the temperature of a vial in transit has exceeded temperature T A  may include sounding an audible alarm to alert a human operator to return the specimen to the bulk or portable cryogenic storage facility. Action initiated may also include issuance of instructions to a robotic arm to return the specimen to a storage location in a bulk or portable cryogenic storage facility, in conjunction with an audible alarm to alert a human operator to the impending automatic operation. In addition to operator or machine action, the temperature excursion may be recorded as an isolated event or as a portion of a complete temperature profile by a microprocessor associated with the bulk cryogenic storage facility (the “mainframe”, although it may in general be a workstation or PC), or temporarily recorded by a microprocessor associated with the portable cryogenic storage facility, for subsequent transfer to the mainframe associated with the bulk cryogenic storage facility. 
     A temperature tracking operation in accordance with the present invention is implemented by a microprocessor or computer and may include extrapolation of a current temperature of a vial or other container exposed to ambient conditions from an initial temperature of the vial as maintained in the storage location. This extrapolation may be based upon an empirical study of a standard vial containing a material with thermal properties similar to those of a representative biological specimen. In that case, the extrapolation consists of a timing operation coupled with a look-up operation on a standard temperature curve. 
     Use may be made in implementation of the present invention of Newton&#39;s Law of Constant Cooling. The Law of Constant Cooling states roughly that heat transfer rate across an insulator is proportional to a temperature difference across that insulator and approximately independent of the absolute temperatures involved. As a consequence of this law, a wide variety of thermal relaxation problems involving the heating or cooling of a sample in an ambient bath will have solutions of the form: 
     
       
           T−T   amb =( T   0   −T   amb ) e   −α(t−t     0   )  (1) 
       
     
     where T 0  is the sample temperature at time t 0 , T amb  is the ambient temperature, and α is a rate constant dependent on the detailed configuration but not on the temperature or time. The rate constant α may be determined empirically for a given sample content and container by a technician without undue experimentation, in a routine measurement operation. Ambient temperature may be monitored by a sensor affixed externally to a static or portable cryogenic storage device, or input by a technician from another source, whereas initial temperature T 0  may either be actively monitored or known from sample storage conditions, e.g. storage under liquid nitrogen. Finally, time t 0 , time of initial exposure to ambient conditions, may be input by an operator or, in a preferred embodiment, automatically detected upon removal of a vial from a storage location in a cryogenic storage facility. 
     Thus a temperature tracking process may be automated, depending on a technician to input a vial type and contents type, amounting to a selection of α, initial and ambient temperatures and time of initial exposure being automatically detected. 
     Triggering of a tracking operation during a cryogenic transfer operation in accordance with the present invention may comprise actuation of a mechanical switch partially in the form of a finger or detent sensing removal of an individual vial or ampule from a storage location, or the commencement of tracking may be signaled by positive feedback from a robotic arm commanded to remove a vial or ampule from a particular storage location, indicating that the ampule has in fact been removed. 
     Alternatively, a continuous monitoring of vial temperature may be effected via a real time temperature measurement of an individual unit or container. Preferably, the measurement is of an internal temperature of a vial or container in a region containing the biological specimen, rather than a surface measurement. Two methods of accomplishing this object are disclosed in connection with the present invention. 
     In the first instance, an electrical temperature sensing element, such as a silicon diode, a ruthenium oxide resistor, a gallium arsenide semiconductor or a thermocouple such as for example a Chromel (TM) Gold thermocouple, may be embedded in an individual storage container, or reference vial. A pair of leads is electrically attached to a pair of relatively rigid protruding studs protruding from the reference vial. Upon engagement by a robotic arm during a transfer operation, positive electrical contact is made with the embedded sensor, and a continuous temperature reading is obtained, corresponding to an internal temperature of the reference vial. 
     In a second instance, a sensor embedded in an individual reference container or vial does not require direct electrical connections for reading. Such a sensor element depends on bulk constitutive properties of a material, such as magnetic or electrical susceptibility, which forms part of an electric circuit together with coils disposed in a robotic arm or gripper, partially mediated by time-varying electromagnetic fields. Temperature at an interior location of the vial or container is then determined by electrical properties of the total circuit, similarly to cases in the first instance, where the variable electrical properties are resistance or electromotive force, but not requiring a direct mechanical connection to complete the circuit. 
