Boule Medical AB CA620-CellGuard Page Usermanual V3.7

Transcription

1 1 File : 620-CellGuard Software version V3.7x Article number of this manual : Table of contents 1 Identification of hazards & interpretation of results Interpretation of results Manufacturer and warning marks on device Intended use Specifications General Parameter ranges Reagents and reagent consumption Limitations Measuring principles RBC,WBC and PLT concentration detection Sizing RBC, WBC and PLT Counting time RBC & WBC WBC differentials MCV (Mean Cell Volume RBCs) RDW (Red Cell Distribution Width) RDW (Red Cell Distribution Width Absolute) HCT (Hematocrit) PLT (Platelets) MPV (Mean Platelet Volume) PDW (Platelet Distribution Width) LPCR (Large Platelets) PCT (Packed Platelet Volume) HGB (Hemoglobin Concentration) MCH (Mean Cell Haemoglobin) MCHC (Mean Cell Haemoglobin Concentration) Parameter flags TU ( Time-out Upper Detector ) TL ( Time-out Lower detector ) SE ( Statistical Error ) DE ( Distribution Error ) FD ( Floating Discriminator ) OF ( Offset error HGB ) LO ( Low blanklevel HGB ) HI ( High blanklevel HGB ) NG ( Negative HGB ) SE ( Statistical error HGB ) TB ( Air bubbles ) NM, OM, TM, BD (WBC Differential Flags) RP ( Red cells Present ) Comments on flagging capabilities...32

2 2 6 Installation Working conditions Mains Supply Environment Location Reagent packages Waste connection Mechanical check and set-up Front panel and command keys Filling the system with reagents Initial system configuration Setting up the instrument for pre-donor screening Setting up the instrument for PLT concentrate Setting date and time Printer configuration Select language Select Units Routine operation Background Analysing the Sample (open tube) Analysing the Sample (Pre-dilute) Analysing the Sample (Micro Pipette Adapter, MPA) Analysing the Sample (Cap Piercing Device) Advanced user options User interface (Menus) Sample memory Setup menu Calibration menu s Set parameter normal ranges Set floating discr. PLT/RBC Set discr. WBC Set default discr. program Print all settings Setup menu Machine ID Blood det. Norm / Blood det. PLT-C Serial port setup Barcode reader PLT offs and High altitude comp Print control blood id:s Reagent type Set PROG names Block parameters Printer setup menu Block pre-dilute start...74

3 3 10 Service menu Pump and valve test HGB photometer adjustment Volume detectors Blood & beaker detectors Motor detectors Noise test Print machine statistics Start a clean cycle Service menu Warning displays Warnings related to the MPA and pre-dilute inlet Stand-by mode Reagent empty Printer and serial output warnings Auxiliary warnings/messages Calibration Use of Commercial Controls and calibrators Calibration on a Known Sample Calibration through a certified Calibrator Calibration on Pre-Diluted Capillary Blood Calibration MPA Calibration of RDW, Lymph and Gran Printer and serial output Selecting the Correct Printer Type The Serial Output / Hardware Connections The Serial Output Format Maintenance & special procedures Daily maintenance Monthly maintenance Three (3) month maintenance Transporting the CA620-CellGuard Switching off the CA620-CellGuard & repackaging Storing the CA620-CellGuard...103

4 4 15 Trouble shooting Counting time 'HI' (TU & TL) Counting time 'LO' HGB value flagged with 'LO' HGB value flagged with 'HI' HGB value flagged with 'OF' High background PLT Indication number XXX displayed Celldiff. only on a few samples Cap piercing device Cleaning / Replacing the needle Clogging in aspiration tube Replacing parts...110

5 5 1. Identification of hazards & interpretation of results Boule Medical incorporates safety features to protect the operator from injury, the instrument from damage and the test results from inaccuracies. A box is used to identify the possible hazards and to call the operators attention to the existence of this condition. The text following the box is in bold-italic. The following hazards could be encountered while operating the system: WARNING! Potentially biohazardous material Caution: Because no test method can offer complete assurance that HIV, Hepatitis B or C viruses, or other infectious agents are absent, these products should be handled at the Biosafety Level 2 as recommended for any infectious human blood specimens in Protection of Laboratory Workers From Infectious Disease Transmitted by Blood, Body Fluids and Tissues - 2 nd Edition, Tentative Guidelines(1991) Document M29-T2 promulgated by the National Committee for Clinical Lab. Standards in the U.S.A. (NCCLS) WARNING! Indicates operating procedures, practices and so on, that could result in personal injury or loss of life if not correctly followed. CAUTION Indicates operating procedures, practices and so on that could result in damage or destruction of equipment if not strictly observed. IMPORTANT Emphasizes operating procedures, practices and so on that must be followed to avoid erroneous results. DANGER Indicates a hazard that is life threatening. This hazard is assigned to substances that are highly toxic, extreme corrosive, extremely flammable or explosive. CAUTION! Indicates a hazard that is not life threatening under ordinary circumstances but might cause illness, burns skin reaction and so on. This hazard is assigned to such substances as diluted acids, mild caustics, mild skin irritants or combustible materials.

6 6 1.1 Interpretation of results System operators and laboratory supervisors are responsible for operating and maintaining the Boule Medical products in accordance with the procedures described in the applicable product labelling (manuals, package inserts and bulletins of any kind) and for determining that product performance conforms to the applicable claims. If, under these prescribed conditions of operation and maintenance, an aberrant or abnormal result occurs, as defined by the laboratory protocol, laboratory personal should first make certain that the system is performing and is being operated in accordance with the Product Labelling then follow the laboratory protocol for advising the clinician of a result that appears to have deviated from the norms established by the laboratory. Boule Medical products do not make diagnoses on patients. Boule Medical intends its diagnostic products (systems, software and hardware) to be used to collect data reflecting the patient s hematological status at a certain point of time. Such data may be used in conjunction with other diagnostic information and with the attending physicians evaluation of the patients condition to arrive at a diagnosis and a clinical course of treatment. Any malfunction of a Boule Medical diagnostic product should be appropriately addressed by the laboratory personnel. Refer to the enclosed section in the Boule Medical manual, bulletins or package inserts to address malfunctions and their possible effects on the results. 1.2 Manufacturer and warning marks on device Name and address of Manufacturer Boule Medical AB POB Västberga Alle Stockholm Sweden * Warning marks on device This marking is an indication of potential hazard due to moving parts.

7 7 2. Intended use The CA620-CellGuard is a fully automated hematology analyser used for in vitro diagnostic testing of whole blood specimens and Platelet concentrates. The CA620-CellGuard is intended to be used especially in blood banks as pre donor screening as well as a quality control tool for PLT concentrates in a bloodbank production facility. During the installation and set-up, the CA620-CellGuard can be configured either as a pre-donor screening or for quality control to measure PLT concentrates. The operator works with a menu from which the desired program is chosen, e.g. discriminator settings, calibration, cleaning etc. Each parameter can be blanked from being reported on the screen and printer. Two external reagent reservoirs are used, one for isotonic diluent and one for hemolyzing agent. Whenever one of these external reservoirs is empty, this is indicated on the display. The CA620-CellGuard also performs a 3-part WBC differential by means of a cyanide-free hemolyzing agent. A sample memory is available for more than 350 samples, protected against mains power failures. Sample search, selective printing and QC options are available from the sample memory. The CA620-CellGuard is a fully automated hematology analyser designed to measure up to 20 parameters using whole blood from an open inlet, closed tubes, micro pipettes 20ul or pre-diluted blood. - The number of Red Blood Cells (RBC) - The number of White Blood Cells (WBC) - The number of Platelets (PLT) - The Mean Cell Volume of red cells (MCV) The Mean Platelet Volume (MPV) - The Haemoglobin Concentration (HGB) The size distributions of PLT, RBC and WBC are measured at the same time as the above-mentioned parameters. - Hematocrit (HCT) - The Mean Cell Haemoglobin (MCH) - The Mean Cell Haemoglobin Concentration (MCHC) - The Red Cell distribution Width (CV) (RDW) - The Lymph. concentration in

8 8 absolute number and percentage (LYMF) - The Mid-sized cells (e.g. Monocytes) in absolute number and percentage (MID) - The Gran. concentration in absolute number and percentage (GRAN) - The Red Cell distribution Width (Absolute) (RDWa) - The Platelet distribution Width (Absolute) (PDW) - The Platelet Macro Range > 12 fl in % (LPCR) - The Platelet Packed Volume (PCT) Note: PCT, LPCR, RDWa and PDW are not established parameters in the United States. Their use should be restricted to research or investigational use only. The CA620-CellGuard employs the electronic impedance principle for cell counting and sizing, and a colorimetric method for measuring hemoglobin. A microprocessor is used to measure the parameters and to size the cells. During the count the processor is checking the analysing process for any irregularities. Size distributions are printed for all populations (RBC, PLT and WBC) The CA620-CellGuard has as standard a parallel and as standard a serial output, which is user programmable. From a built-in selectable program the user can choose between different print formats. A bar-code reader (scanner), available as an option, can be connected to enter the sample ID automatically. The CA620-CellGuard is totally automatic in such way that the system is always powered on and performs automatically check and cleaning cycles to minimize user maintenance. IMPORTANT The MPA is intended to be used for pre-donor screening. Using the MPA for PLT concentrate may reduce the accuracy of the result.

9 9 3. Specifications 3.1 General -Measuring principle RBC,WBC,PLT -Measuring principle HGB -Discriminator -Sampling system Impedance Cyanide free method 540nm Floating programmable Shear valve (maintenance free) -Parameters reported: RBC, MCV, HCT, PLT, MPV HGB, MCH, MCHC WBC, RDW % LYMPH abs, MID abs, GRAN abs. LYMPH %, MID %, GRAN % RDW abs. PDW abs LPCR PCT Note: PCT, LPCR, RDWa and PDWa are not established parameters. Hence, their use should be restricted to research or investigational use only- -Size distributions printed for RBC, PLT and WBC diff. -Aspirated blood volume (open tubes) ca. 125 µl -Aspirated blood volume (closed tubes)* ca. 200 µl *Optional device -Blood volume using the Micro Pipette Adapter 20 µl -Prediluted mode 1:200 to 1:250 using min. 20µl blood e.g. 20 µl to 5 ml diluent 30 µl to 6 ml diluent 40 µl to 8 ml diluent -Screen -Keyboard -Total cycle time -Sample display and print after LCD Numerical ca. 73 seconds ca. 53 seconds

10 10 -Printer* *Printer must conform to standard EN Memory -QC capability -HGB correction on high WBC counts -Warning flags on parameter abnormalities -Floating discriminator RBC/ PLT -Mathematical 3-part diff. WBC calc. external, IBM proprinter, Epson format, HP-PCL or DPU411-2 /DPU414 supplied by Boule-Medical >350 samples SD, CV, Xm Yes Yes Yes (position printed) Yes -Automatic HGB blank on each sample Yes -Carry Over < 1% -Bar-code scanner input Yes -Serial output computer & connection must Yes conform to standard EN Mains voltage / Fuses 230 V Fuse 5x20mm T2A 120 V Fuse 5x20mm T4A -Mains voltage tolerances +15 % / -15% -Power consumption max 250 VA -Power consumption (standby) max 50 VA -Built-in test /adjustment programs Dimensions (mm) Weight (net) Yes H= 350 W=420 D=460 (mm) ca. 22 Kg

