Mutation Research, 234 (1990) 349-354 Elsevier
349
MUTENV 08765
The development of the U.S. EPA health effects research laboratory frozen blood cell repository program G.H.S. Strauss a and S.J. Kelly b U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (U.S.A.) and b Environmental Health Research and Testing, Inc., P.O. Box 12199, Research Triangle Park, NC 27709 (U.S.A.) (Received 21 November 1989) (Revision received 23 April 1990) (Accepted 10 May 1990)
Keywords: Computerized Laboratory Notebook (CLN); Microcomputer; Frozen blood cell repository; Blood cell freezing; Cryovial
Summary We have suggested that proper blood-cell freezing and storage is necessary in longitudinal studies with reduced between tests error, for specimen sharing between laboratories and for convenient scheduling of assays. Our present purpose is to describe the design and use of a prototype computer program, Cryovial, for data-based management of frozen cell repositories. We are pleased to provide, free of charge upon request, a 5.25" (specify 0.36 or 1.2 MB) disk containing all files required to run Cryovial.
An earlier paper (Strauss, in press) presented efforts to characterize the consequences of leukocyte cryopreservation in terms of cellular morphology and function in pertinent assays of the Battery of Leukocyte Tests (BLT). It was suggested that proper blood-cell freezing and storage is necessary in longitudinal studies with reduced between tests error, for specimen sharing between laboratories and for convenient scheduling of assays. The present paper describes the design and
This manuscript has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Correspondence: Dr. Gary H.S. Strauss, U.S. Environmental Protection Agency, Genetic Toxicology Division, MD-68, Research Triangle Park, NC 27711 (U.S.A.).
use of a computer program, Cryovial, for databased management of frozen cell repositories.
Freezer inventory systems With the advent of new methodologies for studying biopsied tissues, genetic toxicology laboratories increasingly are establishing frozen cell repositories. There is a growing need for systems to maintain accurate records of specimens including the identity of each vial and also of information relevant to the future use of each sample. Prior to the completion of the freezer program, Cryovial, our freezer data-base was tracked manually. Manual inventory systems tend to use freezer space inefficiently. New vials are generally stored at random and recovery on demand is slow. The best design for a manual system includes a vial storage location file in which the empty and full
0165-1161/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
kgln Mote Program; End.
Prlnt.Inc Aaaorted.Inc Locotk.Inc Locetlon.Inc Edtt.lnc Steck.Inc itndow.Inc Vtele.Znc F&¥utlle.Inc Moln.lnc
Include Flies
Procedure ZOVtala;
Procedure MolnPrograal;~
I
Procedure ModtfyVlele;
or
Procedure AddVtele;
Pracedure 14atnl4enu; " ~ ] 1 i i i !
IPr,ced.~eckForFt ~a hl.:
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Procedure FlocordEntry;
Vtelm.Inc
l I /
display vtal records
sort. lable, print, end
- other procedures to
Procedure SelectRocord;
[ SSNu/IaP• Inc I
Include Fi]~
NlndoM.Inc
check, delete, end add location 1 to vial location arrays /
i Procedure AutomticSesrch; Procedure Monue|~arch; - other procedures to
Locatlon.Inc
Procedure EditFreeLoc;
Procedure EditSSNo:
Edit.Inc
Fig. 1. The overall program plan. This was prepared prior to encoding and illustrates the total concept and specific functions of Cryovial.
--
Procedure Critical Error Exit; In case of a fetal error, this procedure Mill print the error code on the screen.
Error H o n d l t n l _ _
Access.Box AddKey.Box BotKey.Box DalKe¥.Box
Database Toolbox
InUnlt .Poe Utl IUntt .Pea PrlnUntt .Pu Grephtce .Poe
PrecoapiIed Unite
CRYOVIAL.Pas
351 spaces are indicated. This file is cross-referenced to a file containing information as to the identity of each subject and the data for each subject's vials. In practice, the manual system is time-consuming to manage and prone to error. Computerization of freezer inventories A computerized system can combine cross-referencing, speed, and accuracy. A search for vials with the characteristics needed for particular tests can be completed with relative ease. The physical space needed to store files is, of course, reduced in a computerized system. There are other advantages to a computerized system for vial storage in our laboratory. We continue to develop and upgrade programs for the computerized laboratory notebook (CLN) wherein BLT protocols, data collection from laboratory equipment, data-base storage, data analysis and graphic presentations are computer automated (Strauss et al., 1989). In the completed CLN the freezer data-base program, Cryovial, will be the entry point for data (e.g., cell viability and concentration) relevant to future experiments, but not needed immediately. The ancillary data are stored along with the freeze location in the vial record, and then passed forward to the automated assay protocol program when a vial is used. One hopes to continue the development of these Pascal programs as attempts are made, simultaneously, to demonstrate their utility and to validate the BLT assessment methodologies in terms of their value in predicting ill-health effects of toxic exposures. Materials and methods
Computer hardware IBM AT compatible clone with 640K memory, 30-Megabyte hard drive, 2 floppy disk drives and EGA monitor (Kaypro Corp., Solmia Beach, CA); Laserjet Series II printer (Hewlett Packard, Inc., Rockville, MD). Virtually any microcomputer (IBM compatible personal computer) containing at least 120K available random access memory will run Cryovial. Most CGA, EGA or VGA video adapter cards and most printers will be suitable.