     In a basic embodiment of a method and device in accordance with the present invention, a sample or specimen is initially enclosed within a portable transport container or crystal in an identifiable storage location. A microprocessor attached to the transport container is programmed with the identifier (e.g., bar code) of each specimen associated with a storage location in the transport container. Upon manual withdrawal of a specimen container from the transport container by a technician, a mechanical detent/switch assembly, or other sensing device, delivers a signal to the microprocessor. Using a stored empirically determined temperature curve or equation as discussed previously, an extrapolated temperature of the specimen is determined as a function of elapsed time from withdrawal. The extrapolated temperature may optionally be displayed by a readout attached to the transport container. Upon the extrapolation reaching a preset alarm temperature, T A , an audible alarm is sounded, alerting the technician to the necessity of either returning the specimen to the transport container or depositing the specimen in an alternate cryogenically controlled environment. 
     In the event of insertion of the specimen in a receptacle in a location different from the transport container, the technician presses a button on the transport container, silencing the alarm. Simultaneously, insertion of the specimen in a second location is recorded by an associated microprocessor. Eventually, data from the transport unit associated microprocessor, regarded as a peripheral unit, may be transferred to a microprocessor associated with a second location when that location is a bulk storage location, which microprocessor may be regarded as a mainframe unit; thereby accumulating a complete thermal specimen history on the mainframe. 
     A cryogenic transport device in accordance with the present invention minimally includes at least one identifiable storage location for a vial or ampule, a microprocessor, a means of inputting the identity of a vial inserted into a storage location to the microprocessor, a means of detecting and inputting to the microprocessor a withdrawal of a vial from a storage location located in the cryogenic transport device, and programming enabling the microprocessor to track an internal temperature of the withdrawn vial. 
     In a second embodiment of a method in accordance with the present invention, a specimen is initially stored in an identified location in a bulk cryogenic storage facility. Upon withdrawal of the sample from its storage location by a robotic arm, the withdrawal event is recorded by an associated central microprocessor or mainframe computer. The event may either be identified by actuation of a mechanical switch on withdrawal of the sample, or from proprioceptive feedback from the robotic arm, which, arriving at a known location and grasping with a predetermined force an object of known size is presumed to have grasped a specimen vial. Both techniques may be combined for quality assurance. 
     The withdrawal initiates a tracking process, which associates with the withdrawn specimen at each subsequent time increment a presumed temperature. The temperature is again determined by extrapolation using a pre-recorded empirically determined temperature curve or equation. Alternatively, the instantaneous temperature at an internal point of the vial is determined by the output of one or more sensors attached to or embedded in the vial, and engaged by circuitry in the robotic arm, as discussed above. 
     As before, tracking is continued until the specimen is returned to the original or an alternative storage location in the original or another bulk cryogenic storage unit, or the central microprocessor or mainframe receives input indicating the specimen has been transferred out of the bulk storage system, as for example to a transport unit, or to controlled thawing. As tracking continues a measured or extrapolated alarm temperature T A  may be reached. This event sounds an audible alarm for a storage facility operator, is recorded on a thermal history of the instant specimen by the mainframe, and, if the specimen is still under control of the robotic arm, initiates automatic actions to return the specimen to its original or another available storage location. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a cross-sectional view of a cryogenic transport device in accordance with the present invention. 
     FIG. 2 is a cross-sectional view of a bracket included in the transport device of FIG.  1 . 
     FIG. 3 is an elevational view of another component of the device of FIG.  1 . 
     FIG. 4 is a bottom plan view of the component of FIG.  3 . 
     FIG. 5 is a top plan view of the bracket of FIG.  2 . 
     FIG. 6 is a cross-sectional view of the component of FIG. 3 mounted in the bracket of FIG. 2, showing an instrumented sample container. 
     FIG. 7 is a further elevational view of the component of FIG. 3, showing additional interior detail. 
     FIG. 8 is a schematic of the instrumented sample container of FIG.  6 . 
     FIG. 9 is a top elevational view of the component of FIGS. 3 and 7. 
