Home

C`ENTER

1. NOT 1 42 OK RESULTS OF QC PROCEDURE PERFORM QC LIQ HANDLING amp COLLECT ABS DATA 175 C LIQ HANDLING amp PLATE READING CHECKING REAGENT TITER Fa DO TITER 5B LIQUID HANDLING DO QC LIQ HANDLING amp PLATE READING uo HANDLING QC OPERATOR 10 Qc BROM PROCEDURE FOR ID LOGON SAMPLES 152 153 Sheet 5 of 14 4 873 633 NO BROMELIN PRETREAT PROCEDURE_FOR CHECKING REAGENT CELL SUSPENSIONS 204 202 PROC FOR CHECKING REAGENT CELL SUSP BROMELIN PRETREAT SAMPLE HANDLING RESULTS NOT ACCEPTED ADJUST CELL SUSPENSION 168 C 206 PROC FOR CHECKING REAGENT CELL SUSF REAGENT HANDLING 146 144 BROM PROC FOR QC SAMPLE HANDLING 148 PROC FOR QC REAGENT HANDLING 150 173 PROC FOR CHECKING DONOR CELL SUS PENSION REAGENT HANDLING NDLING PROC FOR SAMPLE HANDLING FOR CHECKING REAGENT TITER 163 FIG SA US Patent 10 1989 Sheet 60f14 4 873 633 TO FROM FIG 5A FIG SA QUALITY CONTROL CONT D 170 ADJUSTING DONOR CELL SUSP HANDLING CURRENT REAGENT DATA TABLE REAGENT DATA FILE 158 ACCESS REAGENT DATA MODULE ACCESS OK LOGON USER PRIVILEGE TABLE FIVE 160 MAIN MENU FIG 6 PRINT US Patent oct 10 1989 Sheet 70f14 4 873 633 PLATE READING MAIN MENU EDIT RESULTS
2. REREAD ROW 176 E AUTOMATICALLY LOGON TO ENTER OR EDIT ABO Rh ENTER OR EDIT RESULTS MANUALLY TEST RESULTS MANUALLY EDIT RESULTS FOR WBN LOGON TO ENTER 72 OR EDIT DU TEST laccess EDIT DU RESULTS RESULTS FROM MANUALLY AE CURRENT LIST LOGON TO ENTER OR EDIT MISC ACCESS g ENTER REMARKS MAIN MENU ENTER RESULTS MANUALLY FOR WBN FIG 7 US Patent Oct 10 1989 Sheet 80f14 4 873 633 WBN LIST FOR SPECIFIED GROUP AND TYPE 213 DISPLAY OF TEST STATUS 215 NT DIRECTORY PLATE INFO TABLE PRINT DISPLAY OF DATA FOR PLATE 3F 217 DIRECTORY Y ANTIBODY SCREEN SCREEN WELL PLATES POSITIONS FOR PLA DISPLAY OF UNVERIFIED Rh NEGATIVE LIST 193 219 221 DISPLAY OF ABO Rh TOTALS 223 225 LIST OF OMITTED CAROUSEL POSITIONS PRINT LAB TEST RECORD Fen FIG 8 US Patent Oct 10 1989 Sheet 90f 14 4 873 633 502 FIG 9A Mees MAIN MENU 506 DETERMINING THRESHOLD VALUES fee 508 BROM PROC FOR LIQ HANDLING No THRESH DETERM PLATES PROC FOR SAMP HANDLING FOR THRESH DETERM PLATES BROM 512 PROC FOR REAGENT HANDLING OF THRESHOLD DETERM PLATES SPECIFY HISTOGRAMS _sl4 TO REVIEW EVAL TEMP THRESHOLD 516 VALUES MENU 526 MANUAL ENTRY OF SAMPLE DISPLAY OF NTDS DERIVED INFO FOR EVAL OF TEMP FROM THRESH DE TERM
3. 5A The system then checks the antisera titer by dis playing an informational screen 163 telling the user what to do to perform this process One of the things the user must do is to make sure the file numbers 14 15 and 16 displayed on the handheld controller of the liquid handler 26 This alerts the host 20 to tag the barcode of the plate that will contain the serial dilutions of the antisera that the plate is a titer plate and that data from this plate is to be collected from the plate reader and stored in the titer file to process and display a graph like FIG 14 The user will then set up the liquid handler in the manner described in the information screen of block 163 and the liquid handler will be downloaded with instructions from the host to do a serial dilution and pipetting of the known donor samples placed by the user in test tubes in the carousel into the diluted rea gents in the 12 well serial dilution plate The system will then do a serial dilution on each reagent to be analyzed using the liquid handling portion of the program and will mix aliquots of the known blood type donor cells supplied by the user with the various dilutions of the reagent so as to generate known positive test results in some of the wells of sufficient concentration as symbol ized by block 167 in FIG 5A The plates are then read and the system looks up the bar codes in the look up table coming in from the plate reader and determines that the absorbance d
4. THRESH VALUES PROCESS BEEN NOT OK 528 SPECIFY 5 DISPLAY OF DISCREPANCIES BETWEEN ENTERED SAMPLE Shee EVEN VALUES AND THRESHOLD OK 53 DETERM PROCESS REVIEW OF EDITED THRESHOLD VERI FY UPDATE OF VALUES MENU 5 VALUES 2 xmas EVI ISTOGRAMS R H US Patent Oct 10 1989 Sheet 100f14 4 873 633 TO FIG FROM FIG 9 9A y FIG 9A 544 SAME REAGENT HANDLING PROCEDURE FOR BROM PLATE READING YES POSITION DETERMINATION PROC FOR PLATE READING POSITION DETERMINATION LIQ HANDLING 548 STEPS OFF CENTER DETERMINATION 554 VERIFY UPDATE OF PLATE READING POSITION FIG 9B US Patent 10 1989 Sheet 110f14 4 873 633 NO LOGON ACCESS MAIN MENU FILE OPERATIONS MENU TRANSFER COMPLETED d pr TO DISK 408 REVISE STORED ER RESET DATA RESET DATA FOR SPECIFIED SPECIFIED WBN ENTRY PLATE ENTRY 410 DELETE DATA ENTRIES DELETE ENTRIES p BY DATE BY PLATE seem FILES 7406 DISPLAY DATA STATISTICS 416 FORMAT FLOPPY DISK 402 MAIN MENU DELETE DELETE IF BY WBN ITRANSFERRED TO MAINFRAME 414 FIG IO US Patent 10 1989 Sheet 120114 4 873 633 LOGON FOR SYSTEM CONFIGURATION ACCESS OK ETE ddl GER 304 306 EST tomar CURRENT MODIFY ABO Rh TEST FORMAT LIQ SANDEN EET PRO NO ACCESS 302 CURRENT TABLE OF
5. 12 channel head The tips are lowered until they are above the meniscus of the wells and reagent is dispensed into the wells without contacting the surface of the liquid they contain This sequence is repeated until the entire plate is filled The tips are not contaminated in the process and they are replaced in the reagent block when the reagent transfer is complete Wash Head The wash head is mounted in the center of the fusion head The wash head allows the user to cycle substan tial amounts of liquid through a row of wells Each of the 12 separate wash cannula groups consists of three cannulas pointing into the well all at different angles One stainless steel cannula pumps solution into the well another keeps the well from overfilling and the third drains the well Each set of cannula has its own sensing apparatus and operates independently This mechanical and electrical configuration enables the liquid handler to cope with considerable variability in the amount of fluid the wells contain initially No mat ter how full they are to start with none will be over filled in the wash process Wash Head Operation The user can program the system to pump anywhere from 10 ul to 10 ml through a well in a continuous washing action and to pump the well dry at the end leaving no more than 3 4 ul behind It is also possible to use the wash head to add liquid stop for incubation then use the wash head to suck the well dry again all un
6. A B Rh Cell Suspension A1 A2 B and Serm Blank step 112 and what test format is being used i e which tests are being carried out in which columns of the microplate to define the format and arrangement of the template i e the pattern of positive and negative reactions which define the blood type step 112 The liquid handling may be done on the plates in any order and the plates may be read in any order The liquid handlers such as device 26 read the bar codes of all the tubes containing donor samples that are pipetted into a particular plate These bar codes are sent to the host computer 20 along with the bar code of the plate into which samples from the identified tubes were placed The host computer knows which rows of each plate contain aliquots of blood from each donor because the control software of the liquid handler always places samples in the plates in the same order relative to the order of the tubes from which the samples were re moved The identification data of the plates and the WBNs that are in each plate are stored by the host computer in a lookup table along with any tag data that the liquid handler appends to the identification data for the plate bar code Such tag data can include data that the plate is a steps off center plate this will be explained more fully below or is a donor cell suspension or rea gent cell suspension plate etc Because this data is per manently stored in a look up table the order in
7. POSITIONS US Patent oct 10 1989 Sheet 1 0414 4 873 633 20 MAINFRAME PRINTER 29 32 22 KEYBOARD Sr FUGE 28 PLATE ORBITAL 34 TERMINAL READER SHAKER 24 LIQUID FIG US Patent 10 1989 Sheet 2 0114 4 873 633 US Patent Oct 10 1989 Sheet30f 14 4 873 633 LOGON FOR SYSTEM STARTUP USER 102 PRIV TABLE FIVE LOGON FOR MAIN MENU 104 SYSTEM START UP REAGENT DATA HANDLING 154 QUALITY CONTROL PROCEDURES LIQUID HANDLING PARAMETER 100 DEFINITION PLATE READING EDIT RESULTS 170 190 RESULTS SUMMARY READER CALIBRATION ac 92 5 5 CONFIGURATION FILE OPERATIONS 400 138 IMMUNOASSAY s S 29 US Patent 10 1989 Sheet 4 of 14 4 873 633 PROCEDURE FOR ABO Rh TESTING REAGENT HANDLING ABO Rh TESTING 108 SAMPLE HANDLING SELECT CRITERIA FOR REPEAT LIQ HANDLING 130 SPECIFYING WBN S OF SAMPLES FOR REPEAT LIQ HANDLING DISPLAY OF NTD S N PROC FOR REPEAT LIQ HANDLING FOR NTD S PROC FOR REPEAT LIQ HANDLING FOR SAMPLES YES LIQ HANDLING FOR_SAMPLES SAMPLE HANDLING 136 SAME REAGENT HANDLING PROC FOR REPEAT ve LIQ HANDLING OF s REAGENT NTD S SAMPLE HANDLING HANDLING FIG 4 US Patent QUALITY CONTROL MENU Oct 10 1989 164 ABSORB OF REAGENT CELL SUSP TABLE amp MENU ACCEPT PRINT LCHART
8. fixed disk The test results the system generates are stored on the fixed disk as testing progresses throughout the day At the close of the day this data is usually transmitted to a mainframe computer or copied onto floppy disk for permanent storage These automatic procedures make space on the fixed disk for the next day s test results The fourth category of information test status has only temporary validity Accordingly it is kept on the fixed disk in its current form but there is no need to archive it onto the mainframe or floppy disks Printing Reports It is crucially important to have hard copy versions of many types of test and quality control reports The system enables the user to produce a wide range of printed reports automatically usually just by pressing a key on the host computer 20 keyboard The user speci fies the formats for the types of reports the user s facility generates On command the system automatically gen erates all pages or selected pages of the reports the user request Accountability Log On and Log Off In a clinical setting like the blood bank it is important to be able to establish which laboratory personnel were responsible for particular test operations The system facilitates accountability several ways Only one person at a time can work with the system The user logs on to the system when the user begins to use it and logs off when the user is done One does so by ident
9. ing positions are then used for all subsequent readings of these type plates including reagent and donor cell sus pensions and histogram plates and quality control plates but not for immunoassay plates More detail is given on this aspect of the invention and the other aspects of the invention in the CETUS PROGROUP SYSTEM USER S MANUAL dated May 28 1986 available from Cetus Corporation in Emeryville Calif which is hereby incorporated by reference Another major advantage of the system over the prior art is the ability to graphically fine tune the sys tems ability to discriminate between positive and nega tive reactions This improves the accuracy of the system and reduces the number of NTD WBN s that result from processing a batch of donor samples The graphic display allows the user to very quickly and easily grasp the statistical results from processing a batch of donor samples This allows the user to graphically adjust thresholds which are used to discriminate between posi 10 20 25 30 35 45 50 55 65 40 tive results negative results and the NTD area This gives the user a very powerful and effective tool to optimize the operation of the system Using the ProGroup System Manager Overview Since the user primarily interact with the host com puter 20 through its software this section focuses pri marily on how the user works with the ProGroup TM automated blood typing system programs These applicati
10. limit for light absorbance for a positive reaction The other threshold is a negative threshold that defines the lower light absorbance limit for a negative reaction The invention allows the user to view histograms for the population of light absorbance readings for reac tions to various reagents or donor cell suspensions or plasma blank and then to graphically adjust the posi tions of the thresholds based on the histogram graphic display This reader calibration process insures good test result interpretation The system automatically prompts the user through the process of doing the liquid handling for the control histograms donor cell suspen sion and plasma blank and the actual forward typing and reverse typing reagent histograms Basically the host computer 20 performs the process of analyzing light absorbance data read by the plate reader from test wells containing donor samples that have been reacted with reagents and processing and storing the data and plotting histograms of that data The user can then graphically locate the two threshold points which de fine positive and negative reactions by the values of absorbance on the histogram These histograms are frequency distributions for the number of samples in the sample population of 48 or 96 which produced particu lar absorbance readings or absorbance ratios On all the histograms the Y axis indicates the total number of samples that had each absorbance or absorbance ratio On th
11. put the cells in suspension after centrifugation to collect all the cells at the bottom of the well The purpose of this procedure is to check the quality of the processing by the liquid handler 26 of the dilution and the quality of the resuspension process in the orbital shaker 34 after centrifugation The shaking process can be done im properly so that the reagent cells are not properly dis persed throughout the solution Also the dilution may have been done improperly Both of these errors will show up as a change of the absorbance of light passed through the solution The points on the graph of FIG 15 represent the absorbance values which result on each day when a new dilution of donor cells is made and the resuspension process is completed of the bovine serum and reagent cells for the QC test done each day in prep aration for that day s processing The maximum and minimum values shown in FIG 15 are constants which are established as the limits of the absorbance range considered acceptable for a reagent cell suspension As can be seen from the drift out of bounds toward the 60 day mark there is some problem which is developing either in the dilution or in the resuspension procedure which is causing the reagent cell suspension test results shown in FIG 15 to become unacceptable thereby signalling the user that a problem is occurring This allows the user to monitor the quality of his reagent cell suspensions to determine that they have bee
12. reagent mixtures that have reached room tempera ture after being refrigerated 2 Place of diluent in the predilution block Insert the Micro Trof to catch the diluent that will be flushed through the system in the priming procedure Fill the carousel with up to 48 bar coded tubes and a corresponding number of long tips 5 Press the RUN key on the hand held controller to start the automatic Home Prime Pump procedure 6 Press the STOP key when the diluent being pumped through the system has flushed out the tubing Empty out the Micro Trof and put it aside Put the reagent and the predilution blocks into posi tion on the liquid handler table 9 Place the carousel on the bed with the 1 position corresponding to the active position for the X Y head 10 Load the plate stacker with six empty bar coded microplates for ABO Rh testing 11 If necessary manually insert a fresh plate for the antibody screen test 12 Respond to the prompts on the controller display to enter the number of tubes in the carousel 13 Press the START key on the controller 14 The liquid handler manifold fills the wells of the predilution block with the appropriate quantity of diluent for the donor cell suspension 15 The liquid handler reads the bar codes on the first eight tubes in the carousel 16 The X Y head picks up the first long tip from the carousel and pipettes donor plasma into 1 2 or 3 wells of the antibody screen plate 17 T
13. USER ID SYSTEM 518 INFO 308 MODIFY MODIFY MAINFRAME HANDSHAKE HANDSHAKE SNA SPECIFY HANDSHAKE EMULATION EMULATION FOR RS 232 PROTOCOL SEE LAB REPORT FORM 310 MODIFY IMMUNOASSAY FORMAT 314 R LIQ HANDLING ERROR HANDLING 512 NU HARDWARE COMPLEMENT MAIN MENU FIG US Patent oct 10 1989 Sheet 130f14 4 873 633 OF TOTAL WELLS HAVING EACH ABSORB ANCE NTD AREA bs 59 4 v ASAMPLE POSITIVE POSITIVE NEGATIVE NEGATIVE REACTIONS THRESHOLD THRESHOLD REACTIONS TOTAL E xxxx 27523 yyy y47 525 272921 ACTUAL ABSORBANCE ACTUAL ABSORBANCE NUMBER OF NTD S AT POSITIVE THRESHOLD NEGATIVE THRESHOLD AT CURRENT THRESHOLD SETTING WELL ABSORB CENTER ANCE POSITIVE R ACTION AGGLUTINA TION ABSORBANCE READING READING POSITION POSITIONS WALL WELL WALL USER DEFINABLE FIG READING POSITIONS FIG I3B US Patent oct 10 1989 Sheet 140f14 4 873 633 ABSORBANCE DILUTION OR TITER ABSORBANCE 123 ee 30 60 90 DAY FIGAS KNOWN EXPERIMENTALLY WBN DETERMINED WEN BLOOD TYPE amp TEMPLATE 123 A POS POS 135 B POS A POS 620 0 0 t 16 16 4 873 633 1 USER CONTROLLED OFF CENTER LIGHT ABSORBANCE READING ADJUSTER IN A LIQUID HANDLING AND REACTION SYSTEM This application is a continuation in part of Ser No 788 998 filed Oct 18 1985 now abandon
14. centrifuged at low speed to cause the cells to settle Then they are resuspended This sequence of steps disperses the cells when the test results are negative but causes the cells in a positive test to clump together in a tight button at the bottom of the well The final step is to load each plate into the automatic plate reader Plate reading can occur no less than three 4 873 633 11 minutes but no more than ten minutes after the final resuspension of the cells The plate reader reads one row of 12 wells at a time making two off center read ings on each well as is known in the art The reading with the higher absorbance is discarded and the lower reading is transmitted to the ProGroup TM automated blood typing system host computer 20 20 for interpreta tion The computer compares the reading for each well with defined threshold values set by the user as de scribed below to determine whether the data point represents a positive or negative result for that test It requires about a minute to read each plate then the plate is removed and placed in a rack for storage The plate can be retrieved if it is necessary to reread a row The tubes from the carousel are also placed in a rack If the results indicate that a given samples might be Rh negative that tube is loaded into a carousel for DU testing If the results for blood typing are internally contra dictory or indeterminate the liquid handling and plate reading must be repe
15. computer For example if a given sample was originally categorized as NTD certain individual test results must be changed after the blood group and type have been positively identified In this situation the user is typing over some or all of the data that already appears on the screen The user changes the screen by 1 Placing the cursor in the right spot 2 Typing in the appropriate characters or pressing the space bar to create a blank then 3 Moving the cursor to the next location that requires a change Changing Default Values In some situations the ProGroup TM automated blood typing system automatically supplies a possible value such as today s date This is called a default value and it essentially represents an educated guess that this value will be correct Usually the default set ting will be appropriate but if it isn t the user simply types over it To create a blank the user presses the space bar When the setting is correct the user presses Return Making Corrections If the user makes an error when typing in or changing information use the BackSpace key This key moves the cursor to the left and deletes the character that had occupied that position Once the user has backed up far enough to erase the error simply enter the correct information In most cases the user presses the Return key after each entry This means that the user can make as many changes as wanted before pressing Return the host compu