Computer software Turbo Pascal 4.0 (or 5.0) (Borland International, Scotts Valley, CA), Turbo Pascal Database Toolbox 3.0 (Borland International, Scotts Valley, CA). This program is now available from Alpha Systems, 711 Chatsworts Place, San Jose, CA 95128.
Cryostorage equipment Liquid Nitrogen Cryogenic Refrigerator (Tank type), Model HC35 (Taylor-Wharton, Indianapolis, IN). Liquid Nitrogen Freezer, K series Kryostorage System (Taylor-Wharton, Indianapolis, IN).
Subjects Normal individuals and patients consented to provide venous blood specimens and personal information, including social security identification numbers (SSN). This arrangement contains the understanding that confidentiality shall be maintained under the purview of the Duke University Institutional Review Board.
Programming languages Turbo Pascal 4.0 was the programming language of choice. Although 'user friendly' programs such as Dbase (Ashton-Tate) and Smart System (Innovative Software) simplify data-base programming, they are comparatively inflexible. Additionally, Pascal presented a number of important advantages. First, the Turbo Pascal 4.0 (or the new 5.0) compiler and Turbo Pascal Database Toolbox 1.21 together cost under $150, less than one fifth the price of the example data-base systems mentioned. Toolbox 1.21, a very useful enhancement, provided procedures to store, retrieve and sort records efficiently. A second advantage of Turbo Pascal is that it is a compiled, rather than an interpreter language. Because Turbo Pascal 4.0 compiles to executable files, the compiler need not be present for the program to run. Systems such as Smart and Dbase require the presence of an interpreter that uses significant memory and tend to be slow. Compiled Turbo Pascal programs are fast and can be further streamlined with machine language subroutines. A third advantage of this compilable language is that it is easy to learn and is currently in use at
352 universities as a teaching language. User support is available both from the supplier, by way of Toolboxes, and from public domain freeware and other after-market sources. Fourth, Turbo Pascal is flexible in terms of possible hardware configurations. Virtually any hardware available for personal computing can be used. This is not necessarily true of systems such as the alternatives mentioned. These justifications notwithstanding, a number of similar languages might have served equally well.
Cryovial program functions The freezer program, Cryovial, maintains a record of each stored vial. Each record identifies a vial by SSN, study title, collection date, and specimen information including viability, suspension volume and cell concentration, date frozen a n d / o r refrozen, and freezer storage location. In addition, the program maintains a file of vial storage locations and whether they are occupied. It automatically finds available storage locations.
Program organization A general overview plan of the Cryovial is presented in Fig. 1. This shows how the program was mapped out prior to encoding and provides information as to its various functions. As different parts of the program were completed, they were separated into include files with the file extension, 'INC'. This left the main file smaller, making programming less complicated. Turbo Pascal reads the include files from the disk and compiles the contents of the file as though they are part of the main program. Further programming details will be made available with the diskette (see below). Programming Cryovial was a useful on-the-job learning experience. Encoding was started before the program was completely planned out and, consequently, the code has been written and rewritten several times. Because the program has not been completely rewritten to comply with the 'top-down' design eventually deployed, there are some poorly written sections of code. There is a possibility that 'bugs' can result, but presumably, thorough testing has flushed them out.
