UK BIOBANK. Biospecimens Manual. Collection of biological samples, processing and storage. Kristian Spreckley 8/4/2011

Transcription

1 UK BIOBANK Biospecimens Manual Collection of biological samples, pocessing and stoage Kistian Speckley 8/4/2011 This manual details the collection of biological samples at UK Biobank Assessment Centes and the subsequent pocessing of those samples at the UK Biobank Cental Pocessing Cente. Biospecimens Manual UK Biobank Page 1 of 123

2 Contents 1 Intoduction Staff Collection of blood samples into Vacutaines Sample stoage and shipping Sample pocessing EDTA (Sample #1 and Sample #2) Plasma Sepaato (Sample #3) Seum Sepaato (Sample #4) Uine (Sample #5) ACD (Sample #6) EDTA (Sample #7, Haematology Sample) Quality Contol Daily Contols Calibation (evey six months) RNA stabilised blood (Sample #8) Saliva sample (sample #9) Sample Stoage... 8 Appendix 1 Beckman Coulte LH700 Seies System Manual... 9 Biospecimens Manual UK Biobank Page 2 of 123

3 1 Intoduction 1.1 This manual details the pocess fo collection, pocessing and analysis of biological samples. The samples ae taken at the 7 th station of the assessment cente visit, shipped and then analysed at UK Biobank cental pocessing cente. The following samples wee collected at the assessment centes befoe being pocessed at the UK Biobank cental pocessing site: 1. Ethylenediaminetetaacetic acid, EDTA (10ml), Puple cap 2. Ethylenediaminetetaacetic acid, EDTA (10ml), Clea cap 3. Plasma Sepaato, PST (10ml), Geen cap 4. Seum Sepaato, SST (10ml), Oange cap 5. Uine (10ml), Gey cap 6. Acid Citate Dextose, ACD (6ml), Pale yellow cap 7. Ethylenediaminetetaacetic acid, EDTA (4ml), Puple cap 8. Applied Biosystems Tempus Tube, RNA Stabilised Blood (3ml), Blue cap 9. 50ml Falcon Tube, Saliva Note: Tubes 8 and 9 wee an enhancement to the study and added fo the last 100,000 paticipants. Please see below fo the sequence of the assessment cente visit. Table 1: sequence of assessment visit Visit Station Assessments Undetaken 1 Reception Welcome & egistation Geneating a USB key fo Paticipants 2 Touch-sceen Section Consent Touch-sceen questionnaie Heaing Test Cognitive function tests (Shape, Pais, Fluid Intelligence, Snap) 3 Inteview & blood pessue Inteviewe questionnaie Blood pessue measuement Measuement of ateial stiffness 4 Eye measuements Visual acuity Auto-efaction Intaocula pessue Retinal image (OCT Scan) 5 Physical measuements Height (Standing and Sitting) Hip & waist measuement Weight and Bio-impedance measuement Hand-gip stength Heel-bone ultasound Spiomety (Lung function Test) Biospecimens Manual UK Biobank Page 3 of 123

4 6 Cadio (Physical fitness) Execise ECG (Cycling) 7 Sample collection & exit Blood samples collected Uine sample sought Saliva sample sought Consent & esult summay pinted Tavel expense claim povided 8 Web-based diet questionnaie Dietay assessment 1.2 Thoughout this document, the tem Paticipant signifies a study paticipant who is taking pat in the Assessment Cente pocess, egadless of whethe they eventually give o withhold consent to take pat in the UK Biobank study. 1.3 The collection of data fom assessment visits uses the diect data enty system of the Assessment Cente Envionment (ACE). 1.4 At the stat of thei visit, each paticipant is issued with a USB Key at the Reception station. This USB Key acts as a paticipant identifie (it contains Paticipant ID, name, date of bith and gende) and as a tempoay stoage device fo the ecoded data. As the paticipant pogesses between stations, the USB key acts as an identifying token and also as a data tansfe mechanism. At the Reception & Exit module, all data on the USB key is emoved, afte it has been backed up to the Assessment Cente head PC. 2 Staff Blood daws ae caied out by egisteed phlebotomists, tained and cetified to conduct blood daws. The Assessment Cente Manage ovesees that all staff wok in accodance with the pocedue. 3 Collection of blood samples into Vacutaines Below is the potocol fo blood sample collection in all Becton Dickinson Vacutaine fomats. 3.1 Hold Vacutaine bael steady with one hand and select, with othe hand, fist Vacutaine tube in sequence (puple cap 10ml EDTA tube) fom pe-pepaed ack. Push Vacutaine tube into bael until shap end of needle within bael pieces ubbe bung of tube; 3.2 Release touniquet as blood begins to flow into Vacutaine tube (most paticipants will not need touniquet afte access to vein has been established), but keep some pessue/tightness on touniquet if flow is slow; 3.3 When Vacutaine 1 is full, blood flow will stop. Remove the Vacutaine tube fom the bael And inset Vactutaine 2. Gently invet Vacutaine 1 ten times while the second bottle is Biospecimens Manual UK Biobank Page 4 of 123

5 filling then etun it to ack. Note: If you ae unable to invet the Vacutaine while the othe bottle is filling then all bottles should be inveted immediately upon completion of blood sampling. 3.4 Repeat this step to fill Vacutaines 2-8 accoding to ode in ack: pay paticula attention to the gentle invesion of the tubes (1 to 6) afte each is filled with blood. Note: It is cucial that the Blue RNA tube must be shaken vigoously fo 20 seconds immediately afte sample is obtained. 1. Puple, EDTA (10ml) 2. Clea, EDTA (10ml) 3. Geen, Plasma Sepaato (10ml) 4. Oange, Seum Sepaato (10ml) 5. Gey, Uine (10ml) 6. Pale yellow, ACD (6ml) 7. Puple, EDTA (4ml) 8. Blue, RNA Stabilised Blood (3ml) 9. 50ml Falcon Tube, Saliva 3.5 If blood flow slows duing collection, e-apply touniquet and ask paticipant to clench and unclench thei fist while keeping am still. When blood flow stats again, elease touniquet and continue collection; Note: If blood flow does not e-stat, explain difficulties to paticipant and ask pemission to epeat venepunctue in othe foeam o, failing that, to collect sample fom veins in back of hand using a Safety Lok buttefly needle (as descibed in Section 8.4 above); 3.6 Afte blood collection is complete (with eithe all equied Vacutaines filled o as many as possible), apply clean cotton wool dessing ove skin at insetion of needle. Remove needle and apply pessue on punctue site (applying pessue befoe needle emoval will cause discomfot); 3.7 Immediately scan each filled Vacutaine using bacode eade. It is vey impotant to check on the compute sceen below that each filled tube has been popely scanned as this links these samples to the paticipant. If the scanne fails to ecognise a bacode, the tube ID should be manually enteed using the 13-digit tube ID; 3.8 If one o moe tubes ae not collected, ecod the eason why not using the dop-down menus fo each specific tube (see below); empty blood tubes do not need to be scanned. 3.9 When all of the filled tubes have been scanned a message is sent to the sample pocessing station and activates a time that ensues that tube 3 [oange cap] is allowed to stand fo 30 minutes pio to centifugation; 3.10 A check is made to ensue that all tubes have been collected (o eason(s) fo not being collected ecoded) Sample collection is complete. Biospecimens Manual UK Biobank Page 5 of 123

6 4 Sample stoage and shipping All samples ae stoed immediately in a efigeato at the clinic. At the end of the day, all samples ae shipped at +4ºC to the centalised pocessing cente in Stockpot, Cheshie. The one exception is the ACD sample which is tanspoted at 18 C. 5 Sample pocessing The samples wee pocessed at the UK Biobank cental pocessing site as follows: 5.1 EDTA (Sample #1 and Sample #2) EDTA samples wee pocessed on an automated system following centifugation. The automated system etieved up to fou 850ul aliquots of plasma, one aliquot of buffy coat and one aliquot of ed blood cells. Samples wee pocessed on a pupose made automated system, chilled to +4 C. 5.2 Plasma Sepaato (Sample #3) Plasma samples wee pocessed on an automated system designed to etieve the plasma laye fom a gel sepaation tube. Up to fou 850ul aliquots of plasma wee ecoveed pe sample. Samples wee pocessed on a pupose made automated system, chilled to +4 C. 5.3 Seum Sepaato (Sample #4) Seum samples wee pocessed on an automated system designed to etieve the seum laye fom a gel sepaation tube. Up to fou 850ul aliquots of seum wee ecoveed pe sample. Samples wee pocessed on a pupose made automated system, chilled to +4 C. 5.4 Uine (Sample #5) Uine samples wee mixed befoe being aliquoted into six 850ul aliquots of uine using an automated liquid handling system. Pocessed samples wee immediately moved to a +4 C incubato. Samples wee pocessed at +4 C. 5.5 ACD (Sample #6) ACD samples wee pocessed by aliquoting with the subsequent addition of RPMI (Roswell Pak Memoial Institute media) media and DMSO (Dimethyl Sulphoxide). Two 700ul aliquots of ACD blood wee aliquoted into 1.2ml Abgene tubes befoe the addition of 150ul of DMSO (30ul) and RPMI (120ul) mixtue, esulting in two 850ul final volume aliquots. Both aliquots wee fozen at a contolled ate down to -196 C. ACD samples wee pocessed at oom tempeatue and the DMSO-RPMI mixtue was cooled to +4 C. The envionment was HEPA filteed. 5.6 EDTA (Sample #7, Haematology Sample) Once eceived at the sample pocessing cente in Stockpot, the samples ae analysed on a Beckman Coulte LH700 System. Samples ae analysed within 24 hous of blood daw. The system detemines the following hematologic paametes of whole-blood specimens: Biospecimens Manual UK Biobank Page 6 of 123

7 WBC White Blood Cell o leukocyte count RBC Red Blood Cell o eythocyte count Hgb Hemoglobin concentation Hct Hematocit (elative volume of eythocytes) MCV Mean Copuscula (eythocyte) Volume MCH Mean Copuscula (eythocyte) Hemoglobin MCHC Mean Copuscula (eythocyte) Hemoglobin Concentation RDW Red Cell (eythocyte volume) Distibution Width Plt Platelet o thombocyte count MPV Mean Platelet (thombocyte) Volume LY% Lymphocyte pecent MO% Monocyte pecent NE% Neutophil pecent EO% Eosinophil pecent BA% Basophil pecent LY# Lymphocyte numbe MO# Monocyte numbe NE# Neutophil numbe EO# Eosinophil numbe BA# Basophil numbe NRBC% Nucleated Red Blood Cell pecent NRBC# Nucleated Red Blood Cell numbe RET% Reticulocyte pecent RET# Reticulocyte numbe *HLR% High Light scatte Reticulocytes % *HLR# High Light scatte Reticulocytes # IRF Immatue Reticulocyte Faction MRV Mean Reticulocyte Volume *MSCV Mean Spheed Cell Volume *Pct Plateletcit *PDW Platelet Distibution Width Fo moe infomation on the calculation of the above haematological paametes, please see the Section 3.7 of the Beckman Coulte LH700 Seies System Manual attached in Appendix Quality Contol Daily Contols The following contols wee un on a daily basis: Laton contols LATRON pime pepaes the tubing and instument components fo the LATRON contol. LATRON contol monitos the pefomance of the volume, conductivity and light scatte measuements. 5C Cell Contol 5C -ES contol monitos the CBC (complete blood count) and diffeential (Diff) paametes. Biospecimens Manual UK Biobank Page 7 of 123

8 Retic C contol Retic-C cell contol monitos the eticulocyte (Retic) paametes Calibation (evey six months) The haematology systems wee calibated evey six months accoding to the manufactue s ecommendations. 5.7 RNA stabilised blood (Sample #8) RNA stabilised blood was initially mixed befoe being aliquoted into 6 aliquots of 850ul. The liquid handling system was HEPA filteed and cooled to +4 C. 5.8 Saliva sample (sample #9) Saliva samples wee mixed and aliquoted into 2 aliquots of 850ul. Saliva samples wee pocessed by an automated liquid handling system in a HEPA contolled envionment at +4 C. 6 Sample Stoage Samples ae all stoed at -80 C o in liquid nitogen. All samples wee taken, pocessed and put into stoage within 24 hous. The vaious sample aliquots ae stoed as follows: Sample Type Aliquot Type Total aliquots No. of aliquots -80 C LN2 EDTA 1 Plasma Buffy Coat 1 1 Red Blood Cells EDTA 2 Plasma Buffy Coat 1 1 Red Blood Cells SST Seum PST Plasma Uine Uine ACD ACD Blood + DMSO+ RPMI Tempus (RNA) RNA stabilised blood Saliva Saliva The -80 C automated feeze is capable of stoing up to 10 million samples and can pick in the egion of 1000 samples pe day. Biospecimens Manual UK Biobank Page 8 of 123

9 Appendix 1 Beckman Coulte LH700 Seies System Manual Biospecimens Manual UK Biobank Page 9 of 123

10 04/30/96 12:30:18 PRIME DRAIN RINSE CLEAR APERT START UP SHUT DOWN *SYSTEM RUN* I II III IV V VI VII VIII READY STOP PREMIX START CONT 3 ID POWER ON E POWER OFF N T E CE 0. R STATUS: READY ALERT: TEST MODE MODE: C/D/R ASPIRATIONS/TUBE 01 MAIN MENU BLOOD DETECT: ON APERT F 1 2 ALARM RESET NEC MultiSync LCD 1700m+ COULTER LH 700 Seies System Refeence (Octobe 2003) Beckman Biospecimens Coulte, Inc. Manual UK Biobank Page 10 of 123 Fulleton, CA 92835

11 WARNINGS AND PRECAUTIONS READ ALL PRODUCT MANUALS AND CONSULT WITH BECKMAN COULTER-TRAINED PERSONNEL BEFORE ATTEMPTING TO OPERATE INSTRUMENT. DO NOT ATTEMPT TO PERFORM ANY PROCEDURE BEFORE CAREFULLY READING ALL INSTRUCTIONS. ALWAYS FOLLOW PRODUCT LABELING AND MANUFACTURER S RECOMMENDATIONS. IF IN DOUBT AS TO HOW TO PROCEED IN ANY SITUATION, CONTACT YOUR BECKMAN COULTER REPRESENTATIVE. HAZARDS AND OPERATIONAL PRECAUTIONS AND LIMITATIONS WARNINGS, CAUTIONS, and IMPORTANTS alet you as follows: WARNING - Can cause injuy. CAUTION - Can cause damage to the instument. IMPORTANT - Can cause misleading esults. BECKMAN COULTER, INC. URGES ITS CUSTOMERS TO COMPLY WITH ALL NATIONAL HEALTH AND SAFETY STANDARDS SUCH AS THE USE OF BARRIER PROTECTION. THIS MAY INCLUDE, BUT IT IS NOT LIMITED TO, PROTECTIVE EYEWEAR, GLOVES, AND SUITABLE LABORATORY ATTIRE WHEN OPERATING OR MAINTAINING THIS OR ANY OTHER AUTOMATED LABORATORY ANALYZER. WARNING Risk of opeato injuy if: All doos, coves and panels ae not closed and secued in place pio to and duing instument opeation. The integity of safety intelocks and sensos is compomised. Instument alams and eo messages ae not acknowledged and acted upon. You contact moving pats. You mishandle boken pats. Doos, coves and panels ae not opened, closed, emoved and/o eplaced with cae. Impope tools ae used fo toubleshooting. To avoid injuy: Keep doos, coves and panels closed and secued in place while the instument is in use. Take full advantage of the safety featues of the instument. Do not defeat safety intelocks and sensos. Acknowledge and act upon instument alams and eo messages. Keep away fom moving pats. Repot any boken pats to you Beckman Coulte Repesentative. Open/emove and close/eplace doos, coves and panels with cae. Use the pope tools when toubleshooting. CAUTION System integity might be compomised and opeational failues might occu if: This equipment is used in a manne othe than specified. Opeate the instument as instucted in the Poduct Manuals. You intoduce softwae that is not authoized by Beckman Coulte into you compute. Only opeate you system s compute with softwae authoized by Beckman Coulte. You install softwae that is not an oiginal copyighted vesion. Only use softwae that is an oiginal copyighted vesion to pevent vius contamination. IMPORTANT If you puchased this poduct fom anyone othe than Beckman Coulte o an authoized Beckman Coulte distibuto, and, if it is not pesently unde a Beckman Coulte sevice maintenance ageement, Beckman Coulte cannot guaantee that the poduct is fitted with the most cuent mandatoy engineeing evisions o that you will eceive the most cuent infomation bulletins concening the poduct. If you puchased this poduct fom a thid paty and would like futhe infomation concening this topic, call you Beckman Coulte Repesentative. Biospecimens Manual UK Biobank Page 11 of 123

12 REVISION STATUS Issue A, 10/01 Softwae vesion 1A. Conveted fom Help Vesion 1A Issue B, Complete Revision, 5/02 Softwae vesion 2A. Conveted fom Help Vesion 2A Issue C, 10/03 Softwae vesion 2B. Conveted fom Help Vesion 2B Changes wee made to: Change the company name fom Coulte Copoation to Beckman Coulte Inc. Change LH 750 to LH 700 Seies. Change all vaiations of ba-code to be consistent. Change 5C-ES to 5C Seies. Note: Changes that ae pat of the most ecent evision ae indicated in text by a ba in the magin of the amended page. This document applies to the latest softwae listed and highe vesions. When a subsequent softwae vesion changes the infomation in this document, a new issue will be eleased. Biospecimens Manual UK Biobank Page 12 of 123 iii