16. plate is lowered to the level of the table and ad vanced onto the table by belts forming a conveyor Each plate holds sample from eight donors Thus the six plate capacity of the stacker matches the 48 tube capacity of the carousel The plate stacker does not handle antibody screening plates These are put in and taken out manually The liquid handler controller prompts the user to remove the antibody screen plate when it is ready for testing CAROUSEL Tube Features The carousel holds up to 48 tubes of sample and an equivalent number of long disposable tips The tubes must be bar coded The Becton Dickinson Vacutainer R Terumo Venoject R Sherwood Medical Mono ject R or comparable blood collection tubes are suit able for use with the system The tubes should contain between 2 mi and 9 ml of sample The donor sample is anticoagulated plasma prepared by standard blood bank procedures Carousel Loading The system uses long disposable Cetus Pro Group tips These are loaded into the carousel before the tubes of sample are inserted The tubes should be loaded into the carousel with the bar code at the top facing the outer edge of the carousel so the code is visible to the scanner The 1 is printed on the carousel to indicate the position of the first tube Subsequent tubes are inserted in a counterclockwise sequence as shown below The carousel rotates clock wise to bring the tubes and tips within range of the X Y head M
17. positions defined above The absorbance results are then automatically organized into histograms by the host computer with the percent of the total number of samples plotted on one axis and the absorbance of the sample plotted on the other axis The horizontal axis is actually the observed absorbance divided by the absorbance of the cell suspension bovine reagent setting maximum possible absorbance i e the absorbance as a percent of the total possible absorbance One histogram is prepared for each type of reagent used anti A anti B etc Such a histogram for a particular reagent is shown in FIG 12 The arrows 517 and 519 represent the temporary thresholds which can be set by the user by manipulating the cursor movement keys on the host keyboard That is the arrows 517 and 519 can each be moved left and right FIG 12 represents the statistical results of the processing of a large number of donor samples The height of the lines represents the percentage of the total population of samples which had the absorbance represented by the horizontal position of each line The horizontal position of each line repre sents the absorbance observed for the number of sam ples represented by the vertical line at that position if any as a percentage of the total possible absorption A donor sample tested in the batch of samples for which FIG 12 is a histogram will be called a positive reaction if its absorbance readings is to the left of the absorban
18. reader in which case the results stored on the computer are updated automatically Alternatively the row of wells can be examined visually or the liquid handling and the reading of that sample can be repeated manu ally In the latter case the user inputs information through the computer keyboard to modify the results of the sample processing procedure Orbital Shaker The ProGroup TM automated blood typing system includes an orbital shaker 34 as part of the system The shaker is used to resuspend the cells after the liquid handling is complete and then again after the plate has been centrifuged The orbital shaker can accommodate up to eight plates Centrifuge The centrifuge 32 can be an instrument such as the Beckman TJ 6R Tabletop Centrifuge It is used in a known manner on the plates from the liquid handler 26 before they are placed in the liquid handler Host Computer The host of the ProGroup TM automated blood typing system is the computer 20 called the host com puter 20 The host computer 20 directs the operation of the liquid handler 26 and the plate reader 28 and man ages the data generated by the other equipment The user primarily interacts with the ProGroup TM auto mated blood typing system through the host computer 20 The user uses the host computer 20 to select the function the user wants the ProGroup TM automated blood typing system to perform and to view stored data in tabular or graphic form The host comp
19. see if an apparent positive result was actually caused by a fibrin clot for example Or the user can perform the liquid handling by hand and read the plate by eye Ei ther way the user enters the test results into the data file by typing the information into the host computer using the process of FIG 7 Additional Test Results The system host computer automatically interprets and stores data for each WBN the ProGroup TM auto mated blood typing system processes This data struc ture accommodates other information as well as the 20 25 30 35 45 50 55 60 65 20 results of the ABO Rh test procedure The user can enter results for the following tests antibody screen Hepatitis HTLV III Cytomegalin Virus CMV Rapid Plasma Reading RMR The invention thus allows the user to organize all the information about a donor sample into a single concise structure Testing Status The system gives the user immediate access to infor mation on test status The user can ask for the WBN of the first NTD the current DU list the tables of ABO Rh results for the day the status of a particular plate etc The system answers these and many other questions with up to the minute accuracy If the user needs to know whether there s any of a particular blood group being tested the user can search by group and type to find the WBNs of samples that fit this profile then determine how far the testing on these sample
20. the user to move the locations of the off center absorbance reading relative to the rest of the graphic display to optimize the discrimi natory powers of the system to distinguish between positive and negative reactions and means to record the user s choice for the position of the off center reading for use in further plate read ing operations 2 The apparatus of claim 1 wherein the means for graphically displaying the absorbance data and for al lowing the user to move the position of the off center reading is a host computer and further comprising means for allowing the user to perform automated liquid handling on each different plate type using a sequence of liquid handling steps which is custom ized for that plate type means for reading a bar code on each plate type and for sending this data to said host system with tag data for plates which have predetermined liquid handling sequences said tag data indicating that the plate has a well bottom geometry for which the plate reader system is not currently optimized means in said plate reader for reading the bar codes on plates placed therein for reading and for sending the bar code data to said host system means in said host system for looking up the bar code data received from the plate reader and determin ing whether the plate is a type for which the plate reader has not been optimized in position of the off 35 center reading means for controlling the plate reader to caus
21. to optimize the absorbance for that reagent and to con serve the amount of reagent used These reagents are often quite expensive Further it is useful to have a quality control and accountability system such the quality of data generated in the testing is consistently high and the persons per forming the testing can be determined For example it is useful to know the expiration data for all the reagents in stock and the associated absorbance values for each reagent lot over the period of its usage It is also useful to compare the test results for known sample types to the resuits that should have been obtained as a check on the accuracy of the system It is also useful to be able to generate reports on daily or monthly activities to deter mine the amount of certain types of blood in stock and where it can be found The number of no type deter mined test outcomes hereafter NTD Thus a large amount of data in the form of process controlling parameters and test results are involved in blood typing and antibody screening operations and a large amount of record keeping for this data is involved Therefore a need has arisen for a system which can perform these thousands of liquid handling steps reli ably and tirelessly and which can handle the thousands of data records which characterize the liquid handling and which constitute the data record for each donor Further such a system should be access controlled and implement accountabili
22. values for the absorbances of these reagent lots over the period of their usage test results including blood group and probable type which are generated automatically The results for the DU test antibody screen and other miscellaneous Hepatitis HTLV RPR can be added manually to the information that is already stored for that donor sample test status for the day in terms of a range of parame ters such as the number of A samples that have been processed already the number of NTDs that have oc curred in the course of the day s testing the status of testing for particular plates etc The section that follows describes each of these four major categories of data Then how data is stored in the system and how the ProGroup TM automated blood typing system safeguards the validity of this information will be described Test Parameters The ProGroup TM automated blood typing system is a programmable system This means that the user only need to specify once how the user wants the ABO Rh test to be carried out and how the user wants the liquid handling performed for the antibody screen Thereafter the user just sets the system up properly and the Pro Group TM automated blood typing system automati cally executes the function just as the user s directed 5 10 25 30 40 45 55 60 65 18 The ProGroup TM automated blood typing system also simplifies the work by making certain c
23. 3A shows a typical well bottom with a positive reaction button FIG 13B shows a typical graph resulting from a positive reaction button 4 873 633 5 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Referring to FIG 1 there is shown a block diagram of the system of the invention The system is comprised of a Digital Equipment Corporation PDP 11 MICRO host computer 20 which coordinates the activities of the system under the control of the resident software in cluded herewith as the appendices and the user not shown who enters commands through a keyboard 22 in response to information displayed by the software on a terminal 24 The host computer 20 runs the commer cially available Micro RSX operating system for the PDP 11 23 11 53 or 11 73 computer versions available from Digital Equipment Corporation The application software which implements the functions described herein is included herewith an appendix The host com puter is coupled by RS232 serial ports and connecting cables to a number of liquid handling and plate handling mechanisms of which liquid handler 26 is an example The preferred embodiment of the system can control up to 8 liquid handlers The details of the liquid handlers and plate handling mechanism are given in a copending U S pat application assigned to the assignee hereof entitled Automated Liquid Handling Apparatus And Process With Plate Handler filed Oct 18 1985 Ser No 789 945 now abandoned and
24. 986 Patel 356 39 4 608 246 8 1986 Bayer et al 424 11 4 665 553 5 1987 Gershman et al 356 39 X 4 678 894 7 1987 Shafer 364 416 X 4 683 120 7 1987 Meserol et al 422 72 4 713 348 12 1987 Ullman 436 501 4 719 087 1 1988 Hanaway 422 102 4 727 033 2 1988 Hijikata et al 356 39 Primary Examiner Clark A Jablon Attorney Agent or Firm Ronald C Fish Kevin R Kaster Albert P Halluin 57 ABSTRACT A host computer controls a plate reader which optically reads the results between donor samples and reagents The system provides the user with the ability to change the position of off center light absorbance readings to maximize the power of the machine to discriminate between positive and negative agglutination reactions In addition the user can tailor thresholds for absorb ances used to distinguish between positive negative and no type determined reactions 7 Claims 4 Drawing Sheets OF TOTAL ELLS HAVING CH ABSORB CE NTD AREA NS 57 E ASAMPLE POSITIVE x POSITIVE NEGATIVE NEGATIVE A m REACTIONS THRESHOLD THRESHOLD REACTIONS TOTAL e xxxx 47923 525 27320321 ACTUAL ABSORBANCE ACTUAL ABSORBANCE NUMBER OF NTD S AT POSITIVE THRESHOLD NEGATIVE THRESHOLD AT CURRENT frt ABSORBANCE READING POSITIONS THRESHOLD SETTING Us DEFINAI READING
25. Head The ProGroup TM automated blood typing system incorporates four methods of liquid transfer All four heads are mounted permanently and operated under software control X Y Head The X Y head is positioned at the left of the instru ment The X Y head travels along a shaft that runs from left to right across the instrument This head picks up a long disposable tip from the carousel aspirates sample from the tube that has been rotated into the correct position then moves gradually across the microplate delivering sample to the specified wells in a given row X Y Head and Liquid Level Sensing The volume of sample in a tube can vary substan tially It is important to be able to determine the liquid level in each tube very precisely to ensure that the tip is lowered to just the right height to pick up first only donor plasma and then only donor cells The ProGroup TM automated blood typing system uses a special liquid level sensing technique that locates the meniscus in each tube with great accuracy The instrument has a hollow piston with a block driving a plunger up and down When the tip is lowered into a test tube the plunger block is pushed down forcing out the air in the tip The pressure rises sharply when the air contacts the surface of the liquid The air contacts the surface of the liquid The pressure transducer registers this sudden increase and this information allows the system to calculate the position of the menisc
26. United States Patent ro Mezei Louis M et al 11 Patent Number 4 873 633 45 Date of Patent Oct 10 1989 54 75 73 21 22 63 51 52 58 56 USER CONTROLLED OFF CENTER LIGHT ABSORBANCE READING ADJUSTER IN A LIQUID HANDLING AND REACTION SYSTEM Inventors Mezei Louis M Fremont Bradley S Albom Richmond Coppock Stan Stephen J Moehle both of Berkeley Brent S Noorda Pleasant Hill Joseph T Widunas Berkeley James Zeitlin Piedmont all of Calif Assignee Cetus Corporation Emeryville Calif Appl No 906 101 Filed Sep 11 1986 Related U S Application Data Continuation in part of Ser No 788 998 Oct 18 1985 abandoned Int CLA G01N 33 48 GOIN 21 01 GOIN 33 80 GOIN 35 02 0 8 Cl 364 413 08 356 39 356 442 422 73 Field of Search 364 416 555 422 13 356 39 442 440 References Cited U S PATENT DOCUMENTS 4 130 395 12 1978 Chryssanthou 424 11 4 253 846 3 1981 Smythe et 436 53 4 255 788 3 1981 Schwartz et al 364 413 07 4 268 268 5 1981 1 436 52 4 319 882 3 1982 Sharma 436 63 4 451 433 5 1984 Yamashita et al 422 63 4 478 094 10 1984 Solomaa et al s 73 863 32 4 556 641 12 1985 et al 422 73 X 4 580 895 4 1
27. agent cell suspension and donor cell suspension against the previously stored values The absorbance of the reagent cell suspensions can be viewed using step 164 of FIG 5A The absorbances of donor cell suspensions can be observed using step 166 in FIG 5B The object of checking donor cell suspensions is to insure that from day to day the same number of red blood cells from the donors is placed in the wells to be reacted with the reagents If different numbers of red blood cells were placed in a well and reacted with the same reagent and both samples were the same type one test might have a different result than the other merely because of the different donor cell concentration This would lead to erroneous test results and should be avoided The same adverse result can occur if the concentration of reagent cell suspensions varies from day to day The reagent cell suspension absorbances can be tested and graphed using the step 164 of FIG 5A Adjustments in either cell suspension amount can be made using the steps 168 and 170 If the graph of the resulting data points shows a shift it indicates a deterioration in reagent quality or in the donor cell suspension concentration Test Results The ProGroup TM automated blood typing system also stores information on each donor sample in a data file The data file is the functional equivalent of a card file except that the information is stored magnetically on the ProGroup TM automated blood typ
28. al is converted to optical den sity units using a logarithmic amplifier The output voltage from the logarithmic amplifier is in turn con verted to a digital value The actual optical density of a row of wells is computed relative to a blanking well designated by the host computer 20 i e the Bovine Serum Albumin containing well A custom programmed EPROM Erasable Program mable Read Only Memory chip commercially avail able added to the plate reader permits very precise control over the motorized carrier that positions the plate in relation to the light source This firmware al lows one to calibrate the plate reader to make two off center readings of each well in the positions that pro vide optimal differentiation between positive and nega tive test results PLATE READER FEATURES Performance Specifications The plate reader reads all 96 wells in a microplate in approximately 60 seconds The measurement range is 0 000 to 2 999 Absorbance Units over wavelengths that can range from 380 nm to 750 nm The narrowband interference filters are mounted on a wheel with a ca pacity of six filters The linearity is plus or minus 1 of the best straight line approximation from 0 000 to 2 000 4 873 633 31 O D The combined accuracy and repeatability is plus or minus 1 and plus or minus 0 010 of the true optical density relative to air on the absorption peak of the solution at a single wavelength Successive readings of a well produce res
29. al to those of the reagent cell suspensions The system will continue to accumulate data on the daily donor cell suspension procedure preceding the day s testing until a reset op tion is exercised The system as in the case of the rea gent cell suspension will give several information screens which give a synopsis of the procedure to be followed to perform the necessary liquid handling and plate reading to check the donor cell suspensions In this procedure the antibody reagents in the ABO Rh block of the liquid handler are replaced with bovine serum albumin 3 BSA This insures that there will be no hemagglutination and that resuspension will be similar to that of a negative reaction The user uses a special ProGroup TM automated blood typing system function to determine the optimal dilution ratio for a specific lot of antibody reagents used in forward typing by selecting option 154 on FIG 3 This selection vectors processing to the process shown in FIG 6 There the user can select option 156 to enter data on the reagents in inventory their lot number and expiration date the date the reagent data was entered into the system and the proper dilution for that particu lar lot of reagent reagents come in varying concentra tions from their manufacturer The proper dilution value is determined experimentally by using the process shown in FIG 5A The user must select the menu op tion check reagent titer shown as block 161 in FIG
