4- Photometer mode operation
4.4 Measuring (2 Run Method)
4.4.1 Methods with Standard
After reading the blank, the Analyzer inquires whether the calibrator serum’s method selected will require the reading of the calibrator serum, or if it should use a stored factor obtained from a previous standard reading:
If YES is selected, a new standard should be read, and the new factor will be calculated and stored for the selected method.
If NO is selected, then the factor calculated during the last reading of the calibrator serum will be used.
Printout of screen values.
Rotates pump backwards (recovery of sample or standard)
Initiates the sipper action, sipping the samples or creating an air gap between samples.
Once the calibrator serum is confirmed, the following prompts are displayed:
Press input the number of replicates of standard.
readings. The instrument will employ the average value of readings.
read water : Suggests reading the coater blank
read Reagent/Blank : Suggests reading the reagent blank
Next message is "Wait"
Afterwards, "reading blank”
Read Standard
#Replicates : 1
Standard.
Read the standard sample by using the pump actuator.
A printout of the standard’s absorbance and the calculated factor Co/Ao is obtained, where Co is the standard’s concentration and Ao its absorbance.
If the standard’s absorbance is lower than the blank’s (negative absorbance), an error message is displayed and the input of a new standard is expected.
To exit, press ESC anytime during the measuring cycle.
Then the reading cycle starts.
Read the following samples by using the pump actuator.
Screen Example
If a change in the sample number is desired, press SAMPLE and enter a new number, the next sample shall retain the new numbering sequence.
If measurement falls within the high and low limits, the diagnostic message will be NORMAL. If value is higher or lower than the fixed limits, the message will be HIGH or LOW.
reading standard
Read next sample
Sample 1
I 35 I 4.4.2 Methods with factor
The measuring cycle with factor omits reading a standard.
Confirm the factor’s value with ENTER or change it with , introduce new factor and press ENTER.
4.4.3 Methods with color blank (color/blk)
The Absorbance value shown on display or in the printout is the difference between the sample absorbance and the absorbance of the corresponding blank.
Abs = Am - Abm Am: sample absorbance
Abm: absorbance blank sample
This kind of technique is used for bi or mono-reagent assays with a high serum/reagent ratio.
Example: Ferremia, Bilirrubin.
Read water Read reagent/Blk Read sample/Blk Factor
Confirm with ENTER or modify with , if standard samples is used.
read water read reagent/blank
Factor
Water
Reagent
read next blank “ 1 “ read next sample “ 1 “
Next, the absorbance reading of the difference between sample and blank is displayed.
Afterwards:
Read next blank “ 2 “ Read next sample “ 2 “
And so until the the whole sample lot is processed.
4.4.4 Kinetic measurements
Kinetic measurements can be performed using a factor, a standard or a calibration curve in analogy with Endpoint methods. Therefore, all considerations of sections 4.4, 4.4.1, 4.4.2 and 4.5 remain valid. However, each read standard (in case a standard is to be read) and every read sample are performed within a cycle that includes:
1) Incubation during the pre-fixed time (LAG TIME) 2) Measuring during the pre-fixed time (MEASURE TIME)
At the end of every measurement, a high pitch sound will tell the operator that the Analyzer is ready for the next sample.
4.4.4.1 First order kinetics
Each measurement consists of 15 readings fixed at intervals; i.e. the total measurement time is automatically divided into 15 equal parts. Measurement is carried out calculating the slope that best fits the 15 read values by following the method of least squares.
Select method to process.
Read blank
factor - confirm factor with Enter Read sample 1
Wait
Remember that kinetics includes a lag time and a measuring time.
The result of the absorbance in the chosen units will be obtained together with the correlation coefficient of the kinetic reaction.
Next, sip consecutive samples.
When operating in kinetic mode, plot of reaction is seen while reaction is in progress.
Absorbance data are also shown.
I 37 I The correlation coefficient written on the printer after the diagnostic is a mathematical measurement of the reaction’s linearity. An ideally linear reaction will have a coefficient of 1.00 (positive or negative depending whether the absorbance grows or decreases during the measuring time). A non-linear reaction will produce values near 0.00. The closer the value is to 1.00, the more linear the reaction will be, hence the more reliable the data will be.
During the incubation readings are performed to warn the user in case the substrate’s consumption is excessive. If so, a message is generated in the printer indicating the need to increase sample dilution.
Dilute Sample
The informed result will only be considered an approximation, and the measuring process shall be aborted.
4.4.4.2 Higher order kinetics (2 points)
Kinetics methods which absorbance rate of change is not constant in time (positive or negative), i.e. that do not produce constant absorbance rates, are usually measured taking readings in two points along their development.