13 REVISION STATUS iv Biospecimens Manual UK Biobank Page 13 of 123

14 CONTENTS WARNINGS AND PRECAUTIONS, ii REVISION STATUS, iii INTRODUCTION, xiii HOW TO USE YOUR COULTER LH 700 SERIES SYSTEM HARD-COPY MANUALS, xiii ABOUT THIS MANUAL, xiv ONLINE HELP SYSTEM, xiv CONVENTIONS, xiv 1 USE AND FUNCTION, INTENDED USE, 1-1 Paametes, QUALITY CONTROL (QC), METHOD HISTORY, 1-3 Development, 1-3 Coected WBC Counts, 1-4 Hemoglobinomety, 1-4 Diffeential Measuement, 1-4 Volume Analysis, 1-4 Conductivity Analysis, 1-5 Light Scatte Analysis, 1-5 Reticulocyte (Retic) Analysis, 1-5 NRBC Enumeation, 1-6 COULTER IntelliKinetics Application, 1-7 XB Analysis, SYSTEM COMPONENTS, 1-7 Powe Supply, 1-7 Dilute, 1-7 Analyze, 1-8 LH 700 Seies Wokstation, 1-8 Handheld Scanne, HARDWARE OPTIONS, 1-8 Gaphic/Lase Pinte, 1-8 LH 700 Seies SlideMake, 1-8 LH 700 Seies SlideStaine, CONTROLS AND CALIBRATOR, 1-9 Contols, 1-9 Calibato, 1-9 Biospecimens Manual UK Biobank Page 14 of 123 v

15 CONTENTS 1.7 REAGENTS, 1-9 Diluent, 1-9 CBC Lytic Reagent, 1-9 LH 700 Seies PAK Reagent System, 1-10 Eytholyse II (PAK LYSE), Diff Lytic Reagent, 1-10 StabiLyse (PAK PRESERVE), Diff Pesevative, 1-10 LH 700 Seies RETIC PAK Reagent System, 1-10 Reagent A, Retic Stain, 1-10 Reagent B, Retic Cleaing Solution, 1-10 Cleaning Agent, MATERIAL SAFETY DATA SHEETS (MSDS), INSTALLATION, GENERAL, SPECIAL REQUIREMENTS: HARDWARE, 2-1 Space and Accessibility, 2-1 Electical Input, 2-1 Ambient Tempeatue and Humidity, 2-2 Ai Conditioning, 2-2 Ventilation, 2-2 Dainage, INTERUNIT CONNECTIONS, 2-3 Powe and Signal Cables, 2-3 Pneumatic/Hydaulic Tubing Connections, OPERATION PRINCIPLES, COULTER METHOD, 3-1 CBC Analysis, 3-1 Diffeential Analysis, 3-1 Effect of Reagents, 3-2 Reticulocyte Analysis, AUTOMATIC ASPIRATION MODE, 3-2 Loading Specimens, 3-2 Tanspoting Cassettes, 3-2 Aspiation, 3-4 Delivey, 3-4 CBC, 3-4 Diffeential (Diff) and Retic, 3-4 CBC Sensing System, 3-5 CBC Analysis in the Baths, 3-5 Diffeential and Retic Multipaamete Sensing System, 3-6 WBC Diffeential Analysis, 3-7 Reticulocyte Analysis, 3-7 Backwash and Rinse, MANUAL ASPIRATION MODE, 3-8 vi Biospecimens Manual UK Biobank Page 15 of 123

16 CONTENTS 3.4 COUNTING AND SIZING, 3-9 Red and White Blood Cell Counting, 3-9 Coincidence Coection, 3-9 Voting, 3-9 Pulse Editing, 3-10 Sweep Flow, 3-10 RBC Size Distibution, 3-10 Plt Count and Size Distibution, 3-11 Plt Fitting Pocess, 3-11 Retic Paametes, MEASUREMENT OF HEMOGLOBIN CONCENTRATION, DATAPLOT DEVELOPMENT, 3-12 Two-Dimensional (2D) DataPlots, 3-13 Thee-Dimensional (3D) DataPlots, PARAMETERS AND THEIR DERIVATION, 3-13 White Blood Cell (WBC) Count, 3-13 Red Blood Cell (RBC), 3-13 Hemoglobin (Hgb) Concentation, 3-13 Mean Copuscula Volume (MCV), 3-14 Hematocit (Hct), 3-14 Mean Copuscula Hemoglobin (MCH), 3-14 Mean Copuscula Hemoglobin Concentation (MCHC), 3-14 Red Distibution Width (RDW), 3-14 Platelet (Plt) Count, 3-14 Mean Platelet Volume (MPV), 3-14 NRBC % (Nucleated Red Blood Cells), 3-14 NRBC # (Nucleated Red Blood Cells), 3-15 Diffeential (Diff), 3-15 Diff Pecentages (DIFF%), 3-15 Diff Absolute Numbes (DIFF#), 3-15 Reticulocyte (Retic) Paametes, 3-16 Retic Pecent (RET%), 3-16 Retic Absolute Numbe (RET#), 3-16 Immatue eticulocyte faction (IRF), 3-16 Mean eticulocyte volume (MRV), 3-16 Summay of Paamete Deivations, 3-17 Measued Diectly, 3-17 Deived Fom RBC o Plt Histogam, 3-17 Deived Fom WBC Histogam, 3-17 Computed, XB ANALYSIS, 3-18 Adjusting Initial XB Taget Values, 3-19 Biospecimens Manual UK Biobank Page 16 of 123 vii

17 CONTENTS 4 SPECIFICATIONS/CHARACTERISTICS, PHYSICAL SPECIFICATIONS, 4-1 Dimensions, 4-1 Powe, 4-1 Input, 4-1 Consumption, 4-1 Tempeatue, 4-1 Humidity, 4-1 Sample Stability, 4-1 CBC/Diff Paametes, 4-1 Reticulocyte Paametes, 4-1 NRBC Paametes, 4-1 Sample Stoage, 4-2 Sample Type, 4-2 Recommended Anticoagulant, 4-2 SAMPLING MODES, 4-2 Aspiation, 4-2 Test/Cycle, 4-2 THROUGHPUT, AUTOMATIC MODE, 4-2 Sample Volume Aspiated, 4-3 Waste, 4-3 Pneumatic Supplies (Intenally Regulated), 4-3 Calibation Stability, 4-3 LH 700 Seies Wokstation Stoage, PERFORMANCE SPECIFICATIONS--LH 700 Seies, 4-3 Pecision, 4-4 Within-Run Pecision, 4-4 Accuacy, 4-4 Accuacy Qualification, 4-4 Accuacy, CBC, 4-5 Accuacy, WBC Diffeential, 4-5 Accuacy, Reticulocyte, 4-5 Lineaity, 4-6 Backgound, 4-6 Cayove, 4-6 Opeating and Repotable Ranges, 4-7 Mode-to-Mode Compaison, 4-7 viii Biospecimens Manual UK Biobank Page 17 of 123

18 CONTENTS 4.3 PERFORMANCE CHARACTERISTICS, 4-8 Sample Stability, 4-8 WBC Diffeential Flagging Stability, 4-11 NRBC Paied Sample Impecision, 4-11 NRBC Accuacy Chaacteistics, 4-11 Platelet Accuacy Chaacteistics, 4-12 Refeence Ranges, 4-12 Known Intefeing Substances, 4-13 CBC, 4-13 NRBC, 4-14 Diffeential, 4-14 Reticulocytes, BAR-CODE SYMBOLOGY OVERVIEW, 4-14 Chaacte Set, 4-14 Symbology Type, 4-14 Fixed o Vaiable Length, 4-15 Self-Checking, 4-15 Stat Code, Stop Code, 4-15 Check Chaacte, Checksum Algoithm, 4-15 Quiet Zone, Quiet Aea, BAR-CODES AND THE LH 700 Seies, 4-15 Checksum Algoithm, 4-15 Ba-code Symbologies Suppoted by the LH 700 Seies, 4-16 Inteleaved 2-of-5, 4-16 Code 39 (Also called 3-of-9 Code), 4-16 Codaba, 4-16 NW-7, 4-16 Code 128/USS 128, 4-16 Ba-code Tips, BAR-CODE LABEL SPECIFICATIONS, 4-17 Handheld Scanne, 4-17 Geneal, 4-17 Optical Chaacteistics at 880 nm ±10% and 633 nm ±10%, 4-17 Pinting Method, 4-17 Label Thickness, 4-17 NE/WE Ratio, 4-17 Label Dimensions and Data, 4-18 Acceptable Ba-codes, 4-20 Checksum Algoithm, 4-21 Inteleaved 2-of-5, 4-21 Codaba and NW7, 4-21 Japan Red Coss NW7 Decoding, 4-23 Code 39 Ba-code, 4-25 Code 128, HAZARDS, LASER SAFETY, 5-1 Biospecimens Manual UK Biobank Page 18 of 123 ix

19 CONTENTS 5.2 RADIATION HAZARDS, LASER WARNING LABELS, BAR-CODE READER, HANDHELD SCANNER, 5-6 REFERENCES, REFERENCES-1 GLOSSARY, GLOSSARY-1 INDEX, INDEX-1 TRADEMARKS x Biospecimens Manual UK Biobank Page 19 of 123

20 CONTENTS ILLUSTRATIONS 1.1 COULTER LH 700 Seies, Coected WBC, Illustation of the ten light scatte egions, NRBC signatue position on Diffeential Dataplot, NRBC location on WBC histogam, Inteunit Powe and Signal Cable Connections, Pneumatic/Hydaulic Connections, Coulte Method of Counting and Sizing, Tanspot System, Tiple Tansduce Module with Potective Housing, Tiple Tansduce Module with Potective Housing Cut Away, Sweep Flow, Ba-Code Label Specifications, Lase Waning Label, Potective Housing Cut Away, Lase Waning Label Locations, Potective Housing On, Ba-Code Reade Lase Waning Label Location, Potective Housing Cut Away, Ba-Code Reade Lase Waning Label Location, Potective Housing On, Handheld Scanne, 5-6 Biospecimens Manual UK Biobank Page 20 of 123 xi

21 CONTENTS TABLES 3.1 Effect of Diectly-Measued Paametes on the Red Cell Indices, Thoughput Sample Citeia, Within-Run Pecision (n = 31), Accuacy, CBC, Accuacy Toleance Limits, WBC Diffeential, Accuacy, Reticulocyte, using NCCLS H16-P o clinical flow cytomete, Accuacy, Reticulocyte, using a pedicate hematology analyze, Lineaity Limits, Opeating and Repotable Ranges, CBC Sample Stability, Room Tempeatue, CBC Sample Stability, Cold Tempeatue, DIFF% Sample Stability, Room Tempeatue, DIFF% Sample Stability, Cold Tempeatue, DIFF# Sample Stability, Room Tempeatue, DIFF# Sample Stability, Cold Tempeatue, RETIC Sample Stability, Room Tempeatue, RETIC Sample Stability, Cold Tempeatue, NRBC Sample Stability, Room Tempeatue, NRBC Sample Stability, Cold Tempeatue, Diffeential Suspect Flagging at Room Tempeatue, Diffeential Suspect Flagging at Cold Tempeatue, NRBC Impecision Chaacteistics,Paied Sample Analysis - Replicate 1 vs. Replicate 2, NRBC Accuacy Chaacteistics, Compaed Samples, Plt Accuacy Chaacteistics vs ICSH/ISLH Platelet Method, Nomal Population Study, Ba-Code Label Specifications, Code-Related Specifications, 4-20 xii Biospecimens Manual UK Biobank Page 21 of 123

22 INTRODUCTION This intoductoy section contains the following topics: How to use you COULTER LH 700 SERIES System had-copy manuals About this manual Online Help System Conventions HOW TO USE YOUR COULTER LH 700 SERIES SYSTEM HARD-COPY MANUALS Use the Getting Stated booklet to see an oveview of the system hadwae and softwae. This document comes with you LH 700 SERIES System. Use the Refeence manual fo in-depth infomation about what the instument does, the methods it uses, its specifications, and infomation on installation, safety and softwae options. The Refeence manual fo the LH 700 SERIES System is included in the online Help system; it is available in had copy by equest. Use the Special Pocedues and Toubleshooting manual to un calibation; to clean, eplace o adjust a component on the instument; and fo toubleshooting the instument. This document is made up of pocedues fom the online Help system; it is available in had copy by equest. Use the Opeato s Guide fo the day-to-day opeation of you instument. This document is made up of pocedues fom the online Help system; it includes Statup, unning contols and samples, eviewing data, Shutdown, and the softwae on the Analyze and the Wokstation. This document is available in had copy by equest. Use the SlideMake Opeato s Guide fo in-depth infomation about what the SlideMake does, the methods it uses, its specifications, and infomation on installation, safety and softwae, as well as day-to-day opeating and toubleshooting you SlideMake. This document is made up of pocedues fom the online Help system; it is available in had copy by equest. Use the SlideStaine Opeato s Guide fo the day-to-day opeating and toubleshooting of you SlideStaine. This document is made up of pocedues fom the online Help system; it includes in-depth infomation about what the SlideStaine does, the methods it uses, its specifications, and infomation on installation, safety and softwae. This document is available in had copy by equest. Use the Maste Index to easily locate a subject in you had-copy Refeence manual, Opeato s Guide o Special Pocedues and Toubleshooting manual. The Maste Index comes with the had copy of both the Opeato s Guide and the Special Pocedues and Toubleshooting manual. Use the Host Tansmission Specification to find the infomation needed to pogam the tansmission inteface between the LH 700 SERIES System and you laboatoy s host compute. This document comes with you LH 700 SERIES System. See the Documentation page on the back cove of this manual fo the contents of each manual. It can help you to detemine quickly in which manual the infomation you need is located. Biospecimens Manual UK Biobank Page 22 of 123 xiii

23 INTRODUCTION ABOUT THIS MANUAL ABOUT THIS MANUAL You LH 700 SERIES System Refeence Guide is a souce of infomation fo the day-to-day opeation of you instument. This infomation is oganized as follows: s s s s s s s s Chapte 1, Use and Function Contains the intended use of the instument, a bief histoy of the methods used, the eagents, calibatos and contols used, and a shot desciption of the majo components. Chapte 2, Installation Contains the instument equiements, and the diagams of the eagent/pneumatic tubing connections and the inteunit cable connections. Chapte 3, Opeation Pinciples Contains desciptions of the Coulte Method, the nomal sample flow though the instument, how counting and sizing ae accomplished, and what the DataPlots show. Chapte 4, Specifications/Chaacteistics Details the instument and pefomance specifications, the pefomance chaacteistics, the intefeing substances, and the ba-code label specifications. Chapte 5, Hazads Descibes lase safety pecautions and the location of the lase-elated labels. Refeences Glossay Index, had copy only ONLINE HELP SYSTEM The LH 700 SERIES Wokstation has a compehensive Online Help System, which includes efeence infomation, all opeating, maintenance and toubleshooting pocedues. On the LH 700 SERIES Wokstation, select to access Help. Select to access the tutoials. CONVENTIONS This document uses the following conventions: indicates a key on the Numeic keypad. indicates a key on the LH 700 SERIES Wokstation keyboad. is the icon fo Patient esults on the LH 700 SERIES Wokstation. is the icon fo the Pinte on the LH 700 SERIES Wokstation. xiv Biospecimens Manual UK Biobank Page 23 of 123

24 5 ; "! ' $ I! & II III IV V 5 6 ) ), ; ) , - VI, - +, 4 ) ) * - VII ) 1-7 *,, VIII GEN S K JE5O? +,% 1USE AND FUNCTION INTENDED USE The COULTER LH 700 Seies, Figue 1.1, is a quantitative, automated hematology analyze Fo In Vito Diagnostic Use in clinical laboatoies. The LH 700 Seies povides automated complete blood count, leukocyte diffeential and Reticulocyte analysis and nucleated ed blood cell (NRBC) enumeation. The pupose of the LH 700 Seies is to sepaate the nomal patient, with all nomal system-geneated paametes, fom the patient who needs additional studies of any of these paametes. These studies might include futhe measuements of cell size and platelet distibution, biochemical investigations, manual WBC diffeential o any othe definitive test that helps diagnose the patient s condition. Figue 1.1 COULTER LH 700 Seies - + Biospecimens Manual UK Biobank Page 24 of

25 USE AND FUNCTION INTENDED USE Paametes The system detemines these hematologic paametes of whole-blood specimens: WBC RBC Hgb Hct MCV MCH MCHC RDW Plt MPV LY% MO% NE% EO% BA% LY# MO# NE# EO# BA# White Blood Cell o leukocyte count Red Blood Cell o eythocyte count Hemoglobin concentation Hematocit (elative volume of eythocytes) Mean Copuscula (eythocyte) Volume Mean Copuscula (eythocyte) Hemoglobin Mean Copuscula (eythocyte) Hemoglobin Concentation Red Cell (eythocyte volume) Distibution Width Platelet o thombocyte count Mean Platelet (thombocyte) Volume Lymphocyte pecent Monocyte pecent Neutophil pecent Eosinophil pecent Basophil pecent Lymphocyte numbe Monocyte numbe Neutophil numbe Eosinophil numbe Basophil numbe NRBC% Nucleated Red Blood Cell pecent NRBC# Nucleated Red Blood Cell numbe RET% Reticulocyte pecent RET# Reticulocyte numbe *HLR% High Light scatte Reticulocytes % *HLR# High Light scatte Reticulocytes # IRF Immatue Reticulocyte Faction MRV Mean Reticulocyte Volume *MSCV Mean Spheed Cell Volume *Pct Plateletcit *PDW Platelet Distibution Width *Fo Reseach Use Only. Not Fo Use In Diagnostic Pocedues. Unless othewise stated, all paamete esults ae shown in a US unit fomat thoughout the manuals. 1-2 Biospecimens Manual UK Biobank Page 25 of 123