30. alculations from the data the user has supplied For example on the basis of the test format and sample volumes the user defines prescribed the ProGroup TM automated blood typing system computes how much donor plasma to pick up and which wells of the ABO Rh plate to pipette it into Because the ProGroup TM automated blood typing system is programmable it may be fine tune the system to optimize its accuracy For example certain test pa rameters must be adjusted over time Each new lot of reagents usually requires a slightly different dilution ratio so the user will need to change the volume of diluent in relation to the volume of donor cells The user makes these changes by entering the new values just once using option 100 in FIG 3 The old values in the program are replaced automatically with the new values by the host computer Quality Control Data ProGroup TM automated blood typing system tracks quality control data to help the user verify that the system is performing properly and recognize when parameters need to change ProGroup TM automated blood typing system stores the lot number expiration dates and dilution ratios for all reagents used in the system The user can use a print out of this information as a convenient way to double check the reagent values at the start of the workday ProGroup TM automated blood typing system also allows the user to compare the mean minimum and maximum absorbance values for each type of re
31. amples of diluted red blood cell samples into multiple wells Multiple diluted reagents are then added to the multiple wells containing plasma and red blood samples and various reactions either occur or do not occur de pending upon the blood type and the reagent in each well Typically these reactions manifest themselves as clumps of protein in the bottom of the well for a posi tive reaction and no clumps for negative reactions the pattern of positive and negative reactions determines the blood type The blood type can be determined by placing the multiple wells under a strong light source and reading the optical absorbence i e the amount of light which gets through the bottom center of the well Typically absorbance readings are taken on both sides of the cen ter bottom of each well and the results are compared to certain threshold criteria for absorbance The com parison of the readings will indicate the presence of a clump at the bottom of the well low absorbance in the off center readings and therefore a positive reaction or the absence of a clump and therefore a negative reaction high absorbance on both off center readings Clearly the process of blood typing of thousands of donor samples involves many thousands of liquid han dling steps and the generation of many thousands of absorbance readings for the multiple wells devoted to each donor Further liquid handling steps are involved in automated antibody screening and some
32. and terminal hardware and the application software The specific functions involved in typing and group ing blood are carried out by a group of application programs developed by the assignee The reset of this section explains how these application programs are structured and how the user interacts with them ProGroup Application Programs Application Program Design ProGroup TM automated blood typing system facili tates the work by using the terminal screen to list all the options at any given point in the program summarize the steps in each procedure such as setting up the liquid handler for quality assurance or sample testing giving the user on screen help to remind the user what keys the user can use at any given time and providing a status line for other relevant information such as error messages or a brief explanation of the task the system is performing 4 873 633 41 Interacting With The System Manager The user interacts with the host computer 20 in two basic ways by supplying new information or updating data that is already stored in the computer or by directing the system to carry out a particular kind of task The user performs these two types of operations in a similar way First the user moves the cursor to the appropriate point on the screen The cursor location shows where the next alpha numeric character the user types will be placed if the user is entering or editing data or which option will b
33. another U S pat application entitled Liquid Manipulation Device and Method filed Jul 5 1985 Ser No 752 449 now aban doned both of which are hereby incorporated by refer ence The object code for the liquid handler disclosed in the above identified patent application has been changed somewhat since the filing date thereof The latest object code is included herewith in the appendices hereto The object code appendices are labelled for the particular microprocessor in the liquid handler for which each is intended The host 20 is also coupled by an RS232 link to a plate reader 28 which optically reads the absorbance values for samples in various wells in plates filled with samples and reagents loaded from the liquid handler The plate reader is commercially available from BioTek Instruments Inc under the designation Autoreader EL309 The host 20 is also coupled by an RS232 link to a printer 30 upon which the host 20 prints the various reports of which the system is capable A centrifuge 32 and an orbital shaker 34 are also part of the system but are not connected to the host 20 The centrifuge and orbital shaker are commercially available off the shelf components and are used to process the plates after the samples and reagents have been put in the wells by the liquid handler 26 and before the plates are loaded in the plate reader 28 Collectively the above described sys tem may hereafter be referred to as the ProGroup TM automate
34. at were omitted from sample processing will be omitted from these totals This report provides an overview of testing results The user can ask for and receive a report of the omit ted carousel positions by selecting the menu option represented by block 225 This selection causes the report generator to collect and process the data regard ing tubes that have not been processed and to generate a report that gives the carousel number and tube posi tion for tubes that were not processed on a given date together with the reasons for the omissions e g a short fill bar code error etc The liquid handler 26 collects this information and sends it to the host for storage in a file dedicated to this type of information As is the case for all the reports generated by the routines symbolized by FIG 8 the omitted carousel positions report may be 4 873 633 33 printed simply by selecting the menu option to print the report All these reports can be generated in real time ie while the user waits Information on test results is displayed in the type of template shown below Instead of being given a series of 5 optical density measurements the following symbols which represent results for individual tests are shown P Positive N Negative Ambiguous results The user can reread a particular row of wells a sec ond time either automatically or by eye If need be the user can repeat the entire liquid handling and plate reading operat
35. ata coming in from that plate is to be stored in a file and processed for display as a titer graph such as that shown in FIG 14 The host knows which dilutions were in each weil because the host instructed the liquid handler to do a serial dilution in a conventional fashion Accordingly the data is sorted and processed according to its well position and the known dilution value for that well The data may then be viewed graphically in the form of a graph like that of FIG 14 by selecting the menu option view titer 5 20 25 35 40 45 55 60 65 16 graphs symbolized by block 162 in FIG 5B The ab sorbance values will be low for all dilutions until the concentration is too diluted to cause a positive reaction to occur At the concentration where no positive reac tion occurs and at more dilute concentrations the ab sorbance will be higher indicating a false negative reac tion such as is shown at 165 in FIG 14 The cursor keys are then used to move the arrow two titers down and that concentration is selected as the optimum concen tration for that reagent lot Then the system automati cally stores the user selected ratio into the reagent data table for that reagent and automatically instructs the liquid handler to dilute that reagent to the user selected dilution every time it is used until the reagent titer is again changed by the user This ratio must be used consistently throughout that reagent lot to obtain rel
36. ated either automatically or manu ally The user selects criteria in step 118 FIG 4 for comparison by the host 20 against test results to deter mine if repeat handling is necessary For NTD s the user can specify the exact procedure to be followed by the liquid handler upon retesting of these donor samples in steps 120 122 124 126 and 128 For other than NTD s for which the user desires retesting he can spec ify the exact procedure to be followed by the liquid handler for this retesting in steps 130 132 134 and 136 Tubes that require repeat processing are loaded into another carousel for further scrutiny Antibody Screening The main menu on FIG 3 has an option 138 for con ducting immunoassays for the presence of some anti bodies such as HTLV and others Because these assays differ widely the software allows the user to link files to perform the assay in pieces By asking the user which assay he wishes to perform and the starting file the user can have flexibility in the types of assays he performs The user can start with the first file customize its pa rameters and put a linking address in the linking field of the first file to the next file he wishes to perform After all the files are properly customized the liquid handler will upon command perform the first file in the se quence and then proceed to the linked file and perform it Many different types of assays can be performed in this manner After the entire anti
37. body screening plate has been filled with sample the 12 channel head picks up a row of short tips from the front row of tips in the reagent MicroTrof TM These tips are used to aspirate sample from the front row of wells in the reagent wells in the reagent MicroTrof 62 which contain reagents necessary for the performance of the antibody screening test The entire plate 58 containing donor plasma samples is filled with the reagents and the user removes the antibody screening plate manually for interpretation of the results manually The ProGroup TM automated blood typing system only delivers donor samples and reagents to the plate for antibody screens and performs no further oper ations Options The user can specify that the antibody screening test be carried out with one reagent with two reagents or with three as test protocol directs 5 15 20 25 30 35 40 45 50 55 60 65 12 Parameters The user defines the volumes of sample plasma and reagent to be pipetted into the microplate Quality Control Quality Control Procedures The ProGroup TM automated blood typing system enhances quality control of test procedures Specific ProGroup TM automated blood typing system func tions are verify test procedures by processing a small number of known samples and checking the experimental data against the known results verify that the dilution of cell suspension reagents and donor cells produce absorban
38. btained by the system That is the user may have the system perform automated liquid handling and automated blood typing of a group of known samples and then may graphically compare the experimentally determined templates to the known templates for the known blood types processed The quality control test by assaying known samples is done by selecting option 140 in FIG 3 which vectors processing to the process flow shown on FIG 5A starting at block 142 Block 142 represents the step of having the quality control opera tor log his identification code into the system The test is done by placing test tubes of known blood types in the carousel and assigning them WBN s on the host com puter and indicating the particular blood group and type for each WBN This process of entering the WBN s of the known blood type samples is symbolized by step 144 Steps 146 148 and 150 represent informa tion screens telling the user how to set up the liquid handler so that the liquid handler has the known sam ples in the carousel and has the proper liquid handling file called up via the handheld controller for execution by the liquid handler The liquid handler then performs 4 873 633 13 the ABO rh type liquid handling procedures described in the U S pat applications describing the liquid han dler which are incorporated by reference The specific file for this quality control procedure includes instruc tions to tag the bar code data from the plates bein
39. ce threshold marked by the arrow 517 If the sample s absorbance is to the right of the absorbance threshold marked by the arrow 519 the sample will be character ized as having had a negative reaction to that reagent If the absorbance falls between the arrows the sample will be characterized as being NTD The total number of for the current threshold settings is shown updated in real time in a legend shown on the graphic display of the histogram as at 521 in FIG 12 When the user changes the positions of the arrows representing the current positive and negative thresholds the num ber of NTD s will be updated based upon the new threshold values Also the host displays the actual ab sorbance value for each position of the positive and negative threshold values As the position of the thresh olds is varied the absorbance values at these threshold locations is given at 523 and 525 When the user selects the menu option represented by block 506 the liquid handler attaches a histogram tag to the bar code of the plates processed according the instructions of screens 508 510 and 512 When these plates are read by the plate reader the absorbance data from these plates is sent to the host computer which collects it in a file for processing into the histogram data Since a histogram is presented for each different reagent type the liquid handler is loaded from the host with instructions as to which reagents to put in s
40. centrifuge for re suspension and re separation of liquids and sam ple cells during some system operations The host software also contains routines for imple menting quality control operations Such operations include verifying test procedures by processing a small number of known blood type samples and checking the experimental data against the known results and verify ing that the dilution of cell suspension reagents and donor cells produce absorbances in the proper range The system can also test the titer of antibody reagents The data management functions of the invention include the management of data in the form of test procedure parameters for liquid handling plate reading and histograms quality control data consisting of rea gent lot numbers and expiration dates with associated absorbances of these reagent lots over the period of their usage test results including blood groups and probable type manually added data from such tests as DU tests antibody screens hepatitis HTLV III CMV RPR and other tests and test status for the day of a range of parameters such as the number of A samples that have been processed the number of NTD s during the day and the status of testing for particular plates BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 is a block diagram of the interconnection of the various components of the system FIG 2 is perspective view of the liquid handler of the system FIG 3 is a flow diagram of the user
41. ces in the proper range and check the titer of antibody reagents to insure that the proper dilution levels are being used for good readings without excessive reagent consumption Testing Known Samples One of the ProGroup TM automated blood typing system s quality control functions enables you to assay known samples The user can thus ensure that the sys tem is functioning correctly in terms of test format that reagents are being placed in the proper rows of the microplate and that donor plasma and cells are being pipetted into the appropriate destination wells This QC procedure also indicates whether the liquid handler and plate reader are working properly and validates the stored thresholds used to interpret test results Further the reagents received from reagent manufacturers such as A1 A2 and B cells vary widely from lot to lot in their dilution strength Manufacturers dilute these reagents for sale but the dilution is not always consistent from lot to lot Further the cells have a shelf life and lose their potency as time goes by To get accurate results the strength and dilution of the reagents must be checked from time to time to insure that the absorbance values obtained from use of the reagents is normalized to some common denominator or reference point Another aspect of the quality control procedures the system is capable of performing is the facility to allow the user to compare known blood type samples to the results o
42. ch plate can hold sample from eight donors Reagents for the test including properly diluted cell suspension and antisera are placed in a row of wells in the reagent MicroTrof 62 along with a set of 12 tips This MicroTrof also contains a row of reagents and a row of tips for the antibody screening test to be de scribed later herein The reagent MicroTrof is placed at the back of the liquid handler bed 74 The predilution block 60 is placed just in front of the reagent Microtrof The block contains a trough of dilu ent and several wells where the donor cells are mixed with diluent ABO Rh Testing Procedure When the liquid handler is set up and ready for oper ation the first step is to carry out the automatic homing and priming procedure Then the user pushes the START key on the hand held controller 94 The plate stacker places a 96 well plate on the liquid handler table and the bar code 59 on that plate is read The bar codes 21 on the first group of eight tubes are also read Then the liquid handler picks up the first ProGroup TM automated blood typing system tip from the carou sel and withdraws the appropriate amount of donor plasma from the top of the tube Aliquots of plasma are placed one by one into the wells of the plate 56 that are designated for reverse typing tests Next the liquid han dler withdraws the required quantity of donor red blood cells for the forward typing tests Aliquots of cells are placed into the wells o
43. ck of how much data is stored on the fixed disk This is important because the fixed disk must al ways have enough space for the data that will be gener ated in the course of the day Transfers test results to the blood bank mainframe computer on a regular basis Archive other important information such as histo grams onto floppy disks for safekeeping Delete unneeded information from the fixed disk on a regular basis The ProGroup TM automated blood typing system software provides convenient functions for carrying out these tasks For information on how to handle the sys tem floppy disks refer to the DEC Micro PDP 11 Operation Manual In addition the Supervisor is re sponsible for developing and implementing a clear sys tem for backup programs and data onto floppy disks so that the contents and the generation of the disk are clearly identified It is claimed 1 An apparatus for optimizing the accuracy of light absorbance readings taken by a plate reader regardless of the type of plate and type of well in said plate com prising 4 873 633 47 means for causing the plate reader to take multiple light absorbance readings at different locations across the bottom of each well means for displaying the absorbance readings in graphical form to a user and for displaying the s current locations of at least one off center absor bance reading used to discriminate positive reac tions from negative reactions means for allowing