The measuring process using these methods in the Analyzer is similar to normal kinetic methods (15 points). The only actual difference is that during the MEASURE TIME two readings are performed instead of 15, one at the beginning (coinciding with the completion of the LAG TIME) and another at the end.
This type of method is also called fixed point.
4.4.5 Recalibration
At any time, the user can recalibrate both the blank or standard.
When the screen shows a message requesting to read sample, if the key BLANK is pressed, the message is replaced by:
Read blank
Place the tube containing blank in the sipper tube and then press the pump actuator.
Once the blank readjustment is performed (including a new reading of "0" transmittance) instrument automatically returns to the sample reading cycle.
If the key BLANK was pressed by mistake, the blank reading can be aborted pressing ESC before confirming the reading.
The same procedure is employed to recalibrate the standard in the case of those techniques programmed with a standard as reference. When pressing STD a message is generated:
Read Standard
Place the tube with standard in the sipper tube and press the pump actuator. The standard’s absorbance reading generates the calculation of a new factor, which will be used
from that moment onwards for future readings. By completing that factor’s calculation the instrument automatically retur ns to the sample reading cycle.
4.4.6 Controls
The Analyzer allows the storage of up to 30 control readings, performs the corresponding statistics and prints them for each of the 130 available methods.
When measuring and storing a control sample is desired, inform to the instrument that the next sample to read is a control. Start the measuring process as usual, according to the method type and references used. A printout will be obtained.
Refer to STATISTICS for viewing and printing controls (section 4.7).
4.5 Operation with calibration curves
When a method does not produce a linear result concentration, measurements must be referred to a calibration curve.
4.5.1 Set Method
Input sequentially the parameters as shown in 4.3.
When reaching reference, press . The message will read:
method reference
Select . The input volume, lag time, place, unit. Then
I 39 I Once completed, press
“ABS 1”____ “ABS2”____
“ABS 3”____ _ _ _ _ _ Press
Read water
Read reagent / Blk
Before reading standard 1, empty cell by using
Read Std 1
Empty cell; pressRead Std 2
And so until all standards are completed.
Press
print factor? Yes No
Press the corresponding key
The calibration curve is shown in the display, and if print out is desired press
Measurements
The effective measurement of samples is carried out in the same way used for methods with factor or standard concentration. Take into account that a method with calibration curve is after all a method with variable factor.
To perform a measurement press key 2 in the main menu (RUN METHOD) and input the selected method number pressing
Read water
Read reagent/Blk
Read Sample
Press the corresponding key
The calibration curve is shown in the display, and if you wish to print it press
4.6 Operation with Cut-off
The cut-off is an absorbance value which establishes a limit between normal and abnormal samples. This value is normally defined with a formula that depends on positive and negative controls.
In order to program the method, proceed as in 4.3, except when defining the REF:
Then press
Values to be entered correspond to the coefficients of the equation:
Cut-off = An • A + Ap • B + C
Where An y Ap are the positive and negative control’s absorbances average, respectively.
The A, B and C values to be entered are supplied by the reagent manufacturer.
Uncertainty: % around the value of the CUT– OFF absorband for which the results are not reliable and should be repeated.
Empty cell and clean sipper
with tissue paper between
readings.
I 41 I Input corresponding values pressing value and ENTER.
Scroll through parameters using Exit the menu using
Input A, B, and C.
4.6.1 Cut-off measurement
Select 2 in main menu, search previously programmed method with
Read blank
Next, the screen will display the message "# negatives", i.e. how many negatives are to be read. Input the corresponding number and press ENTER
Example: 2 negatives
Read negative #1
Read negative #2
PLS verify negatives
Please verify negatives with , scroll through all the read negatives. Using DEL you will be able to eliminate any displayed value. the measuring cycle will be resumed.
Message “#positives” How many positives?
Example: 2 positives.
Read positive #1 Read positive #2
The same procedure is followed. Once finalized press
read next sample (#1)
The printout will indicate if the read value is positive or negative.
4.7 Statistics
Select option 3 from main menu to access statistics.
Statistics correspond to the last 10 controls entered during runs of the selected method.
Statistical data correspond to the last 10 read controls for the corresponding method.
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4.8 Photometry (continuous readings of absorbance and transmittance)
The Analyzer can be used as a direct reading instrument.
Select 4 in the main menu.
Enter wavelength, volume and temperature and then Read Blank
Once the blank is read, read any sample in continuous mode.
Read Sample
4.9 Coagulation
Main menu. Select 5 coagulation.
The system allows the programming and storage of up to 4 methods coagulation.
Inside the parameters’ option, there are two possible selections:
4.9.1 Coagulation Parameters
1-Plot limit 2-Delay
Plot limit: It defines the graphic range in screen of the coagulation process; in order to define the threshold accurately, the graphic is extended from –0.2 to the defined plot limit, in absorbance units.