     FIG. 10 is a schematic elevation of a display and keypad of the device of FIG.  1 . 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     A cryogenic transport container generally shown at  50  in FIG. 1 comprises a lid  64  and a flask  52 . Flask  52  contains a reservoir of LN 2  (liquid nitrogen)  54  or other cryo-coolant, initially filled to a fill line marked by an inside lip or shoulder  60  of a sidewall  56  of flask  52 . A microprocessor  86  mounted in a recess  55  in an outer surface (not designated) of sidewall  56  executes a temperature estimation process when a vial  70 ,  72  is removed from transport container  50 . To that end, microprocessor  86  is wired to a proximity sensor  76  disposed in a pocket  57  in a upper surface of the sidewall. Proximity sensor  76  alerts microprocessor  86  as to the opening of lid  64 , a convenient arbitrary commencement time for a vial removal and transfer operation. To carry out its temperature estimation function, microprocessor  86  also relies on or utilizes initial temperature values provided by an external or room temperature thermocouple  78  and via an electrical penetrator or feed through  90  also wired to the microprocessor. 
     A bracket assembly  62  is disposed in a floor  58  of flask  52  for receipt of a Vial Temporary Storage Holder (VTSH) generally shown at  68 . VTSH  68  is provided with a handle  74  and storage locations or receptacles (not shown in this view) for vials  70 ,  72  etcetera and is removably mounted in bracket assembly  62  inside transport container  50 . An O-ring  92  substantially excludes LN 2  from a recess (not separately designated) enclosing penetrator  90 . 
     Bracket assembly  62  is shown in greater detail in FIG. 2. A bracket body  63  constitutes a principal structural member of bracket assembly  62 . Inside body  63  is an upwardly facing surface  110  which engages a lower face  105  (FIG. 3) of a central body  104  of VTSH  68  to substantially support the VTSH. 
     Electrical penetrator  90  comprises an upper connector  80  and a lower connector  82  joined at a cover plate  94  in turn attached to bracket body  63  via bolts  76 ,  78 . Cover plate  94  does not form a gas tight seal with bracket body  63 , so that any cryo-coolant leaking past O-ring  92  may vent when vaporized and not lift the Vial Temporary Storage Holder from bracket assembly  62 . The primary purpose of the O-ring is to contain the liquid nitrogen, and not to keep the liquid nitrogen away from electrical penetrator  90 , which is unnecessary, as liquid nitrogen forms an excellent insulator. The electrical penetrator however necessarily forms a hot spot in flask  52 , so that it is advantageous to limit the amount of LN 2  present in this area in order to minimize losses by boiling. A standard order of assembly of (discussed below) of a cryogenic transport container  50  (FIG. 1) mounts a VTSH  68  to an instrumented thermally-insulated flask  52  prior to filling the flask with liquid nitrogen, thereby first forming a seal at O-ring  92  and substantially excluding LN 2  from the vicinity of penetrator  90 , as previously indicated. 
     Leads  84  carry a voltage signal to microprocessor  86  from an internal thermocouple (described below) contained in the VTSH  68 . This voltage signal from the internal thermocouple enables microprocessor  86  to monitor pre-transfer or initial temperatures of vials held in the VTSH  68 , while the voltage signal from external sensor or thermocouple  78  enables microprocessor  86  to monitor ambient temperatures. These temperatures are displayed under the control of microprocessor  86  on a combination keypad and display at  88 . In addition to providing microprocessor  86  with an alert signal on removal of lid  64  from flask  52 , the proximity sensor provides the microprocessor with an indication that a vial has been removed from the VTSH  68  when the vial is passed over the sensor by a technician in a procedure to be described hereinafter. 
     FIGS. 3 and 7 respectively show details of a generic temporary vial storage receptacle  114  and a special central storage receptacle  122  in central body  104 . The body  104  of Vial Temporary Storage Holder  68  is conveniently formed by machining from a single block of material, with the exception of studs  96 ,  98 ,  100  and a face plate  102  (FIG.  3 ). Studs  96 ,  98 ,  100  are mounted on lower face  105  of VTSH body  104  and engage in sockets  106 ,  107 ,  109  in upwardly facing surface  110  of bracket body  63  (FIG.  5 ), to ensure positive alignment of an electrical connector  112  mounted on VTSH  68  and lower connector  80  of electrical penetrator  90 . Face plate  102  is provided with a bar code or other machine readable symbols (not illustrated) for enabling automatic identification of a particular VTSH  68  among a plurality of such units. Multiple VTSH are in general interchangeable and may form a repetitive sub-unit in a larger cryogenic mass storage system (not illustrated). 