Operating features The program is installed automatically from the program diskette by typing install. One chooses the drive and subdirectory name to be used. The drive can be either that containing the program diskette or another floppy or hard disk drive. Typing eryovial at the prompt then starts the program. The title screen appears followed by the main menu screen from which a selection is made by entering the number corresponding to the menu item. Menu item 1 is 'Add vials' and the first screen enquires whether there are replicate vials. These are the same in every respect except that they may contain various cell concentrations depending upon numbers of cells required for each test to be applied later. If answered yes, the screen asks for the number of replicates. The next screen presents 3 choices as to the method of selection of storage location. One may choose to have vial positions allocated automatically as a set in the first block of space of suitable size or, the default option, separately and successively in the next available spaces. Manual selection is also possible and, in this procedure, the program will so indicate if a given location is already occupied. Next, under the 'Add vials' label, is entered a set of information descriptive of the alliquoted specimens: (1) SSN; (2) collection date, default value drawn in from computer calendar; (3) freeze date, default = current date; (4) refreeze date; (5) study type, list of 7 choices of active studies in our lab; (6) cell type, list of 9 choices under 4 subtypes used in our lab and (7) % cell viability (prefreeze). The last data entry sequence in this section registers the cell concentration (in millions/cc) and the number of vials for each concentration series. The program checks that the number of replicates originally allocated space and the total vial number correspond at this point. Finally, the program automatically prints a label for each vial. The labels include SSN, collection date, vial number, cell density and storage location. Menu item 2 is 'Vial Data-base' which includes functions for editing, deleting and printing existing vial records. When 2 is entered the screen requests an SSN. This can be entered directly or, if the user wishes to choose the subject from a list
353 11-scrolL (PgUp)&(pgon)-scroti by page - - s h i f t l e f t & r i g h t (ENTER)-setect (Ctrt-(>)-pe@e l e f t & r i g h t ( H ~ ) & ( E n d ) - f i r s t & Lest co|tams (F1) -HELP S.S. Nuld~r CoL|action 053-1~1-4q~. 053-68-4932 053-M-4932 053-68-4932 053-68-4932 053-68-4932 053-68-4932 >053-68-4932 053-68-4932 0S3-68-4932 053-68-4932 053-MI-4932 053-68-4932 053-68-4932 053-68-4932 053-68-4932 053J.R-4932 053-68-4932 053-68-4932
23/ 23/ 231 19/ 191 191 19/ 191 19/ 19/ 19/ 19/ 191 28/ 28/ 28/ 28/ 28/ 281
V1189 VII89 V1189 VIII89 V11189 V11189 VIII89 Vlii89 vii/89 VI1189 V11/89 Vll/89 Viii89 Vii/Sq v11/89 V1|/89 Vil/89 V11/89 V11/89
Freeze 231 231 231 191 191 191 19/ 191 19/
19/ 191 19/ 191 28/ 28/ 281 281 28/ 28/
ReFreeze
V1/89 I V1189 I V1189 I V11/89 / V11189 / V11189 / V11/89 / V11189 / V11189 / v11189 / VI1/89 / VIi/IN I VIII89 i Vli/89 / VII/89 / V11/89 / VIII89 / VIII89 / VII/89 /
/ I / / / / / I / / / / / / / / / / /
Study PUNA PUVA PUVA PUVA PLNA PINA PLNA PtNA P'UVA IqJVA PUVA I~NA PUVA PUVA PUVA PUVA PUVA PUVA PUVA
BLood TyI)e Parl Pert Per1 Per4 Pert Purt Pert Pert Per~ Perl Purl Pars Purl Peri Peri Peri Perl Perl Purl
heraL betel I'm'aL i~erat Steres • erut •orel i~eruL ~herul ~eI"uL dlerai ~ral dlersl Iterll )hers[ d~erat dherut Mrul • eral
iiocd-llCs |iocd-HiCs Blood-Nits |lood-NNCs I|ood-imCe Dtood-NNCe |tood-NNC8 lLuod-NlICe Jiood-HNCs iiOOCI-NNCs IIood-mCs Illood-MNCs Ile~d-MiCs IIo0d-NNCI llom:l-I(NCs Blood-laNes ILoocI-NllCI i[ood-NIICe ILood-NNCs
Cells/co V i a b i L i t y 0.200 0.200 0.200 2.000 2.000 2.000 1.000 1.000 1.000 0.200 0.200 0.200 0.200 2.000 2.000 2.000 1.000 1.000 1.000
96.000 96.000 96.000 97.000 97.000 97.000 97.000 97.000 97.000 97.000 97.000 97.000 9T.OOO 95.500 9S.500 95.500 95.500 95.500 95.500
No of ViaLs VioL no Freezer Location 9 9 9 10 10 10 10 10 10 10 10 10 10 12 12 12 12 12 12
? 8 9 I 2 3 4 5 6 7 8 9 10 1 2 3 /* 5 6
IIlIlIiIIIll1IIII1" 1-
1114&&4444&44777'777-
3A 311 3(; 5C 6A 68 6(: 7A 7B 7C 8A 8B 8C 1A 10 lC 2A 2B 2C
Fig. 2. A representative vial record scroll window. The (19) records shown are for vials with mononuclear leukocytes from 1 PUVA-treated patient. These were prepared and stored on the 3 different occasions listed and had viabilities and densities as shown.