44. creen is reached if in response to a prompt from the host the user indicates the results of the donor cell suspension are not acceptable If the results are accepted block 177 is performed where the donor cell suspension data for that day is permanently recorded as part of the donor cell suspension absorbance data file as a permanent record The user can modify the reagent data table by select ing option 158 if the user has a supervisor status or higher If access is granted after the logon the step 160 can be performed to enter changes in the reagent table ProGroup TM automated blood typing system QC procedures allow you to verify that the dilution of donor and reagent cells are the same and that the ab sorbances of both fall into the appropriate range 1 2 to 1 6 A U A comparable function tests the dilution of the reagents used for the antibody screen testing Data Management Kinds of Information The third major function the ProGroup TM auto mated blood typing system performs is managing the data generated in the course of blood testing proce dures The host computer has software that enables it to manage four main types of information the data governing test procedures such as the pa rameters for liquid handling and plate reading the his tograms that serve as references for interpretation of test results etc quality control data which consists of lot numbers and expiration dates for all reagents with associated
45. ction will occur at some dilutions and not at others The plates are then all read and the absorbance data is plotted The absorbance of the off center readings of the plates will be small for dilutions having sufficient concentrations to react positively However for dilutions of insufficient concentration to cause the reaction the absorbance values for the off center reading will rise as shown at 20 25 35 40 45 50 55 60 65 14 165 The user will know that the optimal dilution to achieve good positive reactions is a dilution slightly more concentrated than the dilution at which the absor bance value starts to rise In the graph of FIG 14 the optimal dilution is 8 since that the most cost effective reagent dilution where a good positive reaction is achieved This number is hypothetical and for illustra tion purposes only FIG 15 shows a graph of the type of quality control graph which the system plots to check the quality of the reagent and donor cell suspensions The graph shows absorbance of the reagent cell resuspension for a nega tive backtyping reaction between the reagent cells of interest and bovine serum albumin 3 BSA on the vertical axis and the day or time of the test on the hori zontal axis Each point represents the absorbance of the suspension of reagents in a well where the reagent has been diluted to the proper dilution ratio in accordance with the above noted procedure and has been shaken to
46. d blood typing system or the system The ProGroup TM automated blood typing system automates many aspects of the blood typing and group ing procedures performed in blood banks This section provides a brief introduction to the functioning of the system ProGroup TM Automated Blood Typing System Functions The major ProGroup TM automated blood typing system functions are broadly defined below ProGroup TM automated blood typing system automates 5 0 5 20 N 5 30 40 45 55 60 65 6 much of the liquid handling involved in processing donor samples for ABO Rh tests and antibody screening sample and plate identification using a bar code reader plate reading for ABO Rh tests record keeping for quality control and test results for individual samples access to information to test status and printing of laboratory records and transmission of infor mation to a mainframe computer for on line storage ProGroup Automated Blood Typing System Benefits The ProGroup TM automated blood typing system is a flexible system designed to adapt to the way work is already organized in the user s laboratory The user defines the way he or she wants particular procedures carried out and ProGroup TM automated blood typing system executes these tasks at command Automating liquid handling makes test results more reliable and allows employment of lab workers having less skill Features like computeri
47. der software control The wash head is designed for use in operations like the ELISA wash used in HTLV III testing Manifold The manifold is used to wash and rapidly fill wells with diluent Pumps The system liquid handler includes two peristaltic pumps The pump 50 feeds the manifold for rapid plate filling and dilution Pump 64 beneath it feeds the wash head The pumps are primed simultaneously under soft ware control They dispense diluent into the predilution block that is positioned near the back of the liquid han dler table PREDILUTION BLOCK Predilution Block The predilution block is a container with a trough at the back The trough holds the diluent either saline or saline bromelin solution that is used to dilute the donor cells to the desired concentration The system uses rea gent cells in a dilution of about 1 3 The optimal dilution is established for a given lot of reagents then the donor blood cells are diluted to the corresponding ratio 4 873 633 25 Diluting Donor Cells At the front of the predilution block are 4 rows of 12 wells The manifold prefills these wells with 900 ul of diluent Later the X Y head aspirates donor cells from the tube and delivers the aliquot of cells to the appro priate well in the predilution block Since the X Y head uses a tip to pipette donor plasma first the tip is pre coated before it picks up the donor cells The result is that very few cells stick to the inside o
48. e 2 is displayed on the handheld controller of the liquid handler before starting this procedure This causes the liquid handler microprocessor to label the bar code of the plates being processed in this procedure with a reagent cell suspen sion tag This is recorded by the host in the look up table so that when the bar code from the same plate comes in from the plate reader the host can look up the bar code and will know that the absorbance data from this plate must be stored in the reagent cell suspension quality control file The data in this file is processed by the host in such a manner so as to be displayed in graph ical format as shown in FIG 15 Step 164 of FIG 5A represents the process of receiving the absorbance data at the host storing it and processing it for display as the QC chart The process of displaying the QC chart is symbolized by block 167 Step 164 also displays the means absorbance and the maximum and minimum ab sorbances observed from the wells read for each differ ent type of reagent Block 168 is an information screen which tells the user the procedures that should be fol lowed to bring the reagent cell suspensions back within accepted tolerance MICROPLATES Plate Types The ProGroup TM automated blood typing system enables you to work with 96 well microplates that are disposable or reusable They can have a U V or flat bottomed configuration And they can be Cetus micro plates or micro plate manufactur
49. e a measurement is taken 9 The optical density i e the absorbance information is then automatically transmitted to the host com puter 20 for analysis and storage 10 When the plate reading is complete after about a minute the shutter will open and the plate carrier will move back out to the starting position 11 Remove the plate by first lifting up the back edge slightly then disengaging the front end 12 When the plate reader is ready to read another plate a message will appear on its display 13 Repeat the above sequence 14 To view the test results select the Results Summary function 190 on FIG 3 through the host computer 20 System Configuration Certain functions of the system can be performed only by the system Supervisor or the system Director 65 These can be performed by selecting option 300 on FIG 3 which vectors processing to the process shown on FIG 11 For example if the format of the test for 10 20 30 35 40 34 ABO Rh is to be changed in that a different pattern of reagents or a different set of reagents is to be used the system Supervisor can make that modification by select ing option 304 This allows the process performed by the liquid handler to be modified using step 306 If the handshake or protocol for information exchange with the mainframe needs to be modified such as when a new mainframe is connected to the system the protocol may be changed by selecting option 308 Also
50. e reader even if the plate contains NTDs or Rh negatives that have yet to be tested for DU The second screen in this report consists of a table of data on the plate identified by the user with the WBN for each row of samples together with the blood group and type for that WBN and the test status displayed The test status is displayed as R for resolved NTD for no type deter mined or H for Rh negative for which a DU result is not yet available This report may be used to determine the test status of a plate if for example the user cannot remember whether a particular plate has been read or not The second screen of the plate testing status report is symbolized by block 217 in FIG 8 A directory of antibody screen plates can be viewed by selecting op tion 219 which shows the WBN antibody screen results for the wells in the plate identified to the system in step 221 This report consists of a table of WBNs for all samples processed on a given date with the results of testing for the antibody screens and for any miscella neous immunoassay tests performed on that WBN which have been done manually and entered into the system manually A display of all ABO Rh testing totals can be viewed by selecting option 223 This report summarizes the number of units of each group and type of blood that were processed on a given date and provides the total number of resolved samples the number of NTD s and a grand total of samples processed Any tubes th
51. e reverse typing and control histograms the X axis shows the actual absorbance reading On the for 4 873 633 35 ward typing histograms the X axis shows the absor bance ratio between the test well and the cell suspen sion FIG 12 shows a forward typing histogram The first histogram in the series is the donor cell suspension histogram It shows one group of readings which are clustered together in some are of the graph The plasma blank histogram is similar in this respect in that it shows only a single distribution of absorbance values These two histograms define the range of ac ceptable values for the concentration of cells in the donor sample and the optical properties of the donor plasma It is necessary to define limits for these ranges to ensure some consistency between donor samples When the cell suspension absorbance reading for a sam ple falls outside the acceptable range all readings for forward typing tests for that sample are designated as questionable results This prevents a very high or very low absorbance reading due to an excess or deficiency of cells in the wells from being confused with a genuine positive or negative test result The plasma blank per forms the same function It screens out samples where the plasma bland well is very clear indicating perhaps that little or no sample was delivered to the well or very cloudy plasma indicating perhaps that the sample is excessively lipemic When the plasma blank ab
52. e said means for causing multiple absorbance readings to be taken to take said readings 3 An apparatus in an automated blood assay system including a plate reader to allow a user to control the position at which the plate reader takes its off well bottom center light absorbance readings comprising means to measure light absorbance along a plurality of paths through the well bottom means to graphically display each absorbance versus the position relative to the well bottom center of the path to which each absorbance corresponds and to display the current path being used for the off center light absorbance reading used in the blood assay and means for allowing the user to redefine the desired path for the off center light absorbance reading by graphically moving a pointer to the desired path position 4 An apparatus for performing automated blood grouping on donor samples comprising a computer directed liquid handler means to perform predetermined automated sequences of operations involving placing aliquots of donor red blood cells and plasma into a plurality of wells in a plurality of assay trays and placing predetermined reagents in predetermined ones of said assay wells a computer directed plate reader to measure light absorbance data for the wells in said trays and a host computer for sending commands to control the operations of said plate reader and said liquid han dler and for collecting analyzing and storing the lig
53. e selected out of a list of alternatives if the user is designating a task The user moves the cursor around the screen using the arrow keys at the right side of the terminal keyboard If the user chooses a task from a list one simply presses the return key when the cursor is in the right spot If the user is entering or modifying data the user types the character or digit then press the Return key when the entry is complete Pressing the Return key tells the computer to act on the input Working with the ProGroup TM automated blood typing system host computer 20 is simply a matter of repeating these two kinds of operations first to program the system to perform the blood grouping and typing procedures in a particular fashion and then to run these tests and quality assurance procedures on a daily basis Controlling ProGroup Activities Multi Tasking Software The host computer 20 controls the ProGroup TM automated blood typing system liquid handler plate stacker bar code reader plate reader orbital shaker terminal and printer The user gives directions to the computer and it in turn issues instructions to all these devices Because the ProGroup TM automated blood typing system has multi tasking software the computer can control a whole range of activities simultaneously The ProGroup TM automated blood typing system can concurrently perform the liquid handling on six plates of sample including automatic insertion and removal o
54. ed CROSS REFERENCE TO MICROFICHE APPENDIX There is included herewith microfiche appendices consisting of 10 microfiche and 461 frames including the hex format object code controlling the various pro cessors in the system BACKGROUND OF THE INVENTION The invention pertains to the field of systems for automatically performing blood typing operations and more particularly to systems for controlling automated liquid handling apparatus to do blood typing and for interpreting the results from a plate reader and for man aging the data generated by the liquid handling appara tus and the plate reader apparatus and for printing vari ous reports Modern blood banks must perform thousands of blood typing and antibody screening operations and manage the data resulting from such tests These opera tions involve the handling of thousands of samples of donor blood the pipetting of reagents into samples of the plasma and red blood cells from the donor blood and the optical reading of the wells containing the donor samples and the reagents to determining the pat tern of positive and negative responses to various rea gents The pattern of positive and negative responses constitutes a template which characterize the blood as being from a particular blood group and having a par ticular Rh factor Each test of each donor s blood in volves pipetting of multiple samples of that donor s plasmas into multiple wells and pipetting of multiple s
55. ed by another vendor For consistent results however plates of a single type from a single source must be used If Cetus microplates are being used the system auto matically calculates the volume of the wells If micro plates from another manufacturer are being used a special procedure must be carried out that allows the system to calculate the weli height This procedure is explained in the Cetus Pro Pette Manual which is in corporated by reference Plate Usage The system liquid handler processes two plates at a time The liquid handling for ABO Rh testing is per formed on the plate at the front of the liquid handler table and the liquid handling for antibody screen test ing is carried out on a plate in the middle of the table If the ProGroup TM automated blood typing system is being used to perform the liquid handling for Hepati tis and HTLV III testing the handler works on pairs of plates The microplates and the reagent and predilution blocks are positioned within a set of rails or positioning guides on the liquid handler table The user only changes the positions of these guides when there is a 4 873 633 27 need to check the titer of the reagents used for antibody screening Plate Stacker The plate stacker consists of two storage hoppers that hold a total of six ABO Rh plates The plate stacker mechanism removes a completed assay plate from the table and loads it into the bay of finished plates Then a fresh
56. ent then pipettes them into the appropriate wells of the microplate 56 Then the ProGroup TM automated blood typing system fills the plate with the reagents from the reagent plate 62 for the assay Options Referring to FIG 3 there is shown the main menu and logon process Referring to FIG 4 there is shown the liquid handling process flow chart for the organiza tion of the software if the liquid handling parameter definition menu selection 100 is selected in FIG 3 FIG 4 would be reached after the user identified himself in the logon step 102 and was accepted as a permissible user Processing would then vector to the main menu screen 104 wherein all the options shown in FIG 3 would be presented to the user If he selected option 100 processing would be vectored to the liquid han dling menu 106 after going through a logon step not shown The liquid handling menu selection allows the 4 873 633 9 user to specify the process parameters of the liquid handling assay for blood typing NTD determination and repeat liquid handling with or without bromelin pre treatment Since these process parameters are criti cal to accuracy of results only supervisors or higher are allowed to log into this function of the system The user specifies the following aspects of liquid handling for the ABO Rh test whether or not a Bromelin pre treatment is being used choice 108 or 110 which blood typing test should be executed the op tions are A B
57. erring multiple reagents to one or more rows of wells a wash head 44 hereafter the wash head for proce dures like ELISA wash and a manifold 46 hereafter the manifold for rapid plate washing and filling The liquid handler includes a two peristaltic pumps 50 and 64 one feeding the wash head and the other feeding the manifold The ProGroup TM automated blood typing system liquid handler makes transfers among several contain ers a block 62 that holds multiple reagents and dispos able tips a block 60 with diluent and 48 wells for mix ing 96 well microplates 56 and 58 and a carousel 20 of 13x100 mm evacuated blood containers The blocks and microplates are placed in a template on the liquid handler tabie 84 hereafter the table This movable table positions the appropriate row of the container beneath the head that is operating in that procedure A plate stacker not shown delivers ABO Rh plates onto 4 873 633 23 the table for processing then back into the rack on completion of the liquid handling The carousel has a capacity of 48 tubes and dispos able tips The carousel rotates past a bar code reader not shown and within range of the liquid head that makes transfer from tubes to plates Another bar code reader scans the bar code on the microplates The user uses a hand held controller to enter certain values and commands and to view some information LIQUID HANDLING UNIT OVERVIEW LIQUID DELIVERY OPTIONS Fusion