     VTSH  68  is provided with a plurality of temporary storage receptacles  114 ,  116 ,  118  et alia (FIGS. 3,  9 ) recessed below an upper surface  120  of body  104 . As shown in FIG. 9, storage receptacles  114 ,  116 ,  118 , etc., are provided with respective numerical designations “ 1 ”, “ 2 ”, “ 3 ”, . . . , “ 30 ” engraved on surface  120  and filled with a low-temperature durable black compound. This enumeration is appropriate for prototyped dimensions and standard available brand of 2.0 ml cryogenic vials. However, an alternative VTSH may be constructed for use with standard 5.0 ml vials. Special central storage receptacle or bore  122  is not so enumerated, and is reserved for holding a reference container  124  (FIG.  8 ). 
     Reference container  124  is optionally formed from a NUNC 5000-0020, 2.0 ml liquid capacity, sterile polypropylene cryogenic vial  126  with a high density polyethylene cap  128 . Vial  126  is filled with a cell sample emulating mixture (not shown) consisting of a cellular medium such as RPMI 1640 and a smaller fraction of a cryoprotectant such as Dimethyl Sulfoxide (DMSO). The sample emulating mixture closely matches the composition and thermal properties of vials containing actual cellular samples and is additionally equipped with a copper-constantan or other cryo-temperature adapted thermocouple  130  inserted through a hole  132  drilled in a base or bottom end  134  of vial  126 , subsequently sealed and pressure tested at hole  132 . The arrangement of reference container  124  in special storage receptacle  122  of VTSH body  104  is illustrated in FIG.  6 . 
     Container or vial assembly  124  is inserted in reserved storage receptacle  122  in body  104 . Reserved receptacle  124 , unlike sample storage receptacles  114 ,  116 ,  118 , et al. (not illustrated in FIG.  6 ), is drilled through a bottom surface (not designated) of body  104 , as well as through top surface  120 . Assembly  124  is inserted and sealed in receptacle or bore  122  with a cryo-adapted adhesive. An aperture  136  is fitted from an underside with a plug or insert  140 , secured from top side or surface  120  with a cap  138 , together securely holding reference container  124  in conjunction with electrical connector  112 . 
     FIG. 6 also illustrates body  104  of VTSH  68  in a partially inserted position in bracket body  63  of bracket assembly  62 , a distance D from a fully engaged configuration. Studs  96 ,  100  are shown partially inserted in sockets  106 ,  109  and VTSH mounted connector  112  is shown engaged with upper connector  80  of bracket mounted electrical penetrator  90 . A positive engagement and resulting defined positions of storage receptacles  114 ,  116  etc. relative to cryogenic transport container  50  facilitates eventual automatic execution or robotization of a transfer process. 
     FIG. 10 shows a detail of key pad and display or interface panel  88  for operator/external interface with microprocessor  86 . The interface panel includes a dot matrix-type liquid crystal display (LCD)  144  and keys or touchpads  146  etc. forming a touch sensitive keyboard or keypad  87 . The term “keypad” is used herein to refer to an array of touch-sensitive keys, while the keys individually are referred to as “touchpads.” Panel  88  also contains an RS-232 jack  142  for connection to an external data-processing device, for example, for transferring thermal history information to an external computer (not illustrated). A battery (not illustrated) provides power for electronics associated with the cryogenic transport device, including microprocessor  86  and display  144 ; the status of the battery is indicated by possible energization or illumination of low-battery status indication  148 . Coincident with illumination of low-battery status indication  148 , detailed instructions for battery replacement are made available to a technician or operator on display  144  following pressure activation of a “Batt.” touchpad (not illustrated). Alternatively, low-battery status could be indicated on LCD matrix display  144 , removing a need for battery status indication  148 . A miniature speaker or tone-generator  150  provides audible status and warning signals under control of microprocessor  86 , operating under a stored machine program. 
     The appearance of the keypad (FIG. 10) is schematic and not to be taken as limiting to the appearance of a particular data-entry pad on a specific embodiment of the invention. 
     A detailed procedure for preserving temperature integrity of cryogenically preserved samples during a transfer operation is described. For the purposes of this disclosure, the term “temperature integrity” refers in a first sense to the maintenance of samples in an acceptable temperature band during a processing operation and in a second sense to the maintenance of an electronic audit trail plausibly assuring an end-user that temperature integrity in the first sense has in fact been maintained. Positive temperature integrity, or the state wherein temperature integrity has not been compromised, thus involves elements of both quality control and of quality assurance; that is, positive temperature integrity means both that physical temperature limits have not been passed and that a sufficiently credible certification of this fact exists for a given set of economic circumstances. 