of all SSNs, this can be accomplished by pressing Enter. The list appears and a choice is made by using an arrow key to highlight an SSN and pressing Enter. A query with a choice of several sort methods appears, and when entered, all records for that subject appear in the vial record scroll window. Fig. 2 represents a vial record scroll window. The actual screen shows only as far as the fight-hand side of the c o m m a n d list. By pressing End or the right arrow key, the user can see the remaining, undisplayed part of the record. The column designations are as follows: SSN; Collection date; Freeze date; Refreeze date, for cases where cells are refrozen for further studies; Study name; Blood-cell type; Cell concentration; % Viable; N u m b e r of vials initially frozen; Individual vial identity number and Freezer location. Under 'Vial Data-base', pressing F1 provides a help screen that lists information as to functions that can be called into play while viewing the vial record scroll window. R resorts all the records by 2 alternative criteria. D deletes the highlighted record. Alt-U moves the highlighted vial record to the ' U s e d Vials' file. (When the C L N is complete, a vial selection procedure at the beginning of each test protocol will automatically select the vials needed for that particular task and move the record to the ' U s e d Vials' file.) P causes the vial
record to be printed. Enter selects the vial record for modification, calling in the 'Vial File Modifier' screen. In this procedure, for any given characteristic entered under Item 1 ('Add Vials'), the old value appears and a new one now can be entered to overwrite it. Menu item 3, 'Identify Vial' allows one to recover the data-base record for a given vial based upon storage location should a vial label be lost or illegible. U p o n entering location parameters, the vial's SSN, collection date, vial number and cell concentration appear on the screen. A query as to whether a new label is to be printed then appears. Menu item 4, 'Unused', was reserved to allow additional menu choices to be added easily. Menu item 5 ends the program and returns to DOS.
CLN integration Within the framework of the CLN, the freezer program will perform two main functions: (1) Addition of new vials and relevant data to the base of available specimens and (2) freezer management. Cryovial will be the first step in C L N handling of information concerning blood specimens being cryopreserved. Occasionally, a frozen specimen is thawed for immediate use, an additional program is being prepared within the C L N to deal with that possibility. When the C L N
354
system is fully operational, users will retrieve sample vials automatically through the various test protocols without having to activate Cryovial directly. To promote cooperation between laboratories involved in direct testing of cryopreserved blood (and other specimen tissues), we are pleased to provide copies of Cryovial on 5.25" floppy disks (specify high or low density), free of charge, on request. This diskette contains 2 program files, Install.exe and Cryovial.exe. It contains 5 precompiled unit files: Bios.tpu, Inunit.tpu, Utilunit.tpu, Printunit.tpu and Graphics.tpu and 11 include files: Print.inc, Assorted.inc, Locstk.inc, Location.inc, Edit.inc, Stack.inc, Window.inc, Vials.inc, F & tutils.inc, Main..inc and Config.inc. Finally, the program source code file, Cryovial.pas, is included. A more detailed description of the programming than offered here will accompany the diskette. The diskette provides cryovial exactly as it is used in our lab. Modification of the program for use in other labs is possible but requires 2 additional, commercially available programs. Customized changes must be accomplished by editing the existing program and compiling by means of Turbo Pascal 4.0 (or 5.0). The Turbo Pascal 3.0 data-base toolbox files Access.box, Addkey.box, Getkey.box and Delkey.box, must be stored in the directory containing the Cryovial source code files to permit compiling. Again, one should like to emphasize that although a particular compiler was chosen, others
might have worked as well. Borland International products are not recommended over those of other suppliers. Further, though reasonable precautions were undertaken in the development of Cryovial, damage to software or hardware owing to its use conceivably could occur. In this event, we would not accept responsibility. It is hoped that, as a result of this effort, interest in the establishment of a network for specimen sharing will be stimulated. With relatively little organizational effort, it should be possible to make aliquotted and cryopreserved cells from large specimen banks accessible to all who wish to participate. Inter-laboratory comparisons of results from testing replicate specimens using both identical test methods and others with a variety of endpoints should contribute to standardization of methods and quality assurance. This 'workshop' type approach would help us in attempts to validate and further calibrate methods for genotoxicity testing in such a way as to provide greater confidence both in the methods and, of no small consequence, in the altruism of our colleagues. References Strauss, G.H.S. (1990) Non-random cell killing in cryopreservation: Implications for performance of the battery of leukocyte tests (BLT), I. toxic and immunotoxic effects, Mutation Res., in press. Strauss, G.H.S., W.L. Stanford and S.J. Berkowitz (1989) The development of an in-laboratory microcomputer system for genetic toxicology experimentation and testing, Mutation Res., 222, 171-189.