11 Parameter ranges -Linearity +/-1 % within the following range: WBC RBC MCV PLT HGB PLT-Concentrate mode * (RBC < 0.3) *Medium, measured 3 times on a Latex dilution -Measuring range: WBC RBC MCV PLT HGB PLT Concentrate mode Correlation * RBC, WBC R > 0.97 PLT R > 0.90 HGB R > 0.99 MCV R > 0.90 * Using Bayer H3 as reference -Reproducibility (typical): Measured as an average of 10 measurements each of 10 different vein K2-EDTA collected normal samples: Parameter Xmean ( CGS units) CV(%) WBC RBC MCV PLT HGB

12 Reagents and reagent consumption CAUTION IMPORTANT Reagents used in the CA620-CellGuard must conform to the specifications of Boule Medical. Erroneous results and damage of the system might occur in case wrong type of reagents are used. The reagent consumption is depending on the total number of samples / day. Using the system for more than ca. 50 samples / day; the reagent consumption will be: -Diluent -Lyzer ca. 19ml /sample ca. 9 ml /sample If the CA620-CellGuard is used for less than 50 samples /day, the approximate consumption can be calculated from the graph below. The figures stated are indicative only and assumes maximum 2 blank counts per day. Diluent consumption CA620 vs # samples Diluent consumption / sampl (ml) Number of Blood_samples/Day Diluent Consumption

13 13 Lyzer consumption CA620 vs # Samples Lyzer consumption / sample (ml) Number of Blood_samples/Day Lyzer consumption For additional information regarding cleaning solutions; please refer to the chapter Maintenance 3.4 Limitations Verification of any abnormal test result (including flagged results or results outside of the normal range) should be performed using reference methods or other standard laboratory procedures for the conclusive verification of results. The section below list known limitations of automated blood cellcounters in general as well as cellcounters using the impedance technology. WBC White Blood Cells (Leukocytes) WBC that exceeds the linearity limits of the system will require dilution of the blood sample. Re-assaying the diluted sample will help to obtain the correct assay value. NRBC Immature Nucleated Red Blood Cells will be counted in the WBC parameter. If the number of the NRBC is sufficient to activate the DE alarm, such interference will be detected. However, the manual differential blood cell count, performed on a stained blood film, will reveal the presence of NRBCs. Unlyzed Red Cells In particularly rare instances, the RBC in the blood sample may not completely lyze. These non-lyzed cells may be detected on the WBC histogram with a DE alarm or as an elevated baseline on the side of the lymphocyte population. Nonlyzed RBCs will cause a falsely elevated WBC count. (See also NRBC above)

14 14 Multiple myeloma The precipitation of proteins in multiple myeloma patients may give elevated WBC counts Hemolyzis Hemolyzed specimen contains red cell stroma (debris), which may elevate the WBC and/or PLT count. Leukemias A spurious low WBC count may result in this disease state because of the possible increased fragility of the leukocytes leading to some destruction of these cells during counting. The cell fragments will also interfere with the white cell partial differential parameters (Lymph, Gran and Mid). A spurious low WBC count may also be seen in patients with lymphocytic leukemias due to the presence of abnormally small lymphocytes, which may not be counted by the instrument. Chemotherapy Cytotoxic and immunosuppressive drugs may increase the fragility of the leukocytes, which may cause low WBC counts. Cryoglobulins Increased levels of cryoglobin that may be associated with myeloma, carcinoma, leukemias, macroglobulinemia, lymphoproliferative disorders, metastic tumours, autoimmune disorders, infections, idiopathic disease, aneurism, pregnancy, thromboembolic phenomena, diabetes etc can elevate the WBC, RBC or PLT counts as well as the HGB. The specimen can be warmed up to 37 C and reanalysed immediately or a manual WBC, RBC or PLT count can be performed. RBC Red Blood Cells (Erythrocytes) The red blood cell dilution contains all the cellular elements of the blood, RBC, WBC and PLT. During the counting platelets are not counted since the size falls below the threshold (discriminator). Leukocytes however, are always included in the RBC count. Since the concentration ratio between RBC and WBC is normally ca. 1000, the introduced error is almost neglectable. In case of high WBC counts with low RBC, the RBC count may be corrected by simply subtracting the WBC from RBC. Agglutinated red blood cells This might cause a falsely decreased RBC count. Blood samples containing the agglutinated red blood cells may be identified by observing abnormal MCH and MCHC values as well as by examination of the stained blood film. Cold agglutinins IgM immunoglobulins which are elevated in cold agglutinin disease may lower RBC and PLT counts and increase the MCV.

15 15 HGB (hemoglobin) Turbidity of the blood sample due to any number of physiologic and/or therapeutic factors may produce falsely elevated HGB results. The CA620 however is compensated for this effect with high WBC counts up to WBC ca e3/µl. In case of extreme WBC counts, the following is recommended: 1. Elevated WBC The diluted sample should be centrifuged and the supernatant fluid checked on a spectrophotometer for turbidity. 2. Elevated lipids Elevated lipids in the blood sample will give the plasma a milky appearance. This condition can occur with hyperlipidemia, hyperproteinemia and hypobilirubinemia. Accurate HGB determinations can be achieved by using reference methods and a plasma blank. Increased turbidity may also be seen in cases where the red blood cells are resistant to lyzing. This condition will cause a falsely elevated HGB result but can be detected by monitoring the MCHC. Fetal blood The mixing of fetal and maternal bloods may produce a falsely elevated HGB value. HCT (hematocrit) As HCT is the product of MCV x RBC, any erroneous result in MCV and/or RBC will produce an equal error in the HCT parameter. Red blood cell agglutination May produce an erroneous MCV value and therefore a false HCT WBC An excessive number of WBCs might cause interference within the RBC population and therefore a false MCV value. PLT Excessive numbers of PLT, in most cases, do not interfere with the MCV parameter due to the use of the floating discriminator technology in the CA620- CellGuard. RDW (Red cell Distribution Width) The red cell distribution width is a function of the RBC count and derived from the RBC histogram. In most cases, any error introduced in the MCV may also cause the RDW to be erroneous. Especially blood transfusions may raise the RDW significantly due to the presence of bi-modal populations.

16 16 PLT (Platelets or Thrombocytes) Very small RBCs might elevate a PLT count. This effect is minimized in the CA620-CellGuard due to the use of a floating threshold (discriminator). By observing the PLT and RBC histograms, this effect is seen as an overlapping PLT/RBC area. Agglutinated RBCs Agglutinated RBCs might trap platelets and may give an erroneous low PLT count. The presence of agglutinated RBCs is detected by monitoring the MCHC parameter and by careful examination of the stained blood film. Giant platelets in excessive numbers This may cause a low PLT count since they might fall within the RBC threshold range. Chemotherapy Cytotoxic and immunosuppressive drugs may increase the fragility of these cells, which may cause low PLT counts. Reference (manual) methods may be necessary to obtain an accurate platelet count. Hemolyzis Hemolyzed specimens contain red cell stroma, which may elevate platelet counts. A.C.D. blood Blood anticoagulated with Acid Citrate Dextrose may contain platelet aggregates, which could depress the platelet count. RBC inclusions Erythrocyte inclusions may also produce a spuriously increased platelet count. (e.g. Howell-Jolly bodies, siderotic and basophilic granules) Platelet agglutination Clumped platelets due to poor collection techniques or platelet satellitosis caused by EDTA activation of immunoglobulins may cause a decreased platelet count and/or an elevated WBC count. The specimen should be recollected in sodium citrate anticoagulant and reanalysed for only the platelet count. The final PLT result must be corrected for the sodium citrate dilution effect. MPV (Mean Platelet Volume) Giant platelets Large platelets counted as RBCs will fall outside the PLT range and therefore lower the MPV.

17 17 Small erythrocytes Very small RBCs might fall into the PLT region and might be counted as PLTs and therefore influence the MPV parameter. (This effect is minimized in the CA620-CellGuard due to its floating threshold system) Agglutinated erythrocytes This may cause trapped platelets and therefore affect the MPV parameter. Note that agglutinated erythrocytes may be detected by carefully examing the MCHC parameter and/or the stained blood film. Chemotherapy May also effect the size of the PLTs EDTA Note that all samples collected in EDTA will not maintain a stable MPV. The PLTs will swell as a function of time and temperature. Lymph (Lymphocytes) The lymphocyte count is derived from the WBC count. The presence of nucleated red cells (NRBC), certain parasites and erythrocytes that are resistant to lyses may interfere with Lymph. Mid (Mid sized area) The mid size area consists mainly Monocytes. The presence of large Lymphocytes, atypical lymphocytes, blasts, and excessive number of basophils may interfere with the Mid area. Gran (Granulocytes) The granulocyte cell count is derived from the WBC count. The excessive presence of metamyelocytes, myelocytes, promyelocytes, blasts and plasma cells may interfere with an accurate granulocyte count.

18 18 4. Measuring principles 4.1 RBC,WBC and PLT concentration detection Detection is accomplished using the electronic impedance principle and occurs in the orifice of the transducer. The blood is diluted to 1:400 (WBC & HGB) and 1:40000 (RBC & PLT) through a precise shear valve system. The shear valve (sampling valve, marked as position 10 in the figure below) cuts a very precise volume (25 ul) from the aspirated blood and dilute with an equal precise volume of diluent (or lyzer) to achieve the final dilution rates. Two separate measuring chambers and transducers are used, one for RBC/PLT and one for WBC/HGB analysis. This excludes any possibility of cross contamination between the lyzer and the RBC/PLT dilution. A pressure is applied on top of the diluted sample and the diluted sample is pressed through an orifice (aperture) of 80 um diameter. Each side of the orifice is equipped with a platinum electrode and an electrical current is applied between the electrodes. When a cell is drawn into a constant current, flowing from an electrode through the orifice to a second electrode, the electrical conductivity changes. This generates an equivalent voltage pulse. The amplitude of the pulse is directly proportional to the volume of the represented cell. The number of pulses corresponds to the number of cells detected. Coincidence corrections are made within the software of the CA620-CellGuard, giving the CA620-CellGuard a full linear range within the specifications of the instrument.

19 19 The PLT, RBC and WBC parameters are measured on a precise aliquot of the sample. The amount of sample measured is determined by the volume of a precise glass column; called a metering tube. Two optical detectors are used for start and stop detection. The start detector is activated by the flow of the isotonic diluent through the metering tube. As the meniscus passes the optical path it causes a voltage change that activates the count and sizing circuitry of the system. As the isotonic diluent continues to flow upward into the metering tube it passes the stop detector. This action stops the count and analysing process and the parameters and distribution curves are displayed (and automatically printed if so programmed by the user). Due to this principle, the CA620-CellGuard series performs absolute counts related to fixed volumes. Re-calibration on the counted parameters is therefore unnecessary.