     An operator is charged with the responsibility of safe-handling a cryogenically processed cellular sample and is provided with means to ensure maintenance of temperature integrity of the sample while moving it through a non-cryogenic, e.g., room-temperature, environment from one cryogenic storage location to another. The temperature estimation process described herein utilizes the physical relationship which exists between elapsed time and accumulated temperature rise of a cellular sample vial, such as a 2 milliliter or 5 milliliter vial, when the vial is removed from a cryogenic temperature and placed into a room temperature environment for an indeterminate time period. It is contemplated to provide both a warning that an extrapolated sample temperature is about to pass a critical temperature threshold and a permanent record of a temperature excursion during a transport event. 
     In a preparation stage of a Standard Operating Procedure (SOP) for executing a controlled cryogenic transfer utilizing cryogenic transport container  50 , an operator or cryotechnician will assemble or gather the following components: the VTSH  68 , the instrumented thermally-insulated flask  52  and lid  64 , and a pair of metal ratcheting forceps (not illustrated) for grasping, holding and releasing a vial. VTSH  68  is introduced into the flask  52 , as illustrated in FIGS. 1 and 6. The VTSH may accommodate either 2 ml or 5 ml sample vials in a standard embodiment. For fully automated operation a height detection device (not shown) would be incorporated in the VTSH to detect vial size, whereas in a partially manual implementation of a cryogenic transfer operation, a technician can determine vial size by visual inspection or record consultation and press an appropriate button. A simplified operation would avoid mixing vial sizes in a single VTSH, although it is not inconsistent with the objects of the invention to do so. 
     A vial size selection is subsequently input via keypad  87 , allowing microprocessor  86  to select a correct time vs. temperature relationship. Subsequently, with lid  64  removed, the flask  52  is filled with liquid nitrogen to the inside lip or fill line  60  (FIG.  1 ). Lid  64  is attached to flask  52  to close container  50  following filling thereof. According to the SOP, the technician shall keep transport container  50  within reach during a cryogenic transfer procedure. 
     During a first stage of a cryo-preservation procedure, a source of a cellular material may either be at or above room temperature, as from a fresh tissue preparation, or be packed in ice, thereby exhibiting an initial temperature in an approximate range of 0° to 37° C. A cellular sample prepared from the source of cellular material is subsequently encapsulated in a cryoadapted vial nominally of either 2 ml or 5 ml capacity, the vial subsequently being provided with a unique Sample Identification Number (SIN) which may be affixed to the vial in the form of a bar code. The vial is additionally filled with a mixture consisting substantially of a cellular nutrient medium such as RPMI 1640 and a smaller fraction of a cryoprotectant compound such as Dimethyl Sulfoxide (DMSO), as is known in the cryogenic arts. The encapsulated sample is placed in a programmable controlled cooling rate freezer (not illustrated) for a defined regimen of cooling from an initial sample temperature to an intermediate processing temperature of −95 degrees C. 
     In a second stage of a cryo-preservation procedure, sample capsules or vials pre-cooled in the controlled cooling rate freezer are transferred to a cryogenic transport device for transport to a long-term cryogenic storage facility. Prior to commencement of a transport operation, the cryogenic transport device is moved to the location of the controlled cooling rate freezer. Following attainment of the intermediate processing temperature in the controlled cooling rate freezer, lid  64  is removed from flask  52 , exposing VTSH  68  which is pre-positioned and pre-cooled within the cryogenic transport device. The vial is placed in the Vial Temporary Storage Holder (VTSH)  68 , in one of a set of vial hole receptacles or temporary storage receptacles  114 , etc. The temporary storage receptacles are identified with temporary storage receptacle numerals, as described above with reference to FIG.  9 . The relationship of a sample identification number (SIN) to a vial temporary storage receptacle numeral is recorded by entry on the touch pad and display  88 . Removal of lid  64  from flask  52  transmits a ready signal to microprocessor  86  via actuation of proximity switch or sensor  76 . Following receipt of the ready signal, microprocessor  86  drives LCD  144  to display ancillary instructions necessary to effect vial transition. Multiple vials may be inserted in the VTSH  68  following removal of the flask lid  64 . 