26 USE AND FUNCTION QUALITY CONTROL (QC) QUALITY CONTROL (QC) You laboatoy can use these QC techniques with the LH 700 Seies: Daily instument checks Commecial contols Delta checks Patient contols XB Analysis Intelaboatoy Quality Assuance Pogam (IQAP) Quality Assuance includes outine maintenance and sevice in conjunction with the use of contols and calibatos. The combination of these methods povides the assuance of complete quality contol and should be applied sepaately o in combination, in accodance with you laboatoy, state and fedeal potocols. 1.3 METHOD HISTORY Development W.H. Coulte (1956) descibes the Coulte Pinciple: 1 A suspension of blood cells is passed thu [sic] a small oifice simultaneously with an electic cuent. The individual blood cells passing thu the oifice intoduce an impedance change in the oifice detemined by the size of the cell. The system counts the individual cells and povides cell size distibution. The numbe of cells counted pe sample is appoximately 100 times geate than the usual micoscope count to educe the statistical eo by a facto of appoximately 10 times. This substantial impovement in pecision ove pevious methods helped to establish the eythocyte count as a sensitive index of eythopoietic dyscasia, paticulaly when consideed togethe with Hct and Hgb measuements. 2 The COULTER COUNTER Model S analyze was the fist instument that automated simultaneous multipaamete measuements on blood. Bittin et al., Gottmann, and Hamilton and Davidson, eviewed the pefomance and clinical value of the Model S. 3, 4, 5 Refinements of the COULTER COUNTER analyze to povide accuate size (volume) distibution data led to a eawakening of inteest in pathological eythocyte size distibution, fist spaked by Pice-Jones. 6, 7 Among the advantages offeed by the Coulte method of counting and sizing was the ability to deive an accuate Hct measuement by summing the electonic volume of eythocytes. England et al. speculated that electonic Hct measuements did not contain the tapped plasma eo of centifugal Hct measuements. 8 Bull et al. descibed the use of a COULTER COUNTER analyze fo counting thombocytes.9 This method, useful as it was, depended on pepaing thombocyte-ich plasma to avoid counting eythocytes as thombocytes. Mundschenk et al. and Schulz and Thom discussed the possibility of counting thombocytes in the pesence of eythocytes and classifying them by size. 10, 11 Electonic efinements in the Model S-PLUS enhanced the accuacy of the Biospecimens Manual UK Biobank Page 26 of

27 USE AND FUNCTION METHOD HISTORY hydodynamic method. Von Behens and Paulus have also cited the feasibility of counting thombocytes by the Coulte method. 12, 13 Coected WBC Counts White Blood Cell count esults fom the CBC analysis. The WBC count is adjusted fo intefeing substances when appopiate. If thee is a population of cells in the fa left of the WBC histogam, the numbe of cells is deived and the WBC count is coected. No futhe coection of WBC is equied. Figue 1.2 Coected WBC When WBC coection has occued, the uncoected WBC will appea in the pintout Comments field as UWBC = "value". The "Cellula Intefeence" suspect message is displayed and the coected WBC count is epoted. The uncoected WBC can be found on the CBC data tab. When the sepaation between the WBC populations is pooly defined on the histogam, WBC coection will be pefomed and the coected WBC will have an R flag. Hemoglobinomety The lytic eagent used fo the complete blood count (CBC) paametes pepaes the blood so the system can count leukocytes and measue the amount of hemoglobin. The lytic eagent apidly and simultaneously destoys the eythocytes and convets a substantial popotion of the hemoglobin to a stable pigment while it leaves leukocyte nuclei intact. The absobance of the pigment is diectly popotional to the hemoglobin concentation of the sample. The accuacy of this method equals that of the hemiglobincyanide method, the efeence method of choice fo hemoglobinomety ecommended by the Intenational Committee fo Standadization in Hematology. 14 Diffeential Measuement The COULTER VCS established WBC diffeential technology using thee measuements: individual cell volume, high-fequency conductivity and lase-light scatte. The combination of low-fequency cuent, high-fequency cuent and light-scatteing technology povides abundant cell-by-cell infomation that is tanslated by the instument into conventional stained-film leukocyte categoies. Volume Analysis Electonic leukocyte volume analysis, using low-fequency cuent, has been used since It has been evaluated as a possible adjunct to the diffeential white cell count. 16,17,18, Biospecimens Manual UK Biobank Page 27 of 123

28 USE AND FUNCTION METHOD HISTORY 1 Conductivity Analysis Cell walls act as conductos to high-fequency cuent. The cuent, while passing though the cell walls and though each cell inteio, detects diffeences in the insulating popeties of cell components. The cuent chaacteizes the nuclea and ganula constituents and the chemical composition of the cell inteio. 20,21,22 Light Scatte Analysis Coulte's expeience in flow cytomety dates back decades to Fulwyle's pioneeing use of light scatte fo cell analysis. 23 Loken et al. and Jovin et al. discuss the elationship of paticle size and efactivity to the angle of light scatteed fom a lase beam. 24,25 Reticulocyte (Retic) Analysis Reticulocytes ae immatue, nonnucleated eythocytes etaining a small netwok of basophilic oganelles, consisting of RNA and potopophyin. The enumeation of eticulocytes povides a simple, effective means to detemine ed cell poduction and egeneation. 26,27,28,29 The most common means of measuing eticulocytes is to use supavital dyes, such as New Methylene Blue o Billiant Cesyl Blue. These dyes pecipitate and aggegate the basophilic substances within the eticulocyte, esulting in a ganula, staining patten easily seen with light micoscopy. 30 Reticulocyte immatuity is elated to cell volume and light scatte. Since moe immatue eticulocytes ae lage, contain moe RNA and cause inceased light scatte, the cell volume and light scatte will incease with immatuity of the cell. Figue 1.3 illustates the IRF and MRV algoithms. This figue is a epesentation of the VCS data that is shown on the thee-dimensional and/o two dimensional analyze displays. Figue 1.3 Illustation of the ten light scatte egions 4 A JE? I = JK H A 9DEJA A I? A I 0 ECD ECDJI?=JJAHHAJE?K?OJAI 8 K A 2 =JA EC D JI? = JJA H = JK H A A I! " # $ % & ' 4 AJE?K?OJAI The detected light scatte intensity of the eticulocyte population is divided into the 11 equal egions as shown above. Afte the eticulocyte population is measued and identified as RET% (egions 0 to 10), the spectum of light scatte intensity fo the identified eticulocyte population is analyzed algoithmically. The RET% is calculated as the atio of eticulocytes to Biospecimens Manual UK Biobank Page 28 of

29 USE AND FUNCTION METHOD HISTORY the total numbe of ed cells. The IRF paamete is calculated as the atio of the total numbe of eticulocyte events in the outemost 8 egions (egions 3 to 10) to the total numbe of eticulocytes (defined as egions 0 to 10). The MRV paamete is calculated as the aveage volume of all eticulocytes; o the mean volume of all etic events. NRBC Enumeation The NRBC Enumeation is achieved though the combined use of impedance and VCS technology and a popietay algoithm. The fist step in NRBC enumeation is the identification of paticles in the NRBC signatue position in the diffeential data plot. This infomation is geneated fom VCS analysis of the cells. Figue 1.4 NRBC signatue position on Diffeential Dataplot Once cells have been identified in this egion, the LH 700 Seies examines the fa left egion of the WBC histogam fo the pesence of cells. Figue 1.5 NRBC location on WBC histogam If the VCS dataplot and the WBC histogam both indicate the pesence of NRBCs, then the combined infomation is futhe evaluated fo special data pattens -- such as small lymphocytes, giant platelets, and aging blood. If the combined infomation fom the VCS dataplot and the WBC histogam ae consistent with NRBCs, the NRBC count is deived fom the WBC histogam. 1-6 Biospecimens Manual UK Biobank Page 29 of 123

30 USE AND FUNCTION SYSTEM COMPONENTS 1 COULTER IntelliKinetics Application The LH 700 Seies utilizes the COULTER IntelliKinetics application. Contol of eaction kinetics is extemely impotant to ensue the best pefomance of the automated white cell diffeential and eticulocyte analysis. The IntelliKinetics application is a management tool fo the key step of system optimization when fluctuations in extenal vaiables in the laboatoy, such as tempeatue, occu. The IntelliKinetics application management ensues consistent eaction kinetics. This application intelligently manages vaiations in ambient laboatoy tempeatue though automatic adjustments to eagent eaction tempeatue, exposue time and delivey volumes. Enhancements in instument electonics, such as impoved signal-to-noise atio, wok with the IntelliKinetics application to povide bette data signals fo the system algoithms to analyze. Reagent tempeatue contol helps to incease the speed of dye uptake, theeby impoving instument thoughput. Analysis occus unde contolled conditions. The LH 700 Seies with the IntelliKinetics application shows impoved sepaation of populations, both fo the white cell diffeential and eticulocytes. Cell populations made available fo analysis by the algoithms ae in a moe consistent location in thee-dimensional space. The IntelliKinetics application, woking in concet with new algoithms, povides the instument with the best signals fo analysis, even when the laboatoy envionment vaies thoughout the day. XB Analysis Dennis B. Dosey, MD, poposed in 1963 that the elatively constant blood cell indices could be used to follow the pefomance of hematology instumentation. 32 Bian Bull, MD, impoved the technique and it is temed XB Analysis. 32 XB Analysis uses a "weighted moving aveage" of patient sample esults because Koepke and Potexto said that QC mateials "ideally should be simila in stuctue and in eactivity to the patient constituent being measued. Theefoe feshly dawn patient blood samples seem to be the most appopiate [QC mateial]." 33 Bull explains, "The analyse [sic] is consideed to be in contol when mean MCV, MCH, and MCHC detemined on a batch of 20 patients by use of the algoithm XB ae within 3% of the expected mean indices of the population." SYSTEM COMPONENTS The LH 700 Seies is a modula system that consists of the following units. Powe Supply This unit consists of two assemblies. The Electonic Powe Supply assembly povides the egulated and unegulated voltages equied by the cicuity of the system. The Pneumatic Powe Supply assembly is the souce of ai pessue and vacuum. Dilute This unit is the pimay opeating unit of the system. It pefoms the mixing, tanspoting, pipetting, diluting, lysing, and sensing functions. Biospecimens Manual UK Biobank Page 30 of

31 USE AND FUNCTION HARDWARE OPTIONS Analyze This unit contols the electonic sequence of each opeating cycle and calculates the esults. It eceives count and size infomation diectly fom the Dilute while the sample is being cycled; then it counts, measues, and computes the paametes. The Analyze then sends this infomation to the Wokstation. Many of the contols and indicatos needed fo nomal daily opeation ae on the font of the Analyze. LH 700 Seies Wokstation The LH 700 Seies Wokstation holds the data algoithms used to pocess the List Mode Data supplied by the Analyze. Fom the List Mode Data, the Wokstation computes Diff and Retic esults, develops the histogams and DataPlots, and displays the esults. The Wokstation stoes the data and tansmits it to the Pinte and Host compute. The LH 700 Seies Wokstation is equipped with a mouse that allows opeato inteaction with the softwae. CAUTION System integity can be compomised and opeational failues can occu if: This equipment is used in a manne othe than specified. You intoduce softwae that is not authoized by Beckman Coulte into you compute. You install softwae that is not an oiginal copyighted vesion. Opeate the instument as instucted in you poduct documentation. Only opeate you system s compute with softwae authoized by Beckman Coulte. Only use softwae that is an oiginal copyighted vesion to pevent vius contamination. Handheld Scanne Use the handheld scanne to manually ead ba-code labels. 1.5 HARDWARE OPTIONS Gaphic/Lase Pinte You can use any pinte that is suppoted by the Micosoft WindowsÒ 2000 opeating system. The Pinte pints the data displayed on the LH 700 Seies Wokstation sceen, including paamete data and gaphics. LH 700 Seies SlideMake The LH 700 Seies SM SlideMake makes blood smeas fom samples as they ae being analyzed, accoding to use-defined citeia. LH 700 Seies SlideStaine The SlideStaine stains blood smeas geneated by the LH 700 Seies SlideMake o by manually pepaed blood smeas intoduced into the SlideStaine. 1-8 Biospecimens Manual UK Biobank Page 31 of 123

32 USE AND FUNCTION CONTROLS AND CALIBRATOR CONTROLS AND CALIBRATOR Contols Use stable efeence contols to monito the instument pefomance as pat of you quality contol and to veify calibation. Refe to the package inset fo detailed infomation befoe using a contol. 5C -ES contol monitos the CBC and diffeential (Diff) paametes. LATRON pime pepaes the tubing and instument components fo the LATRON contol. LATRON contol monitos the pefomance of the volume, conductivity and light scatte measuements. Retic-C cell contol monitos the eticulocyte (Retic) paametes. Calibato The S-CAL calibato kit is an acceptable altenative to the whole-blood efeence method of calibation. S-CAL calibato is taceable to efeence methods and mateials. Use S-CAL calibato to ensue accuate instument measuements. Refe to the package inset fo detailed infomation befoe use. The diffeential and eticulocyte measuement devices ae set fo optimum pefomance at the factoy. 1.7 REAGENTS Beckman Coulte ecommends these eagents o thei equivalents. All stated pefomance chaacteistics in this manual ae based on the use of the LH 700 Seies with these eagents. Refe to the containe's label fo detailed infomation befoe using the eagent. Diluent LH 700 seies diluent is an isotonic electolyte solution that: Dilutes the whole-blood samples. Stabilizes cell membanes fo accuate counting and sizing. Conducts apetue cuent. Rinses instument components between analyses. Caies and focuses the sample steam in the flow cell to diect the blood cells though the apetue. Since cell size (volume) is measued, the effect of diluent on osmosis o othe phenomena must be tightly contolled. The diluent must not contain paticles and must not suppot gowth of bacteia o molds. CBC Lytic Reagent LYSE S III Diff lytic eagent: Rapidly lyses eythocytes (RBCs), feeing hemoglobin (Hgb) and educing the size of cellula debis to a level that does not intefee with leukocyte (WBC) count. Causes a substantial convesion of the Hgb to a stable cyanide-containing pigment, the absobance of which is diectly popotional to the Hgb concentation ove the clinical ange. Biospecimens Manual UK Biobank Page 32 of

33 USE AND FUNCTION MATERIAL SAFETY DATA SHEETS (MSDS) LH 700 Seies PAK Reagent System The LH 700 Seies PAK eagent system contains Eytholyse II (PAK LYSE) and StabiLyse (PAK PRESERVE). Eytholyse II (PAK LYSE), Diff Lytic Reagent Eytholyse II eythocyte lytic eagent: Dilutes the blood samples. Rapidly lyses eythocytes (RBCs). Reduces cellula debis to an insignificant level. StabiLyse (PAK PRESERVE), Diff Pesevative StabiLyse leukocyte pesevative: Maintains leukocytes (WBCs) in thei nea-natual state. Allows the leukocytes to be diffeentiated into thei subpopulations though the volume, conductivity and light-scatte measuements. LH 700 Seies RETIC PAK Reagent System The LH 700 Seies RETIC PAK contains Reagent A and Reagent B. Reagent A, Retic Stain Reticulocyte staining solution is a specially fomulated, New Methylene Blue (NMB) dye that stains the eticulum. Reagent B, Retic Cleaing Solution Reticulocyte cleaing solution is a cleaing eagent that emoves hemoglobin fom the eythocytes (RBCs) without emoving the pecipitated dye-rna complex, keeping the cell and its membanes intact. Cleaning Agent COULTER CLENZ cleaning agent cleans and inses the intenal sufaces of the instument components. Daily use pevents potein buildup and eliminates the need fo outine apetue bleaching. 1.8 MATERIAL SAFETY DATA SHEETS (MSDS) To obtain an MSDS fo Beckman Coulte eagents used on the LH 700 SeiesLH 700 Seies: 1. On the intenet, go to and select MSDS fom the Custome Suppot dopdown menu. 2. If you do not have intenet access: In the USA, eithe call Beckman Coulte Custome Opeations ( ) o wite to: Beckman Coulte Inc. Attn: MSDS Requests Biospecimens Manual UK Biobank Page 33 of

34 USE AND FUNCTION MATERIAL SAFETY DATA SHEETS (MSDS) 1 P.O. Box Miami, FL Outside the USA, contact you Beckman Coulte Repesentative. Biospecimens Manual UK Biobank Page 34 of

35 USE AND FUNCTION MATERIAL SAFETY DATA SHEETS (MSDS) Biospecimens Manual UK Biobank Page 35 of

36 2INSTALLATION GENERAL CAUTION Possible system damage can occu if you uncate the instument, install it o set it up. Keep the instument in its packaging until you Beckman Coulte Repesentative uncates it fo installation and setup. You instument is tested befoe it is shipped fom the factoy. Intenational symbols and special handling instuctions pinted on the shipping catons tell the caie how to handle this electonic instument. Caefully inspect all catons when they aive. If you see any sign of mishandling o damage, file a claim with the caie immediately. If the shipment is sepaately insued, file a claim with the insuance company. 2.2 SPECIAL REQUIREMENTS: HARDWARE Install and opeate this instument in a conventional clinical laboatoy envionment. Since the individual units ae all inteelated, you must detemine the system location and layout befoe you Beckman Coulte Repesentative aives to install the instument. Conside the following special equiements. Space and Accessibility In addition to the space equied fo the individual components, conside: Comfotable woking height. Access to pefom sevice pocedues. Allow at least 46 cm (18 in.) fo the ea doos plus sufficient oom fo wok space. Units can be moved to obtain additional wok space. Electical Input CAUTION Intoduction of electical intefeence causing the instument to lock up o eset fequently can occu if you do not plug the pimay powe cables diectly into an electical outlet. Oveheating, melting and buning of the powe lines can occu if you use an extension cod with the pimay powe cables. Plug the pimay powe cables diectly into an electical outlet. Place the instument close enough to an electical outlet that an extension cod is not needed. This instument equies: An independent potected cicuit A thee-wie outlet funishing the applicable line voltage, single-phase input powe A gound path capable of caying the full cuent of the cicuit (confimed thidwie eath gound) That the 3-m (10-ft) pimay powe cod on the ea of the Powe Supply be plugged diectly into the electical outlet. Do not use an extension cod. Biospecimens Manual UK Biobank Page 36 of