20 20 The CA620-CellGuard uses a lower discriminator level at ca. 2.5 fl. All cells over this level are analysed and stored. The user defines, by means of setting the discriminator level(s), which cell is seen as a PLT or as RBC cell. A red blood cell is defined as a cell with a volume larger than the upper PLT discriminator level as set in the Setup Menu. All cells smaller than the upper PLT discriminator level will not be recognised as red cells. In case the user has selected a 'floating' discriminator; RBC cells are counted from the set-point of this variable discriminator, which varies from sample to sample between the volume limits set by the user. The number of cells for determining RBC is counted from a suspension of 1:40,000 dilution ratio of whole blood. Let us suppose that a sample contains 5,000,000 cells/µl. A dilution of 1:40,000 then give a final concentration of 5,000,000 divided by 40,000 = 125 cells/µl, so each µl drawn through the aperture generates 125 pulses. The measured volume drawn through the aperture is 270 µl (factory calibrated), so the system will count 270*125 = pulses. The analyser uses a fixed division factor of 67.5, so the display will show 33750/67.5 = 500, which is the correct value. With the size of the orifice and the concentration of cells, there will be a certain coincidence in the orifice system. However this is a constant system factor and compensated within the software by a correct algorithm to make the RBC count linear within the stated specifications. Hence, the total number of cells passing through the aperture when determining the RBC is the value on the display multiplied by the factor Therefore, a sample which gives 5.00 in the RBC display field has been analysed by measuring 500 * 67.5 = 33,500 cells. The reproducibility is directly related to the total number of cells entering the orifice. The higher the concentration, the better the reproducibility. In the CA620- CellGuard with the dilution ratio for RBC of 1:40,000 the CV will therefore be less than 1% for samples with an RBC number within the normal range.

21 Sizing RBC, WBC and PLT The sizing is done in a matrix with the volume on the horizontal (x-) axis and the number of cells on the vertical (y-) axis. (The x-axis is divided into 2048 channels) As stated above, a size distribution is performed for both PLT and RBC simultaneously. The 'RBC' distribution however, is the distribution with a higher volume than the upper discriminator level which is marked on the display and printer as a dotted vertical bar. The maximum (RBC) cell size that can be analysed is 250 fl. Clumps of cells larger than this volume are analysed as being 250 fl and are 'collected' in the highest channel. As the curves are 'normalised', the height of the distribution curve is not necessarily related to the number of RBC cells counted. The reproducibility of the curve is also dependent upon the concentration of cells in the sample. On a low number of RBC cells there might be some slight differences in the shape of the curve from one count to another due to statistical sampling. Sizing of the WBC population is done simultaneously in an equal second matrix. The highest channel represents a volume of ca. 400 fl. As with the RBC/PLT sizing, the reproducibility of the curve is dependent upon the concentration of (WBC) cells in the dilution.

22 Counting time RBC & WBC The microprocessor system checks the counting time for the RBC and PLT counting process. The count time is defined as being the time needed for the sample to be drawn from the start- to the stop detector in the metering unit (see 4.1 above). A counting time, which is marked as 'LO', means (usually) air in the system and the PRIME program should be executed, selected from the SYSTEM FLOW MENU. A counting time, which is HI, may be caused by a blockage in the aperture. In such case, the system tries to rinse the aperture (orifice) automatically. It is advisable to check the counting time by using isotonic diluent as a new sample. (To save the original blood sample.) If the counting time remains 'HI', it is advisable to execute the CLEAN ORIFICE program from the SERVICE MENU. The counting time limits on the RBC/PLT process are 10.5 and 17.5 seconds respectively. Normal counting time is 13.5+/-1.5 seconds. If the counting time is outside the limits, the text 'LO' or 'HI' will be displayed. The counting time can be displayed using the left/right arrow keys and is shown as: RBC-Time= Note that the Counting time is not related to the actual result. Atmospheric pressure variations, protein built-up within the orifice (aperture) and other secondary effects that might cause pressure changes will NOT influence the counted parameters RBC, PLT and WBC.

23 WBC differentials Within the 3-part diff. technology a general problem is found how to define the correct settings of the discriminators for the Lymphocyte, Mid sized cell (mainly Monocytes) and Granulocytes areas. Many analysers are using a fixed discriminator analogue where fixed thresholds are used to separate the 3 populations. The 3 part. diff. technology is based on the reaction of the lyzer reagent(s) to each specific cell type. Lymphocytes are more lyzed compared to the Granulocytes, resulting in 2 main populations whereof the Lymphocytes will be the smallest. The main problem is related to the Granulocyte population. The cytoplasma surrounding the cells is slowly dissolved in the surrounding plasma in whole blood. This means that the Granulocyte population is slowly collapsing and finally interferes with the Lymphocytes. Refer to the figure below where the same sample is analysed after ca. 4, or more, hours. It is clearly seen that a fixed discriminator technology is unreliable whenever analysing blood exposed to time, high temperature or other factors that reduce the cytoplasma surrounding the Granulocytes. To overcome the above problem, the CA620-CellGuard utilizes a so-called mathematical differential where the curves are analysed within the software and 3 separate curves are built through a curve fitting method. Hence, the software of the CA620-CellGuard is building an artificial matching distribution around the main populations. To do so, after the analysing process, the CA620-CellGuard finds 2 main modes (= peaks) within the total distribution. Then matching of the 2 main populations takes place including extrapolation to the base line. The remaining area that was not covered by the 2 main populations is now classified as being the Mid cell area which mainly consists of the Monocytes. A third population is now calculated, representing this area.

24 24 The final picture below consists of 3 curves and will be printed exactly this way. It is obvious that this approach is not dependent on the actual position of the 2 main populations and therefore superior to any system using a fixed discriminator technology. 4.5 MCV (Mean Cell Volume RBCs) The MCV parameter is derived from the RBC distribution curve. As the distribution curve has a maximum volume range of 250fl, the maximum channel also contains clumps of cells that are larger than this volume. Therefore this channel is excluded from the MCV calculation. The MCV is calculated from the volume position of the discriminator to 249 fl. Be aware that the discriminator might be 'floating' or fixed by the user in the 'Discriminator set-up program' In general, RBC counts that are lower than 0.60 (displayed value) do not give a MCV/HCT value due to low statistical significance. If the MCV is calibrated by using the 'calibration' program, found in the set-up menu; the whole curve is recalculated and moved in a correct way that reflects the new calibration setting. The printed curve will therefore always be correct in respect to the actual MCV value. 4.6 RDW (Red Cell Distribution Width) The RDW parameter is calculated from the RBC distribution curve. The CV of the curve is calculated. However, the CV is only calculated on a portion of the curve. This avoids that other populations might interfere. The RDW value is therefore only measured on a portion of the RBC size distribution curve. Hence, not all particles are included in the RDW calculation. The RDW parameter is only valid if the MCV value is not zero.

25 RDW (Red Cell Distribution Width Absolute) The RDW parameter (absolute) is calculated from the RBC distribution curve at a fixed level. In contrast to the RDW parameter expressed as CV, this RDW parameter measures the absolute width of the curve. The RDW parameter is only valid if the MCV value is not zero. 4.8 HCT (Hematocrit) The HCT is defined as being the packed volume of red cells in whole blood and is calculated through MCV * RBC. If no MCV is derived from a sample due to too low a number of RBC cells, no HCT is calculated. 4.9 PLT (Platelets) Platelets are defined (for the purpose of discrimination) as cells in a range from 2.5fl to the discriminator level that is either set on a fixed volume or 'floating' and determined by the software on each sample. The setting of the upper discriminator is done in the set-up menu. See chapter 'Installation' for further explanation. The platelets are determined from the same dilution as the RBC, in fact, the system is counting just 'cells' during the RBC/PLT counting process. The determination of which cell is a PLT or RBC is done at the end of the counting procedure and fully determined by the setting of the user defined discriminator behaviour ( floating or fixed) Let us suppose that a sample contains 200,000 platelets/µl in whole blood. After a dilution of 1:40,000 the sample contains 200,000 divided by 40,000 = 5 cells/µl. So, each µl drawn through the aperture gives 5 pulses. As the counting volume (the volume of the metering glass tube) is 270 µl, the total number of cells that are analysed will be 5*270=1350 cells. In other words, the total number passing through the orifice when determining the PLT is the value shown on the display screen without decimals multiplied by the division factor 6.75 The reproducibility is directly dependent on the total number of cells entering the orifice. In the CA620-CellGuard, measuring PLT from the same dilution as RBC, the CV will be less than 3.5% for samples in the normal range. The CV will be lower for most samples, but on some samples it might be slightly higher. A 'mean' CV of about 3.2 % is expected for well-treated fresh EDTA whole blood samples within the range of e3/ul.

26 26 As the system uses an orifice size of 80 µm diameter, coincidence losses will take place with extreme sample RBC/PLT counts. The system has a well-balanced mathematical correction algorithm for these effects within the software. Please note that if a floating discriminator is used and no well-defined minimum is found between the RBC and PLTs the reproducibility of mainly the PLT is affected. To check the reproducibility of the low PLTs, it might be wise to put the analyser in a fixed discriminator mode to exclude any error introduced by a not well-defined RBC- PLT population. In case the CA620-CellGuard is programmed and used for PLT concentrates, the reproducibility is better than the stated CV figures above. Note that in case high numbers of RBC are present using the PLT concentrate mode, PLTs will show a flag RP (Red cells Present) to warn the operator that the PLT parameter may not be correct analysed MPV (Mean Platelet Volume) The mean cell volume of the platelets is determined from the PLT size distribution curve. The MPV is defined as being the mean value of the PLT size distribution curve from the lower discriminator (2.5 fl) to the position of the upper discriminator which might be programmed as 'floating' or fixed as set in the 'Discriminator set-up menu' PDW (Platelet Distribution Width) The PDW parameter is calculated from the PLT distribution curve. The SD is calculated of the PLT size-distribution curve up to the actual setting of the (upper) PLT discriminator. The PDW parameter is only valid if the MPV value is not zero LPCR (Large Platelets) The LPCR (Large Platelets Concentration Ratio) parameter is calculated from the PLT distribution curve. Platelets that are larger than 12fl up to the (floating) discriminator are expressed in % of the total PLT count. The LPCR parameter is only valid if the MPV value is not zero PCT (Packed Platelet Volume) The PCT is calculated as being PCT= PLT x MPV and expressed in %.