     Following insertion of a last vial into VTSH  68  in flask  52 , an action subsequently signaled by reinstallation of lid  64 , touchpads or keys  146 , etc., associated with vial transition are deactivated for a 15 minute wait period to allow inserted vials to cool to temperature of the liquid nitrogen or other cryo-coolant  54 . During this 15 wait minute period, microprocessor  86  causes a visible countdown to be displayed on LCD  144  and also sounds an audible signal signifying the end of the countdown period and accordingly readiness for vial transfer. Transport container  50  may be moved from a vicinity of the controlled cooling rate freezer to a vicinity of the long-term cryogenic storage facility during the wait period, in preparation for a transfer of sample vials from the transport device to the long-term storage facility. 
     During a third and final stage of a cryo-preservation procedure, transport container  50  is positioned within arm&#39;s length of a cryogenic operator in turn positioned within arm&#39;s length of an access port of the long term storage facility (not illustrated). The pair of metal ratcheting forceps is also positioned within reach of the operator. Following expiry of the 15 minute wait period, the microprocessor driven LCD  144  will alternately display the temperature of the vials  70 ,  72  contained within VTSH  68  and the temperature of the ambient air. These temperatures must be within prescribed limits prior to initiation of a second vial transfer. Microprocessor  86  will advise the cryogenic operator, via LCD  144 , when either the vial temperature or the ambient temperature is outside of the prescribed limits and optionally suggest corrective action. 
     Prior to removal of a vial  70 ,  72  or lid  64  from the transport container  50 , the operator depresses a TS (transition start) touchpad (not designated) on keypad  87 . Detailed step by step instructions for commencement of a vial transfer operation are displayed on LCD  144  as an aid to the cryogenic operator. The identity of a given vial as either a 2 ml or a 5 ml vial remains stored in microprocessor  86  from the required preparation stage of a controlled cryogenic transfer utilizing cryogenic transport container  50 . Following activation of the TS touchpad, the operator then removes and sets aside the flask lid  64 , triggering a first signal from proximity sensor  76 , and subsequently manipulates the aforementioned forceps to grasp a vial of choice from VTSH  68 . The metal forceps are passed over the proximity sensor in the course of a removal of the vial  70 ,  72  from the transport container  50 , which triggers generation of a second signal inducing microprocessor  86  to start either the 2 ml or the 5 ml countdown period. Successful activation of the proximity sensor is signaled to the operator by an audible signal or “beep” emitted by speaker or tone generator  150 . In the event of a failure of audible confirmation of a sensor activation, the operator must by procedure try again until a signal is heard, or, after a predetermined number of trials, return the vial to the VTSH. 
     During the selected countdown period, microprocessor  86  executes an automatic process tracking the temperature of the removed vial  70 ,  72 . In general, the temperature of a removed vial  70 ,  72  will rise following removal from portable cryogenic container  50 . Prior to the attainment by the removed vial  70 ,  72  of a critical temperature T c , as estimated by microprocessor  86 , the microprocessor undertakes preventative action at an alarm temperature T A =T c −Δ, where Δ is a temperature increment pre-chosen to allow time for corrective action to be completed prior to incipient accumulation of irreversible specimen damage at temperature T c . 
     Microprocessor extrapolates a current temperature of the removed vial  70 ,  72  from an initial temperature of the vial as maintained in storage and transport container  50 . This extrapolation may be based upon empirical data or upon a mathematical formula. Empirical data is obtained using a standard vial which contains a material with thermal properties similar to those of a representative biological specimen. In that case, the extrapolation or estimation process executed by microprocessor  86  includes a timing operation coupled with a look-up on a standard temperature curve, table or other data storage device. A sample mathematical formula utilizable by microprocessor  86  is Newton&#39;s Law of Constant Cooling. This law states roughly that heat transfer rate across an insulator is proportional to a temperature difference across that insulator and approximately independent of the absolute temperatures involved. The law takes the form: 
     
       
           T−T   amb =( T   0   −T   amb ) e   −α(t−t     0   )  (2) 
       
     
     where T 0  is the sample temperature at time t 0 , T amb  is the ambient temperature, and α is a rate constant dependent on the detailed configuration but not on the temperature or time. The rate constant α may be determined empirically for a given sample content and container by a technician in a routine measurement operation. As discussed above, ambient temperature T amb  is monitored by external or room temperature thermocouple  78 , whereas initial temperature T 0  is monitored via thermocouple  130  in reference container  124 . Exposure onset time t 0  is automatically detected by microprocessor  86  in response to a signal transmitted from proximity sensor  76  upon removal of a vial  70 ,  72  from transport container  50 . 