37 INSTALLATION SPECIAL REQUIREMENTS: HARDWARE Cuent-caying capacity of 20 A is ecommended, although the actual powe consumption is only 2080 W as shown in the table below: Instument Components Building outlets must be popely gounded and tansients potected. Ambient Tempeatue and Humidity Opeate the system in a oom with a tempeatue of 15.5 to 32 C (60 to 90 F) and humidity up to 95% without condensation. If the aveage oom ambient tempeatue changes moe than 5.5 C (10 F) fom the calibating tempeatue, veify calibation and ecalibate if necessay to ensue confomance to specifications. Ai Conditioning In ai-conditioned envionments, an additional 5,500 Btu is equied to compensate fo the heat the system geneates. Ventilation All ventilation fans must be at least 25 cm (10 in.) away fom walls o obstuctions that could intefee with the flow of ai. Dainage Watts Analyze, Dilute, Powe Supply 860 SlideMake 430 SlideStaine 430 Compute 300 Monito CAUTION Incomplete waste chambe dainage and eventual waste chambe oveflow into the vacuum system can occu if the waste line is too long. Contact you Beckman Coulte Repesentative if you need to incease the length of the waste line supplied with the instument. WARNING Biohazadous contamination could occu fom contact with the waste containe and its associated tubing if not handled with cae. Avoid skin contact. Clean up spills immediately. Dispose of the contents of the waste containe in accodance with you local egulations and acceptable laboatoy pocedues. The maximum waste line length is 3.7 m (12 ft). The waste dain tubing (ea panel of the Dilute) supplied with the system can be connected to eithe: An open dain, suitable fo biohazadous waste, less than 76 cm (30 in.) above the floo A waste containe with a minimum capacity of 20 L (5 gal.). 2-2 Biospecimens Manual UK Biobank Page 37 of 123

38 .7 4) / - 9 ) 4 1 / INSTALLATION INTERUNIT CONNECTIONS 2 When using an open dain instead of a waste containe: Mechanically secue the waste tube into the dain so the tube cannot accidentally come out of the dain. This pevents spillage. Be sue to dispose of waste in accodance with envionmental potection egulations. 2.3 INTERUNIT CONNECTIONS Powe and Signal Cables Figue 2.1 shows the inteunit connections of the powe and signal cables that ae supplied with the instument. You Beckman Coulte Repesentative makes these connections when installing the instument. Figue 2.1 Inteunit Powe and Signal Cable Connections ) = O A H 9 H IJ=JE K I A A O > = )+ F M AH 5 E K JA H 8 =? #FIE!FIE )+ F M AH + = > A =@=FJAH EJ H I?= AH 6 FHE JAH 1 I JHK A J 5 E@ A 5 J= E A H 0 I J+ F K JA H " "&8F M AH #? JH IEC = I 2 M A H 5KFF O 2 H E = H O F M AH? H@ Biospecimens Manual UK Biobank Page 38 of

39 INSTALLATION INTERUNIT CONNECTIONS Pneumatic/Hydaulic Tubing Connections CAUTION Possible eagent siphoning effect and piming poblems can occu if a eagent containe is placed above the level of the Analyze. Do not place eagent containes above the level of the Analyze. IMPORTANT Placing Reagent Paks in any location othe than on the counte next to the instument may cause eoneous esults. Figue 2.2 shows the tubing connections between the Dilute and the: Reagent containes Waste containe Powe Supply pessue and vacuum supplies. Figue 2.2 Pneumatic/Hydaulic Connections B A WASTE LEVEL SENSOR DILUENT WASTE CBC LYSE RETIC CLEAR RETIC STAIN PACK PRESERVE PACK LYSE CLEANER VAC 30 PSI 5 PSI Back of dilute Diluent Waste Cleaning agent 2 Retic clea 3 Retic stain 1 CBC 4 lyse Pak peseve Powe supply A 5 Pak lyse 30 PSI 5 PSI VAC B Retic pak Scatte pak 2-4 Biospecimens Manual UK Biobank Page 39 of 123

40 3OPERATION PRINCIPLES COULTER METHOD CBC Analysis The Coulte method counts and sizes cells by detecting and measuing changes in electical esistance when a paticle (such as a cell) in a conductive liquid goes though a small apetue. See Figue 3.1. Figue 3.1 Coulte Method of Counting and Sizing VACUUM APERTURE CURRENT INTERNAL ELECTRODE EXTERNAL ELECTRODE SAMPLE BEAKER BLOOD CELL SUSPENSION DETAIL OF APERTURE APERTURE APERTURE TUBE Each cell suspended in a conductive liquid (diluent) acts as an insulato. As each cell goes though the apetue, it momentaily inceases the esistance of the electical path between two submeged electodes, one located on each side of the apetue. This causes an electical pulse that can be counted and sized. While the numbe of pulses indicates paticle count, the size of the electical pulse is popotional to the cell volume. 35,36,37,38 Diffeential Analysis WBC diffeential analysis and classification occus in the flow cell, whee: Low-fequency cuent measues volume, High-fequency cuent senses cellula intenal content though measuing changes in conductivity, Light fom the lase bouncing off the individual WBC cells chaacteizes cellula suface, shape and eflectivity. Biospecimens Manual UK Biobank Page 40 of

41 OPERATION PRINCIPLES AUTOMATIC ASPIRATION MODE Effect of Reagents The conductive diluent must affect cells minimally, if at all. Both lytic eagents must destoy eythocytes without significantly affecting leukocytes. They must wok apidly to satisfy the speed with which the system woks. The leukocyte pesevative must Povide clea sepaation of the white blood cell populations, and Peseve leukocytes in thei nea-natual state fo accuate cytometic measuement. Reticulocyte Analysis A supavital dye, New Methylene Blue, is incubated with whole-blood samples. The dye pecipitates the basophilic RNA netwok found in eticulocytes. Hemoglobin and unbound stain ae emoved by adding a cleaing eagent, leaving clea spheical matue RBCs and dakly stained eticulocytes. Stained eticulocytes ae diffeentiated fom matue ed cells and othe cell populations by light scatte, diect cuent measuements, and opacity chaacteistics. 3.2 AUTOMATIC ASPIRATION MODE The system automatically tanspots, mixes, aspiates and pocesses specimens. Loading Specimens 1. The opeato places specimen tubes, which can be identified by ba-code labels, into cassettes. Each cassette and the tube positions in the cassette ae identified by ba-code labels. 2. You can load up to 12 standad o Hemogad cassettes with 144 samples into the loading bay at one time. Figue 3.2 shows the loading bay filled with cassettes. Tanspoting Cassettes The system tanspots each cassette fom the loading bay to the sampling station. 1. The ight lift platfom (Figue 3.2) ises beneath the stacked cassettes in the loading bay. 2. The bottom cassette is deposited on the platfom. 3. The platfom lowes the cassette to the level of the ocke bed. 3-2 Biospecimens Manual UK Biobank Page 41 of 123

42 4 - ), ; I II III IV V VI VII VIII OPERATION PRINCIPLES AUTOMATIC ASPIRATION MODE 3 Figue 3.2 Tanspot System 5 ; "!! & 5 6 ) ), ; ) , -, - +, 4 ) ) * - ) 1-7 *,, ) 4, 4 ) ) ) ) , 9 " % + -! # $ & ' 5 6 ) 4 6) ) : , ) = O AH AOF=@ =@E C>=O BE M EJD?=IIAJJAI =@E C =HA= 4? AH>A@ F =JB H 4? A H >A@ 4 EC D J EBJ F =JB H 4. The cassette moves onto the ocke bed whee it ocks back and foth, mixing the specimens. 5. The cassette moves towad the sensing station until it eaches the tube senso. 6. When the fist tube is sensed, the stippe plate locks onto the tube. 7. Afte at least 14 ocks fom the time the cassette was loaded, the ocke bed locks in a 45-degee fowad position. 8. At the sampling station, the tube is locked in position. 9. The tube am pushes the tube out fom the cassette, causing the needle to piece the tube stoppe. 10. The ba-code eade scans the cassette and tube labels on both its fowad and etun passes; an audible indicato can be enabled to indicate each coectly-ead ba-code. If the two ba-code scans ae identical, the bed continues to ock, and the cassette moves fowad until the next available tube eaches the sampling station. If the ba-code eade detects a discepancy between the fowad and etun eadings, it makes an additional pass. If the ba-code eade cannot ead the cassette ba-code label, the Dilute stops, posts a message on the Analyze, Dilute and Wokstation displays, backwashes the system, then etuns to the Ready state. If Cass/Pos is the Positive Identifie, and the ba-code eade cannot ead the tube ba-code label, dashes (-----) appea in the Sample ID field on the Wokstation with the sample esults. 11. Afte the last tube in the cassette is aspiated, the cassette continues along the ocke bed and is deposited onto the unloading bay platfom. The left platfom lifts the cassette into the exit bay, whee up to 12 cassettes can be stacked. Biospecimens Manual UK Biobank Page 42 of

43 OPERATION PRINCIPLES AUTOMATIC ASPIRATION MODE Aspiation At the sampling station, afte the cap is pieced: 1. A pump daws a maximum of 300 µl (LH 700 Seies only) o 550 ul (LH 700 Seies SM SlideMake) of sample though the needle and though the Blood Sampling Valve (BSV). 2. The blood detectos monito the passage of sample though the BSV and aspiation lines. 3. The needle is withdawn and the sample tube is eseated in the cassette. Delivey CBC Afte the sample is aspiated: The cente section of the BSV otates and segments the sample into two sepaate volumes. Beginning a few seconds befoe the delivey of the dilutions to the appopiate baths, 5 psi of pessue is sent to the WBC bath. This pessue allows dainage of any esidual liquid in the WBC bath, thus peventing cayove. The pessue continues duing delivey and foms bubbles that mix each cell suspension befoe sensing begins. At the beginning of the delivey, any esidual inse in the Hgb cuvette dains into the waste chambe and the waste chambe dains. Diluent fom the diluent dispenses dives the sepaated volumes of sample fom the BSV to the baths. One volume of sample, 1.6 µl, is deliveed with 10 ml of diluent to the RBC bath. This dilution is used fo RBC/Plt counting and MCV/Plt sizing. The othe volume, 28 µl, is deliveed with 6 ml of diluent to the WBC bath. This dilution is used to count WBC and develop Hgb. Duing delivey to the WBC bath, 1 ml of lytic eagent is added to the dilution to lyse the ed cells and convet Hgb. At the same time the lytic eagent is dispensed, 5 ml of diluent fom the backwash tank is tansfeed into the Hgb cuvette fo the Hgb blank eading. The final dilution in the WBC bath is 1 pat whole blood in a total volume of 251 pats. The final dilution in the RBC bath is 1 pat whole blood in a total volume of 6250 pats. The vent section of the piecing needle is insed, then died by high vacuum. The cente section of the BSV etuns to the aspiate position. Diffeential (Diff) and Retic At the same time as the segmented pats of the sample ae being deliveed to the baths, the Diff and Retic segmenting modules segment additional 31 µl samples of the blood fo analysis. Fo the WBC diffeential, the sample and appoximately ml of heated Diff lytic eagent ae deliveed to the mixing chambe, which agitates to mix them thooughly. 3-4 Biospecimens Manual UK Biobank Page 43 of 123

44 OPERATION PRINCIPLES AUTOMATIC ASPIRATION MODE 3 Duing the mixing pocess, appoximately 0.2 ml of Diff pesevative entes the mixing chambe to peseve the leukocyte populations, and the instument initiates the sheath steam of diluent in the tiple-tansduce flow cell. Fo the Retic analysis, the sample and appoximately 166 µl of Retic stain ae deliveed to the Stain chambe, whee they ae mixed, heated and incubated fo 31 seconds. A 2-µL segment of the stain/blood mixtue and 2.00 ml of heated o cooled Retic cleaing solution ae deliveed to the Retic chambe, whee they ae mixed and incubated fo 25 seconds. The sample line between the Retic chambe and the flow cell is pimed with the stain/blood/cleaing solution befoe cell counting begins. Afte the sample line is pimed, the flow cell is cleaned and pimed with diluent, and the sheath steam of diluent begins. Fo both the WBC diffeential and the Retic analysis, the instument injects the sample into the cente of the sheath steam and activates the flow cell apetue cuent. The lamina flow guides the sample though the cente of the flow cell apetue; the sheath steam on the exit side of the flow cell apetue pevents the sample's cells fom e-enteing the apetue. CBC Sensing System Vacuum, equal to 6 in. of mecuy, daws a pecise volume of suspension fom each bath though the thee apetues. At the same time, sweep flow is dawn behind the RBC apetues to pevent cells fom e-enteing the sensing zone. When the vacuum stats to daw the suspension, cuent is supplied to the electode. The electical path allows sensing of the numbe and volume of each cell pulled though the apetues. While the sample in each bath is sensed, the photomete eads the Hgb-blank and the Analyze etains this efeence voltage. CBC Analysis in the Baths The RBC and Plt data is geneated by the RBC bath. The WBC and Hgb data is geneated by the WBC bath and Hgb cuvette. In the analyze the R/W Poc counts and sizes the RBC and WBC data. The PLT Poc sizes the PLT data. The Comm Intfc measues the HGB blank and sample. The RBC and WBC aw data consists of counts, waittime counts, count time, and channelyze time, as well as histogams fo each of the thee apetues. The PLT aw data consists of the histogam and channelyze time fo each apetue. The HGB aw data consists of two voltage measuements fo the blank and two measuements fo the sample. The Analyze then sends the aw data to the wokstation. The wokstation then: Wait time and Coincidence coects the RBC and WBC aw counts Fit the PLT histogam and Coincidence coect the PLT histogam count. Calculate HGB fom the blank and sample eadings Scale fo calibation and dilution Biospecimens Manual UK Biobank Page 44 of

45 5 ; "! I! & II III IV V 5 6 ) ), ; 6-5 6, - VI ) ) ) * - VII, - +, 4 ) 1-7 VIII *,, ) ) 4 6) ) 4, 4 ) ), ; : ) ) ) ! 1, , 9 " # $ % & ' OPERATION PRINCIPLES AUTOMATIC ASPIRATION MODE Pefom Voting on the thee apetues fo RBC, WBC, PLT. Deives the MCV and RDW paametes fom the RBC histogam; Deives the MPV and PDW paametes fom the Plt histogam. Displays esults and histogams. Diffeential and Retic Multipaamete Sensing System Fo the WBC diffeential and fo the Retic analysis, the multipaamete sensing system poduces the thee measuement signals. Figue 3.3 shows the Tiple Tansduce Module and its potective housing as it esides in the Dilute. WARNING The lase beam can cause damage to you eyes and to the instument. Do not attempt to emove the lase fom the Dilute module. If emoval is equied, it must be done only by a Beckman Coulte Repesentative. Tampe-poof scews secue the potective housing; they can only be emoved with a special tool. The lase is a helium-neon lase that complies with the United States' pefomance standad fo lase poducts, Title 21 Code of Fedeal Regulations and Figue 3.4 shows the lase module without its potective housing to display the flow cell and label locations. Figue 3.3 Tiple Tansduce Module with Potective Housing?=JA@ >=? BK EJ ) , * + 0 ) " ), " + ) 5 5 ) , = K B =? J K H ) 6 - ' > O ) 6 1 E= E. =!! ' $ ) ), 1 ) ), , -. - ) 6 -, ) 8 1,, ; - - : Biospecimens Manual UK Biobank Page 45 of 123

46 OPERATION PRINCIPLES AUTOMATIC ASPIRATION MODE 3 Figue 3.4 Tiple Tansduce Module with Potective Housing Cut Away LASER ON LAMP LASER RADIATION WHEN OPEN AND INTERLOCK DEFEATED. AVOID DIRECT EYE EXPOSURE. ELECTROMAGNETIC SHIELD LASER RADIATION IS EMITTED FROM THIS APERTURE AVOID EXPOSURE FLOW CELL 1096 Mellon Avenue Manteca, CA MODEL MANUFACTURED SERIAL NO. THIS LASER DOES NOT COMPLY WITH 21 CFR USE ONLY AS A COMPONENT. SEE INSTALLATION INSTRUCTIONS. PATENT NOS 4352, , , ,583 MADE IN USA TAMPER-PROOF SCREWS LASER Note: As installed in the Tiple Tansduce Module (TTM) safety fixtue, the lase pesents no adiation hazad to uses and complies with 21 CFR WBC Diffeential Analysis The LH 700 Seies makes thee measuements (volume, conductivity, and scatte) as each cell passes though the flow cell. The low-fequency impedance measuement defines cell volume. The high-fequency conductivity measuement indicates the intenal conductivity. The light-scatte measuement indicates the stuctue and shape. Thee aw analog signals ae sent to the Analyze fo amplification and signal pocessing. The Analyze sends this aw data to the Wokstation fo computation and Data Plot geneation of the five diffeential paametes. The Analyze sends the CBC esults to the Wokstation. All esults ae displayed at the same time. Reticulocyte Analysis Reticulocyte samples ae un in one of the thee RETIC modes. The modes, which ae set at the Wokstation, ae: RETIC CBC/RETIC CBC/DIFF/RETIC Biospecimens Manual UK Biobank Page 46 of