27 HGB (Hemoglobin Concentration) The hemoglobin is determined from the same dilution as the WBC. For each sample a blank is measured as a reference, this means that any drift in reagent-, cuvette-absorption, or lamp is eliminated. The photometer system consists of a tungsten lamp, a cuvette with a length of 15 mm and a filter at a wavelength of 535 nm (bandwidth 20 nm). The HGB readings are slightly corrected for turbidity in case of extreme WBC counts. The lamp is switched off if the CA620-CellGuard is in standby-mode, giving it an extended lifetime MCH (Mean Cell Haemoglobin) The MCH is a calculated value and is defined as HGB/RBC giving the mean HGB concentration in each red cell MCHC (Mean Cell Haemoglobin Concentration) The MCHC is a calculated value and is defined as HGB/HCT. The MCHC is calculated from 3 measured parameters and therefore an excellent instrument stability check. MCHC=HGB/HCT=HGB/(MCVxRBC). In general it could be stated that if a daily mean value is found outside the range g/dl, the instrument has been wrongly calibrated! The daily mean value of the MCHC parameter should always be /- 1.5 g/dl.

28 28 5. Parameter flags The CA620-CellGuard system has several indication- and warning flags related to the measured parameters. The flags are shown on the display as well as printed on the connected printer. An error flag should be seen as parameter value error. The sample should be re-analysed. A warning flag might indicate a pathological sample. If on the same parameter both an error and a warning flag occurs; the error flag has always a priority and is displayed and printed in the first place. Note that a parameter that is outside the Normal Range which is set in the Set-Up Menu is either marked with H or L on the connected printer to indicate if the value is Higher or Lower than the pre-set Normal Range values. The display will in such case show a * in front of the parameter value. The following flags are seen as 'Error flags' or ' Warning flags' 5.1 TU ( Time-out Upper Detector ) The error flag TU might be displayed on the following parameters: RBC, PLT and/or WBC. This error is related to the counting time. It means that the counting time is outside the limits and displayed as 'HI' or 'LO'. Note: The CA620-CellGuard performs an auto-clean of the orifice after a TU is displayed; a re-analyse of the sample might cancel this warning in most cases. See also chapter Maintenance & Trouble shooting 5.2 TL ( Time-out Lower detector ) The error flag TL might be displayed on the following parameters: RBC, PLT and/or WBC. If the system is unable to move the liquid level in the metering unit up to the lower detector, this error is reported. No counting time is displayed in this case. Most probably this is caused by a clogged aperture (orifice). Note that the orifice is cleaned automatically and that in most cases a re-analyse of the sample will cancel this clogging. See also chapter Maintenance & Trouble shooting 5.3 SE ( Statistical Error ) The error flag SE might be displayed on RBC/PLT /WBC and/or HGB. During the analysing process, clumps of cells, partial blockages or other disturbances might affect the statistical accuracy of the displayed number of cells. Therefore the analyser checks the count/time ratio continuously and if this number/time ratio is strongly fluctuating, the parameter will be flagged with the SE mark.

29 29 SE on the HGB parameter is related to an unstable extinction during the blank and/or sample measurement. Most probably the shown result will not be correct and the sample should be reanalysed. See also DE ( Distribution Error ) This warning flag is related to the floating upper discriminator of the PLT size distribution curve. The software analyses the number of cells on the right-hand side of the position of the upper discriminator in respect to the total number within the size distribution curve. If a large population of cells is found in this area, it usually means that no correct minimum can be found between the RBC and PLT distribution. The reason for this might be an incorrect setting of the 'floating' discriminator or an incorrect setting of a fixed discriminator as done in the 'Discriminator set-up' in the set-up menu. However, on pathological samples with low PLT values this effect might occur even if the analysis is correct. The PLT values are probably correct within 20 % of the actual value. In case of extremely low PLT values, this DE mark can be seen as a warning. In case of normal PLT values, it should be seen more as an error flag. A DE flag is also possible on the WBC parameter. In such case the size-distribution curve is too far shifted left, probably due to pathological sample. Another possibility is the presence of aggregated PLTs, which might interfere with the LYMF region. See also chapter Limitations 5.5 FD ( Floating Discriminator ) If the discriminator, between the upper volume of PLT and the lowest volume of RBC, has been set as 'floating' (see set-up menu) the CA620-CellGuard tries to find a minimum on the PLT / RBC curve. At this point, the 'floating' discriminator will be set. If no minimum can be found between the two limits that have been set in the 'discriminator set-up' menu; the 'floating' discriminator will locate itself on the limit where the lowest number of cells are found. This is a warning and NOT an error flag. On low PLT values, where often a non logfitting PLT curve is found, this warning might occur frequently. Usually, with correct settings of the discriminator limits, the PLT parameter is still measured correctly.

30 OF ( Offset error HGB ) This error flag is related to the HGB parameter only. During a Power On or an exit from the Stand-by mode the offset voltage in the photometer system is measured. If the offset limits are violated, this flag is set. The OF flag might also occur during the installation process. In such case, the analyser was probably exposed to high temperature changes and condensation may have occurred. Let the analyser warm up ca. 1-2 hours and proceed with the installation procedure. 5.7 LO ( Low blanklevel HGB ) This error flag is related to the HGB parameter. During each sample a blank is measured as a reference, if the blank level is too low in intensity, due to a bad lamp or a contaminated cuvette, the automatic blank adjustment cannot work reliably. Go to the Service Menu and enter HGB Photometer Adjustment. Press digit 1. The HGB photometer will now perform a self-adjustment. This process takes ca. 5 minutes. See also section HI ( High blanklevel HGB ) During each sample a blank is measured as a reference, however if the blank level is too high in intensity, due to a bad lamp or a previously cleaned cuvette, the automatic blank adjustment cannot work reliable. See 5.7 above on how to correct the HGB photometer. 5.9 NG ( Negative HGB ) This error flag is related only to the HGB parameter. During each sample a blank is measured as a reference, however if a sample has a lower absorption than the measured auto-blank; this error flag is set. The reason might be: a. Heavy fluctuations in stray light b. Strongly-coloured lyzer-reagent To cancel this error, first be sure that the system is filled with correct diluent and lyzer reagents and that the cover and 'front door' are closed. Go to the Service Menu and select 'HGB Photometer Adjustment. Press digit 1 on the keyboard and the photometer will perform all necessary adjustments automatically concerning offset and gain settings. The measured values are automatically stored and the photometer will be fully operational.

31 SE ( Statistical error HGB ) This error flag is related to a statistical check that is made on the HGB parameter. The blank and sample is measured 25 times and the SD is calculated from the photometer output values. If the SD exceeds a certain limit, the SE flag is displayed on the HGB parameter. The reason might be: 1. Bad mixing in the WBC/HGB cuvette. 2. Air bubbles or large particles in the cuvette. 3. Heavy mains disturbance (see service manual) In case this warning occurs occasionally, just re-analyse the sample TB ( Air bubbles ) This error flag is related to the start detector. If during the raise of the diluent column in the glass tube, an air bubble should pass the start detector; this warning is displayed. Select ' Start a Prime Cycle ' from the System Flow Menu to cancel this error NM, OM, TM, BD (WBC Differential Flags) If no WBC Differential is displayed; the following warning flags are valid: NM No Mode No significant population was found. There is no valid WBC population. OM One Mode Only one population is found This flag might be displayed in case of Granulocytosis or Lymphocytosis or if the Granulocytes are collapsed into the Lymphocytes (old blood). TM Triple Mode There are more than 2 main populations. This flag might be displayed in case of Monocytosis, heavy mains disturbance or wrong installation due to poor grounding of the instrument and/or connected printer. BD Bad Distribution The Lymf and Gran population are too much overlapped. This flag is displayed if the Granulocyte population is collapsed into the Lymphocyte area. The reason might be: Too old blood or extremely fragile Granulocytes Refer also to section Limitations WBC

32 RP ( Red cells Present ) This flag is only displayed in case the CA620-CellGuard is used for PLT concentrates. If the concentration of Red Blood Cells is > 0.40 in the PLT concentrate, both RBC and PLT are flagged with RP to indicate that Red Cells are present and that the PLT value may not be correct Comments on flagging capabilities All anomalies and/or abnormal distributions signalled by the CA620-CellGuard should be verified manually for the presence of pathological elements. As a result of the differences in stability towards lyzing of cytoplasmic membranes in the different cell types, pathological elements can be found in a number of different zones. This also applies to the presence of normal or non-pathological cells that have been subject to chemotherapy or some other form of treatment. This might result in false flagging. See also chapter Limitations

33 33 6 Installation CAUTION IMPORTANT The following procedures must be followed exactly. Boule Medical has no responsibility in case of faulty or erroneous installation. The CA620-CellGuard is packed as standard in a wooden box. Before the box is opened check for any physical damages on the outside and notify your carrier immediately in case of such. Unpack the instrument and check that the following items are included: Users Manual 1 Waste tube 1 Inlet tube with detector for isotonic diluent 1 Inlet tube with detector for pre-diluted hemolyzing agent 1 Mains cable (U.S. or EU) 1 Check that the CA620-CellGuard and the accessories are not physically damaged. If there is any damage or accessories are missing, contact distributor and carrier immediately. 6.1 Working conditions Mains Supply Environment The CA620-CellGuard should be operated at an indoor location only. The instrument is designed to be safe for transient voltages as defined in IEC In case higher transient voltages, or mains voltages that exceed + 10% of the marking at the serial number plate are expected (e.g. within tropical areas), a CVT (Constant Voltage Transformer, also called Magnetic Stabilizer or Ferro-Resonant Transformer ) must be installed to protect the CA620-CellGuard against damage. In case a printer and/or computer is connected to the CA620-CellGuard in such areas, the system must be connected as shown below. Avoid using so-called UPS as they might not protect the CA620-CellGuard against high mains voltage transients in all cases.

34 34 An abrupt interruption of the mains supply will not damage the CA620-CellGuard as all calibration constants and other parameters necessary for the correct working of the instrument are protected against mains supply loss. The CA620-CellGuard must only be connected to a grounded mains supply. Violating this, might result in injuries and/or loss of life and/or erroneous parameter results Location The CA620-CellGuard should be placed on a clean and level table or workstation. Avoid exposure to sunlight. Be sure that the CA620-CellGuard has access to proper ventilation and that at least ca. 10 cm free space must be left at the rear of the analyser. The CA620-CellGuard can work correctly between C with a maximum relative humidity of 80%. To minimize the risk of bacterial growth in the reagent packages however, it is strongly recommended to keep the laboratory at ca. 22 C only. Operating the instrument in a 32 C environment increases service-needs as well as degradation of sample specimen within a short period of time Reagent packages Reagent packages are preferably placed at the same level as the CA620-CellGuard. (e.g. the same table). In case such conditions cannot be met, place the Diluent container at the floor level (max 1 meter below the instrument level) but keep the Lyzer container at the same level as the instrument (e.g. the same table).