349
MUTENV 08765
The development of the U.S. EPA health effects research laboratory frozen blood cell repository program G.H.S. Strauss a and S.J. Kelly b U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (U.S.A.) and b Environmental Health Research and Testing, Inc., P.O. Box 12199, Research Triangle Park, NC 27709 (U.S.A.) (Received 21 November 1989) (Revision received 23 April 1990) (Accepted 10 May 1990)
Keywords: Computerized Laboratory Notebook (CLN); Microcomputer; Frozen blood cell repository; Blood cell freezing; Cryovial
Summary We have suggested that proper blood-cell freezing and storage is necessary in longitudinal studies with reduced between tests error, for specimen sharing between laboratories and for convenient scheduling of assays. Our present purpose is to describe the design and use of a prototype computer program, Cryovial, for data-based management of frozen cell repositories. We are pleased to provide, free of charge upon request, a 5.25" (specify 0.36 or 1.2 MB) disk containing all files required to run Cryovial.
An earlier paper (Strauss, in press) presented efforts to characterize the consequences of leukocyte cryopreservation in terms of cellular morphology and function in pertinent assays of the Battery of Leukocyte Tests (BLT). It was suggested that proper blood-cell freezing and storage is necessary in longitudinal studies with reduced between tests error, for specimen sharing between laboratories and for convenient scheduling of assays. The present paper describes the design and
This manuscript has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Correspondence: Dr. Gary H.S. Strauss, U.S. Environmental Protection Agency, Genetic Toxicology Division, MD-68, Research Triangle Park, NC 27711 (U.S.A.).
use of a computer program, Cryovial, for databased management of frozen cell repositories.
Freezer inventory systems With the advent of new methodologies for studying biopsied tissues, genetic toxicology laboratories increasingly are establishing frozen cell repositories. There is a growing need for systems to maintain accurate records of specimens including the identity of each vial and also of information relevant to the future use of each sample. Prior to the completion of the freezer program, Cryovial, our freezer data-base was tracked manually. Manual inventory systems tend to use freezer space inefficiently. New vials are generally stored at random and recovery on demand is slow. The best design for a manual system includes a vial storage location file in which the empty and full
0165-1161/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
kgln Mote Program; End.
Prlnt.Inc Aaaorted.Inc Locotk.Inc Locetlon.Inc Edtt.lnc Steck.Inc itndow.Inc Vtele.Znc F&¥utlle.Inc Moln.lnc
Include Flies
Procedure ZOVtala;
Procedure MolnPrograal;~
I
Procedure ModtfyVlele;
or
Procedure AddVtele;
Pracedure 14atnl4enu; " ~ ] 1 i i i !
IPr,ced.~eckForFt ~a hl.:
l a l n . Inc
Procedure FlocordEntry;
Vtelm.Inc
l I /
display vtal records
sort. lable, print, end
- other procedures to
Procedure SelectRocord;
[ SSNu/IaP• Inc I
Include Fi]~
NlndoM.Inc
check, delete, end add location 1 to vial location arrays /
i Procedure AutomticSesrch; Procedure Monue|~arch; - other procedures to
Locatlon.Inc
Procedure EditFreeLoc;
Procedure EditSSNo:
Edit.Inc
Fig. 1. The overall program plan. This was prepared prior to encoding and illustrates the total concept and specific functions of Cryovial.
--
Procedure Critical Error Exit; In case of a fetal error, this procedure Mill print the error code on the screen.
Error H o n d l t n l _ _
Access.Box AddKey.Box BotKey.Box DalKe¥.Box
Database Toolbox
InUnlt .Poe Utl IUntt .Pea PrlnUntt .Pu Grephtce .Poe
PrecoapiIed Unite
CRYOVIAL.Pas
351 spaces are indicated. This file is cross-referenced to a file containing information as to the identity of each subject and the data for each subject's vials. In practice, the manual system is time-consuming to manage and prone to error. Computerization of freezer inventories A computerized system can combine cross-referencing, speed, and accuracy. A search for vials with the characteristics needed for particular tests can be completed with relative ease. The physical space needed to store files is, of course, reduced in a computerized system. There are other advantages to a computerized system for vial storage in our laboratory. We continue to develop and upgrade programs for the computerized laboratory notebook (CLN) wherein BLT protocols, data collection from laboratory equipment, data-base storage, data analysis and graphic presentations are computer automated (Strauss et al., 1989). In the completed CLN the freezer data-base program, Cryovial, will be the entry point for data (e.g., cell viability and concentration) relevant to future experiments, but not needed immediately. The ancillary data are stored along with the freeze location in the vial record, and then passed forward to the automated assay protocol program when a vial is used. One hopes to continue the development of these Pascal programs as attempts are made, simultaneously, to demonstrate their utility and to validate the BLT assessment methodologies in terms of their value in predicting ill-health effects of toxic exposures. Materials and methods
Computer hardware IBM AT compatible clone with 640K memory, 30-Megabyte hard drive, 2 floppy disk drives and EGA monitor (Kaypro Corp., Solmia Beach, CA); Laserjet Series II printer (Hewlett Packard, Inc., Rockville, MD). Virtually any microcomputer (IBM compatible personal computer) containing at least 120K available random access memory will run Cryovial. Most CGA, EGA or VGA video adapter cards and most printers will be suitable.