47 OPERATION PRINCIPLES MANUAL ASPIRATION MODE The LH 700 Seies uses the Tiple Tansduce Module to measue these paametes: Reticulocyte pecent (RET%) - the numbe of eticulocytes pe 100 RBCs, diectly measued and epoted as a pecentage of RBCs. Reticulocyte numbe (RET#) - the absolute numbe of eticulocytes, calculated fom RET% and RBC numbe. Expessed and epoted as 10^6 cells/µl o 10^9 cells/l (US units). Mean eticulocyte volume (MRV) the aveage cell volume of the eticulocytes, detemined by the VCS technology algoithm and epoted in femtolites (fl). Immatue eticulocyte faction (IRF) - a calculated atio using the count of the highest light scatte eticulocytes to the total count of eticulocytes. The highe the light scatte the moe immatue the eticulocytes ae. The degee of stained eticulum (using New Methylene Blue) is popotional to the degee of the RNA pesent in the cell and thus the amount of light scatte poduced. It is epoted as a decimal. Backwash and Rinse The Dilute pefoms backwash of aspiation pathways and inses its components. Appoximately 0.5 ml of Eytholyse II eythocyte lytic eagent is deliveed to the mixing chambe to emove esidual mateial fom the pevious cycle. 10 ml of diluent inse fo the WBC bath comes fom the RBC diluent dispense, and 6 ml of diluent inse fo the RBC bath comes fom the WBC diluent dispense. The WBC bath needs a lage inse volume to Remove RBC cell stoma afte lysing Remove emaining lytic eagent Rinse above the 7 ml fill line Rinse the hemoglobin cuvette. 3.3 MANUAL ASPIRATION MODE IMPORTANT Clots in the specimen can cause misleading esults. The blood detectos ae not active in the Manual mode. Be sue to inspect the specimen fo clots, and use good laboatoy pactices to veify esults. The Manual mode of opeation is like the Automatic mode except: Befoe you un the sample, you ente the sample identification numbe by pefoming any of the following-- t Scanning the ba-code label on the tube t Enteing the ID numbe on the Numeic Keypad t Enteing the ID numbe in the ba-code field on the LH 700 Seies Wokstation fom its keyboad. You use an open vial and intoduce the sample at the aspiato tip. You begin the cycle by eithe t Obstucting the optical senso s beam 3-8 Biospecimens Manual UK Biobank Page 47 of 123

48 OPERATION PRINCIPLES COUNTING AND SIZING 3 t Pessing the panel behind the tip. The system aspiates a maximum of 200 µl of sample. The blood detectos ae not active in the Manual mode. 3.4 COUNTING AND SIZING Red and White Blood Cell Counting The RBC and WBC baths each have thee discete apetues that function as independent systems. The six apetue cuents ae individually adjusted duing calibation. When the Apetue Cuent/Signal Geneato (API-SIG GEN) cad applies cuent to the apetues, thee is a delay. Duing this delay, the system conditions the electonics to pefom the counting and sizing of the sample. Regulated vacuum daws a pecise volume of sample dilution though the apetues. At each apetue, the system gathes pulses fo 4 seconds. The RED/WHITE PRE-AMP cads amplify these pulses and the Analyze sceen displays them. The system sends these pulses to the RED/WHITE PROCESSOR cad. The RED/WHITE PROCESSOR cad counts and sizes the RBC and WBC data. The RBC and WBC aw data consists of counts, waittime counts, count time, and channelyze time, as well as histogams fo each of the thee apetues The Analyze then sends the aw data to the wokstation. The wokstation then: Wait time and Coincidence coects the RBC and WBC aw counts Fit the PLT histogam and Coincidence coect the PLT histogam count. Calculate HGB fom the blank and sample eadings Scale fo calibation and dilution Pefom Voting on the thee apetues fo RBC, WBC, PLT. Deives the MCV and RDW paametes fom the RBC histogam; Deives the MPV and PDW paametes fom the Plt histogam. Displays esults and histogams. Coincidence Coection Occasionally, moe than one cell passes though the apetue at the same time. When cells coincide, the Analyze counts only one pulse. As the fequency of coincidence is popotional to the actual count, the system automatically coects esults fo coincidence. Voting To pevent data eos due to statistical outlies o obstuctions that may block an apetue, the Analyze votes on the data fom all of the apetues, and ejects any questionable data. Fo the WBC count, RBC count, MCV, RDW, Plt count, and MPV, the Analyze compute compaes the data fom the thee apetues. It veifies that at least two apetues have poduced data within an established statistical ange of each othe. Biospecimens Manual UK Biobank Page 48 of

49 OPERATION PRINCIPLES COUNTING AND SIZING If the data fom one apetue is outside the established statistical ange, the compute votes out the data and histogams fom that apetue. The compute deives the affected paamete by aveaging the data fom the two emaining apetues. The data fom at least two of the thee apetues must be within an established statistical ange of each othe, o the system totally votes out the paamete and histogams. When a paamete totally votes out, the system does not give any esults fo the affected paamete o fo any paametes that ae deived fom it. See you Online Help System o Opeato s Guide fo codes and messages that appea in these cicumstances. Pulse Editing When cells pass though the apetue nea the edge o at an angle athe than at the cente, they ceate atypical pulses. The RED/WHITE PROCESSOR cad edits RBC and WBC pulses to exclude these atypical pulses fom analysis because they distot the tue size of the cell. This pevents the atypical pulses fom influencing size measuement. Each of the six apetues has an edito. Sweep Flow The sweep flow is a steady steam of diluent that flows behind the RBC apetue duing the sensing peiod. This pevents cells fom e-enteing the sensing zone and being counted as platelets. See Figue 3.5. Figue 3.5 Sweep Flow A B TO WASTE SENSING ZONE CELL CELL SENSING ZONE SWIRLING EFFECT NO SWEEP FLOW SWEEP FLOW RBC Size Distibution Afte editing, the RED/WHITE PROCESSOR convets the thee sets of both the RBC and WBC pulses. It convets each pulse to a numbe that coesponds to the size of the cell. The Analyze Micocontolle cad (AMC) eads this data and geneates histogams. The digital infomation fom each apetue is stoed accoding to volume in 256-channel, size-distibution histogams. To ensue that the size-distibution cuve accuately eflects the tue cell population, the system extends RBC sensing fo up to eight additional 2-second sensing peiods wheneve Biospecimens Manual UK Biobank Page 49 of

50 OPERATION PRINCIPLES COUNTING AND SIZING 3 the RBC data accumulations ae below a pedetemined value. The RBC size distibution cuve eflects the total data accumulated in all of the sensing peiods. Afte the sensing peiods ae completed, the system sends these histogams though the Communication Inteface (COMM INTFC) cad to the Wokstation fo display. Plt Count and Size Distibution Pulses epesenting cells fom 2 to 20 fl ae classified as platelets in the PLATELET PROCESSOR cad. The system digitizes the Plt pulses. The AMC cad eads the digital infomation and channelyzes it into thee 64-channel size-distibution histogams.the PLT channelyzing time is 2 seconds minimum, up to 20 seconds maximum, in 1 second incements. PLT channelyzing stops when thee ae at least 1500 cells channelyzed in each of the 3 histogams, o the 20 second maximum time is eached. Afte the sensing peiods ae completed, the system sends these histogams and channelyzing time though the COMM INTFC cad to the Wokstation. In the Wokstation, an initial platelet count is deived fom the fitted o aw histogams and the channelyzing time. Then, coincidence coection is applied. MPV and PDW ae deived fom the histogams. Pct is calculated. The compute votes on the paamete esults fom each histogam (fitted o not fitted). Results ae scaled fo epoted unit and calibation, and the paametes displayed on the Wokstation. Plt Fitting Pocess The Platelet Algoithm smooths the platelet histogam fom each apetue. The algoithm then finds a maximum (mode) point and two minimum points (valleys) in each smoothed histogam. The minimum points ae located to the left and the ight of the maximum point. The algoithm uses all the infomation in the aw histogam within the two minimum points to fit an extapolated cuve to the log-nomal smoothed cuve, using a least-squaes fit method. The Algoithm veifies that each of the fitted cuves: 1. Is positive and unimodal 2. Has a mode between 3 and 15 fl 3. Has a PDW less than 20. (NOTE: This is an appoximate value since the measuement is made befoe esults ae scaled fo calibation.) 4. Has a atio between the aw and fitted histogams mode amplitudes of less than Has a atio between channels 0 and 1 and the mode amplitudes of less than Has a plt count appoximately geate than 20. If any of these conditions ae not met, the algoithm deives the platelet count fom the potion of the aw histogam between the two minimum points. Condition 5 above causes geneation of a Plt no-fit condition whee the Plt is flagged with a Review (R) flag, and the 'Low Plt Intefeence' message is displayed on the Reseach sceen. Additionally, Plt esults ae Review (R) flagged when the 'High Plt Intefeence' flag occus (the text message is displayed on the Reseach sceen). Biospecimens Manual UK Biobank Page 50 of

51 OPERATION PRINCIPLES MEASUREMENT OF HEMOGLOBIN CONCENTRATION Retic Paametes The system makes thee measuements as each cell passes though the flow-cell apetue: The low-fequency impedance measuement, which defines the cell s volume The high-fequency impedance measuement, which indicates the cell s intenal conductivity The light-scatte measuement, which indicates the cell s stuctue and shape. 3.5 MEASUREMENT OF HEMOGLOBIN CONCENTRATION Afte the WBC count, the lysed WBC dilution dains into the hemoglobin cuvette fo Hgb measuement. A beam of white light fom an incandescent lamp goes though the cuvette and then though an optical filte that has a cente tansmission wavelength of 525 nm. Light passing though the filte falls on a photocell. The photocuent thus geneated is popotional to the tansmittance of the contents of the cuvette at the chosen wavelength. It is sent to the COMM INTFC cad whee it is digitized. The digital infomation is sent to the Analyze compute, then to the Wokstation. A significant efinement of the Beckman Coulte systems is the intoduction of a eagent blank into the cuvette duing each opeating cycle. Afte the pecent tansmittance is conveted to absobance, the eagent-blank signal level povides a efeence to which the sample signal is compaed. 3.6 DATAPLOT DEVELOPMENT The Wokstation pefoms a seies of opeations on the stoed digital aw values eceived fom the flow cell to identify membeships (subpopulations) and calculate the fequency of cells within each membeship goup. It also poduces the DataPlot displays fo visual epesentation of the WBC Diff and Reticulocyte/RBC membeship and density. In the LH 700 Seies, the Beckman Coulte AccuGate algoithm uses adaptive contouing methods designed fo finding optimal sepaation between ovelapping clustes of data. The AccuGate algoithm povides a statistical analysis tool that discoves the ovelapping populations using nonlinea sepaation techniques. The newly developed adaptive gating techniques use multidimensional data to distinguish the pesence of even the faintest subpopulation. The AccuGate algoithm can adapt to unusual population shifts and ovelaps. It can define highly iegula sepaation, and signal the need fo futhe analysis. It can then make a subsequent analysis of the identified egions and coect deficiencies in sepaation. In the DataPlots, diffeent colos epesent diffeent membeships (types of cells). Shades of colos epesent density (concentation): dak colos fo low density, bight colos fo high density. DIFF: Lymphocytes Blue Neutophils Puple Eosinophils Oange Monocytes Geen Basophils White Non-white debis Red RETIC: RBCs Red Reticulocytes Blue Platelets Geen WBCs Puple Biospecimens Manual UK Biobank Page 51 of

52 OPERATION PRINCIPLES PARAMETERS AND THEIR DERIVATION 3 Two-Dimensional (2D) DataPlots The 2D WBC Diff DataPlot shows the five membeships: lymphocytes (LY), monocytes (MO), neutophils (NE), eosinophils (EO) and basophils (BA), plus the non-white cell populations. Cell volume (VOL), detemined by the low-fequency impedance measuement, is plotted on the Y-axis; otated light scatte (RLS) is plotted on the X-axis. The Reticulocyte DataPlot shows matue ed cells and Reticulocytes. Cell volume is plotted on the Y-axis, and linea light scatte (LLS) is plotted on the X-axis. Thee-Dimensional (3D) DataPlots The 3D DataPlot view classifies by density, light scatte and opacity. The axes ae colo coded. On the WBC diffeential DataPlot the axes ae: Volume = geen RLS (otated light scatte) = ed OP (opacity) = blue On the Retic DataPlot the axes ae: Volume = geen LLS (linea light scatte) = ed OP (opacity) = blue 3.7 PARAMETERS AND THEIR DERIVATION Mathematical expessions in this section ae in U.S. units of measuement. Paamete units can be changed to othe Intenational System of Units (SI) though Setup on the LH 700 Seies Wokstation. White Blood Cell (WBC) Count WBC is the numbe of leukocytes measued diectly, multiplied by the calibation facto. This numbe is expessed as WBC = n 10 3 cells / µ L Red Blood Cell (RBC) RBC is the numbe of eythocytes measued diectly, multiplied by the calibation facto. This numbe is expessed as RBC = n 10 6 cells / µ L Hemoglobin (Hgb) Concentation The tansmittance of light (525 nm wavelength) though the lysed WBC solution in the hemoglobin cuvette is compaed to the tansmittance of the same light though a eagent blank. The system convets this atio to an Hgb value in g/dl using a calibation facto. Weight (mass) of hemoglobin detemined fom the degee of absobance found though photocuent tansmittance is: Biospecimens Manual UK Biobank Page 52 of

53 OPERATION PRINCIPLES PARAMETERS AND THEIR DERIVATION HGB (g / dl) = Constant x log 10 Refeence %T Sample %T Mean Copuscula Volume (MCV) MCV is the aveage volume of individual eythocytes deived fom the RBC histogam. The system multiplies the numbe of RBCs in each channel by the size of the RBCs in that channel. The poducts of each channel between 36 fl and 360 fl ae added. This sum is divided by the total numbe of RBCs between 36 fl and 360 fl. The analyze then multiplies by a calibation constant and expesses MCV in femtolites. Hematocit (Hct) Hct is the elative volume of packed eythocytes to whole blood, computed by the fomula: HCT (%)= (RBC x MCV) / 10 Mean Copuscula Hemoglobin (MCH) MCH is the weight of hemoglobin in the aveage eythocyte, computed by the fomula: MCH (pg) = (HGB/RBC) x 10 Mean Copuscula Hemoglobin Concentation (MCHC) MCHC is the aveage weight of hemoglobin in a measued dilution, computed by the fomula: MCHC (g/dl) = (HGB/HCT) x 100 Red Distibution Width (RDW) RDW is the size distibution spead of the eythocyte population deived fom the RBC histogam. It is the coefficient of vaiation (CV) expessed in pecent of the RBC size distibution. Platelet (Plt) Count Plt is the numbe of thombocytes deived fom the Plt histogam and multiplied by a calibation facto. This numbe is expessed as: PLT = n 10 3 cells / µ L Mean Platelet Volume (MPV) MPV is the aveage volume of individual platelets deived fom the Plt histogam. It epesents the mean volume of the Plt population unde the fitted Plt cuve multiplied by a calibation facto. This numbe is expessed in femtolites. NRBC % (Nucleated Red Blood Cells) NRBC enumeation is achieved though the combined use of VCS technology along with an advanced algoithm applied to the WBC count. NRBC% is defined as the numbe of nucleated ed blood cells pe 100 WBC. Biospecimens Manual UK Biobank Page 53 of

54 OPERATION PRINCIPLES PARAMETERS AND THEIR DERIVATION 3 NRBC # (Nucleated Red Blood Cells) A paamete that is calculated fom the NRBC % and the total WBC count. NRBC # epesents the total numbe of nucleated Red Blood Cells. NRBC (103 cells µl) = NRBC% x WBC count Diffeential (Diff) Diff Pecentages (DIFF%) The pecentages of leukocytes fom each categoy ae deived fom the DataPlot. NE% = no. of cells inside NE aea no. of cells inside NE + LY + MO + EO + BA 100 LY% = no. of cells inside LY aea no. of cells inside NE + LY + MO + EO + BA 100 MO% = no. of cells inside MO aea no. of cells inside NE + LY + MO + EO + BA 100 EO% = no. of cells inside EO aea no. of cells inside NE + LY + MO + EO + BA 100 BA% = no. of cells inside BA aea no. of cells inside NE + LY + MO + EO + BA 100 Diff Absolute Numbes (DIFF#) The absolute numbes of leukocytes in each categoy ae computed fom the WBC count and diffeential pecentage paametes. NE (10 3 cells / µ L) = NE% 100 LY (10 3 cells / µ L) = LY% 100 WBC count WBC count MO (10 3 cells / µ L) = MO% 100 EO (10 3 cells / µ L) = EO% 100 WBC count WBC count BA (10 3 cells / µ L) = BA% 100 WBC count Biospecimens Manual UK Biobank Page 54 of

55 OPERATION PRINCIPLES PARAMETERS AND THEIR DERIVATION Reticulocyte (Retic) Paametes Retic Pecent (RET%) Numbe of eticulocytes pe 100 RBCs. This paamete is diectly measued and epoted as %, a pecentage of RBCs.The RET% is calculated as the atio of eticulocytes to the total numbe of ed cells; this calculation is shown below: Retic% = Retics Red_Cells Retic Absolute Numbe (RET#) Absolute numbe of eticulocytes computed fom the eticulocyte pecent (RET%) multiplied by the RBC count. RET# = RET% x RBC Count 100 Immatue eticulocyte faction (IRF) The pecentage of the count of the highest light scatte eticulocytes elative to the total count of the eticulocytes is calculated and is epoted optionally as a decimal atio. The IRF paamete is an indication of new eticulocyte synthesis. It is calculated fom the RET% as the total numbe of eticulocyte events in the outemost light scatteing egion, coesponding to immatue eticulocytes, elative to the total numbe of eticulocytes and is epoted as this atio. The algoithm fo calculating the IRF paamete is shown below: IRF = Region - 10 Retics Region - 3 Region - 10 Region - 0 Retics Mean eticulocyte volume (MRV) The aveage volume of individual eticulocytes deived using VCS eticulocyte algoithm. It is calculated and is epoted in femolites. The MRV paamete is the aveage volume of all eticulocytes (o the mean volume of all etic events). It is calculated fom the RET% and epoted in femolites (fl). The MRV algoithm is shown below: MRV = Region - 10 Retics_Volume Region - 0 Region - 10 Region - 0 Retic Biospecimens Manual UK Biobank Page 55 of