58. ers provide The user establishes the optimal dilution ratio using the pro cedure of option 140 on FIG 3 This dilution ratio the lot number the expiration date and the length of time a given lot of reagents has been used in the system are all included in a screen display that can be viewed on the host computer 20 or printed for inclusion in laboratory records Daily quality control procedures indicate whether each of the reagents is continuing to produce absor bance readings in the acceptable range This informa tion is provided in both tabular and graphic form in the form of a Lange Levy graph displaying daily absorb ances for the reagents plotted with the absorbance on the Y axis and the day on the X axis for easy interpreta 5 20 25 35 45 50 60 65 26 tion This feature is embodied in the procedure of steps 202 204 and 206 on FIG 5A Step 202 represents the information screen given the user on the procedure used for non bromelin pretreated reagent cell suspension quality control procedures Step 204 gives the procedure for quality control reagent cell suspension checks telling the user to place 3 BSA solution in the carousel test tubes instead of actual sam ples from donors to guarantee an emulated negative reaction Step 206 tells the user the procedure for rea gent handling for the reagent cell suspension quality control procedure One of the things the user is in structed to do is to make sure the fil
59. f in response to a prompt from the host the user indicates the results of the donor cell suspension are not accept able If the results are accepted block 177 is performed where the donor cell suspension data for that day is permanently recorded as part of the donor cell suspen sion absorbance data file as a permanent record The user can modify the reagent data table by select ing option 158 if the user has a supervisor status or higher If access is granted after the logon the step 160 can be performed to enter changes in the reagent table ProGroup TM automated blood typing system QC procedures allow you to verify that the dilution of donor and reagent cells are the same and that the ab sorbances of both fall into the appropriate range 1 2 to 1 6 A U A comparable function tests the dilution of the reagents used for the antibody screen testing Data Management Kinds of Information The third major function the ProGroup TM auto mated blood typing system performs is managing the data generated in the course of blood testing proce dures 4 873 633 17 The host computer has software that enables it to manage four main types of information the data governing test procedures such as the pa rameters for liquid handling and plate reading the his tograms that serve as process of instructing the user as to changes that can be made to bring the absorbance of the donor cell suspensions back into tolerance This information s
60. f ABO plates and bar code reading on both ABO and antibody screening plates read the optical absorbance results on a series of plates as they are inserted and remove them from the plate reader print a designated document such as a list of NTDs for the day s testing and allow the user to interact with the ProGroup TM automated blood typing system programs or the data stored in the system for example to edit the test results for a WBN or to check the status of testing on a given carousel of samples Once the user has directed the host computer 20 to start a particular task such as liquid handling on a stack of plates the computer will carry that operation through to completion With that task under way the user can use the terminal to select and initiate another function such as printing a given document In this way the user can use the computer to execute multiple tasks in the background while the user interacts with the terminal in the foreground 15 20 25 45 50 55 65 42 Selecting A Function The various ProGroup TM automated blood typing system functions are grouped into lists of options These lists are known as menus The menus are organized into the hierarchy shown on the next page from the most general choices on the first or main menu of FIG 3 to the more specific choices on the subordinate menus of FIGS 4 11 When the user selects a menu option by moving the cursor to tha
61. f the predilution block 60 which already contain the required amount of dilu ent This diluent had been placed in the wells by the microprocessor of the liquid handler previously in ac cordance with data received from the host regarding how much dilution the supervisor wants in the test procedure for red blood cells The diluent and cells are mixed according to data regarding how many mixes that are desired which was specified by the user and sent from the host to the liquid handler and then the mixture is pipetted into the forward testing wells of the microplate The used ProGroup TM automated blood typing system tip is then ejected into a used tip con tainer not shown When the microplate 56 is filled with donor samples the 12 channel head picks up reagents from the reagent Micro trof 62 and fills the row of the plate 56 containing the first donor s samples with the pattern of reagents specified by the user in the liquid handling procedure set up step previously described the user must fill the reagent trof 62 wells with the proper reagents in accor dance with the desired pattern The row of Pro Pette tips used for this operation is then ejected back into the Microtrof At this point the liquid handling on the plate is fin ished The plate is loaded back into the plate stacker to await completion of the remaining five plates in that group When all six are done they are moved to the orbital shaker for resuspension Next they are
62. f the tip a prob lem that often occurs with manual dilution of blood cells The cells are mixed with diluent by sucking in and blowing out the tip repeatedly as the tip is raised This procedure mixes the fluids thoroughly with a minimum of cell damage The diluted ceils are then pipetted into the forward testing wells of the ABO Rh plate REAGENT BLOCK Reagent Block Functions The reagent block is a container with two rows of wells that hold various reagents and two rows of short disposable tips The back row of tips and the back row of wells are used to fill the antibody screen plate with one two or three types of reagents The front row of wells and tips are used to deliver up to 10 different reagents to the ABO Rh testing plate The reagent block is refilled after every two carousels of samples have been processed The block is washed and the tips are discarded at the end of every day If the user is performing the full range of ABO Rh tests the reagent block contains 2 5 ml each of the fol lowing immunoglobulin solutions for forward tests Anti A Anti B Anti A B Anti Rh Bovine Serum Albumin Cell suspension solutions for reverse tests Al A2 B Saline solution Using Reagents with ProGroup TM Automated Blood Typing System The cell suspension reagents and antisera and the antibody screen reagents are most effective in the Pro GroupT automated blood typing system at lower concentrations than the manufactur
63. from known samples or checking the resulting number of NTD s that result from processing real donor sam ples If comparison to known data is selected step 518 allows the user to type in the known data regarding the WBN and each blood group and type for each WBN Once this is done the discrepancies between the known data and the test results obtained using the temporary thresholds being evaluated are displayed in step 520 Blood typing requires the test results from all the differ ent reagents to match certain known templates Thus step 520 represents the process of comparing the known template for the type of blood the system has been told by the user it is processing against the experimentally determined template derived using the histograms and thresholds established in step 514 The WBN of each manually entered known sample will be displayed and its known blood type will be shown next to it in step 520 The positive or negative reaction to each reagent will then be displayed in the row next to each WBN as determined from the new thresholds established in step 514 so that the user can compare the template for each sample which is experimentally determined using the temporary thresholds being evaluated against the known template for that type of blood In this manner the thresholds can be adjusted to their proper values and the new thresholds set in the system via steps 522 524 and 532 These steps overwrite the old threshold values with
64. g processed for this procedure with a data tag that is stored with the bar code in a look up table The plates with the reagents and donor samples are then removed by the operator and put on the plate reader and the absorbance readings of the various wells containing the various reagents are taken These steps are symbolized by the block 153 in FIG 5A The plate reader also reads the bar codes from the plates it reads and sends them to the host computer 20 The host looks up the bar code data and finds that the absorbance data is intended for a quality control chart The data is then stored in a file that stores this type of quality control data and it is processed for graphical display FIG 16 is an example of the type of quality control graphic display which will be displayed by the host computer Block 153 represents the process of storing the absorbance data received from the plate reader and processing it into the graphi cal display shown in FIG 16 Block 152 represents the process of actually displaying the data in the graphical format shown in FIG 16 As seen in FIG 16 the known blood type and group for the WBN s tested as entered by the user is displayed in one column Another column displays the experimentally determined blood types and the experimentally determined blood typing templates i e the pattern of positive and negative reac tions to the various reagents used in the forward and reverse typing process The user can compa
65. g the bar codes on each plate These absorbance readings for each well on each plate are sent to the host with the bar code of the plate for interpretation The host knows which wells of each plate contain samples from which donors having obtained this infor mation by downloading the bar code data read by the liquid handler from the donor test tubes and the plates that received samples from each tube The absorbance values are interpreted by plotting histograms for the number of samples having each absorbance for each different type of reagent The user is then allowed to define the threshold values between the positive and negative reactions for each reagent by viewing the histogram and graphically moving an arrow to the de sired threshold value A no man s land region where the absorbance does not clearly indicate either a posi tive or a negative value is also defined and samples having absorbance values falling in this region are tagged as NTD The user defined thresholds for each type of reagent define templates or patterns of posi tive and negative reactions which will define each par ticular blood type The absorbance values for each sample are then compared to these templates and typed as to their blood group type The host is also coupled to a printer and the user can request various pre formatted management reports The 10 20 25 30 35 40 45 55 65 4 host is also coupled to an orbital shaker and a
66. hard disk and a VT220 terminal The host system 20 runs a commercially available operating system The control software is resident in the host It is coupled to a custom designed liquid handling system which has several mi croprocessors resident therein which control various stepper motors bar code read heads and monitor vari ous sensors The process controlling parameters to con trol operations of the liquid handler come from the host computer after being defined by the user at the host terminal Once the process parameters are loaded the resident software in the liquid handler controls various transfers of donor blood plasma and red blood cells to various wells in plates loaded by an automated plate reader The resident software also controls dilution of the red blood cells before depositing them in wells and the transfer of specified quantities of various types of reagents into the wells containing the donor s blood plasma and diluted red blood cells Each plate can hold samples from eight donors and the donor samples are stored in test tubes in a circular rotating lazy susan like device which is controlled by the software of the liquid handler The plates so filled are then unloaded by a plate han dler device and manually transferred to a plate reader Model Autoreader EL309 manufactured by Biotech Instruments which is also coupled to the host The plate reader then obtains absorbance readings on the wells in the plates after readin
67. he X Y head then returns to aspirate more donor plasma and places it in the reverse testing wells of the ABO Rh plate 18 Next the X Y head transfers donor cells into the appropriate wells of the predilution block The cells and diluent are mixed then the resulting cell suspen sion is pipetted into the forward testing wells 19 The X Y head then ejects the used tips into the waste receptacle 20 Next the X Y head picks up the next fresh tip and pipettes plasma and celis from the next tube repeat ing the above sequence 21 This process is repeated until the first group of eight samples has been pipetted into the microplate 22 The liquid handler then uses the 12 channel head to pick up the appropriate number of short tips from the front row of the reagent block 23 The 12 channel head then aspirates aliquots of rea gent from the front row of wells on the reagent block and fills the ABO Rh plate with these reagents 24 The 12 channel head then returns the tips to the back row of the reagent block 25 If all the wells on the antibody screen plate are now full the 12 channel head also picks up the back row of tips from the reagent block gt 4 20 25 35 45 55 60 65 30 26 The 12 channel head then aspirates reagents from the back row of the reagent block and fills the entire antibody screen plate 27 The tips in the 12 channel head are then replaced in the reagent block 28 The plate stac
68. hreshold values or modifying stored threshold values or setting liquid handling procedures for determining plate reading posi tions or modifying plate reading positions The option to set proper thresholds in histograms to distinguish between positive and negative reactions requires that the user select option 506 in FIG 9A The user is then instructed at steps 508 or 510 of the liquid handling procedure to use for sample handling for this process Basically the user is instructed to fill 48 tubes having bar codes with donor samples of known group and type in a distribution of approximately 30 A 30 O 25 B and 15 AB Two of the samples should be limpemic and two should be slightly hemolysed The user is then instructed to use file 12 for control of the liquid handler This file automatically prepares the 10 15 20 25 30 35 40 45 55 60 65 36 donor cell suspension and the plasma blank Step 508 gives the instructions for tests that do not use bromelin pre treatment while step 510 gives instructions for bromelin treated samples The liquid handling instruc tions for the reagents necessary to perform this process are then given in step 512 and information on how to set up the liquid handler is given Then 6 12 plates are processed in the instructed manner and are available to be read by the plate reader The user then physically moves the plates to the plate reader where they are read using the plate reading
69. ht absorbance data from said plate reader to determine the blood group and type of each do 10 15 25 45 50 60 65 48 nor s blood said host computer also having means therein for allowing the user to define graphically the optimum position for shining light through the bottom of one of said assay wells to measure light absorbance so as to maximize the accuracy of the host computer in determining blood group and type including means to cause said plate reader to measure the light absorbance along a plurality of different paths through the bottom of a well con taining a positive reaction agglutination button and to send said data to said host computer and includ ing means to display the light absorbance along said plurality of different light paths through said well as a graph of light absorbance versus steps off center of well bottom center for each said path and including means for graphically displaying the current position for at least one off well bottom center light absorbance reading used for measuring light absorbance in automated blood assay and for allowing the user to graphically alter the position at which this off well bottom center light absor bance reading is taken by moving the marker on the light absorbance graph 5 The apparatus of claim 4 further comprising means in said liquid handler to read the bar codes of test tubes containing donor samples and the bar codes of trays into which the donor samp
70. i able test results The control software for the host follows a similar process for donor cell suspensions The procedures for doing the donor cell suspension are specified in informa tion screens symbolized by blocks 169 171 and 173 on FIG 5A for bromelin pretreatment and no bromelin pretreatment Step 175 represents the process of down loading of liquid handling instructions for the database of the specific file the user has been instructed to select on the liquid handler handheld controller to do the liquid handling for the donor cell suspension Step 175 also represents the liquid handling by the liquid handler 26 and the process of receiving data from the plate reader when the plates having the donor cell suspen sions are read Block 175 also represents the process of storing the received data in the donor cell suspension file and processing the data for display as a graph like that of FIG 15 Block 166 on FIG 5B represents the process of dis playing the results of the donor cell suspension quality control procedures numerically on the terminal of the host If the user wishes to review the donor cell suspen sion data graphically in the format of FIG 15 the host performs step 175 to display the results graphically on the terminal Block 170 represents the process of in structing the user as to changes that can be made to bring the absorbance of the donor cell suspensions back into tolerance This information screen is reached i
71. ifying oneself to the host computer 20 typing in the users initials and a designated password The user s initials are then printed automatically on the re cord of samples that are processed while the user logged onto the system Manual Data Entry Whenever information is entered manually or is ed ited through keyboard input or whenever a sample is retested the laboratory report forms indicate these procedures in a special column on the printed page This arrangement increases accountability for work done with the system Double Keying of Data The system was designated to accommodate the dou ble key procedure some facilities use to increase the accuracy of manual entry of crucial information First 20 25 30 45 55 60 65 22 the user types in the values then the screen goes blank and the user types in the values a second time If the two entries match the system accepts this as valid input If there s a discrepancy the user is notified immediately so that the user can correct the error Levels of Access When the user logs on to the system the password the user types in identifies the system functions the user is authorized to work with The system has three access levels Level 1 is the most restrictive level 2 is less restrictive and level 3 allows access to all system func tions The Technician who runs the tests on a daily basis has access to level 1 The system Supervisor has access to leve