Computer software Turbo Pascal 4.0 (or 5.0) (Borland International, Scotts Valley, CA), Turbo Pascal Database Toolbox 3.0 (Borland International, Scotts Valley, CA). This program is now available from Alpha Systems, 711 Chatsworts Place, San Jose, CA 95128.
Cryostorage equipment Liquid Nitrogen Cryogenic Refrigerator (Tank type), Model HC35 (Taylor-Wharton, Indianapolis, IN). Liquid Nitrogen Freezer, K series Kryostorage System (Taylor-Wharton, Indianapolis, IN).
Subjects Normal individuals and patients consented to provide venous blood specimens and personal information, including social security identification numbers (SSN). This arrangement contains the understanding that confidentiality shall be maintained under the purview of the Duke University Institutional Review Board.
Programming languages Turbo Pascal 4.0 was the programming language of choice. Although 'user friendly' programs such as Dbase (Ashton-Tate) and Smart System (Innovative Software) simplify data-base programming, they are comparatively inflexible. Additionally, Pascal presented a number of important advantages. First, the Turbo Pascal 4.0 (or the new 5.0) compiler and Turbo Pascal Database Toolbox 1.21 together cost under $150, less than one fifth the price of the example data-base systems mentioned. Toolbox 1.21, a very useful enhancement, provided procedures to store, retrieve and sort records efficiently. A second advantage of Turbo Pascal is that it is a compiled, rather than an interpreter language. Because Turbo Pascal 4.0 compiles to executable files, the compiler need not be present for the program to run. Systems such as Smart and Dbase require the presence of an interpreter that uses significant memory and tend to be slow. Compiled Turbo Pascal programs are fast and can be further streamlined with machine language subroutines. A third advantage of this compilable language is that it is easy to learn and is currently in use at
352 universities as a teaching language. User support is available both from the supplier, by way of Toolboxes, and from public domain freeware and other after-market sources. Fourth, Turbo Pascal is flexible in terms of possible hardware configurations. Virtually any hardware available for personal computing can be used. This is not necessarily true of systems such as the alternatives mentioned. These justifications notwithstanding, a number of similar languages might have served equally well.
Cryovial program functions The freezer program, Cryovial, maintains a record of each stored vial. Each record identifies a vial by SSN, study title, collection date, and specimen information including viability, suspension volume and cell concentration, date frozen a n d / o r refrozen, and freezer storage location. In addition, the program maintains a file of vial storage locations and whether they are occupied. It automatically finds available storage locations.
Program organization A general overview plan of the Cryovial is presented in Fig. 1. This shows how the program was mapped out prior to encoding and provides information as to its various functions. As different parts of the program were completed, they were separated into include files with the file extension, 'INC'. This left the main file smaller, making programming less complicated. Turbo Pascal reads the include files from the disk and compiles the contents of the file as though they are part of the main program. Further programming details will be made available with the diskette (see below). Programming Cryovial was a useful on-the-job learning experience. Encoding was started before the program was completely planned out and, consequently, the code has been written and rewritten several times. Because the program has not been completely rewritten to comply with the 'top-down' design eventually deployed, there are some poorly written sections of code. There is a possibility that 'bugs' can result, but presumably, thorough testing has flushed them out.