56 OPERATION PRINCIPLES PARAMETERS AND THEIR DERIVATION 3 Summay of Paamete Deivations Paametes ae eithe: Measued diectly. Deived fom the RBC o Plt histogam. Computed. Measued Diectly Paamete RBC WBC Hgb DIFF% paametes RET% Method Coulte Pinciple Coulte Pinciple Photometic Measuement VCS Technology VCS Technology Deived Fom RBC o Plt Histogam Paamete MCV RDW Plt MPV Deived Fom RBC Histogam RBC Histogam Plt Histogam Plt Histogam Deived Fom WBC Histogam Paamete NRBC% Deived Fom WBC Histogam and VCS Technology Computed Hct MCH MCHC DIFF# paametes RET# IRF MRV NRBC# Biospecimens Manual UK Biobank Page 56 of

57 OPERATION PRINCIPLES XB ANALYSIS 3.8 XB ANALYSIS Studies (Bull 1974, Koepke 1981) indicate that the ed cell indices (MCV, MCH, and MCHC) of patient populations ae stable ove time. 39,40 This stability chaacteistic of the indices is the basis of a quality-contol technique called XB Analysis. In a manually implemented system, population means (taget values) ae established by analyzing as lage a sample as possible, at least 250, but ideally 1,000 blood samples. (The XB Analysis used in the Wokstation does all the calculating automatically.) Once the taget values have been established, the XB Analysis can be applied using quite small batches fom the patient population. A 20-patient sample batch is a typical size, and can be used in the LH 700 Seies Wokstation. Hee is the XB fomula. N X( B,) i = X( B, i 1) + SGN SGN[ X(,) j i X( B, i 1)] X(,) ji X( B, i 1) F j= 1 F = N SGN X ji XBi X ji Bi (,) (, 1) (,) (, 1) j= 1 2 N 2 Whee: X(B,i) X(B, i-1) X(j,i) SGN N x = ith XB value = (i-1)th XB value = the jth X value in the ith batch = the aithmetic sign of numbe in paentheses = numbe of samples in the batch = symbol used to epesent multiplication The fomula is easily implemented with a compute. Its function is to enable eliable estimates of the values fo these paametes to be made fo a population fom small samples of that population. It is supeio to the taditional moving aveage because it eacts quickly to changes. Small batch sizes allow fo moe fequent, theefoe tighte quality contol. The fomula both tims the data by giving less weight to outlies, and smoothes it by incopoating infomation fom the pevious patient batch in the analysis of the cuent batch. As each sample is pocessed, the mean of the pevious set of samples is subtacted fom each of the ed cell indices. The squae oot of this deviation (diffeence between the means) is stoed. Afte 20 samples have been pocessed, the sum of the squae oots is divided by 20. The esult is squaed to ecove the mean (aveage) deviation. The individual deviations cay a positive o negative sign, so then it can be added to o subtacted fom the coesponding pevious means. The esulting new mean is then used fo the succeeding batch of 20 samples. The hematology system is consideed "in contol" when the batch means ae within established limits of the taget values. Using the XB Analysis, the diection and amount of change due to the instument, the eagent, flagged samples o sample handling can be detected. Because of the chaacteistic appeaance of the gaphs of the XB esults, it is also often possible to identify changes. Biospecimens Manual UK Biobank Page 57 of

58 OPERATION PRINCIPLES XB ANALYSIS 3 The LH 700 Seies Wokstation calculates and displays the pecent diffeence between each batch mean and its coesponding peset taget value. The pecent diffeence is deived as follows: MCV MCV Batch Mean pecent diff = MCV T et Value ag MCH MCH Batch Mean pecent diff = MCH T et Value ag MCHC MCHC Batch pecent diff = Mean MCHC T et Value ag Adjusting Initial XB Taget Values The ecommended taget values fo initial enty ae: MCV 89.5 MCH 30.5 MCHC 34.0 As samples ae un and laboatoy values established, the ecommended taget values can be adjusted to fit you laboatoy's population. Afte 20 XB batches have been analyzed, calculate the mean and CV% fo each of the XB indices. The mean values should not diffe fom the taget values by moe than 3%, and the CV should be less than 1.5%. If the CVs ae less than 1.5% and the means ae less than 3% diffeent fom the taget values, use the calculated means as new taget values. If the CVs ae geate than 1.5%, o the mean values ae geate than 3% diffeent fom the ecommended taget values, thee may be an instument o population poblem. In this case, epeat this pocedue using the next 20 XB batches. If the indices themselves ae stable in a hospital population, then any deviation fom the Taget Values and Action Limits may point to an instument o eagent poblem. These poblems would involve the paametes diectly measued by the instument and used to calculate the ed cell indices. Table 3.1 lists the diectly measued paametes that would be involved with out-of-limits XB batch values fo each of the ed cell indices. If the XB indices ae still out-of-limits, you should investigate the instument and eagent systems associated with the diectly-measued paamete(s) as indicated by Table 3.1 and call you Beckman Coulte Sevice Repesentative. Biospecimens Manual UK Biobank Page 58 of

59 OPERATION PRINCIPLES XB ANALYSIS Table 3.1 Effect of Diectly-Measued Paametes on the Red Cell Indices Index Diectly-Measued Paamete MCV RBC HGB Inceased Deceased Inceased Deceased Inceased Deceased MCV HIGH LOW NORMAL NORMAL NORMAL NORMAL MCH NORMAL NORMAL LOW HIGH HIGH LOW MCHC LOW HIGH LOW HIGH HIGH LOW See the Glossay fo tems used with the XB Analysis. Biospecimens Manual UK Biobank Page 59 of

60 4SPECIFICATIONS/CHARACTERISTICS PHYSICAL SPECIFICATIONS Dimensions Unit Height* Width* Depth* Weight Analyze/Dilute 88.9 cm cm 61 cm 93.2 k (35 in.) (40 in.) (24 in.) (205 lb) Powe Supply 59 cm 35.5 cm 60 cm 56.7 k (23.3 in.) (14 in.) (24 in.) (125 lb) * ±5.1 cm (2 in.) Powe Input Powe Supply: Wokstation: Vac, Hz Vac, Hz o Vac, Hz Consumption 2080 W (5500 BTU/h) maximum Installation Categoy: pe IEC , Categoy II Tempeatue Ambient opeating ange: 15.5 to 32 C (60 to 90 F) Humidity 0 to 95% without condensation Sample Stability Refe to Heading 4.3, PERFORMANCE CHARACTERISTICS on page 8. CBC/Diff Paametes Stoed at oom tempeatue (23.9 C o 75 F), up to 24 hous afte collection Stoed between 2 and 8 C (35.6 and 46.4 F), up to 48 hous afte collection Reticulocyte Paametes Stoed at oom tempeatue (23.9 C o 75 F), up to 24 hous afte collection Stoed between 2 and 8 C (35.6 and 46.4 F), up to 72 hous afte collection NRBC Paametes Stoed at oom tempeatue (23.9 C o 75 F), up to 24 hous afte collection Stoed between 2 and 8 C (35.6 and 46.4 F), up to 24 hous afte collection Biospecimens Manual UK Biobank Page 60 of

61 SPECIFICATIONS/CHARACTERISTICS PHYSICAL SPECIFICATIONS Sample Stoage Room tempeatue (23.9 C, 75 F) up to 24 hous Sample Type Anticoagulated human whole blood Recommended Anticoagulant K 2 EDTA o K 3 EDTA SAMPLING MODES Aspiation Automatic cap-piecing, whole blood Manual, open vial, whole blood and diluted specimens. Test/Cycle CBC CBC/DIFF CBC/RETIC CBC/DIFF/RETIC RETIC THROUGHPUT, AUTOMATIC MODE Typical thoughput pefomance is descibed as "aveage" fo samples exhibiting paamete levels within the nomal ange, and "maximum" fo samples with elevated paamete levels. Appoximate thoughput pefomance data, not including sample pepaation, is: LH 700 Seies Maximum Sample/hou CBC 110 CBC/Diff 110 CBC/Diff/Retic 45 LH 755 (LH 700 Seies with SlideMake and Maximum Sample/hou SlideStaine) CBC 105 CBC/Diff 100 CBC/Diff/Retic 45 Thoughput equiements ae based on the sample citeia in Table Biospecimens Manual UK Biobank Page 61 of 123

62 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE SPECIFICATIONS--LH 700 Seies 4 Table 4.1 Thoughput Sample Citeia Paamete RETIC modes CBC CBC/DIFF WBC N/A > 7.0 x 10 3 /µl > 7.0 x 10 3 /µl RBC > 5.0 x 10 6 /µl > 5.0 x 10 6 /µl > 5.0 x 10 6 /µl Plt N/A > x 10 3 /µl > x 10 3 /µl Sample Volume Aspiated Automatic, cap-piecing mode: maximum 300 µl Manual, open-vial mode: maximum 200 µl With optional LH 700 Seies SlideMake 550 ul Waste 20-lite waste containe Pneumatic Supplies (Intenally Regulated) Pessue = 60 psi (pounds pe squae inch) Vacuum = 22 in. Hg (inches of mecuy) minimum at sea level Calibation Stability Electonic measuement system: < 1% pe month Vaiation with tempeatue: If ambient oom tempeatue changes by less than 5.5ºC (10ºF) fom the calibating tempeatue, and the tempeatue is within the tempeatue specifications, then the LH 700 Seies does not equie calibation. Unde these conditions, the expected vaiation in calibation factos is: WBC <1.25% MCV <1.18% RBC <0.70% Plt <2.70% Hgb <0.78% MPV <5.00% IMPORTANT The opeating tempeatue influences the ate of kinetic eactions. The online help states that the LH 700 SERIES should be ecalibated wheneve the ambient tempeatue changes by 10 degess Fahenheit. If you have to ecalibate due to a lage change in laboatoy ambient tempeatue, you should e-evaluate the diffeential flagging sensitivity settings fo you typical patient population. LH 700 Seies Wokstation Stoage Patient esults, up to 20,000 with numeic, gaphic and list mode data. 4.2 PERFORMANCE SPECIFICATIONS--LH 700 Seies The LH 700 Seies consists of thee subsystems, designated as "CBC" (Complete Blood Count), "WBC Diffeential" and "Retics." The CBC subsystem is based on the established Coulte pinciples of automated cell counting. The WBC diffeential subsystem is based on Biospecimens Manual UK Biobank Page 62 of

63 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE SPECIFICATIONS--LH 700 Seies the Coulte pinciples of leukocyte diffeential counting as embodied in the COULTER VCS. The Retics subsystem is based on the Coulte volume, conductivity and light scatte technology. Pefomance specifications stated apply only to an instument that has been popely maintained as indicated in the COULTER LH 700 Seies documentation, using the ecommended eagents. If the aveage oom tempeatue should change moe than 5.5 C (10 F) fom the calibating tempeatue, veify calibation and ecalibate if necessay to ensue confomance to specifications. Pecision Within-Run Pecision Within-un Pecision is based on at least 31 deteminations of the same sample. See Table 4.2. Table 4.2 Within-Run Pecision (n = 31) Appoximate Level Limit WBC 9 to 11 x 10 3 cells/µl 1.7% CV RBC 4.5 to 5.5 x 10 6 cells/µl 0.8% CV Hgb 14 to 16 g/dl 0.8% CV MCV 80 to 90 fl 0.8% CV RDW 12 to 14% 2.2% CV Plt 280 to 320 x 10 3 cells/µl 3.3% CV Plt 90 to 110 x 10 3 cells/µl 6.6% CV Plt 10.0 to 15.0 x 10 3 cells/µl 14.0% CV MPV 8 to 10 fl 2.2% CV NE% 50 to 60% 2SD 3.0 LY% 25 to 35% 2SD 3.0 MO% 5 to 10% 2SD 2.0 EO% 2 to 5% 2SD 1.0 BA% 0.5 to 1.5% 2SD 1.0 RET% 0.00 to 0.49% 1SD 0.23 o 16.5% CV RET% 0.50 to 1.49% 1SD 0.23 o 14.5% CV RET% 1.50 to 4.00% 1SD 0.68 o 11.0% CV RET% 4.01 to 15.00% 1SD 0.68 o 5.5% CV Accuacy Accuacy Qualification Accuacy is tested on a minimum of 150 non-flagged, mophological nomal samples fo CBC and Diff, and 50 non-flagged samples fo Retics. Distibutional abnomal samples should be 4-4 Biospecimens Manual UK Biobank Page 63 of 123

64 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE SPECIFICATIONS--LH 700 Seies 4 included. NCCLS H20-A 42 citeia should be used in the sample selection and manual diffeential examination. Accuacy, CBC Fo the CBC paametes, the LH 700 Seies can be adjusted within the esolution of the eadout to agee with a pedetemined efeence value at any point in the opeating ange. Accuacy was tested against a pedicate hematology analyze. Table 4.3 lists the specifications fo the mean diffeence and mean pecent diffeence. Table 4.3 Accuacy, CBC Paamete Mean Diffeence Mean Pecent Diffeence WBC 0.00 to ± % to N/A 9.0% RBC ± % Hgb ± % MCV N/A 2.0% RDW ± % Plt ±10 7.0% MPV N/A 5.0% Accuacy, WBC Diffeential Accuacy of the WBC diffeential should be detemined by compaison against eithe o both of two compaato methods. Accuacy can be tested against the manual diffeential efeence method (NCCLS H20-A [n = 400]). Accuacy was tested against a pedicate hematology analyze. When the LH 700 Seies is compaed to eithe method, using non-flagged, mophological nomal samples, expect esults within toleance limits listed in Table 4.3. Table 4.4 Accuacy Toleance Limits, WBC Diffeential Cell Type Mean Diffeence % using NCCLS H20-A Lymphocyte ±3.0 ±1.5 Monocyte ±3.0 ±1.0 Neutophil ±2.0 ±2.0 Eosinophil ±1.0 ±0.5 Basophil ±1.0 ±0.5 Mean Diffeence % using pedicate analyze Accuacy, Reticulocyte Reticulocyte paamete (RET%) accuacy can be detemined against any of thee compaato methods. Accuacy can be tested against a clinical flow cytomete following the guidelines of NCCLS H44-P. Accuacy was tested against the manual method using NCCLS H16-P. See Table 4.4. Accuacy was tested against a pedicate hematology analyze. See Table 4.5 Biospecimens Manual UK Biobank Page 64 of

65 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE SPECIFICATIONS--LH 700 Seies Table 4.5 Accuacy, Reticulocyte, using NCCLS H16-P o clinical flow cytomete Paamete/Population Mean Diffeence SD of Diffeence RET% Range 0.00 to 15.00% <30% have a RET% >4.00 RET% Range 0.00 to 30.00% <50% have a RET% >4.00 <10% have a RET% >20.00 ± ± Table 4.6 Accuacy, Reticulocyte, using a pedicate hematology analyze Paamete/Population RET% ±0.5 Lineaity When tested using a stable mateial having no intefeing substances, the LH 700 Seies value should be equal to the expected value within the limits given in Table 4.7. To minimize the effects of impecision, take multiple eadings at each dilution point. Subtact the backgound count fom the values obtained. Table 4.7 Lineaity Limits Paamete Lineaity Range WBC x 10 3 cells/µl 0.00 to to Mean Diffeence Limits (mean diffeence o % diffeence, whicheve is geate) ±0.2 o 3.0% ±9% RBC x 10 6 cells/µl 0.00 to 8.00 ±0.05 o 2.0% Hgb g/dl 0.0 to 25.0 ±0.2 o 3.0% Plt x 10 3 cells/µl 0 to 3000 ±10 o 7.0% Backgound Backgound counts fo CBC paametes ae pefomed duing the Statup cycle. The backgound limits ae WBC 0.20 RBC 0.01 Hgb 0.20 Plt 3.00 DIFF RETIC 100 events 600 events Cayove High-to-Low Cayove on the LH 700 Seies should meet these limits: 4-6 Biospecimens Manual UK Biobank Page 65 of 123

66 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE SPECIFICATIONS--LH 700 Seies 4 WBC RBC Hgb Plt DIFF RETIC 2.0% 1.0% 2.0% 2.0% 200 events 600 events Opeating and Repotable Ranges The opeating anges eflect the ange of values ove which the instument displays, pints and tansmits esults. Values that ae between the linea ange and the opeating ange, and values outside the epotable ange, ae displayed, pinted and tansmitted with an ove linea ange flag (+). Values that ae above the opeating ange ae inhibited, and the value is eplaced by pluses (+++++). The epotable ange identifies the values whee the instument is accuate, and eflects the ange studied in accuacy testing. See Table 4.8. Table 4.8 Opeating and Repotable Ranges Paamete Opeating Range Repotable Range WBC 0.00 to x 10 3 cells/µl 0.00 to x 10 3 cells/µl RBC 0.00 to x 10 6 cells/µl 0.00 to 8.00 x 10 6 cells/µl Hgb 0.0 to 99.9 g/dl 0.0 to 25.0 g/dl Hct 0.0 to 99.9% N/A MCV 0.0 to fl 0.0 to fl MCH 0.0 to 99.9 pg N/A MCHC 0.0 to 99.9 g/dl N/A RDW 0.0 to 99.9% N/A Plt 0.00 to 5000 x 10 3 cells/µl 0.00 to 3000 x 10 3 cells/µl Pct 0.0 to 9.999% N/A MPV 0.0 to 99.9 fl N/A PDW 0.0 to 99.9% N/A NE%, LY%, MO%, EO%, BA% 0 to 100% 0 to 100% NE#, LY#, MO#, EO#, BA# 0.00 to x 10 3 cells/µl 0.00 to x 10 3 cells/µl RET% 0.00 to 100% 0.00 to 30.0% RET# 0.00 to x 10 6 cells/µl to x 10 6 cells/µl NRBC% 0.0 o 2.0 to 600 % 0.0 o 2.0 to 600 % Mode-to-Mode Compaison Automatic (closed vial) and Manual (open vial) mode-to-mode diffeences of the means of 10 hematologically "nomal" blood specimens, measued in tiplicate, o 50 samples fom accuacy testing should be less than o equal to the following: Biospecimens Manual UK Biobank Page 66 of