35 Waste connection The CA620-CellGuard has an open waste outlet and can be connected to a central waste system within the laboratory. The waste outlet, or container, must always be at a lower level than the instrument. National and/or local regulations must be followed in all cases. WARNING! Potentially biohazardous material Because no test method can offer complete assurance that HIV, Hepatitis B or C viruses, or other infectious agents are absent, the waste should be handled at the Biosafety Level 2 as recommended for any infectious human blood specimens in Protection of Laboratory Workers From Infectious Disease Transmitted by Blood, Body Fluids and Tissues- 2 nd Edition, Tentative Guidelines(1991) Document M29-T2 promulgated by the National Committee for Clinical Lab. Standards in the U.S.A. (NCCLS) 6.2 Mechanical check and set-up Instrument installation Lift the instrument and place it on the chosen location as shown below. Be careful not to stress the front door of the instrument.

36 36 Open the front door of the CA620-CellGuard, and locate the cover plate of the tubing system. Unlock the screw at the left hand-side as shown below. Lift up the screen slightly and remove it forwards. Remove the grounding wire at the rear of the cover plate.

37 37 Remove all transport guidings at the pinch valves. These guidings are red coloured plastic clamps inserted in each valve. Save them for later use in case of a deinstallation. IMPORTANT One valve is located behind the pre-dilute start lever. Do not forget to remove the transport guiding in this valve. Re-assemble the front cover in reverse order. Be sure to reconnect the groundwire! Reagent pack installation Place the reagent pack in the proper place, see also 6.1.3, and put the probes as shown below (the example shows the lyzer container)

38 38 Locate the reagent inlets at the rear of the instrument, see picture below. Connect the corresponding tubes from the reagent probes and the level detector cables as shown in the picture below. Connect the waste outlet to a proper container or open drain, see also CAUTION The end of the waste tube must be at a lower level than the instrument itself.

39 39 Final set-up Locate the serial number plate at the rear and check that the mains voltage and frequency corresponds to your local supply. Insert the power cable and connect to the mains supply. A short beep is heard where after the instrument will perform a self-check that normally takes 2 minutes. (Max. 15 minutes in case of condensation) CAUTION In case the local mains supply is expected to be non-conforming to international standards regarding transients, bursts, voltage variations etc. Refer to section 6.1.1

40 40 Printer installation (optional) In case the DPU411-2/DPU414 printer is used as supplied through Boule Medical, connect the printer to the print outlet as shown below. Follow the instructions as shown in the DPU411-2/DPU414 user manual how to insert paper and how to set the printer on-line In case an IBM compatible printer Epson or HP printer is used, please refer to section Printer and Serial Output 6.3 Front panel and command keys The front panel of the CA620-CellGuard consists of a numerical keyboard, display and a power-on indicator as well as a Print and Menu button. See picture below.

41 41 Using the keyboard Numerical keys are used to enter ID, Calibration factors, Normal ranges and other entries that need numerical identification. CE always clears a previous entry unless the Enter validation key has been pressed. Print will send the sample data to either the printer and/or serial output depending on how the instrument is configured by the end-user. Menu-Operate is used to switch between Menus and the operational mode. The operational mode is defined as the instrument status where the next sample can be entered at the aspiration pipette. The arrow keys are used to scroll through the Menu system, switch between different display modes and to display alternative parameters. The right arrow key is also used to enter either a + or a within the calibration menu.

42 42 Note that the power-on indicator is an indicator only. Note: Aspiration command keys are not located on the keyboard. Aspiration of a sample is started by pressing the start-lever behind the aspiration needle. 6.4 Filling the system with reagents After power-on, the CA620-CellGuard will perform a self-check which lasts for ca. 2 minutes. During this period, no key entry is accepted by the CA620-CellGuard. Press Menu until the main menu is displayed on the LCD screen. Scroll with the up-arrow key to System flow menu and press Enter. Scroll to Start filling system and press Enter. The system is now filled with reagents. This cycle lasts for ca. 6 minutes. When the system is finished with this cycle, the cursor field returns to the field Start Filling system. Press Menu to return to the main menu.

43 43 7. Initial system configuration The CA620-CellGuard is configured as default to the most common use. However the end-user must decide if the CA620-CellGuard is used for pre-donor screening or if it is mainly used to monitor PLT concentrates. This chapter gives guidelines how to install the CA620-CellGuard under these different conditions. 7.1 Setting up the instrument for pre-donor screening The CA620-CellGuard used for pre-donor screening accepts whole blood at the open aspiration pipette, micro capillary collected blood through the MPA device or prediluted blood entered at the predilute aspiration probe. In case the optional cap-piercing device is installed, closed tubes can be entered as well. Normal range values, floating discriminator settings are set to default to the most common and correct settings. All available parameters are set to default to be reported on the LCD screen as well as on the connected printer. Hence, no extra settings are needed except that the instrument is programmed to default in blood mode To do so, press Menu until the main menu is displayed and scroll with the up-down arrow keys to the line Set up menu and press Enter. Scroll to the line Set default discr. Programs and press Enter Press digit 1 and press Enter. This sets the CA620-CellGuard to blood operational mode as default. 7.2 Setting up the instrument for PLT concentrate The CA620-CellGuard used as a monitor for PLT concentrates, accepts the sample at the open aspiration pipette Normal range values and floating discriminator settings are as default the most common and correct settings.

44 44 Parameters that are not valid are blocked from the display (e.g. HGB and related parameters are not displayed and printed). Hence, no extra settings are needed except that the instrument must be programmed as default in PLT concentrate mode To do so, press Menu until the main menu is displayed and scroll with the up-down arrow keys to the line Set up menu and press Enter. Scroll to the line Set default discr. Programs and press Enter Press digit 2 and press Enter. This sets the CA620-CellGuard to PLT concentrate operational mode as default. 7.3 Setting date and time Enter the Setup menu as described in 7.2 above and scroll to the line Set Date/time and press Enter As seen on the LCD screen, 4 different date formats can be set. For EU, set date format 0, for the U.S. set date format 2. Use the up-down arrow keys to scroll to the date format field and enter the correct setting. Scroll to the line Date and enter the correct date using the previous set date format. Scroll to the line Time and set the correct time in 24 hours notation. In case a different date separator is required, scroll to the line Date separator en press Enter. From the following menu, different date separator signs can be chosen. Use the scroll arrow keys to select and press Enter to validate.

45 Printer configuration In case a printer is connected to the CA620-CellGuard, which is strongly recommended, the print command as well as the print format needs to be set. Press Menu until the main menu is displayed. See below. Point 2, 3 and 4 describes the settings of the print commands and format. Be sure that the printer is connected and put on-line. Scroll to the line Set auto-print-mode and press the key Print A list is printed with several options. In case an automatic printout after each sample is required including the size histograms, select 2 followed by Enter. Setting 0 will disable the auto-print of the CA620-CellGuard. Scroll to the line Set print-key function. This entry selects what and where to print the parameter results if the Print key is pressed. Press Print again, an equal list is printed. Select 2 followed by Enter to activate the Print key function to print all parameters including the histograms. Scroll to the line Set print format and press Print. A list is printed with available pre-programmed formats. In case a DPU411-2/DPU414 printer is connected (recommended) only formats can be chosen which are indicated as DPU411. The print format function describes the order of parameters on the printout as well as the font style. As default, format 8 is set (DPU411). IMPORTANT If no external computer is connected to the instrument, do not select settings 3-8. For detailed info regarding printers and printing options, please refer to section and section 13, Printer and serial output

46 Select language The CA620-CellGuard can be programmed to show the displayed text in different languages, e.g. English, Swedish etc. (depending on program version) To change language, scroll to Setup menu from the main menu and press Enter Scroll (up) to Setup menu2 and press Enter. Type the figure corresponding to the selected language and press Enter to validate to new setting. (e.g. # 1 = English ) 7.6 Select Units The CA620-CellGuard has four different unit modes, based on SI- and CGS units. To change, choose Select Units from the Setup Menu2 and select the required unit expression from the table below.

47 47 SELECT : HGB expr. g/dl mmol/l g/l g/l HCT expr. % l/l % l/l WBC 10 3 /mm /l 10 9 /l 10 9 /l RBC 10 6 /mm /l /l /l HGB g/dl mmol/l g/l g/l HCT % l/l % l/l MCV µm 3 fl fl fl MCH pg fmol pg pg MCHC g/dl mmol/l g/l g/l PLT 10 3 /mm /l 10 9 /l 10 9 /l MPV µm 3 fl fl fl RDW(CV) % % % % RDW fl fl fl fl PDW fl fl fl fl PCT % % % % LPCR % % % % LYMF 10 3 /mm /l 10 9 /l 10 9 /l GRAN 10 3 /mm /l 10 9 /l 10 9 /l MID 10 3 /mm /l 10 9 /l 10 9 /l LYMF % % % % GRAN % % % % MID % % % %

48 48 8. Routine operation The CA620-CellGuard should always be connected to the mains supply and then automatically performs an auto-checking/cleaning cycle every fourth hour. This unique feature protects the instrument from bacterial growth, checks all electronical and mechanical settings automatically and cancels almost all user maintenance. If the CA620-CellGuard is not used within 35 minutes (no key has been pressed or no sample has been run), the instrument will automatically enter a Standby-mode Press Menu and the instrument is operational after the auto prime cycle is finished. Press Menu again to enter the operational mode and the last run sample will be displayed. In case the instrument was powered-on and a Fill system or Prime system was performed, the sample screen will display no parameter results. The operation is identical if the system is programmed for pre-donor screening or used as a PLT concentrate control, however the displayed number of parameters will be different if used in PLT concentrate mode. 8.1 Background The following sequence is performed to check that the background is low enough, before each series of counts: Use isotonic diluent as the sample and aspirate by pressing the whole blood start lever which is located behind the whole blood aspiration needle. Note that the aspiration time of diluent will be ca.10 seconds in case the CA620-CellGuard is set-up for blood specimens and ca. 2 seconds in case the instrument is setup for PLT concentrate. The CA620-CellGuard applies a blood detector, which is an optical device, that stops the blood flow when blood is detected after the shear valve system. Therefore, running in blood mode and entering diluent as a sample, the system will time-out after ca. 10 seconds and continues its cycle. If

49 49 the instrument is used for PLT concentrate, a timer is used to aspirate the sample. This timer is programmable and set as default at ca. 2 seconds. The background should not be higher than: (assuming CGS units) RBC 0.01 WBC 0.1 HGB 0.1 PLT 10 Example:

50 Analysing the Sample (open tube) Choose the operational mode with Menu-Operate so that the last run sample is displayed. Aspirate the sample through the aspirating pipette by pressing the start lever behind the aspiration needle. See picture below. (Hand gloves are not shown for clarity only) The display shows the following sequences: Last Sample (= blank) Aspirating sequence (ca. 3 sec.) The CA620-CellGuard switches now to the following menu. Although not mandatory, enter the ID of the sample with the numerical keypad. Note that a positive identification of a sample is highly recommended to avoid erroneous patient parameter reporting. A maximum of 15 digits are allowed in the ID field. In case the optional Bar-Code reader is installed, simply scan in the ID barcode from the sample tube.