72. ing system hard disk instead of on paper The data file for each sample is identified by a Whole Blood Number WBN which is derived from the bar code on the sample tube The carousel number and the position 1 48 pinpoint the location of the tube The bar code on the plate and the number of the row specify the location of the test 4 873 633 19 wells for that sample This information makes it easy to locate the wells and the tube should the user need to Categories of Test Results The results of the ABO Rh tests are entered automat ically in the appropriate data file These results can fall into two broad categories Resolved and NTD Resolved Results If the blood group is positively identified the sample is considered to be resolved When the Rh factor is positive the ABO Rh testing for that sample is complete If the Rh factor is negative the sample is added to a DU list which is a list of WBN s where the presence of the D antigen on the red blood cells is unknown indicating a possible Rh negative blood type If the results for the Rh factor fall into a no man s land between the clearcut positive and nega tive results the sample is treated as if it were Rh nega tive The WBN of this sample is added to the DU list so that further testing can determine the status of the Rh factor NTD Results If the blood group cannot be identified the sample is categorized as an NTD The ProGroup TM automated blood typing system a
73. ion Status And Error Messages A single line at the bottom of the screen is reserved for error messages and for information about the host computer 20 s activities For example if the user selects the function results summary 190 in FIG 3 and display of WBN test results 202 in FIG 8 the headings for the table appear on the screen first then the menu line is displayed Then there is a short pause and the status line provides the message Reading In Data A few sec onds later the stored data is incorporated into the screen image and the status message disappears Error messages are presented in English and the error message disappears a short time later 4 873 633 45 System Security ProGroup TM automated blood typing system has a security arrangement that prevents unauthorized access to system functions When the ProGroup TM auto mated blood typing system system is installed in a blood bank laboratory one individual assumes the responsibil ity of acting as its Director This person creates a table of ProGroup TM automated blood typing system users identifying each by means of their initials and a defined password and assigning each a security level of 1 2 or 3 Logging On The user logs onto the system by entering the infor mation request The user then types in his password but it is not displayed on the terminal screen to maintain its confidentiality The system matches the initials and password the user give
74. ions either manually or automatically The final results are entered into the system data base 15 using the host computer 20 keyboard and display The user can print the test results using a function on the host computer 20 The user obtains a hard copy from a printer attached to the host computer 20 and the format for the report is predefined through the host computer 20 OPERATING SEQUENCE Using the Plate Reader The user should read a plate no fewer than three and no more than ten minutes after the final resuspension for best results 1 When the liquid handling on a plate is complete select the plate reader function from the ProGroup TM automated blood typing system host computer 20 2 Position the plate so the bar code is facing the plate reader keyboard 3 Load the plate into the motorized carrier by tipping the front of the plate down slightly 4 When the front of the plate is firmly seated push the back edge straight down to engage the spring 5 Press the START key on the plate reader keyboard 6 The motorized carrier will move the plate into the instrument and the shutter will close behind the plate The plate reader displays the bar code that identifies it 7 The first information obtained from the plate is the bar code that identifies it 8 If you listen closely you can then hear the sound of 45 the plate being positioned for each of the 16 series of readings and the clicking of small relays each tim
75. ker then removes the finished ABO Rh plate from the table and loads it into the full plate storage hopper 29 A fresh ABO Rh plate is rolled out stacker and onto the table 30 If the antibody screen plate is complete an audible beep and a message on the controller display prompt you to remove the plate manually and put in a fresh plate 31 At this point the system is ready to begin the liquid handling on the next group of eight samples USING THE PROGROUP PLATE READER Overview of the plate This section describes the major features of the sys tem plate reader 28 and how it operates in context of the system Plate Reader Functions The system plate reader measures the optical density by way of absorbance of solutions in the wells of 96 well microplates In the context of the system the plate reader makes two off center readings for each well using a single wavelength Plate Reader Technology A tungsten halogen lamp provides light that is col lected and passed through a narrowband filter with a wavelength of 410 nm The filtered light beam illumi nates the large end of a fiber optic bundle Light from the 12 fiber ends passes simultaneously through the bottom of all 12 wells in a row and exits from the top of the wells Then it falls onto the surface of 12 photo detectors The photo detectors provide an electrical signal that is directly proportional to the amplitude of the light This linear electrical sign
76. l 2 and the system Director has level 3 privileges Individuals at all access levels can display and print daily test results Only those with level 2 or 3 access can manually change ABO Rh test results Only those with level 3 privileges can change the threshold values that determine how data points from the plate reader will be converted into blood grouping results USING THE PROGROUP LIQUID HANDLER Overview The ProGroup TM automated blood typing system System provides a wide range of liquid handling capa bilities This system was designed as a dedicated instru ment for blood typing and grouping If the user chooses however he can also use the ProGroup TM automated blood typing system s liquid handler for other analytical procedures ELISA wash plate to plate transfers plate empty fill etc These other capa bilities are described in the ProPette Manual that is incorporated herein by reference This section describes the most features of the liquid handler 26 hereafter the liquid handler and explains how these are used in ABO Rh testing and antibody screening Overview of ProGroup Liquid Handler Referring again to FIG 2 the ProGroup TM auto mated blood typing system liquid handler has four heads that perform specialized kinds of liquid transfer an X Y head 28 hereafter the X Y head for transfers from tubes to individual wells in a microplate a 12 channel head 42 hereafter the 12 channel head for transf
77. les are deposited and for sending this data to the host computer for storage in a look up table and further comprising means for appending either a steps off center code or a histogram code to bar codes for trays whose light absorbance data is to be graphi cally displayed to the user means in said plate reader for detecting these histo gram or steps off center codes and for sending same to said host computer along with light absorbance data from each well in each tray along with data labelling the light absorbance data for each well from each tray with identifying information locat ing the well and tray from which the data came and means in said host computer for storing and analyzing the data from the plate reader to determine the blood group and type of each user including means for processing the data from each well in which the same type reagent was placed into histogram for mat and for graphically displaying same to the user along with the absorbance values currently set as the positive and negative reaction discrimination thresholds and for allowing the user to graphically adjust the positions and absorbance values for said thresholds 6 The apparatus of claim 5 further comprising means in said host computer for allowing the user to put a known blood group and type into the liquid handler and for displaying the known reaction template for that known blood group and type on a display and for using the new thresholds to experimen
78. lood typing system host com puter 20 is by typing in data for the system to act on Sometimes this is a fill in the blank operation At other times the user supplies information as part of a list or table In both cases the user follows the general sequence given below 1 Position the cursor at the spot on the screen where the user wants to place the first character the user will input 2 Type in the appropriate sequence of alpha numeric characters and spaces These will appear on screen as the user enters them The user can type upper or lower case characters as both are acceptable 3 In most cases the user presses Return after each indi vidual entry 4 873 633 43 4 The cursor then moves to the next position where data can be entered or modified and the user repeats the process The information the user gives the ProGroup TM automated blood typing system host computer 20 serves one of the following purposes providing new data modifying existing data or altering a default value Entering New Data In many situations the host computer 20 requires input from the user to carry out a task For example when the user wants to check the test results for a par ticular WBN the user must type in the digits that iden tify that sample The position for the entry is indicated by a colon after the heading or by a pair of brackets Modifying Data Alternatively the user may be revising data that is already stored in the
79. n properly diluted into the proper absorbance range and that the resuspension process is being done properly to maintain the quality of test results on donor blood samples using the reagent cell suspensions tested The donor cell suspension quality control process is similar and the system makes a similar graph to that shown in FIG 15 The donor cell suspension quality control checking procedure is important because the positive negative decision process involves a ratio of the forward typing test absorbances to the donor cell suspension In order for these ratios to be valid the donor cell suspension needs to be within the range of sensitivity and linearity of the plate reader It is impor tant that there be enough cells to give a large enough absorbance to give a reliable separation between the ratios of a positive and a negative reaction The absor bance of the donor cell suspension is a function of the volume of cells withdrawn from the sample tube the volume of diluent in the predilution block the effi ciency of the mixing during the cell dilution the force at which the donor sample tubes and the ABO Rh plates are centrifuged and the efficiency of resuspen 4 873 633 15 sion Thus the donor cell suspension is a sensitive indi cator of the quality of all these steps or how closely to being correctly done these steps are being performed The system keeps donor cell suspension records and quality control graphs which are identic
80. of the two off center readings to maximize the discrimination between posi tive and negative reactions then the host vectors pro cessing on line 546 to a step 548 where specific com mands are to the plate reader 28 These commands 4 873 633 39 order the plate reader to set the wavelength of the light being used for the absorbance readings and orders the plate reader 28 to take 36 absorbance readings across each well bottom at each of 36 different positions across the bottom of the well These absorbance readings are sent to the host from the plate reader The host then performs some calculations on the data to prepare it for display and then the data is graphically plotted with absorbance on one axis and angle of each reading on the other axis The plot will show high absorbance readings at angles that pass through the positive reaction button in the middle of the bottom of the well On either side of this button will be angles having low absorbance FIG 13A shows a typical well bottom with a positive reac tion button and FIG 13B shows a typical graph which results from this process reading process for a positive reaction button Note the high absorbance for the read ings taken where the beam passes through the center of the well bottom where the positive reaction agglutina tion button is located The absorbance readings are lower for readings on either side of the positive reaction button in the center of the well because the light
81. on programs have been created specif ically for use in blood banks The software is quite self explanatory System Manager Summary Computer Features The computer has a number of serial and parallel ports that allow it to interact with external devices in the system the terminal printer liquid handler plate reader and orbital shaker These devices are largely or entirely controlled through the computer and the data they generate is stored in the computer Terminal Features The terminal consists of a monochrome green dis play with two modes a standard matrix of 80 characters by 24 lines for conventional screens and a high density matrix for screens that contain very large amounts of data The detached keyboard includes a set of standard typewriter type keys plus special keys for cursor con trol the numeric key pad etc Most of the function keys at the top of the keyboard are not used in the ProGroup TM automated blood typing system applica tion System Manager Software The Micro PDP 11 host 20 runs the commercially available operating system called RSX11 that enables the ProGroup TM automated blood typing system host computer 20 to carry on several operations at the same time The operating system provides a set of standard operating system management processes to allocate the machines resources among the various tasks or applica tions running on the system It also serves as an interme diary between the computer
82. options for vec toring processing to various sub functions that the sys tem can perform FIG 4 is a flow diagram for steps performed in al lowing the user to customize the process parameters controlling liquid handling operations FIGS 5A and 5B are a flow diagram of the process of allowing the user to monitor and control various quality control functions FIG 6 is flow diagram for the process of allowing the user to create and access various reagent data for use in insuring proper processing and improving reliability of test results FIG 7 is a flow diagram of the plate reading function of the system and a flow diagram of the process fol lowed in allowing the user to edit or enter various re sults from testing FIG 8 is a flow diagram for the process followed in allowing the user to specify results summaries the user would like to see FIGS 9A and 9B are a flow diagram of the process that the system follows in allowing the user to calibrate the plate reader and to adjust the thresholds used by the host computer in interpreting test results FIG 10 is a flow diagram of the process used by the system in performing various file operations FIG 11 is a flow diagram of the process used by the system in allowing the user to configure the system for a specific installation FIG 12 is an illustrative histogram for a single rea gent illustrating the meaning of adjusting the thresholds used in interpreting test results FIG 1
83. ore than one carousel accompanies the system so you can prepare the next group of samples while the system is carrying out the liquid handling on the cur rent group When the user is ready to process a new carousel it is installed on two aligning posts in the motor drive mechanism for the carousel If a carousel contains fewer than 48 samples one en ters the actual number of tubes using the liquid handler controller This prompts the system to stop processing samples after finishing work with the last tube present BAR CODE READER Two Bar Code Readers The system liquid handler includes two identical bar code readers One at the left side of the instrument reads the bar code on each sample tube The other at the right side of the unit reads the bar code on the microplates Function of the Tube Bar Code The 7 digit bar code on the sample tube is the basis for the Whole Blood Number that identifies the sample The liquid handler reads the bar codes on a group of eight tubes pipettes the sample in these tubes then reads the codes on the next group of eight tubes The bar code together with the carousel number and the position in the carousel identify each tube This infor 10 20 25 30 40 45 35 60 65 28 mation enables the user to locate the tubes that require DU testing or need repeat liquid handling or other attention If the scanner cannot read the bar code on a particu lar tube the system does not car
84. ousel num ber and tube position for the tubes This report may be used to determine which tubes to pull for further DU testing 5 10 5 20 25 40 45 50 55 60 65 32 If he wishes to view the list of all WBN s of a speci fied group and type he selects option 211 This report allows the user to search for all samples processed on a given user defined date with a specified blood group and type The report lists the WBNs in numerical order and gives the carousel number and tube position of each tube This report may be used to identify which samples to pull to expedite testing of blood with a particular profile If he wishes to view the current test status he selects option 213 This report gives the user an overview of how far testing for a given user defined day has prog ressed The report provides a count of the number of completed plates and the plates yet to be read and provides a count of the NTDs and unverified Rh nega tive test results determined up until the time of the report This report may be used to assess test status at any given moment If he wishes to view the directory of plate informa tion he selects option 215 The first screen of this report lists all plates processed on a given user defined date and supplies the test status of whichever plate the user identifies as to whether the plate is tested or untested The status of the plate is tested after it has been read in the plat
85. passes to one side of the button and not much is absorbed by the clear fluid as the light travels to the photosensitive detector above the well At angles that pass through points on the bottom very near the wells high absorbance values will exist caused by the lens action of the walls and the bottom shape Two arrows 550 and 552 are displayed These arrows represent the current position of the two readings that are taken off center in addition to the readings taken in the center of the weil bottom These arrows can be graphically moved by the user through use of the arrow keys on the keyboard of the host 20 The positions of the arrows 550 and 552 define the step position or position off center at which the two off center readings are taken After the user has viewed the graphs for each of the positive anti Rh 1 A2 and B reactions and se lected the arrow positions for arrows 550 and 552 which present the best compromise to discriminate for positive reactions for each type of reaction the host then re cords the arrow positions and verifies the user s desire to use these arrow positions for future readings in the step symbolized by block 554 in FIG 9B The host computer will then instruct the plate reader to use these angles for absorbance readings for these types of wells for all subsequent readings of all wells in ABO plates if the user instructs the host computer to replace the cur rent angles with the newly selected angles These read