     A Touchpad Access Time-Delay feature inhibits LCD  144  and touchpad keys  146  for approximately 15 seconds after a vial  70 ,  72  is removed from the VTSH  68  for vial transfer. After initiation of and during this delay, microprocessor  86  generates a 3 digit random set of numerals, which are not displayed until the time delay is over. A flashing line of dashes is displayed by LCD  144  until the programmed 15-second delay period has elapsed. When the vial transfer operation is complete, the cryogenic operator presses and holds down a TC (transition complete) touchpad (not designated) on keypad  87  and keys in the 3 digit random set of numerals in sequence, which stops the countdown period and induces a display on LCD  144  of the warmest temperature attained by the vial during the transition or transport operation. The cryogenic operator records this temperature against the vial&#39;s predetermined identification, or SIN, and this information is also stored in electronic form in a memory of microprocessor  86  for possible subsequent download via a data cable (not shown) attached to RS-232 jack or port  142 . Detailed step by step procedures for completion of a vial transfer operation are displayed on LCD  144  following pressure activation of the TC touchpad, similarly to display of instructions for commencement of a vial transfer operation following pressure activation of the TS touchpad. 
     In addition to visual indications appearing on LCD  144 , audible indications are provided on speaker or tone-generator  150  as an aid to the cryogenic operator or technician. Once a transition operation countdown is commenced, following activation of the proximity sensor by lid removal and a sequential activation by proximal passage of sample bearing forceps, a steady tone is initiated and persists on speaker  150 . A pulsed tone is generated when an extrapolation by microprocessor  86 , based on vial size, internal and ambient temperatures, indicates 5 seconds remain to attainment of a critical vial temperature of −130° centigrade. At the critical temperature, the sound changes to a swept tone, indicative of an alarm condition, which persists until actuation of the TC keypad by the operator. 
     Behavior of visual display or LCD  144  mimics audible indications, beginning to flash 5 seconds before estimated attainment of a critical temperature by the sample. Since, by procedure, the transport device  50  with removed lid  64  and exposed VTSH  68  remains at arm&#39;s length of the technician during a vial transfer procedure, a 5 second warning provides adequate time for the technician to abort the transfer operation and return the  70 ,  72  vial to the safe environment of the VTSH in the event that an unforseen difficulty prevents completion of the transfer to the long term bulk cryogenic storage facility. 
     Further automation of a vial transfer procedure would include greater utilization of the RS-232 data port to communicate vial removal and receipt events between microprocessor  86  and an off-device computer; for example, a computer integrated with robotic operation of a bulk cryogenic long-term storage facility (not illustrated). Bar codes or other machine readable codes placed on individual vials  70 ,  72  as well as the VTSH  68  or VTSH&#39;s, provide further automation opportunities. It may also be envisaged that individual sample vials may carry respective sensors, for example, solid state devices having electromagnetic properties which change as a function of temperature. In that case, the sensors are queried by ancillary sensors or pick-ups located in a robotic arm functioning as a component of a bulk-cryogenic storage facility or located in an alternative instrumented version of a racheting forceps for use by a cryogenic operator. The instrumented forceps would communicate with either a cryogenic transfer device or a bulk cryogenic storage facility via a data cable or other data transfer means. 
     Although the present invention has been described in terms of specific embodiments and procedures to facilitate understanding and demonstrate feasible enablement, the person skilled in the art will readily conceive of equivalent embodiments and methods. For example, microprocessor  86  may utilize either a partially calculated cooling (warming) curve based on Newton&#39;s law of constant cooling, or a fully empirically evaluated cooling curve. Similarly, the invention may be understood to be equally applicable to a transfer of a vial from a portable device to a bulk storage facility, or from a bulk storage facility to a portable device, or other similar operations; the functionality inherent in microprocessor  86  or the cryogenic transport container  50  being fully or partially transferred to devices ancillary to the bulk storage facility or to other cryogenic storage containers, or a centralized location in a particular operating environment populated with both fixed and portable instrumented storage devices. 
     Accordingly, the embodiments and methods specifically disclosed herein are therefore not to be understood to be limiting to the scope of the invention as claimed.