51 51 After ca. 30 seconds; the CA620-CellGuard will switch to the INTRA mode as shown below: The display shows the counting rate on each counted parameter in real-time. The horizontal lines in the boxes represent the parameter normal range as set for PROG1 in menu 5.4.1, which is assigned to blood mode. The drawn line during the analysis is slightly irregular due to the counting statistics. However if the drawn line is within the parameter limit lines, the sample is probably within the normal range. In case the drawn line is outside the limits, the sample is probably pathological. With this tool, the operator may be alerted of a suspected pathological case before the sample is analysed completely. The vertical dotted lines represent the expected normal counting time. See below. Sample displayed after ca. 53 seconds from aspiration:

52 52 IMPORTANT The CA620-CellGuard is ready to accept the next sample after the flashing display cursor in the upper left display corner disappears. 8.2 Analysing the Sample (Pre-dilute) The CA620-CellGuard system gives the operator the ability to choose between using whole blood (or PLT concentrate) or pre-diluted samples. To use pre-diluted samples, proceed as follows: Dilute the sample externally with Diluent using one of the following options: 20 ul and 5 ml Diluent to 40 ul and 8 ml Diluent This means that any dilution rate between 1:200 and 1:250 can be used under the condition that the minimum total volume is 5ml and the maximum volume is 8 ml. It is obvious that always the same dilution rate must be chosen, as any reproducibility error in an external dilution will directly affect the counted parameters. The sample should be analysed as soon as possible. Prolonged waiting increases the inaccuracy of the MCV and WBC Differential parameters. Place the cup with the pre-diluted sample under the aspiration pipette for pre-diluted samples and press the lever. Note that the CA620-CellGuard aspirates more than 5 ml, however volumes over 5ml are not specified. This means that in most cases the whole sample (volume) is aspirated into the analyser. Remove the sample cup after aspiration. The display sequence on the LCD screen is identical to the description in 8.1 above. If no sample was entered and the predilute-start lever was pressed anyhow, the system will recognise this and restore the liquid flow system automatically.

53 Analysing the Sample (Micro Pipette Adapter, MPA) The Micro Pipette Adapter MPA is a device that allows the operator to use direct capillary samples without any pre-dilution. This is suitable in case the patient is nearby the CA620-CellGuard, like small care-stations or private doctor s offices and especially when used as a pre-donor screening device at blood banks. Capillary tubes used are high precision 20 µl (+/- 1 %) EDTA tubes supplied by Boule-Medical. a. Press Menu so that the CA620-CellGuard is in operational mode. (The previous sample is displayed). b. Pull out the MPA adapter, remove the previous sample capillary pipette and place the adapter on the table in the supplied MPA holder. The display shows: c. Puncture, using the Boule Medical Micro Lancet d. Aspirate the sample as shown below.

54 54 e. Insert the micro pipette (capillary) in the longest tube of the MPA device. f. Insert the MPA into its holder and the CA620-CellGuard will automatically start the analysing sequence. IMPORTANT Detailed movie shots are available on our support server how to use the MPA system in detail. Please put your internet browser to the following address and download the MPEG movie shots. The movie shot shows also how to clean the Micro Pipette before it is loaded into the MPA Analysing the Sample (Cap Piercing Device) The cap piercing device model 220 is an optional built-in unit that enables the CA620-CellGuard to be used with several types of closed tubes. A tube selector is used with 6 positions giving the operator the ability to use 6 different kinds of tubes. A motor driven needle is penetrating the closed tube and aspirating the sample where after the needle and aspiration tube is cleaned and dried. The closed tube might be of a vacuum or zero pressure type. The adapter handles even a slight overpressure in the closed tube in a correct way. The minimum volume in the closed tube should be ca. 1 ml using a standard B&D tube with a length of ca. 73 mm.

55 55 a. Rotate the wheel to a position that fits your used tube dimensions. The positions can hold auxiliary adapter devices that can be ordered from Boule Medical to fit your local needs. b. Push (rotate) the protection cover to the left and insert the tube up-side down.

56 56 c. Push (rotate) the protection cover to the right. The aspiration cycle is now started, following the sequence as described in section 8.2. Remove the tube after the sample parameters are displayed in reversed order. The adapter is ready to accept the next tube when the sample parameters are displayed and the flashing cursor in the parameter-field of the display disappears. 8.5 Advanced user options In section 8 above the normal use of the instrument is described. The system was either used for Blood mode or PLT concentrate mode It is possible however to switch between these 2 modes without altering the menu Set default discr. Program as found in the Set up menu. Suppose the following: In case the CA620-CellGuard was set up for PLT concentrate mode as default but it is required to analyse some whole blood samples. Proceed as follows. 1. Press the keys until PROG 1 is displayed within the Operational mode Note that the text PROG1 might have been replaced with the text Blood. See section

57 57 The LCD display shows the following. 2. Aspirate the sample by pressing the start lever or pull out the MPA 3. After the aspiration of the sample or insertion of the MPA, enter the sample ID if required. The sample is now processed as Blood all PROG 1 settings are applied to this sample. Note that the next sample will fall back to the default PROG mode unless changed by the user!

58 58 9. User interface (Menus) This section describes the function of each available menu (except the Service menu) in the CA620-CellGuard that is not described in any previous section of this manual. The main menu is used to directly select the most commonly used functions. Direct access to the Sample memory, as well as easy access to the System flow menu is provided. 9.1 Sample memory Entering position 1 from the main-menu gives access to the sample memory. The memory can store more than 350 samples including the size-distribution curves (Current program version 359 samples). Search, print Q/C and delete functions are selectable by ID#, SEQ #, Date or PROG in any combination. In case the memory is full, the oldest sample is automatically deleted. This means that the last 359 samples always are memorised. As default, the current date is used, giving direct access to all samples during the current day. In the display example above it is seen that a total of 19 samples are in memory but none (0) during the current date. The whole memory, in this example 19 samples, is selected by just pressing Enter at the Date menu.

59 59 Use the arrow down key to select other condition-selections. The selected number of samples is always displayed as well as the total number in memory. If PROG=0, all specimen types are selected. In the CA620-CellGuard, PROG 1 is assigned to blood and PROG 2 is assigned to PLT concentrate. Please find below some examples how the sample memory can be used. Question 1 (example): To-days date is 26 March We have a sample in memory tagged as ID = This sample was analysed 9 times during this day. We want to determine the CV of this sample. Answer: Enter the Sample Memory. Scroll down to the line ID = and type 1021 followed by Enter. Now, the top line will change to: 9 OF 359, confirming that 9 samples with this ID were found in memory. Scroll further down to DATE= 26/03/1999. It is seen that this is the current day and no input is necessary. Scroll down to Q/C Calculations and press Enter and Print if a printout is required. Note: Q/C calculations are done on both Normal+Abnormal and Normal parameter values only. Where Abnormal parameter values are defined as being all values except 0 or out of measuring range. Normal parameter values are values within the parameter normal range as set in the Setup Menu line Set parameter normal ranges. SD, CV, X and n are displayed for each parameter. Use the / keys to scroll between the parameters and use the keys to select SD, CV, X or n for NORM+ABN or NORM values only. Pressing <PRINT> will print all available data to the selected printer. This is the recommended way of listing the statistical calculations.

60 60 Below is an example of a Q/C display with no specific ID selection: The example above shows that on RBC, 16 samples are present in the selected memory whereof 9 within the normal range. Question 2 (example) We want to have access to all samples in memory and scroll backwards through all samples. Answer: Enter the Sample Memory. Scroll down to the line Date= and press Enter Now, the top line will change to 359 OF 359, confirming that all samples in memory are selected. (the figure 359 corresponds to a full memory) Scroll down to Search Stored Samples and press <ENTER>. The display shows now the last sample in memory. Use the keys to scroll through the sample memory and the / keys to display different parameters. The Print key can be used to print a specific sample. The letter M is displayed in the upper right corner to indicate that the display shows a memorised sample and not the last run. Question 3 (example) We want to check the mean value of all PLT concentrate samples in memory. Answer: Enter the Sample Memory. Scroll down to the line Date= and press Enter. Now, the top line might change to: 359 OF 359, confirming that all samples in memory are selected. (the figure 359 corresponds to a full memory)

61 61 Scroll down with the key to the line PROG and select 2. As 2 is assigned to PLT concentrate mode. The display might change to 22 of 359 confirming that 22 samples were tested in PLT concentrate mode. Scroll down to Q/C calculations; press Enter and Print to get a print out of the statistics. 9.2 Setup menu Within the setup menus all user settings are defined, calibration, normal range settings etc. Select Set up menu from the main menu and press Enter. Scroll with the up-down arrow keys to display the second page of the set-up menu Calibration menu s The CA620-CellGuard has 3 calibration menus, one for each sample inlet. This means that each sample entry can be matched to show the same results independent at which inlet the sample was run. The recommended way of calibrating the system is therefore to use the whole blood inlet as a reference for Predilute-calibration and Capillary blood calibration (MPA). Press Enter to select Whole blood calibration. Arrow key, used to enter + or -

62 62 Type in the numerical value in %, with one decimal accuracy, but without entering the decimal point to adjust the required parameter Example 1: - The RBC shows 5.00 and we want to adjust to Place the cursor with the keys on the RBC field and type The decimal point is automatically added. - Press Enter and the RBC is adjusted with +2.0 % Example 2: - MCV shows 90.0 and must be adjusted to Move to the MCV field by using the keys - Type 56 and press the key to set a (minus) sign - Press Enter - The MCV is now adjusted with 5.6% which reflects the difference between 90 and 85 fl. Example 3: - PLT concentrate shows 1200 and must be adjusted to Move to PLT within the PLT-Conc. field by using the keys - Type 5 - Press Enter - The PLT in PLT-Concentrate mode is now adjusted with +5% which reflects the difference between 1200 and In general: The + or - is entered with the second function of the key. Numerical values are entered within the ---- field and validated with the Enter key. Key in the calibration, than press the key to toggle between + or - and confirm with Enter Note that only the current sample is re-calibrated whenever changing / leaving this menu. The sample memory is not recalculated, except the last run sample. Notes: RDW and PDW calibration is done by entering an absolute value instead of a percentage difference. E.g.: a sample with a known RDW and/or PDW must be analysed first, than key in the known value(s) and confirm with ENTER: Therefore, RDW and PDW cannot be calibrated if the last sample run was a blank. Also, RDW and PDW cannot be calibrated if the sample has too low a count (RBC < 2.00 and PLT < 200) or if there were any error flags like TU, TL etc. on the last analysed sample. The balance between Lymf and Gran can be changed at the last line of the calibration Menu. Entering a + % or -% calibration factor will move a percentage from the Gran to the Lymf or vice versa. Note that the % calibration here is related to the Absolute number of Lymf / Gran. Leave these calibration factors at 0% unless the CA620-CellGuard is used to match another method or analyser. The Lymf / Gran calibration is the same ( global ) for all 3 calibration menus.