86. pecific wells The bar code information from the plates so loaded is then associated with the template of reagents for forward and reverse typing used for that plate This allows the absorbance data coming from specific wells 4 873 633 37 of a particular plate to be collected in the proper file for a histogram for each specific reagent The host does this sorting by looking up the template of reagents used i e which reagents were put in which wells for the plate having the bar code sent by the plate reader In other words the absorbance data from all the A wells is col lected and processed into histogram form as is all the data from the B wells the A B wells and the Rh wells In step 514 the user specifies which of these above noted histograms he wishes to view and the histogram is displayed graphically in a display like that of FIG 12 The user is then allowed to move the threshold arrows and the host stores the updated values for the positive and negative threshold values in a temporary file for each histogram The user is then afforded an opportu nity to review the results that will be obtained with the new threshold values to evaluate the new thresholds for quality control purposes In step 516 the user is allowed to choose the manner in which he will evaluate the effectiveness of the thresholds he will set His options are to evaluate the temporary threshold values by com paring experimentally determined data against data
87. re these experimentally determined templates to the known tem plate for each blood group and type Checking Dilution Ratios The ProGroup TM automated blood typing system carries out ABO Rh tests using reagents at a fraction of their standard concentrations Positive and negative test results on the ProGroup TM automated blood typing system are distinguished more or less sharply depend ing on the concentration of the reagent and donor cell suspensions This differentiation results because a sub stantial excess of antigens or antibodies causes the red blood cells to return into suspension In general the optimal dilution for these assays is around 1 3 FIG 14 shows a graph of the typical absorbance for various levels of dilution of an antibody reagent with a sample which is known to cause a positive reaction with that antibody reagent Antibody reagents are very expen sive and their concentrations from the manufacturer vary considerably These reagents are reacted with donor cells in forward typing to determine the type of blood the patient has in a process which is well under stood by those skilled in the art Thus at times it is necessary to dilute the antibody reagents to get uniform results and to not waste money The absorbance graph of FIG 14 is derived by taking a serial dilution of an antibody reagent and mixing the reagent with 3 bo vine serum albumin and reacting it with samples from known AB positive donors A positive rea
88. rein The user responds to prompts on the controller display by giving commands and entering values through the key board The Controller and The System Manager When using the system liquid handler for blood grouping and typing the user programs the system almost entirely through the host computer 20 as de scribed in this manual There are several operations such as pushing the START key when ready to begin the liquid handling on a new microplate when using the hand held controller The other blood testing situa tions when the controller is used are homing and priming the pump when a key on the controller is pressed to initiate the process checking the titer of reagents for the antibody screen tests when the Plate to Plate transfer capability is used to carry out a serial dilution using the controller fairly extensively establishing tip height when user is using non Cetus microplates and must establish the volume of the wells and The controller is an important adjunct to the host computer 20 but the latter programs and controls 99 of the operations involved in grouping and typing blood 4 873 633 29 LIQUID HANDLING FOR ABO RH AND ANTIBODY SCREENING Operating Procedures The liquid handling for blood typing and grouping consists of the following sequence of steps At the start of every other new carousel the user should do the following steps 1 Fill the reagent block with 2 5 ml volumes of diluted
89. rocess shown on FIG 10 The user can then select option 402 and format the floppy dis kette The user inserts a floppy disk in the drive when the user wants to use the information it contains and the user removes the floppy disk when the user is finished with its contents Step 404 in FIG 10 allows the user to transfer completed results onto the hard disk Option 406 allows the user to back up data on the hard disk to a floppy diskette Option 408 allows the user to reset data for a specified WBN or plate while option 410 allows the user to delete data by any of the criteria specified in options 411 414 Data statistics can be dis played by selecting option 416 The fixed disk offers much faster access to data as well as greater ease of use For this reason the fixed disk is used for primary storage of programs and data The floppy disks are primarily used to backup informa tion to facilitate recovery in the event of a problem with the hard disk and to archive data that is no longer actively needed on the hard disk Working With The ProGroup Automated Blood Typing System Disks The only ProGroup TM automated blood typing system user who is directly involved with the fixed disk and floppy disk drives is the system Supervisor This person has a range of important responsibilities for in formation storage which are summarized below The Supervisor performs the following ProGroup TM auto mated blood typing system functions Keeps tra
90. ructional screens are symbolized by the blocks 540 542 and 544 in FIG 9B and the menu option is symbolized by the block 504 on FIG 9A The plate is then processed using the ABO Rh testing pro cedure following the directions given on these screens The host CPU stores predetermined liquid handling routines for each different type of plate that will be used in the system These routines are stored as files When the user loads the liquid handler with a particular type of plate the user will signal this fact to the host CPU by ordering from the handheld controller that liquid han dling is to be done on that plate using the file designated for that type of plate The host CPU then downloads the liquid handling machine instructions to the liquid handler 26 The liquid handler then reads the bar code on the plate and tags that bar code with a steps off center tag This data is then sent to the host CPU and stored there in a look up table of plate bar codes When the plate is ready to be read the plate is put in the plate reader The plate reader reads the bar code of the plate again and this is sent the host CPU 20 hereaf ter called the host The host then looks up the plate s bar code from the stored bar codes in the look up table and determines if the plate is a steps off center plate If the plate is a steps off center plate and the user selects a menu option telling the host that the user wishes to adjust the plate reading positions
91. ry out liquid handling on that sample Instead it skips the tube and includes that carousel number and position on a list of excep tions The tube can them be moved to a new carousel to see if this solves the problem If the system is still unable to read the bar code the user can do the blood grouping and typing manually and enter the results into the sys tem through the system option 170 in FIG 3 Plate Bar Code The sample destination is identified in terms of plate number and row number This information enables one to locate a given row of wells if one needs to recheck the test results for any reason The bar codes on the ABO Rh plate and the anti body screen plate are read as part of the automatic Homing and Priming operation at the start of the liquid handling sequence The liquid handler table moves rap idly back and forth homing all the stepper motors in the unit and running the plates in front of the scanner LIQUID HANDLER CONTROLLER Controller Function The system liquid handler includes a hand held con troller with a LED display and a 19 character mem brane keyboard This controller is primarily used for functions like Plate Empty Fill and Well To Well transfer that are not an integral part of the blood testing process If the user wants the Liquid Handler to carry out any of the operations described in the Pro Pette manual he programs the instrument as described in that manual which is incorporated by reference he
92. s against the information stored in the user privilege table that is set up by the system director The system director sets up the user privilege table by selecting option 300 in FIG 3 which vectors processing to the process shown in FIG 11 Then the user selects step 302 and enters the data in the table of user ID s and system security information This matching of ID s to entries in the system user privilege table serves the following purposes Identifying Users The user responses to the logon screen establish whether or not the user is entitled to use the system If the user enters his initials or password incorrectly the screen will display an error message The system will allow retries in case of a typographical error The way the user logs on also defines to the Pro Group TM automated blood typing system which sys tem functions the user is authorized to use Certain functions such as calibrating the plate reader i e the process shown in FIG 9 are very critical to the Pro Group TM automated blood typing system s successful operation Consequently only the system Director has access to this function If the user has inadvertently selected a function that the user is not authorized to use the user will see an error message The user may then pick another func tion Accountability for Work The further purpose of the logon procedure is to make it possible to identify which user is responsible for the work the sy
93. s displayed on the host computer s termi nal 24 when the user starts processing samples Storing Data On The Fixed Disk All the above categories of data are stored on the fixed disk in the host computer The fixed disk provides extensive storage capability Working with the fixed disk is considerably more convenient than using floppy disks The user doesn t have to remove and insert multiple disks as one does with floppy disks and the user can store very large files without having to worry about exceeding the storage capability of the floppy disk 4 873 633 21 The host computer 20 includes two floppy disk drives but these are primarily used for backing up the information stored on the fixed disk Other Forms Of Information Storage The system has extensive but not unlimited storage capability Most blood banks process large quantities of information so data is only stored on the fixed disk as long as it is needed Some information remains valid over the long term For example parameters like the test format are proba bly changed rarely if ever This information is kept on the fixed disk on a permanent basis Other information must be updated at regular inter vals For example every time a reagent lot changes the plate reader must be recalibrated and a new histogram generated for that test The current version of this infor mation is always kept on the host computer s hereafter referred to as the host computer 20
94. s has progressed Working With ProGroup Automated Blood Typing System Data The information the user inputs and the data the system generates are displayed on the host computer 20 in lists or in graphical format For example the WBNs that require DU testing are displayed as a list by select ing option 190 on FIG 3 which vectors processing to FIG 8 where step 192 is performed The results of quality control tests for reagent cell suspension are graphed as a series of values for daily absorbance over the interval of that reagent lot The user enters new information or modifies existing data through the host computer keyboard by selecting option 170 in FIG 3 This is an interactive process If the user makes a mistake it s usually easy to correct And keyboard input is immediately stored on the fixed disk The user doesn t have to remember to tell the system to save data because it happen automatically Differentiating Between QC and Data The system can automatically distinguish between tests that the user runs on actual samples and the proce dures which are used to develop or modify quality control data The same kind of liquid handling is in volved whether the user is testing a new donor sample or checking the reagent cell suspension But the plate reading techniques and data management techniques are different The system is programmed to recognize which type of work the user is doing on the basis of which screen i
95. s to minimize cross contamination The ProGroup TM automated blood typing system accurately transfers volumes between a minimum of 10 ul and a maximum of 200 ul Two identical bar code readers not shown are built into the liquid handler one for reading the bar code 21 on the sample tube actually located above the top sur face of the carousel 20 and the other for reading the bar code 57 and 59 on the microplates actually located on the other sides of the plates The bar code on the sam ple tube is the basis for the Whole Blood Number WBN that identifies the donor sample The bar code on the microplate allows the system to pair the test results from the plate reader with the WBN of the sam 4 873 633 7 ple donor The bar code readers can recognize Code Bar or Code 39 labels Plate Stacker not shown holds six 96 well micro plates for ABO Rh testing Each plate can accommo date samples from eight donors These plates are fed into the liquid handler 26 one by one for processing Once filled a plate is automatically transferred off the bed 74 of the liquid handler into the other rack of the plate stacker i Plate Reader The Plate Reader 28 reads ABO Rh plates automati cally and transmits data points to the ProGroup TM automated blood typing system host computer 20 for interpretation The results are reported by group and type or as NTD No Type Determined readings An NTD sample can be reread automatically with the plate
96. sor bance reading for a sample falls outside the acceptable range all readings for reverse typing tests for that sam ple are designated as questionable results This prevents distorted absorbance readings from being interpreted as legitimate test reactions The histograms for the various reagents used in the forward and reverse typing tests have a different con figuration These histograms show two distributions of absorbances One group of low absorbances generated by the positive sample readings and another group of high absorbances generated by the negative sample readings These two clusters are separated by a buffer area An example of such a histogram is shown in FIG 12 One histogram is prepared by the host computer 20 for display on the terminal for each different reagent used in the forward and reverse typing The user may then calibrate the plate reader by setting the positions of the thresholds by graphically moving the threshold arrows on the histogram display This calibration process is performed by selecting option 500 on FIG 3 which vectors processing to the routine symbolized by FIG 9 The user then logs on the reader calibration function and the system checks his ID against the user privilege table to determine if this user may have access to this function If access is granted in step 502 the reader calibration menu is dis played in step 504 This menu gives the user the option of determination and setting of proper t
97. special tests may have to be done by hand on the blood which are 10 15 20 25 35 40 45 55 60 65 2 not subject to automated liquid handling Test results from these antibody screen tests and other tests done by hand must be recorded for each donor s blood for which these tests are done Further testing procedures for blood typing can change over time in that different dilution values are often needed for different batches of reagents to get the proper absorbance readings Fur ther the amount of dilution of the red blood cells should be optimized The data for the optimal dilution values must be recorded Further other process parameters of the blood typing sequence should be subject to customization to enable the various users to customize their testing procedures to conform to local practice This data defining the characteristics of each step such as the amount of each sample to be placed in each well which wells in which to place samples the amount of dilution in certain steps the wells to place reagents in the number of mixes at various points in the process and various other criteria should be subject to customization should be easily changeable and should be remembered by some mecha nism so that it can be automatically invoked each time a procedure is performed without having to look it up every time Of particular importance is the amount of dilution of each particular reagent which should be used
98. ssigns a NTD if the pattern of positive and negative reagent reaction results for a row of wells does not match any of the stored templates for known blood types Most commonly an NTD reflects contradictory results among the various tests A sample is also designated as an NTD if a test result falls into no man s land between the Thresholds for the positive and the negative results An NTD can also occur because the cell suspension or the plasma blank for that row of samples was out of the acceptable range Resolving The Sample The processing of a sample is not complete until the blood group and type have been established If a WBN is added to the DU list as a potential Rh negative the results of the DU test must be entered manually in the data file to complete the sample profile This is done by selecting option 170 on FIG 3 which vectors processing to FIG 7 and selecting option 172 there If the user logon ID indicates the user is autho rized to enter DU results then access to step 174 will be granted and the user will be allowed to enter the test results If the automatic test procedure produces an NTD the row can be reread automatically This can be speci fied by selection of option 176 in FIG 7 Or the entire liquid handling and plate reading procedure can be repeated automatically In either case the new results automatically replace the previous results Alterna tively the user can inspect the row of wells visually to
99. stem carries out during a given interval Only one person can be logged on to ProGroup TM automated blood typing system at a time If the user is logged onto the system for two hours in the morning for example the user s initials will be printed on the laboratory data records for samples tested during that period Likewise if the user is authorized to enter or edit data manually the user s initials will be printed on the labo ratory data record for any test results the user works with manually The ProGroup TM automated blood typing system only identifies the system user who worked with the data most recently however Conse quently if the user changes a given test result and another system user changes these same results again later their initials will replace the user s on the data record 20 25 30 35 45 50 55 60 65 46 Thus the logon procedure enhances the security of the blood bank operation by limiting access to the sys tem as a whole and to crucial functions in particular and by increasing the accountability for work per formed using the system Storing Information On The System Manager Storing Data On Disk The fixed disk can hold 10 Mbytes of data and it is permanently resident in the computer The floppy disks hold 360 Kbytes of data and these are interchangeable The user can format disks in preparation for storage on them by selecting option 400 on FIG 3 which vectors processing to the p