Operating features The program is installed automatically from the program diskette by typing install. One chooses the drive and subdirectory name to be used. The drive can be either that containing the program diskette or another floppy or hard disk drive. Typing eryovial at the prompt then starts the program. The title screen appears followed by the main menu screen from which a selection is made by entering the number corresponding to the menu item. Menu item 1 is 'Add vials' and the first screen enquires whether there are replicate vials. These are the same in every respect except that they may contain various cell concentrations depending upon numbers of cells required for each test to be applied later. If answered yes, the screen asks for the number of replicates. The next screen presents 3 choices as to the method of selection of storage location. One may choose to have vial positions allocated automatically as a set in the first block of space of suitable size or, the default option, separately and successively in the next available spaces. Manual selection is also possible and, in this procedure, the program will so indicate if a given location is already occupied. Next, under the 'Add vials' label, is entered a set of information descriptive of the alliquoted specimens: (1) SSN; (2) collection date, default value drawn in from computer calendar; (3) freeze date, default = current date; (4) refreeze date; (5) study type, list of 7 choices of active studies in our lab; (6) cell type, list of 9 choices under 4 subtypes used in our lab and (7) % cell viability (prefreeze). The last data entry sequence in this section registers the cell concentration (in millions/cc) and the number of vials for each concentration series. The program checks that the number of replicates originally allocated space and the total vial number correspond at this point. Finally, the program automatically prints a label for each vial. The labels include SSN, collection date, vial number, cell density and storage location. Menu item 2 is 'Vial Data-base' which includes functions for editing, deleting and printing existing vial records. When 2 is entered the screen requests an SSN. This can be entered directly or, if the user wishes to choose the subject from a list
353 11-scrolL (PgUp)&(pgon)-scroti by page - - s h i f t l e f t & r i g h t (ENTER)-setect (Ctrt-(>)-pe@e l e f t & r i g h t ( H ~ ) & ( E n d ) - f i r s t & Lest co|tams (F1) -HELP S.S. Nuld~r CoL|action 053-1~1-4q~. 053-68-4932 053-M-4932 053-68-4932 053-68-4932 053-68-4932 053-68-4932 >053-68-4932 053-68-4932 0S3-68-4932 053-68-4932 053-MI-4932 053-68-4932 053-68-4932 053-68-4932 053-68-4932 053J.R-4932 053-68-4932 053-68-4932
23/ 23/ 231 19/ 191 191 19/ 191 19/ 19/ 19/ 19/ 191 28/ 28/ 28/ 28/ 28/ 281
V1189 VII89 V1189 VIII89 V11189 V11189 VIII89 Vlii89 vii/89 VI1189 V11/89 Vll/89 Viii89 Vii/Sq v11/89 V1|/89 Vil/89 V11/89 V11/89
Freeze 231 231 231 191 191 191 19/ 191 19/
19/ 191 19/ 191 28/ 28/ 281 281 28/ 28/
ReFreeze
V1/89 I V1189 I V1189 I V11/89 / V11189 / V11189 / V11/89 / V11189 / V11189 / v11189 / VI1/89 / VIi/IN I VIII89 i Vli/89 / VII/89 / V11/89 / VIII89 / VIII89 / VII/89 /
/ I / / / / / I / / / / / / / / / / /
Study PUNA PUVA PUVA PUVA PLNA PINA PLNA PtNA P'UVA IqJVA PUVA I~NA PUVA PUVA PUVA PUVA PUVA PUVA PUVA
BLood TyI)e Parl Pert Per1 Per4 Pert Purt Pert Pert Per~ Perl Purl Pars Purl Peri Peri Peri Perl Perl Purl
heraL betel I'm'aL i~erat Steres • erut •orel i~eruL ~herul ~eI"uL dlerai ~ral dlersl Iterll )hers[ d~erat dherut Mrul • eral
iiocd-llCs |iocd-HiCs Blood-Nits |lood-NNCs I|ood-imCe Dtood-NNCe |tood-NNC8 lLuod-NlICe Jiood-HNCs iiOOCI-NNCs IIood-mCs Illood-MNCs Ile~d-MiCs IIo0d-NNCI llom:l-I(NCs Blood-laNes ILoocI-NllCI i[ood-NIICe ILood-NNCs
Cells/co V i a b i L i t y 0.200 0.200 0.200 2.000 2.000 2.000 1.000 1.000 1.000 0.200 0.200 0.200 0.200 2.000 2.000 2.000 1.000 1.000 1.000
96.000 96.000 96.000 97.000 97.000 97.000 97.000 97.000 97.000 97.000 97.000 97.000 9T.OOO 95.500 9S.500 95.500 95.500 95.500 95.500
No of ViaLs VioL no Freezer Location 9 9 9 10 10 10 10 10 10 10 10 10 10 12 12 12 12 12 12
? 8 9 I 2 3 4 5 6 7 8 9 10 1 2 3 /* 5 6
IIlIlIiIIIll1IIII1" 1-
1114&&4444&44777'777-
3A 311 3(; 5C 6A 68 6(: 7A 7B 7C 8A 8B 8C 1A 10 lC 2A 2B 2C
Fig. 2. A representative vial record scroll window. The (19) records shown are for vials with mononuclear leukocytes from 1 PUVA-treated patient. These were prepared and stored on the 3 different occasions listed and had viabilities and densities as shown.