63 Set parameter normal ranges In this menu the normal range for each parameter can be defined. Parameter values outside these limits are marked with * on the LCD display and H (high) or L (low) on the printout. Normal ranges for each parameter might vary considerably between populations and should be established using local population mean values. The default values are set according to medium values within the EU. The CA620-CellGuard has the possibility to set 9 different normal range settings assigned to PROG1-9. Within the CA620-CellGuard, PROG 1 is assigned to blood mode and PROG 2 is assigned to PLT concentrate mode. PROG 3 to 9 are not assigned to any particular application. IMPORTANT Note that each PROG has its own set of Normal Range settings, Floating PLT/RBC discriminator settings and WBC differential settings. The Calibration setting, see above, is the same for all PROGs. Scroll with the arrow keys to Set parameter normal ranges and press Enter. The following is displayed. Enter PROG 1 to set normal ranges for Blood mode or enter PROG 2 to set normal ranges for PLT concentrate mode The example above displays the settings for PROG 1. Enter the required ranges by using the numerical keypad and validate with Enter. Scroll through this menu using the arrow keys and press Menu to return. The following example sets the normal range limits for PLT concentrate mode.

64 64 Enter the required ranges the same way as described above. Note that several parameters do not have normal ranges set as they are blocked from being displayed and printed. See also the set-up for Block parameters Set floating discr. PLT/RBC Scroll with the arrow keys to Set floating discr. PLT/RBC and press Enter In this menu the floating range for the discriminator (also called threshold) is set for each PROG. It is not advisable to alter these settings as the default settings for PROG 1 and PROG 2 ( blood mode and PLT concentrate mode ) are set to values that are strongly recommended. Blood mode In the example below, PROG 1 is shown by pressing Enter and the settings for the floating discriminator are displayed. The displayed fields are: PLT-L. The lowest possible setting for the floating threshold between PLT and RBC in fl (= um3). As default this is set to 15 fl. PLT-H. The highest possible setting for the floating threshold between PLT and RBC in fl (=µm3). As default this value is set to 30 fl, which is the maximum level available in this menu. PLT conc. Mode is set to 0 as PROG 1 is assigned to Blood mode only. PLT concentrate mode The example below shows the default values set for PROG 2, which is assigned to PLT concentrate mode.

65 65 The displayed fields are: PLT-L. The lowest possible setting for the floating threshold between PLT and RBC in fl (=µm3). As default this is set to 15 fl PLT-H. The highest possible setting for the floating threshold between PLT and RBC in fl (=µm3). As default this value is set to 60 fl, which is the maximum level available in this menu. PLT conc. Mode is set to 1 as PROG 2 is assigned to PLT concentrate mode only. Setting this menu to 1 enables the PLT-H setting to a maximum level of 60 fl. This setting (60fl) is chosen as default in PLT concentrate mode to include possible macro PLTs in the total PLT count. This setting 1 also enables the aspiration mode to switch to a time aspiration instead of using the Blood detector. See also section Also, if PLT-H is set to 30 fl instead of 60 fl, all PLTs larger than 30 fl will be reported by the system as RBCs, which might introduce a slightly false RBC parameter value. Therefore, it is recommended not to change this setting without a compelling reason. IMPORTANT Note that each PROG has its own set of Normal Range settings, Floating PLT/RBC discriminator settings and WBC differential settings. The Calibration setting, see above, is the same for all PROGs Do not alter PLT Conc. Mode. This must be 1 to enable the correct sample aspiration mode Set discr. WBC Scroll with the arrow keys to Set discr. WBC and press Enter In this menu the total range in fl for which the mathematics are applied to check for a valid Lymph and Gran population within the 3 part differential. The settings in all PROGs are the same and set as default to fl. Hence, the software will look for a valid population where 2 main modes (peaks in the histogram) is found within this volume range. It is not recommended to alter these settings without instructions from Boule Medical. The following is displayed.

66 66 The displayed fields are: LYM-L. The lowest possible mode (peak on the histogram) where Lymphocytes might be detected. GRAN-H. The highest possible mode (peak on the histogram) where Granulocytes might be detected. In case any of the modes (peaks on the histogram) are outside these limits, the 3-part differential is aborted and a NM or OM differential flag is displayed. See further section Parameter Flags Set default discr. program This menu sets the default mode of the CA620-CellGuard, which PROG setting is used whenever a sample is aspirated. As described in 7.1 and 7.2 in section 7 Initial system configuration, the CA620- CellGuard is configured to be used as a pre-donor screener or as a PLT concentrate tool. Setting the Setting default discr. Program to 1 will select all PROG 1 settings, which equals Blood mode as default with any aspiration of a sample. Setting the Setting default discr. Program to 2 will select all PROG 2 settings, which equals PLT concentrate mode as default with any aspiration of a sample. See further section 7 for detailed information. IMPORTANT Note that each PROG has its own set of Normal Range settings, Floating PLT/RBC discriminator settings and WBC differential settings. The Calibration setting, see above, is the same for all PROGs

67 Print all settings Scroll with the up-down arrow key to Print all settings and press Enter. The connected printer will list all settings that are user programmable. It is advisable to do so after the installation procedure is finished to backup and save the original settings of the instrument. Detailed information of each printed value is listed in the CA620 (CA530) service manual Setup menu 2 Scroll with the up-down arrow key to Setup menu 2 and press Enter. The following is displayed. Within setup menu 2, less common functions are displayed and altered, such as language, units etc. To set language and units; please refer to section 7 Initial system configuration Machine ID Refer to above and scroll down with the arrow keys to the line Machine ID This menu is only of use in case the CA620-CellGuard is connected to a computer system. On the serial output a Machine ID number is send to the connected computer to identify the CA620-CellGuard in case several CA620-CellGuard instruments are used at a lab. In such an environment the Machine ID can be set to 1 9 to give the host a positive identification from which CA620-CellGuard system data was sent.

68 Blood det. Norm / Blood det. PLT-C Refer to above and scroll down with the arrow keys to the line Blood det. Norm, see below. The CA620-CellGuard aspiration of samples in Open tubes and Cap piercing is stopped when the sample (blood) reaches a Blood detector, which is located after the shear valve system. This blood detector consists of a (green) LED and a photocell. Within 10 seconds after the aspiration command, this device should detect blood. In case no blood is detected, the system will proceed anyhow. Setting this to 0 will enable the blood detector function of the CA620-CellGuard. This means that if the CA620-CellGuard is used in Blood mode, the setting must be 0 at this line. Scroll down with the arrow key to menu line In this menu line Blood det. PLT-C the aspiration time is set in seconds. As the Blood detector cannot detect PLT concentrate, this function is disabled and the aspiration of the sample is based on a time set in this menu. To check that this time is correct, observe the flow of the PLT-concentrate during the sample aspiration. The aspiration should stop when the PLT concentrate has passed the blood detector with at least 3 cm. If not, adjust the setting accordingly. An aspiration time of 1.5 seconds is set as default as seen above. Hence, the text 0=ON means that if set to 0, the blood detector is enabled Serial port setup In case the CA620-CellGuard is connected to an external computer, the serial data flow has to be set. Scroll to this menu line with the arrow keys and press Enter. The following is displayed.

69 69 Change the setting, if required, by using the arrow keys and jump to another line within this menu with the arrow keys. See further section 13. Printer and serial data output Barcode reader In case a Barcode reader is connected (available from Boule Medical as optional device), this is enabled within this menu line. Scroll with the arrow keys to this line. If no Barcode reader is connected, this must be set to 0. The available Barcode reader from Boule Medical is enabled by setting this line to PLT offs and High altitude comp In menu PLT offset, the PLT background can be set. This entered value is subtracted from all PLT readings. This menu should be handled with care and only used if the diluent background on PLT is stable and not higher than 10. The menu High altitude comp is used whenever the instrument is installed at levels higher than 1500 meter above MSL (Medium Sea Level) Scroll down to this line using the arrow keys and set 1 in case the CA620- CellGuard is located above 1500 meters MSL. This setting will only extend some of the washing sequences in the CA620- CellGuard due to the capacity reduction of the waste (drain) pump when used at high altitudes. No other instrument specifications will be affected.

70 Print control blood id:s This menu prints out a list of defined quality control-blood types. Type Enter at this menu line to print out the list. As quality control-blood is not comparable with human blood regarding the structure of the WBC differential mainly, certain discrepancies in the total WBC count and/or differential count might occur whenever using such fixed cells as a normal sample. Entering the defined ID numbers as obtained from the list, terminated with a + sign (right arrow key), will instruct the CA620-CellGuard to shift it s floating discriminator range automatically so that it fits the specific controlblood type. As of the current software program status, the following controls are defined. Note that all defined control bloods are of a WBC 3 part differential type! Baker Low Baker Norm Baker High 777+ Boule Low 888+ Boule Norm 999+ Boule High So, entering one of the above defined control-blood Product-numbers as ID#, will be shown on the display (only) in clear text as a confirmation that the quality controlblood mode was selected. E.g.: 888+ will display Control Norm., but is stored in memory as 888+ Thus, enter such control blood by typing 888 and press the arrow key. IMPORTANT Note that entering a specific control blood ( e.g to 3784+), the parameter values are stored under PROG 9. Using 777+ to 999+ Boule control bloods, are stored in PROG 8 This enables the operator to set Normal range value in PROG 8 or PROG9 that reflects the control blood parameter range.

71 Reagent type This menu line is only used to initialise the basic set up at the factory. It defines for which purpose the instrument is used and stores the correct default values. This menu is password protected and cannot be altered by the end-user. It should show 15 in all cases Set PROG names As described in several previous sections, the CA620-CellGuard is equipped with 9 preset- table programs which are linked to Floating discr. RBC/PLT, Set parameter normal range and WBC discr. Program. Assigning the PROG numbers to a name, that is shown on the display as well the printout at each sample, will simplify the identification of in which mode the sample was run. The CA620-CellGuard has 2 defined programs PROG1 and PROG2. PROG1 is assigned to Blood and PROG 2 is assigned to PLT concentrate. Therefore, it is advisable to set the name for PROG1 to Blood and the name for PROG 2 to PLT-C. To do so, scroll down with the arrow keys to the line Set PROG names and press Enter. The following is displayed: In the above example, we want to set the name Blood for PROG1. Press Enter and the sub menu is displayed asking to define the text to be shown on the display and printer. Press Enter to set the required text on the display or scroll to the next line to alter the printed name on the printout instead.

72 72 The following is displayed. Scroll with the arrow keys through the displayed field and press any digit to select the required character. When finished, press Enter to return. Changing both PROG1 and PROG2; the list will now show. Note that the name PROG1 and PROG2 are not changed in any menu to Blood or PLT-C but clarified only as shown above. The display and printout however will show Blood or PLT-C instead of PROG1 or PROG Block parameters The CA620-CellGuard allows the operator to block certain parameters to be displayed and printed. This is especially useful when the instrument is used for PLT concentrate control only. Several parameters are of no interest in such case, like HGB, HCT, MCHC etc. To do so, scroll with the arrow keys to the line Block parameters and press Enter The following is displayed.