100. t point and pressing the Return key that indicator is highlighted for a moment then the appro priate screen appears on the display Often the next Screen is another menu showing the alternatives within that overall category Menu Arrangements Vertical Menus Higher level ProGroup TM automated blood typing system menus are vertical lists of up to nine options as shown below The only information that appears on these screens are the menu choices Horizontal Menus The lower level menus include several options listed horizontally across the bottom of the screen These menus are included on screens that present program data of various kinds Printing is a function that always appears on this kind of low level horizontal menu Selecting Menu Options Making Choices Both vertical and horizontal menus respond in the same basic fashion 1 use the right and left arrow keys to position the cur sor at the option the user is choosing The right arrow key moves the cursor to the right or down The left arrow key moves the cursor to the left or up The up and down arrow keys do not move the cursor when the user is working with menus When the cursor is in the right spot press the Return key 3 The indicator for that option will be highlighted momentarily then the next appropriate screen will appear on the display Entering And Editing Data Typing Input The other major way the user interacts with the Pro Group TM automated b
101. tally determine a reac tion template for reactions to all the conventional rea gents used in blood typing and for displaying the experi mentally determined reaction template on the same display with the known template for that blood group and type for comparison by the user 7 The apparatus of claim 6 further comprising means in said host computer for allowing the user to change the thresholds on the histograms until the number of no type determined donor samples or the matching be tween the known templates and the experimentally determined templates using the new thresholds is ac ceptable
102. ter 20 will only act on the final version on your input In some situations when the user is supplying a series of values the cursor automatically moves to the next data entry position after the user completes the current entry Here the user only presses Return after the addi tions or changes to the entire screen are complete If the user finds an error before pressing the Return key use the arrow keys to move the cursor back to the location that needs correction If the user doesn t notice a mistake until after the user has pressed the Return key the solution is to choose the Return to This Screen option on the horizontal menu at the bottom of the screen This option returns you to 10 15 20 25 30 35 40 45 50 55 60 65 44 the data portion of the screen so the user can make whatever changes are necessary If the program has already advanced to the next screen the user can usu ally select the option called Return to Previous Screen This achieves the same object placing the user again at the top of the screen where the correction is needed Moving the Cursor Screen Types The way the user moves the cursor around the screen depends on the type of screen the user is working with The host computer 20 uses field screen and scrolled screens Field Screens If a single screen can present all the pertinent infor mation in a single display the screen is called a field Here the user moves
103. the cursor around using just the right and left arrow keys as one would do within a menu The right arrow moves the cursor to the right or down and the left arrow moves the cursor to the left or up Scrolled Screens Other screens include more data than can be pres ented in a single display These screens are called scrolled screens and they are identified by a bold barid at the top and bottom of the data area An example is shown below Here the complete screen contains a predetermined number of lines of data and the display can only present a few lines at a time On scrolled Screens the user uses the up and down arrow keys to move the cursor from one line to the next and to essen tially pull the data up or push it down so that a different portion will be visible in the display Here again the right and left arrows move the cursor horizontally but for vertical movements the user uses the up and down arrows Composite Screens Many screens that solicit or present data include a horizontal menu of options These screens are essen tially a composite of a field or a scrolled area and a menu line If as shown above the screen contains a field area the cursor will automatically move to the first menu selection after the user supplies any data that is re quired If the screen contains a scrolled area as shown be low the user must press Return to move the cursor from the data area to the menu area Other On Screen Informat
104. the format for lab reports may be set up by selecting option 310 and the error handling method used by the system may be modified by selecting option 312 The liquid handling procedure for immunoassays may be modified by select ing option 314 Reader Calibration Reader calibration is a task which is important to the successful operation of the system Blood group and type assays involve reacting donor red blood cells with antisera or antibody reagents forward typing and re acting reagent cells of known type with donor plasma reverse typing These reactions produce templates of positive and negative reactions to each of the various multiple reagents used on the blood of each donor The template is comprised of a string of positive and nega tive reactions that define the blood group and type according to which reagents elicited positive reactions and which elicited negative reactions The positive reactions are characterized by agglutination buttons in the bottom center of the test wells The plat reader shines a light beam through the bottom of the well at an off center position that should clear the button and pass through the liquid with less absorbance if a positive reaction occurred than if a negative reaction occurred The light absorbance readings from each well are then interpreted by the host computer 20 according to two thresholds One threshold is the positive threshold which defines a light absorbance value that is the upper
105. the new threshold values If the NTD approach is selected for quality control of the newly established thresholds the step 526 displays the number of NTD s experimentally determined using the new thresholds An NTD is determined by taking into account the positive and negative results to all the reagents using the new thresholds established in step 514 If the number of NTD s is acceptable step 522 is performed to allow the user to update the old thresh olds to the new thresholds established in step 514 If the number of NTD s is not acceptable in either step 526 or step 520 a step 528 is performed where the user is given the chance to specify which reagent histogram to re view Processing then proceeds to step 530 where the user has 5 options He can review the effects of the edited thresholds by comparing the results of previ ously run samples with their entered known values which vectors processing back to step 520 He can 20 25 30 40 45 50 55 60 65 38 review the effects of edited threshold values by check ing the resulting number of NTD s which vectors pro cessing back to step 526 He can decide to update all or some of the thresholds by vectoring to step 522 or he can refuse to update any thresholds and review the histograms vectoring processing to a step 532 Finally he can go back to the menu of step 504 The system allows different manufacturer s plates to be used in the plate reader Different man
106. to the host computer 20 There the higher value is dis carded and the lower value is stored on the computer s hard disk The computer interprets this value in relation to user defined thresholds that differentiate between positive negative and ambiguous test results The user can view these test results at his conve nience on the host computer 20 screen by selecting the Results Summary function 190 in FIG 3 Processing is then vectored to the process shown in the flow chart of FIG 8 where the user can select which test results he wishes to review If he wishes to view the results for a particular WBN he selects option 201 and can have the results printed by selecting option 203 If he wishes to view the NTD list he selects option 207 This produces a report of all the WBNs process on a given date de fined by the user which produced no type determined results together with the carousel number and test tube position of the NTD tube in that carousel and the plate number and row number of the wells containing sam ples from the NTD tube This use can be used to dete mine which plates or sample tubes need to be pulled from storage for retesting or rereading This list can be printed by selecting option 209 If the user wishes to view the DU list he selects option 193 This causes the report generator routines in the host processor to gener ate a list of all the unverified Rh negative test results processed on a given data along with the car
107. ty It should also ease quality control operations and maintain data useful in quality control efforts Such a machine should also maintain all test results and be able to communicate them to a main frame computer for permanent storage or allow them to be archived onto permanent magnetic storage Such a machine should also enable the automated generation of reports for management SUMMARY OF THE INVENTION The invention is a system for performing a host of functions which aid in the operational work and man agement of a blood bank The invention consists of various off the shelf components and a custom designed liquid handler combined in one system all controlled by a comprehensive software resident in a host computer 4 873 633 3 which allows the user to control blood typing and other liquid handling operations in the liquid handler and to manage the data which results therefrom The system automates much of the liquid handling involved in processing donor samples for ABO Rh blood typing tests and antibody screening sample and plate identifi cation using a bar code reader plate reading for the ABO RRh tests record keeping for quality control data and test results for individual samples access to infor mation on test status and printing of laboratory records and transmission of information to a mainframe com puter for on line storage The physical system includes a Digital Equipment Corporation PDP 11 MICRO with a 10 megabyte
108. ufacturer s plates have different well bottom configurations These different well bottom configurations cause different meniscus lens effects and other optical effects which can adversely affect the quality of absorbance readings unless steps are taken to adjust the operation of the system to account for the peculiarities of a particular plate The CPU 20 controls the plate reader to take multiple absorbance readings by shining a light up through the bottom of the well at a wavelength controlled by a command from the CPU 20 The beam is shined up through the bottom of the well at each of several angles both in the center of the bottom of the well and at posi tions off center If a positive reaction bottom is settled in the bottom center of the well the absorbance readings will be different at different angles If a negative reac tion exists the absorbance will be more or less constant at all angles Thus the system can discriminate between positive and negative reactions Therefore the best angle to take the absorbance readings must be deter mined to optimize the discriminatory powers of the system The menu option for setting liquid handling proce dure for determining plate reading position displays screens of information which instruct the user to put known type O blood in 8 donor well positions To these samples are added the proper reagents to cause positive reactions for anti Rh and reverse typing reactions A1 A2 and B These inst
109. uld be interpreted The Director modifies this framework as necessary to re flect changes in system users test protocol etc Security Precautions The system utilizes a user privilege table and user identification at logon time to guarantee accountability and to insure that only users with the proper authoriza tion have access to certain portions of the program and certain data files The following publicly available documents are in corporated by reference for support material MICRO PDP 11 System Owner s Manual Digital Equipment Corporation Pro Pette Liquid Handling System User s Manual Cetus Corporation Automated Microplate Reader Model EL309 Opera tor s Manual Bio Tek Instruments Inc Installing and Using the LA50 Printer Digital Equip ment Corporation LASO Programmer s Reference Manual Digital Equipment Corporation Orbital Shaker User Manual ABO Rh Testing ProGroup TM automated blood typing system auto mates the typing and grouping of donor samples This section describes the liquid handling resuspension and plate reading tasks the ProGroup TM automated blood typing system performs for ABO Rh testing Liquid Handling Functions The ProGroup TM automated blood typing system liquid handler 26 transfers donor plasma out of the tubes 22 into designated wells of a microplate 56 It also trans fers donor red blood cells into a special predilution block 60 mixes the cells with a defined quantity of dilu
110. ults that are repeatable within plus or minus 5 and plus or minus 0 0050 D when measure ments are made at a single wavelength and blanked on air Plate The Bio Tek plate reader used in the system System can operate in a variety of modes You can program it directly using the 31 character keyboard and the 16 character display that are built into the plate reader You can also output data directly from the plate reader to a parallel printer In the context of the ProGroup TM automated blood typing system however the plate reader operates en tirely under computer control of the host computer 20 This option allows the plate reader to accept commands from an external computer in this case the host com puter 20 and to transmit the optical measurement data it generates back to the computer for analysis there A serial I O link permits the plate reader to operate in this fashion How the ProGroup Uses the Plate Reader When using the plate reader for ABO Rh testing the instrument is calibrated and controlled through the ProGroup TM automated blood typing system host computer 20 The user gives the initial directive to start the plate reader by selecting the plate reader function on the host computer 20 terminal After mounting the plate in the reader as described below you simply press the START key on the plate reader keyboard The readings are made automatically and the optical density measurements are immediately transmitted
111. us When the liquid handler is aspirating plasma for the antibody screening plate or ABO Rh plate the end of the tip is placed just 1 16 below the meniscus The height of the original meniscus and the setting for the hematocrit level enable the system System to calculate the height of the red blood cells in the tube So as long as the tube contains at least a certain amount of sample when the liquid handler tip withdraws sample for trans fer to the forward testing wells it will aspirate only red blood cells After depositing the cells in the appropriate wells the X Y head ejects the used tip into a receptacle at the right side of the table It then repeats this cycle Trans ferring sample from eight donor to a 96 well plate takes about 30 minutes Fusion Head The fusion head provides the mounting for the 12 channel head the wash head and the manifold The 12 channel head delivers from 1 to 12 reagents simulta neously to a row of wells on a microplate The micro plate can be moved steadily forward for rapid filling of one row after another 10 20 25 35 40 45 50 55 65 24 12 Channel Head The 12 channel head starts by picking up a row of short disposable tips from the reagent block at the back of the liquid handler table The head then aspirates the desired reagents for a row of wells in this block Next the table moves the first row of the ABO Rh microplate or the antibody screening plate under the
112. uter 20 allows the user to modify data that has been created through automatic procedures The user can also enter addi tional information pertaining to donor samples such as antibody screen results or Hepatitis and HTLV III results The host computer 20 is a DEC MICRO PDP 11 computer with a 10 Mbyte hard disk and a VT 220 terminal with graphics capability The computer 20 runs the software included herewith as Appendix A on the RSX11 operating system which is commercially available from Digital Equipment Corporation Printer The ProGroup TM automated blood typing system also includes an LASO dot matrix printer 30 from Digi tal Equipment Corporation The printer is connected to 10 20 25 35 45 50 65 8 aa the computer so the user can quickly generate hard copies of laboratory records The ProGroup TM automated blood typing system is used by laboratory personnel at three levels The Technician who operates the machine on a daily basis running test and printing results _ The Supervisor who performs quality control proce dures adds to or revises test data as necessary handles the storage of information on the ProGroup TM auto mated blood typing system host computer 20 and data transfers data to a mainframe computer The Director who creates the initial framework for the ProGroup This includes tasks such as defining who will be using the system how the tests will be carried out and how test results sho
113. which the plates are read by the plate reader is unimportant The plates can be loaded in the plate reader in any order The plate reader then reads the bar code for each plate and sends it to the host computer 20 The host computer then looks up the bar code in the look up table and determines if any special instructions need to be sent back to the plate reader e g as to wavelength to be used for the light or whether or not to do a steps off center series of light absorbance readings Parameters The user defines the test protocol by specifying in steps 114 and 116 for no bromelin and bromelin pre treatment respectively the volume of donor plasma to be pipetted into the wells of the microplate volume of donor cells the volume of diluent to be mixed with the cells thus the concentration of the donor cell suspension and the number of mixes concentration of the reagent cell suspension and titer of the reagent antisera Liquid Handler Set Up Bar coded tubes containing properly prepared donor blood samples are loaded into the ProGroup TM auto mated blood typing system carousel The carousel holds 48 tubes and 48 ProGroup TM automated blood typing system disposable tips The carousel mounts at the side of the ProGroup TM automated blood typing system 10 20 25 35 45 50 55 65 10 liquid handler A set of six 96 well microplates is loaded into the plate stacker on the other side of the liquid han dier Ea
114. zed data management techniques and the built in bar code reader reduce the incidence of operator error ProGroup TM automated blood typing system does not require any previous experience with automated liquid handling equipment or with computers ProGroup System Overview There follows more detail on the individual compo nents of the system Liquid Handler The liquid handling portion of the ProGroup TM automated blood typing system consists of the follow ing items ProGroup TM automated blood typing system liquid handler 26 shown in more detail in FIG 2 automates the pipetting of donor plasma directly from a carousel 20 of tubes 22 into designated wells of a microplate The ProGroup TM automated blood typing system mixes donor red blood cells with diluent in designated propor tions before transferring the cells to the wells of the microtitre plates 56 and 58 ProGroup TM automated blood typing system fills the ABO Rh plate 56 and the antibody screening plate 58 with the appropriate rea gents from a reagent trough 62 The ProGroup TM automated blood typing system liquid handler includes four permanently mounted liquid delivery heads X Y head 28 12 channel head 42 fill manifold 46 and wash head 44 One or several of these can be used in the course of a single procedure since there is no require ment to remove one head to install another The X Y head and 12 channel head use disposable pipette tips 24 and 63 of several size

C`ENTER

Contents

Download Pdf Manuals

Related Search

Related Contents