67 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE CHARACTERISTICS WBC ± 0.4 x 10 3 cells/µl o 5%, whicheve is geate RBC ± 0.2 x 10 6 cells/µl o 2%, whicheve is geate Hgb ± 0.3 g/dl o 2%, whicheve is geate Plt ± 20 x 10 3 cells/µl o 7%, whicheve is geate In addition, 95% of the individual diffeences should be within the stated limits. 4.3 PERFORMANCE CHARACTERISTICS The infomation in this section descibes typical pefomance fo an instument that has been popely maintained as indicated in the COULTER LH 700 Seies documentation, using the ecommended eagents. Sample Stability The following tables show the aveage esults fo specimens fom five nomal donos collected in K 3 EDTA. The specimens wee stoed at oom tempeatue and cold tempeatue. Fo this study, oom tempeatue was 72-73º (22-23ºC) and cold tempeatue was 37-39ºF (3-4ºC). Room tempeatue specimens wee mixed fo 22 invesions and then immediately analyzed. Upon emoval fom the efigeato, the cold specimens wee mixed fo 22 complete invesions and analyzed within five minutes Table 4.9 CBC Sample Stability, Room Tempeatue Hous WBC RBC Hgb MCV RDW PLT MPV Table 4.10 CBC Sample Stability, Cold Tempeatue Hous WBC RBC Hgb MCV RDW PLT MPV 1* *Results Room Tempeatue 4-8 Biospecimens Manual UK Biobank Page 67 of 123

68 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE CHARACTERISTICS 4 Table 4.11 DIFF% Sample Stability, Room Tempeatue Hous NE LY MO EO BA Table 4.12 DIFF% Sample Stability, Cold Tempeatue Hous NE LY MO EO BA 1* *Results Room Tempeatue Table 4.13 DIFF# Sample Stability, Room Tempeatue Hous NE LY MO EO BA Table 4.14 DIFF# Sample Stability, Cold Tempeatue Hous NE LY MO EO BA 1* *Results Room Tempeatue Biospecimens Manual UK Biobank Page 68 of

69 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE CHARACTERISTICS Table 4.15 RETIC Sample Stability, Room Tempeatue Hous RETIC% RETIC# MRV IRF Table 4.16 RETIC Sample Stability, Cold Tempeatue Hous RETIC% RETIC# MRV IRV 1* *Results Room Tempeatue Table 4.17 NRBC Sample Stability, Room Tempeatue Hous NRBC% NRBC# Table 4.18 NRBC Sample Stability, Cold Tempeatue Hous NRBC% NRBC# 1* Results Room Tempeatue Biospecimens Manual UK Biobank Page 69 of

70 . SPECIFICATIONS/CHARACTERISTICS PERFORMANCE CHARACTERISTICS 4 WBC Diffeential Flagging Stability The tables below indicate flagging at timed intevals using mid-level flagging sensitivity with NRBC enumeation enabled. Fo additional infomation egading flagging sensitivity, efe to Flagging Pefeences section of the LH 700 SERIES System Opeato s Guide. Table 4.19 Diffeential Suspect Flagging at Room Tempeatue Sample # 1 hou 4 hou 8 hou 16 hou 24 hou 1 Imm Ne 1 Imm Ne 1 Imm Ne 1 Imm Ne 1 Mo Blast, Imm Ne 1, Imm Ne 2 2 * * * * Imm Ne 2 3 * * * * * 4 * * * * * 5 * * * * * *Indicates that thee wee no flags pesent. Table 4.20 Diffeential Suspect Flagging at Cold Tempeatue Sample # 4 hou 8 hou 16 hou 24 hou 36 hou 48 hou 1 Imm Ne 1 * Imm Ne 1 Imm Ne 1 Imm Ne 1 Imm Ne 1 2 * * * * * * 3 * * * PLT Clumps * * 4 * * * * * * 5 * * * * * * *Indicates that thee wee no flags pesent. NRBC Paied Sample Impecision Table 4.21 shows the NRBC esults fo 60 eplicate deteminations of whole blood in K 3 EDTA, Automatic Mode The individual diffeences fo each specimen wee calculated (un1 - un 2) and the Mean and Standad Deviation of the Diffeences wee detemined Table 4.21 NRBC Impecision Chaacteistics,Paied Sample Analysis - Replicate 1 vs. Replicate 2 Paamete Units N Population Minimum Population Maximum Mean Diffeence NRBC % SD of Diffeence NRBC Accuacy Chaacteistics NRBC Accuacy Chaacteistics was defined as the ageement between the LH 700 Seies and the esults given by the efeence method (NCCLS H20A) and a pedicate hematology analyze. All NRBC % esults geate than o equal to two and less than o equal to fifteen will be flagged with an R symbol. Beckman Coulte, Inc. ecommends a slide eview as pe you Biospecimens Manual UK Biobank Page 70 of

71 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE CHARACTERISTICS individual laboatoy potocol. The pesence of this flag does not affect the coection of the white blood cell count in the pesence of NRBC's. Table 4.22 shows the NRBC esults fo whole blood specimens with and whtout NRBC collected in a salt of EDTA. Table 4.22 NRBC Accuacy Chaacteistics, Compaed Samples Paamete Units N * NCCLS ** pedicate hematology analyze Population Minimum Population Maximum NRBC* % NRBC** % Mean Diffeence Platelet Accuacy Chaacteistics Table 4.23 shows the Plt esults fo whole blod specimens collected in a salt of EDTA. Table 4.23 Plt Accuacy Chaacteistics vs ICSH/ISLH Platelet Method Paamete Units N Population Minimum Population Maximum Mean Diffeence Mean % Diffeence PLT x 10 3 cells/l Refeence Ranges A Nomal Range study was conducted to assess the Refeence Ranges fo the LH 700 Seies. Whole-blood samples wee collected fom 131 donos (males and females). The selection of donos was consistent with guidelines stated in NCCLS, C28-A. Table 4.24 Nomal Population Study Paamete Units Gende Mean 95% Confidence Low Limit WBC x 10 3 cells/µl M/F RBC x 10 6 cells/µl M/F Hgb g/dl M/F Hct Ratio M/F MCV fl M/F MCH pg M/F MCHC g/dl M/F RDW % M/F Plt x 10 3 cells/µl M/F MPV fl M/F NE % M/F % Confidence High Limit Biospecimens Manual UK Biobank Page 71 of

72 SPECIFICATIONS/CHARACTERISTICS PERFORMANCE CHARACTERISTICS 4 Table 4.24 Nomal Population Study (Continued) LY % M/F MO % M/F EO % M/F BA % M/F NE x 10 3 cells/µl M/F LY x 10 3 cells/µl M/F MO x 10 3 cells/µl M/F EO x 10 3 cells/µl M/F BA x 10 3 cells/µl M/F RET % M/F RET x 10 6 cells/µl M/F MRV fl M/F IRF Ratio M/F Known Intefeing Substances CBC All All All WBC RBC Hgb MCV RDW Misleading esults can occu if the specimen is not popely collected, stoed o tanspoted. Beckman Coulte ecommends that you follow NCCLS o equivalent pocedues to ensue pope specimen collection, stoage and tanspot. Always follow manufactue s ecommendations when using micocollection devices fo capillay specimen collection. Misleading esults can occu if specimens contain clots. Always use good laboatoy pactices fo inspecting specimens fo clots and veifying esults. Misleading esults can occu if the specimen is not popely mixed. Always use good laboatoy pactices to ensue specimens ae appopiately mixed. Do not bypass o cicumvent the automated mixing pocess used on the LH 700 Seies. NRBCs, giant platelets, platelet clumps, malaial paasites, pecipitated elevated poteins, micolymphoblasts, vey small lymphocytes, fagmented white cells, agglutinated white cells, lyse esistant ed cells, unlysed paticles > 35 fl in size. Vey high WBC count, high concentation of vey lage platelets, auto-agglutination. Vey high WBC count, sevee lipemia, hepain, cetain unusual RBC abnomalities that esist lysing. Vey high WBC count, high concentation of vey lage platelets, auto-agglutination. Vey high WBC count, high concentation of vey lage platelets, auto-agglutination. Biospecimens Manual UK Biobank Page 72 of

73 SPECIFICATIONS/CHARACTERISTICS BAR-CODE SYMBOLOGY OVERVIEW Plt Hct MCH MCHC NRBC Diffeential Reticulocytes Giant platelets, platelet clumps, white cell fagments, electonic noise, vey small ed cells, ed cell fagments Known intefeences elated to RBC and MCV. Known intefeences elated to Hgb and RBC. Known intefeences elated to Hgb, RBC and MCV. Known intefeences elated to lyse esistant ed cells, platelet clumps, giant platelets, malaial paasites, vey small o multi-population lymphocytes, pecipitated elevated poteins. NRBC > 35 fl may not be counted and may esult in a false negative condition. If an NRBC% is epoted as zeo and thee ae any suspect messages o paamete codes, Beckman Coulte, Inc. ecommends a slide eview pe you laboatoy potocol. Cod blood is a ecognized souce of high numbes of NRBC. Howeve, due to the possible matix effect of the sample constituents that may be intoduced duing sample, cod blood is not ecommended fo NRBC studies. Hypoganula ganulocytes, aganula ganulocytes, lyse-esistant ed cells, vey small o multi-population lymphocytes, elevated tiglyceides, pecipitated elevated poteins. Eythocyte inclusions stained by New Methylene Blue, if sufficiently numeous within a sample, and some hemoglobinopathies (SS, SC) might affect the accuacy of the eticulocyte enumeation BAR-CODE SYMBOLOGY OVERVIEW A ba-code symbol is a seies of adjacent bas and spaces. The widths of the bas and spaces epesent the actual data encoded within the symbol. A device, usually called a scanne, o eade is used to decode this data into human eadable infomation. A ba-code symbology, such as Inteleaved 2-of-5, descibes the unambiguous conventions o ules used fo the way in which data is encoded into the aangement of the adjacent bas and spaces. A symbology is defined by the following chaacteistics: Chaacte Set This efes to the ange of data chaactes that can be encoded fo a specific symbology. Thee ae essentially thee types: Numeic The symbology can encode only numeic chaactes. Alphanumeic Can encode the full alpha and numeic chaactes of the ASCII chaacte set. 128 Can encode all the 128 available ASCII chaactes. Symbology Type Thee ae essentially two types: Discete Each chaacte is sepaated fom the next by an intechaacte gap. This allows each chaacte to be decoded independently. Evey chaacte has a ba on each end. Continuous Has no intechaacte gaps. Evey chaacte begins with a ba and ends with a space. Biospecimens Manual UK Biobank Page 73 of

74 SPECIFICATIONS/CHARACTERISTICS BAR-CODES AND THE LH 700 Seies 4 Fixed o Vaiable Length Some symbologies can only encode messages of a fixed length. Some can be used to encode vaiable length data. Some should only be used as a fixed length to incease the secuity of data decoding. Self-Checking A self-checking symbology has the ability to pevent chaacte tansposition due to a single pinting defect. Stat Code, Stop Code Stat and Stop Codes ae ba and space pattens that ae placed at the beginning and end of the encoded data. A stat code indicates the beginning of the symbol, and indicates the stat of the encoded infomation. The stop code indicates the end of the symbol and maks the end of the encoded infomation. Check Chaacte, Checksum Algoithm Vitually all ba-code symbologies use a check chaacte. The check chaacte is deived by a mathematical calculation using the chaactes in the encoded infomation. It is used by the scanning device to veify that the coect infomation has been decoded. It is highly ecommended that a check chaacte, also called a checksum algoithm, is used. Quiet Zone, Quiet Aea The Quiet Zone is a clea aea on eithe side of the stat and stop codes. It helps the scanning device establish the eflectivity chaacteistics of the label. 4.5 BAR-CODES AND THE LH 700 Seies The LH 700 Seies suppots a wide vaiety of ba-code symbologies. When choosing a symbology to use in you laboatoy, it is impotant to undestand the diffeent ba-codes available. Some ba-codes ae moe stable, and theefoe less pone to miseads than othes. The following infomation is intended as a guide to help you in you selection pocess. IMPORTANT Risk of misidentification. Use of poo quality, dity, impopely placed o damaged ba-code labels could keep the instument fom eading the ba-code labels. Ensue the ba-code labels ae undamaged. Ensue the ba-code labels confom to the specifications povided in Chapte 4 of the Refeence manual. Checksum Algoithm Beckman Coulte stongly ecommends the use of ba-code checksums to povide automatic checks fo ead accuacy. IMPORTANT Use of ba-codes is an extemely accuate and effective method of positive patient identification. Cetain featues, such as checksum digits, maximize accuacy in eading Codaba, Code 39 and Inteleaved 2-of-5 labels. In one study, the use of checksum digits detected 97% of misead eos. Use checksums to povide potection against occasional misead eos caused by poblems such as damaged o misapplied labels. If you must use ba-codes without checksums, Beckman Coulte ecommends that you veify each ba-code eading to assue coect patient identification. Biospecimens Manual UK Biobank Page 74 of

75 SPECIFICATIONS/CHARACTERISTICS BAR-CODES AND THE LH 700 Seies Ba-code Symbologies Suppoted by the LH 700 Seies Inteleaved 2-of-5 Inteleaved 2-of-5 is a high density, continuous numeic symbology. It is self-checking. Evey chaacte in the symbology encodes two digits, one in the bas and one in the spaces. This symbology is susceptible to an incoect ead due to a patial scan (a scanning path that does not include both leading and tailing quiet zones). The most common incoect ead is a shote, but valid decoding of the infomation. The pesence of a checksum does not eliminate this isk. It is ecommended that any Inteleaved 2-of-5 label contain Beae Bas. Altenatively, this label should be used with a fixed length only, with the scanning devices set to ecognize labels of a specific length (fo example 12 digits). Code 39 (Also called 3-of-9 Code) Code 39 was the fist alphanumeic symbology developed. It is a self-checking, discete and vaiable length symbology. The Health Industy Ba-code Council (HIBCC) has adopted the use of a check chaacte fo health cae applications. This is encouaged, paticulaly whee pint quality is less than optimum. Codaba Codaba has a chaacte set of 16 chaactes. It is a discete, self-checking symbology and is most commonly used in libaies and blood banks. NW-7 NW-7 is vey simila to Codaba. It uses the same chaacte set and check chaacte. The diffeence is in the ba and space stuctue. Code 128/USS 128 Code 128 is a continuous, vaiable length symbology. This symbology has 106 diffeent pinted chaactes. Code 128 is chaacte dependent. See AIM Unifom Symbol Specification (USS) Rev fo additional equied dimensional toleances. You must use and pint a checksum chaacte, and it must confom to the AIM USS 128 checksum geneation pocedue. IMPORTANT If a laboatoy uses Code 128 ba-code fomat, it must have checksum enabled. If the checksum is disabled, the LH softwae poduces a ba-code that does not comply with the Code 128 standad and cannot be ead by ba-code scannes. Do not use these values: Code set A - 0, 64 though 102 Code set B - 0, 95 though 102 Code set C though 102 Do not use leading o tailing spaces in the ID. Biospecimens Manual UK Biobank Page 75 of

76 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS 4 Ba-code Tips No ba-code symbology is pefect. You will occasionally get a ead eo. Below ae some tips to help you get the best pefomance fom the symbology you choose: Use high quality labels. Always use a check chaacte. Use fixed length ba-code infomation. Use Beae Bas with Inteleaved 2-of-5. When using the handheld scanne, check that the label was decoded coectly. 4.6 BAR-CODE LABEL SPECIFICATIONS Handheld Scanne Infomation about configuing the handheld scanne is located elsewhee. Geneal A ba-code consists of black lines (bas) and white lines (spaces), which ae called elements. Thee ae naow elements (NE) and wide elements (WE); thei aangement is detemined by the code. IMPORTANT Possible misidentified esults. Fo accuate eading by the scanne, it is impotant that ba-code labels fo specimen tubes adhee stictly to the specifications given in this section. Labels that meet these specifications ae available fom Beckman Coulte. Optical Chaacteistics at 880 nm ±10% and 633 nm ±10% Pint Contast Signal (PCS): 80% min. Reflectivity of Media (RW): 80% min. Reflectivity of Ink (Rb): 16% max. No spots o voids; no ink smeaing. Edge oughness is included in the ba and space toleances. Measuement method is accoding to Ameican National Standads Institute's MH10-8M Pinting Method Photogaphic, o themal tansfe. Label Thickness Maximum label thickness must be such that: The tube's oute diamete including the label is not geate than 13.3 mm. The label including adhesive = ±0.003 in. NE/WE Ratio Must emain constant ove code length. Biospecimens Manual UK Biobank Page 76 of