73 73 Scroll down to the line PROG 2 in case some of the parameters in the PLT concentrate mode should be blocked. See below. Press Enter and the next sub menu is displayed. In this menu, setting a 1 to a parameter means that it will be displayed and printed. Setting 0, will block the parameter from the display and printout. In the above example it is seen that all parameters are blocked except RBC, PLT, MPV, WBC and LPCR whenever the CA620cellGuard is used in PLT concentrate mode Printer setup menu This menu is used for advanced printer options and user definable printout programming. Detailed information is available in appendix

74 Block pre-dilute start This menu is used to delay the pre-dilute start switch with 5 seconds. Set 1 to enable this function. To minimize the risk that a user enters whole blood at the predilute inlet, this mode might be enabled. In such a case, one must keep the pre-dilute start lever pressed for 5 seconds to start the pre-dilute aspiration.

75 75 10 Service menu The CA620-CellGuard is equipped with an extended service menu. In this menu several functions of the instrument can be tested as well as adjusted if necessary. Detailed description of the menus is described in the Service Manual. Press Menu until the main menu is displayed and scroll with the arrow keys to Service menu and press Enter. See below Pump and valve test Press Enter at the line Pump & Valve test chosen with the arrow keys. The LCD display will show the following. Valve= 0 is initially shown on the display. Pressing the Whole Blood start lever located behind the whole blood aspiration pipette will start the waste (drain) pump. Select with the keys Valve 2-21 and press the Whole Blood start lever to test the pinch valves. Note that 19 and 20 are not valid valves in case the optional Cap piercing device is not installed HGB photometer adjustment In this menu, the HGB photometer setting can be checked and altered. Note that this menu has nothing to do with the actual HGB calibration!

76 76 Scroll with the arrow keys to the line HGB photometer adjustment and press Enter. See below. This menu is used to set the lamp intensity to the correct medium level only. In case an HGB flag is displayed LO or HI, use this menu for corrective action. Press digit 1, and the photometer will adjust itself to its optimum performance. The auto adjust cycle will take ca. 5 minutes Volume detectors This menu is used in service procedures described in the service manual. The current status of the optical liquid detectors in the metering units are displayed. Scroll with the arrow keys to the menu line Volume detectors and press Enter. See below. In the above example, it shows that: The lower detector in the RBC metering column (RBC-L) as well as both WBC column detectors do see liquid. The upper RBC metering column detector (RBC- H) shows air. Note that the above is just an example and is not necessarily displayed as such in all cases Blood & beaker detectors This menu shows the current status of the external reagent containers, the built-in first dilution cup as well as the status of the blood detector located after the shear valve.

77 77 Scroll to this line with the arrow keys and press Enter. See below. The display shows: Diluent = 1 which indicates that Diluent is detected by the reagent probe in the diluent container. Lyzer = 1 which indicates that Lyzer is detected by the reagent probe in the lyzer container. Mix. Cup = 0. This indicates that the internal mixing cup is empty which should always be the case in a well functioning CA620-CellGuard instrument. Blood detector = 0.5 V. This value must always be lower than 2 V. In case of higher values read, the blood detector might not work properly and a service call is needed to readjust the blood detector. It might also indicate that the aspiration tube is highly contaminated with proteins. Use a 4% hypochlorite solution as sample several times at the open tube aspiration inlet and check this voltage again. Another possibility of high readings in this menu might be caused by a malfunction of the aspiration probe-washing device. Call your service centre Motor detectors This menu is used in service procedures described in the service manual or attached bulletins. It displays the status of the shear valve as well as piston position. Scroll with the arrow keys to the line Motor detectors and press Enter. See below.

78 78 The display shows: Turnv. Detector = 2265 mv, this displays the current position of the shear valve Piston detector = 0 displays the position of the optical piston position detector. Note that the displayed values above are just examples. Different values for each individual instrument are common and correct. The Turnv. Detector should never show 0 however Noise test This menu is used to determine if the mains supply and grounding conforms to the standard IEC 801 in respect to transients and bursts. Scroll to the line Noise test, using the arrow keys, and press Enter. The following is displayed. The noise test menu must show 0 in all fields. In case any value is displayed, refer to section to correct the mains supply Print machine statistics This menu is used by service engineers and prints several statistics regarding the use of the instrument (e.g. number of samples, errors, total running time etc.). Detailed instructions are found in the Service manual. Scroll to the line Print machine statistics and press Enter to print the list.

79 Start a clean cycle This menu is only used in case a TL or TU indication occurs at the RBC, PLT or WBC parameter and does not vanish if the sample is re-analysed several times. Scroll to this line using the arrow keys and press Enter to activate this cycle. See below. This cycle lasts for ca. 3 minutes. CAUTION Only activate this cycle in case TU or TL is displayed and if re-analysing of the sample will not correct this error indication. Excessive use of this cycle might reduce the lifetime of the orifice transducer! 10.9 Service menu 2 In Service menu 2 several functions are available to adjust the CA620-CellGuard analyser. These adjustments are password protected and can only be altered by Service engineers certified by Boule Medical. Scroll to the line Service menu 2, using the arrow keys, and press Enter. The following is displayed. Turning valve Calib is related to the shear valve system. Syringe calib. is used to adjust the piston system Closed tube adapter calib. is used to check the Cap piercing device (optional) Procedures to follow are shown in the Service manual.

80 80 11 Warning displays Warnings are displayed in clear text in case of any abnormal situation. This section lists all available warning texts Warnings related to the MPA and pre-dilute inlet This is a normal warning and indicates that the MPA was pulled out. The instrument is waiting for the MPA to be inserted where after a count cycle starts automatically. The MPA cycle was aborted by press the CE key. Push back the MPA. The system will not start a count cycle. The MPA was pulled out before the cycle was finished. Push back the MPA adapter and the CA620-CellGuard will restore the flow system with a prime cycle. See menu message above.

81 81 The MPA was pulled out in a wrong mode. (a menu was displayed). The MPA may only be pulled out in Operational mode where the last run sample is displayed. The operational mode is selected by pressing the Menu/Operate key. The above warning indicates that the Predilute Sample inlet was activated but no sample was aspirated. This might be caused by: a. No sample is entered b. An attempt is made to run on distilled water in the pre-dilute inlet 11.2 Stand-by mode This warning is displayed after a 35 minutes idle situation. The instrument will go to stand-by mode after further 2 minutes. The operator may cancel this by pressing Menu. If the instrument is in Stand-by mode, the text displayed might not be clear as the back lighter of the LCD display is switched off. The CA620-CellGuard will now perform an auto check and cleaning cycle every 4 th hour.

82 82 In case one of the reagent containers are empty within the Stand-by mode, one of the above warnings will be displayed. It is essential that new reagent containers are installed, as the 4-hour checking cycle cannot be performed by the CA620- CellGuard. This warning indicates that an error indication occurred and it was not cleared by the user before the system entered the Standby mode. Restart the CA620-CellGuard, cancel indications by pressing CE and perform a Start prime cycle from the System flow menu. When the CA620-CellGuard is in Standby mode, and the Menu key is pressed, the system will perform a Prime cycle to restore the flow system. This lasts for ca. 2 minutes.

83 Reagent empty The above warnings are displayed in case of an empty reagent container. Replace and perform a Start prime cycle from the System flow menu Printer and serial output warnings The warning messages above are related to the connected printer. Either the printer is not connected or busy. The above warnings indicate that no printer is connected or no computer is available on the serial output. The Print-key functions were incorrectly programmed. E.g. the serial output was selected but no computer was connected.

84 84 The above warning is displayed in case the connected computer serial line is busy and not ready to accept data Auxiliary warnings/messages The above messages are displayed from the sample memory only. Print of 19 samples is selected. The second warning shows a question in case a deletion of 19 samples is requested from memory. The above is displayed during a Power-on. The CA620-CellGuard performs a selfcheck, which might last for maximum 15 minutes in case the instrument was exposed to extreme temperature changes. Normal self check time is 2 minutes. The indicated figures are examples only.

85 Calibration Introduction The CA620-CellGuard has been calibrated at the factory. This applies to all measured parameters. As the CA620-CellGuard has a mechanical fixed metering unit and fixed mechanical micro-volumes; the numerical calibration for the RBC/WBC and PLT are stable at all time. Good laboratory practice however, requires regular checks and calibration of the measured parameters. The software calibration factors for all measured parameters can be changed by the user within a large range; mainly done to match other analysers or methods in the laboratory. It is advisable that the performance of the CA620-CellGuard system is checked daily (not calibrated) with a certified control blood. Special attention should be paid to the calculated MCHC parameter (MCHC = HGB/(MCV*RBC)). Recommended calibration periods are twice a year with an Hematology Blood Calibrator certified by Boule Medical and using a certified PLT Concentrate Calibrator to calibrate the CA620-CellGuard in the PLT concentrate mode. The MCHC parameter can be used as an excellent check on the relation between HGB and HCT. This parameter should be in the range of g/dl. Daily mean values of the samples should always be g/dl. RBC, WBC and PLT Calibration of the above-mentioned parameters is done at the factory with reference standard instruments and volumetric measuring methods. The metering tube is mechanically constant and will remain stable. MCV Calibration of the MCV parameter may be necessary if the temperature environment of the laboratory has changed significantly. (> 10 C) IMPORTANT Never calibrate the MCV parameter using commercial controls with values given for other analysers, unless approved by Boule Medical.

86 Use of Commercial Controls and calibrators The calibration of the CA620-CellGuard can be verified by counting pre-assayed commercial reference control samples or by counting retained patient samples with known reference values from another reference instrument. To ensure the accuracy of the values obtained whenever commercial controls are used: -Always re-suspend according to the manufacturer's recommendations. -Never use an open vial longer than recommended by the manufacturer or subject any vial to excessive heat or agitation. -Verify the condition of controls when received. Make sure that they are cold and not leaking. -Do not use a commercial control for calibrating the CA620-CellGuard, use only Calibrators IMPORTANT Use the special ID function to identify the specific control blood. See section

87 87 IMPORTANT In order not to contaminate the control with cleaning solution, be sure to dry the aspiration pipette manually on the outside at each control blood run. Not following this discipline might give dropping values over time on the concentration parameters like RBC, HGB, WBC and PLT. Also, do not push the control or calibrator tube against the upper washing device. See pictures below: Dry the aspiration pipette manually. Do not push the sample tube against the washing device. Correct sample handling IMPORTANT Note that blood controls for cell differential have fixed cells, they do not behave as fresh blood in a diluted sample. WARNING! Potentially biohazard us material Caution: Because no test method can offer complete assurance that HIV, Hepatitis B or C viruses, or other infectious agents are absent, these products should be handled at the Biosafety Level 2 as recommended for any infectious human blood specimens in Protection of Laboratory Workers From Infectious Disease Transmitted by Blood, Body Fluids and Tissues - 2 nd Edition, Tentative Guidelines(1991) Document M29-T2 promulgated by the National Committee for Clinical Lab. Standards in the U.S.A. (NCCLS)