of all SSNs, this can be accomplished by pressing Enter. The list appears and a choice is made by using an arrow key to highlight an SSN and pressing Enter. A query with a choice of several sort methods appears, and when entered, all records for that subject appear in the vial record scroll window. Fig. 2 represents a vial record scroll window. The actual screen shows only as far as the fight-hand side of the c o m m a n d list. By pressing End or the right arrow key, the user can see the remaining, undisplayed part of the record. The column designations are as follows: SSN; Collection date; Freeze date; Refreeze date, for cases where cells are refrozen for further studies; Study name; Blood-cell type; Cell concentration; % Viable; N u m b e r of vials initially frozen; Individual vial identity number and Freezer location. Under 'Vial Data-base', pressing F1 provides a help screen that lists information as to functions that can be called into play while viewing the vial record scroll window. R resorts all the records by 2 alternative criteria. D deletes the highlighted record. Alt-U moves the highlighted vial record to the ' U s e d Vials' file. (When the C L N is complete, a vial selection procedure at the beginning of each test protocol will automatically select the vials needed for that particular task and move the record to the ' U s e d Vials' file.) P causes the vial
record to be printed. Enter selects the vial record for modification, calling in the 'Vial File Modifier' screen. In this procedure, for any given characteristic entered under Item 1 ('Add Vials'), the old value appears and a new one now can be entered to overwrite it. Menu item 3, 'Identify Vial' allows one to recover the data-base record for a given vial based upon storage location should a vial label be lost or illegible. U p o n entering location parameters, the vial's SSN, collection date, vial number and cell concentration appear on the screen. A query as to whether a new label is to be printed then appears. Menu item 4, 'Unused', was reserved to allow additional menu choices to be added easily. Menu item 5 ends the program and returns to DOS.
CLN integration Within the framework of the CLN, the freezer program will perform two main functions: (1) Addition of new vials and relevant data to the base of available specimens and (2) freezer management. Cryovial will be the first step in C L N handling of information concerning blood specimens being cryopreserved. Occasionally, a frozen specimen is thawed for immediate use, an additional program is being prepared within the C L N to deal with that possibility. When the C L N
354
system is fully operational, users will retrieve sample vials automatically through the various test protocols without having to activate Cryovial directly. To promote cooperation between laboratories involved in direct testing of cryopreserved blood (and other specimen tissues), we are pleased to provide copies of Cryovial on 5.25" floppy disks (specify high or low density), free of charge, on request. This diskette contains 2 program files, Install.exe and Cryovial.exe. It contains 5 precompiled unit files: Bios.tpu, Inunit.tpu, Utilunit.tpu, Printunit.tpu and Graphics.tpu and 11 include files: Print.inc, Assorted.inc, Locstk.inc, Location.inc, Edit.inc, Stack.inc, Window.inc, Vials.inc, F & tutils.inc, Main..inc and Config.inc. Finally, the program source code file, Cryovial.pas, is included. A more detailed description of the programming than offered here will accompany the diskette. The diskette provides cryovial exactly as it is used in our lab. Modification of the program for use in other labs is possible but requires 2 additional, commercially available programs. Customized changes must be accomplished by editing the existing program and compiling by means of Turbo Pascal 4.0 (or 5.0). The Turbo Pascal 3.0 data-base toolbox files Access.box, Addkey.box, Getkey.box and Delkey.box, must be stored in the directory containing the Cryovial source code files to permit compiling. Again, one should like to emphasize that although a particular compiler was chosen, others
might have worked as well. Borland International products are not recommended over those of other suppliers. Further, though reasonable precautions were undertaken in the development of Cryovial, damage to software or hardware owing to its use conceivably could occur. In this event, we would not accept responsibility. It is hoped that, as a result of this effort, interest in the establishment of a network for specimen sharing will be stimulated. With relatively little organizational effort, it should be possible to make aliquotted and cryopreserved cells from large specimen banks accessible to all who wish to participate. Inter-laboratory comparisons of results from testing replicate specimens using both identical test methods and others with a variety of endpoints should contribute to standardization of methods and quality assurance. This 'workshop' type approach would help us in attempts to validate and further calibrate methods for genotoxicity testing in such a way as to provide greater confidence both in the methods and, of no small consequence, in the altruism of our colleagues. References Strauss, G.H.S. (1990) Non-random cell killing in cryopreservation: Implications for performance of the battery of leukocyte tests (BLT), I. toxic and immunotoxic effects, Mutation Res., in press. Strauss, G.H.S., W.L. Stanford and S.J. Berkowitz (1989) The development of an in-laboratory microcomputer system for genetic toxicology experimentation and testing, Mutation Res., 222, 171-189.