77 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS Label Dimensions and Data The dimensional and data specifications ae illustated in Figue 4.1. Table 4.13 explains the specifications called out in Figue 4.1 Figue 4.1 Ba-Code Label Specifications LEADING EDGE OF LABEL A B FIRST BAR LINE (SEE SPEC. 1) ALL SUBSEQUENT BAR LINES (SEE SPEC. 2) HUMAN-READABLE CODE (SEE SPEC. 3) " 0.055" " LABEL WIDTH (SEE SPEC. 9) 0.300" MIN. PLACEMENT INDICATOR (SEE SPEC. 8) " * " 0.062" CODE AREA (SEE SPEC. 6) 0.250" MIN. LEADING QUIET ZONE (SEE SPEC. 7) LABEL LENGTH (SEE SPEC. 5) 0.250" MIN. TRAILING QUIET ZONE (SEE SPEC. 4) Table 4.25 Ba-Code Label Specifications Specification Called Out in Figue 4.1 Explanation 1 The fist ba of the code (B) must be paallel to the label edge (A) within 0.002". 2 All subsequent ba lines must be paallel to (B) within 0.001". 3 The human-eadable code (HRC) does not include the checksum; the dash in the HRC is not encoded in the ba-code. 4 The tailing quiet zone must be 0.250" minimum. 5 The maximum label length is detemined by the tube length. The scanne can accommodate labels up to 2.35". With HEMOGARD tubes, the maximum label length is 2.04". 6 The ba-code aea contains the stat chaacte, data digits, checksum, and stop chaacte. 7 The leading quiet zone must be 0.250" minimum. Biospecimens Manual UK Biobank Page 77 of

78 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS 4 Table 4.25 Ba-Code Label Specifications 8 The placement indicato shows you which end of the label goes next to the tube stoppe. This is an optional featue, not a mandatoy one. 9 The width of the label must leave at least a 1/8" window fo viewing the contents of the tube. The maximum label width fo a 10-mm diamete tube is 1.1". The minimum label width is 0.400". Acceptable Ba-codes Within the given specifications, the scanne automatically distinguishes the following ba-codes. Inteleaved 2-of-5 Code 39 Codaba NW7 Code 128/USS 128 The following table summaizes the code-elated specifications. Table 4.26 Code-Related Specifications Code Naow element (NE) width Wide element/ naow element atio (WE/NE) Inteleaved 2-of-5 Codaba Code 39**** NW " ±0.001" 0.010"* Scaling Facto = 1.538* Code 128**** USS " ±0.001" " ±0.001" 0.010" ±0.001" (2.2 to 3): 1 N/A (2.21 to 3): 1 (2.2 to 3): 1 (2 to 4): 1** Intechaacte gap No 0.010" Min. >=NE 0.010" Min. No Data digits 3 to 11 3 to 9 3 to 9 (3 to 8 with HEMOGARD tubes) 3 to 9 LH 700 Seies, 3 to 11 data digits SlideMake, 3 to 9 data digits Checksum always pinted*** * Accoding to Ameican National Standad fo ba-code specifications that yield 10 chaactes pe inch at NE = ". ** Code 128 is chaacte dependent. See AIM Unifom Symbol Specification Rev fo additional equied dimensional toleances. *** You must use and pint a checksum chaacte, and it must confom to the AIM USS 128 checksum geneation pocedue. IMPORTANT If a laboatoy uses Code 128 ba-code fomat, it must have checksum enabled. If the checksum is disabled, the LH softwae poduces a ba-code that does not comply with the Code 128 standad and cannot be ead by ba-code scannes. Do not use these values: Code set A - 0, 64 though 102 Code set B - 0, 95 though 102 Code set C though 102 **** Do not use leading o tailing spaces in the ID Biospecimens Manual UK Biobank Page 78 of

79 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS Checksum Algoithm Beckman Coulte stongly ecommends the use of ba-code checksums to povide automatic checks fo ead accuacy. IMPORTANT Use of ba-codes is an extemely accuate and effective method of positive patient identification. Cetain featues, such as checksum digits, maximize accuacy in eading Codaba, Code 39 and Inteleaved 2-of-5 labels. In one study, the use of checksum digits detected 97% of misead eos. Use checksums to povide potection against occasional misead eos caused by poblems such as damaged o misapplied labels. If you must use ba-codes without checksums, Beckman Coulte ecommends that you veify each ba-code eading to assue coect patient identification. The algoithm fo detemining the checksum fo each code is given below. Inteleaved 2-of-5 This code equies 3 to 11 data digits plus a checksum. To detemine the value of the checksum chaacte: 1. Identify even- and odd-positioned chaactes in the message with the ight-hand message chaacte always defined as an even-positioned chaacte. 2. Sum the numeic values of the odd-positioned chaactes. 3. Sum the numeic values of the even-positioned chaactes and multiply the total by 3. Sum the odd and even totals fom steps 2 and Detemine the smallest numbe which, when added to the sum in step 4, esults in a multiple of 10. This numbe is the value of the checksum chaacte. 5. Detemine whethe total numbe of chaactes (message plus checksum) is odd o even. If odd, add a leading nonsignificant zeo to the message to poduce an even numbe of chaactes as equied by the symbology. Example: MESSAGE PARITY 0 E 0 E 0 E STEP 2: 1+5+7=13 STEP 3: (2+6+8)x3=48 STEP 4: 13+48=61 STEP 5: 61+9=70 Theefoe, the checksum is 9, and the final decoded message is Codaba and NW7 These codes use thee to eight data digits. Note: Codaba and NW7 codes have the same chaacte set and the same checksum algoithm. The diffeence between these two codes is that Codaba has 18 diffeent ba and space dimensions, and NW7 has only NE and WE stuctue. Biospecimens Manual UK Biobank Page 79 of

80 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS 4 The value assigned to each of the chaactes is pesented in the following table. CHARACTER VALUE CHARACTER VALUE $ : / A B C D 19 The checksum technique is: The chaacte value of a message is obtained fom the above table and added togethe. This sum is divided by 16, and the emainde coesponds to the value of the checksum chaacte. Examples: MESSAGE VALUE = = 1, REMAINDER 4 The value 4 coesponds to chaacte 4; theefoe, the checksum is 4 and the final decoded message is MESSAGE $ $ / / VALUE = = 6, REMAINDER 12 The value 12 coesponds to chaacte :, theefoe, checksum is :, and the final decoded message is: $$//++++: Biospecimens Manual UK Biobank Page 80 of

81 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS Japan Red Coss NW7 Decoding This code uses thee to nine data digits. Japan Red Coss Hospitals use the following NW7 values: CHARACTER VALUE The checksum technique is: The data digit value that is the diffeence between 11 and the Mod 11 sum of the weighted values of the data digits is used as the check digit. The stat and stop digits ae not used as pat of the checksum calculation. NW7 is made up of 1 stat digit, 9 data digits and 1 stop digit. The checksum digit immediately pecedes the stop digit. WEIGHTED MODULUS 11: DIGIT POSITION (Right Justified) WEIGHT (1) WEIGHT (2) The fist 9 digits fom the ight ae used fo the calculation of the check digit. To detemine the value of the checksum chaacte: 1. Detemine the Modulus 11 value coesponding to each chaacte in the message. 2. Multiply each chaacte in the message by the coesponding Mod 11 value. 3. Add the esulting values and divide by Subtact the emainde fom Detemine the chaacte that coesponds to the esult fom step 4. This is the checksum chaacte. Biospecimens Manual UK Biobank Page 81 of

82 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS 4 Examples: 1. MESSAGE USE WEIGHT (1): DIGIT POSITION (Right Justified) WEIGHT (1) Result = = 11, REMAINDER 3 When the REMAINDER IS 0, 0 is the check digit = 8 The value 8 coesponds to chaacte 8, theefoe the checksum is 8 and the final decoded message is MESSAGE USE WEIGHT (1): DIGIT POSITION (Right Justified) WEIGHT (1) Result = = 11, REMAINDER 1 When the REMAINDER is 1, the calculation must be epeated using weight (2): DIGIT POSITION (Right Justified) WEIGHT (2) Result = = 14, REMAINDER 6 When the REMAINDER is 0, 0 is the check digit = 5 The value 5 coesponds to chaacte 5, theefoe the checksum is 5 and the final decoded message is Biospecimens Manual UK Biobank Page 82 of

83 : SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS Code 39 Ba-code This code uses thee to nine data digits. The value assigned to each of the chaactes is: CHARACTER VALUE CHARACTER VALUE CHARACTER VALUE 0 0 F 15 U G 16 V H 17 W I 18 X J 19 Y K 20 Z L M N 23 SPACE O 24 $ 39 A 10 P 25 / 40 B 11 Q C 12 R 27 % 42 D 13 S 28 E 14 T 29 The checksum technique is: The chaacte values of the message ae obtained fom the above table and added togethe. This sum is divided by 43, and the emainde coesponds to the value of the checksum chaacte. Example: CHARACTER S T U V W X Y F VALUE = = 5, REMAINDER 17; 17 = H = CHECKCHARACTER The value 17 coesponds to chaacte H; theefoe, checksum is H, and the final decoded message is: STUVWXYFH. Biospecimens Manual UK Biobank Page 83 of

84 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS 4 Code 128 This code uses thee to eleven data digits on the LH 700 Seies and thee to nine data digits fo the SlideMake ba-code label pinting. The checksum chaacte immediately pecedes the stop chaacte. The checksum chaacte used with Code 128 must confom to the AIM USS 128 checksum geneation pocedue. Do not use these values: Code set A - 0, 64 though 102 Code set B - 0, 95 though 102 Code set C though 102 The checksum value (see the following table) is equal to the modula 103 sum of the value of the stat chaacte and the weighted values of the data/special chaactes. The weights ae one fo the fist data/special chaacte and continuing with two, thee, fou and so foth fo the following data/special chaactes. Fo example, a label contains a START chaacte (Code C), Data (25), a Check chaacte and a STOP chaacte. The value of the Stat chaacte C is 105, and the data chaacte fo 25 is 25. The weight of the fist data chaacte is one, so the check chaacte value is calculated as follows: (25 x 1) = 130 whee 105 and 25 ae the values and 1 is the weight. The checksum is equal to 130 modula 103 (the emainde of 130 divided by 103): = 1, REMAINDER 27 Theefoe the check chaacte equals chaacte value 27, which is ; in Code Set A. Fo additional infomation on this pocedue, efe to AIM USS-128 Rev. 1986, published by AIM, Inc., 1326 Feepot Road, Pittsbugh, PA VALUE CODE A CODE B CODE C 0 SP SP 00 1!! 01 2 " " 02 3 # # 03 4 $ $ 04 5 % % 05 6 & & 06 7 ' ' 07 8 ( ( 08 9 ) ) * * ,, Biospecimens Manual UK Biobank Page 84 of

85 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS / / : : ; ; < < = = > > 30 31?? A A B B C C D D E E F F G G H H I I J J K K L L M M N N O O P P Q Q R R S S 51 Biospecimens Manual UK Biobank Page 85 of

86 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS 4 52 T T U U V V W W X X Y Y Z Z [ [ \ \ ] ] NUL ` SOH a STX b ETX c EOT d ENQ e ACK f BEL g BS h HT i LF j VT k FF l CR m SO n SI o DLE p DC1 q DC DC3 s DC4 t NAK u SYN v ETB w CAN x EM y 89 Biospecimens Manual UK Biobank Page 86 of

87 SPECIFICATIONS/CHARACTERISTICS BAR-CODE LABEL SPECIFICATIONS 90 SUB z ESC { FS GS } RS ~ US DEL FNC 3 FNC FNC 2 FNC SHIFT SHIFT CODE C CODE C CODE B FNC 4 CODE B 101 FNC 4 CODE A CODE A 102 FNC 1 FNC 1 FNC START (CODE A) 104 START (CODE B) 105 START (CODE C) Biospecimens Manual UK Biobank Page 87 of

88 5HAZARDS LASER SAFETY WARNING Possible ham to opeato. Do not use any contols, make any adjustments, o pefom any pocedues othe than those specified heein. To do so may esult in hazadous adiation exposue. The Tiple Tansduce Module and Ba-Code Reade contain lases. A lase is a unique light souce that exhibits chaacteistics diffeent fom conventional light souces. The safe use of the lase depends upon familiaity with the instument and the popeties of coheent, intense beams of light. The beam can cause eye damage and instument damage. Thee is enough powe fom the lase to ignite substances placed in the beam path, even at some distance. The beam might also cause damage if contacted indiectly fom eflective sufaces (specula eflection). The lases on the LH 700 Seies ae coveed by potective housings that ae held in place by tampe-poof scews. WARNING Possible ham to opeato. Do not attempt to emove the lase o to open it. Failue to comply can esult in hazadous adiation exposue. If emoval is equied, it must be done only by a Beckman Coulte Repesentative. All sevice and maintenance of the lase must be done at the Beckman Coulte factoy by tained pesonnel. If emoval is equied, it must be done by a Beckman Coulte Repesentative. 5.2 RADIATION HAZARDS In the design and manufactue of the LH 700 Seies, Beckman Coulte Inc. has complied with the equiements govening the use and application of a lase as stipulated in egulatoy documents issued by the U.S. Depatment of Health and Human Sevices, and Cente fo Devices and Radiological Health (CDRH). In compliance with these egulatoy documents, evey measue has been taken to ensue the health and safety of uses and laboatoy pesonnel fom the possible danges of lase use. 5.3 LASER WARNING LABELS WARNING WARNINGPossible ham to opeato. This instument contains components dangeous to the opeato. If any attempt has been made to defeat a safety featue, o if this instument fails to pefom as listed in this manual, disconnect powe and call you Beckman Coulte Repesentative. CDRH-appoved labels ae placed nea o on those coves that, when emoved, might expose lase adiation. Figue 5.1 shows the lase cove and the potective housing cut away. This illustation is intended only to show you what the system looks like, in compliance with CDRH. See Figue 5.1 fo the labels and thei locations on the lase head. See Figue 5.2 fo the label location on the beam cove between the lase head and the sampling compatment. Figue 5.1 and Figue 5.2 show cetification labels. Biospecimens Manual UK Biobank Page 88 of

89 HAZARDS LASER WARNING LABELS Figue 5.1 Lase Waning Label, Potective Housing Cut Away LASER ON LAMP LASER RADIATION WHEN OPEN AND INTERLOCK DEFEATED. AVOID DIRECT EYE EXPOSURE. ELECTROMAGNETIC SHIELD LASER RADIATION IS EMITTED FROM THIS APERTURE AVOID EXPOSURE FLOW CELL 1096 Mellon Avenue Manteca, CA MODEL MANUFACTURED SERIAL NO. THIS LASER DOES NOT COMPLY WITH 21 CFR USE ONLY AS A COMPONENT. SEE INSTALLATION INSTRUCTIONS. PATENT NOS 4352, , , ,583 MADE IN USA TAMPER-PROOF SCREWS LASER Note: As installed in the Tiple Tansduce Module (TTM) safety fixtue, the lase pesents no adiation hazad to uses and complies with 21 CFR Biospecimens Manual UK Biobank Page 89 of 123

90 5 ; "! I! & II III IV V 5 6 ) ), ; 6-5 6, - VI ) ) ) * - VII, - +, 4 ) 1-7 VIII *,, ) ) 4 6) ) 4, 4 ) ), ; : ) ) ) ! 1, , 9 " # $ % & ' HAZARDS LASER WARNING LABELS 5 Figue 5.2 Lase Waning Label Locations, Potective Housing On?=JA@ >=? BK EJ ) , * + 0 ) " ), " + ) 5 5 ) , = K B =? J K H ) 6 - ' > O ) 6 1 E= E. =!! ' $ ) ), 1 ) ), , -. - ) 6 -, ) 8 1,, ; - - : Biospecimens Manual UK Biobank Page 90 of

91 HAZARDS BAR-CODE READER 5.4 BAR-CODE READER Figue 5.3 Ba-Code Reade Lase Waning Label Location, Potective Housing Cut Away C A U T I O N LASER RADIATION DO NOT STARE INTO BEAM THIS LASER DOES NOT COMPLY WITH 21 CFR 1040 CLASS II LASER PRODUCT OPTICAL GLASS LENSES Note: As installed in the Ba-Code Reade Assembly, the lase pesents no adiation hazad to uses and complies with 21 CFR Biospecimens Manual UK Biobank Page 91 of 123

92 5 ; "! I! & II III IV V 5 6 ) ), ; 6-5 6, - VI ) ) ) * - VII, - +, 4 ) 1-7 VIII *,, ) 4 6) ) ), ; : ) ) ) ! , 9 " # $ % & ' LASER LIGHT - DO NOT STARE INTO BEAM 670nm DIODE LASER 1.0 MILLIWATT MAXIMUM CLASS II LASER PRODUCT HAZARDS BAR-CODE READER 5 Figue 5.4 Ba-Code Reade Lase Waning Label Location, Potective Housing On = I A E= JE A EIIE H -,, 4 ) ) 4 1, CAUTION Lase adiation when open and intelock defeated. DO NOT STARE INTO BEAM. ) 5-4 CAUTION + ) = I A H H E = J E M D A F A E J A A B A = J ) * - ) 6 = F A H F H BI? HA M I + ) ) 5-4 1/ 0 6, ) * - ) $%, 1, - ) , 7 4 ) 6 1 I # 1 19 ) 6 6 ) : 1 + 7) ) , Note: As installed in the Ba-Code Reade Assembly, the lase pesents no adiation hazad to uses and complies with 21 CFR Biospecimens Manual UK Biobank Page 92 of

93 HAZARDS HANDHELD SCANNER 5.5 HANDHELD SCANNER Figue 5.5 illustates the label on the PSC handheld scanne. Figue 5.5 Handheld Scanne 5-6 Biospecimens Manual UK Biobank Page 93 of 123