Delay: It is the time interval from the moment when the time measurement is started till the beginning of the absorbance measurement. As during the first seconds absorbance remains practically unchanged, it is convenient to start the reading only after a few seconds, hence preventing the turbulence effects of the mixing and exterior light from creating a fake threshold.
A five-second delay (for example) is enough to dispense, mix and close the cell’s cover without altering the absorbance measures. Nevertheless, actual time measure starts when Sample button is pressed.
4.9.2 Preparing a coagulation method
Select 1 Set method.
I 45 I Measure time: Maximum time during which coagulation may occur; between 15 and 300 seconds.
Higher limit: Upper range in seconds.
Lower limit: Lower range in seconds.
Threshold: Threshold absorbance that defines the time of coagulation (not larger than 2 absorbance units).
Correction: It is an additive or subtractive value in seconds measured to create compatibility with data produced by other measuring methods.
4.9.3 Coagulation measurement
Set the instrument in the cuvette measuring mode. Select 2 Run Method.
Coagulation measurements are done in disposable semi-micro 10 x 10 x 45-mm cuvettes. Do not use the flow-cell. This could result in permanent damages to the instrument.
Follow the instructions on screen.
Read next sample “1”
Place the reactive with serum in the cuvette inside the cuvette tray, simultaneously pressing SAMPLE. After the “delay” time defined in 4.9.1, the analyzers will start to examine the absorbance variation of the sample. Once the measurement is finalized, a beep will indicate that the threshold plus the correction have been surpassed. Press any key and the coagulation time will be displayed on screen.
4.10 Utilities
Press 7 in the main menu. The following will be displayed:
4.10.1 Date & Time
Press 1 and the screen will display:
After pressing the screen will show:
Month: Input the number of the month, confirm with ENTER.
Afterwards, Day will appear: Enter the day and press ENTER.
Year will also be shown on screen: input the last two digits of the year and confirm with ENTER.
When is pressed, the following is displayed:
I 47 I 4.10.2 Calibration
There is a function that performs the complete fully automatic calibration of the instrument . It is recommended to run this function every time the lamp is replaced.
Do not forget to fill the cell with distillated water. Keep the cuvette tray empty.
Press the key dot (·) to perform this calibration.
The instrument requests the input of the number of consecutive calibration runs. At least two calibrations is recommended.
In case the FLOWCELL position was not selected, the Analyzer will give a sound signal.
The amplifier calibration is started, after which the calibration of the cell’s lamp tension is carried out.
The instrument requests a change into CUVETTE position.
The calibration of the lamp is now performed for this position.
Next, the calibration for each of the filters takes place.
The instrument requests once again a change of position, this time into FLOWCELL.
Last, the calibration for each one of the filters in the flow-cell position is performed.
4.10.3 Filter selection table
In the main menu, press 7 and enter the Utility Menu and press 6.
Press the number of the filter you wish to change or add and enter the wavelength, then press ENTER.
Once the filters’ data are introduced, press ESC.
If you wish to print the table, press 4.10.4 Delay ready Led
Led indicates when capillary tube can be removed from sample vial.
Default value is set to 2 seconds. This value can be modified in
Main > Menu 2 > 7. Utilities > Main 2 > 4. Delay Ready Led
If too long time is required before solution stops movin in capillary tube, replace
sample filter.
4.10.5 Automated cleaning system.
To access to automated cleaning menu press:
Menu 2 > 7 UTILITIES > Menu 2 > 5. FLOW CELL CLEANING Screen shows:
Capillary intake tubing must be immersed in cleaning solution for about 5 minutes.
Solution will be aspirated and released to vial. This will enhance cleaning effectiveness.
When cleaning is finished, in screen it will be read instructions for positioning a distilled water reservoir. Intake will be 10 aspiration volumes, approximately.
Cleaning operation will be automatically requested if more than 12 hours elapsed from the last cleaning operation. This will occur whenever attempting to run a method defined with micro cell operation.
Operator can select not cleaning this time but message will show up again and again each time a method is selected for running.
Additional recommendations related to cleaning:
•
Perform cleaning operation when daily work is finished.
•
Do not reject instrument cleaning warnings
•
Verify effectiveness of cleaning solution. Always keep vial capped.
Avoid direct sunlight. Take a small amount for the day and discard it at the end.
•
Automatic method does not request the use of rinsing solution.
Nevertheless, after each cleaning cycle load cell with it, leave for about two minutes and flush with distilled water.
•
If regular cleaning is not enough for cell cleaning and bubbles still produce anomalous values, fill cell with a 10% in water Nitric Acid and leave cell loaded for about 15 minutes. Repeat operation 4 times and flush cell with water.
FLOWCELL CLEANING
Put cleaning solution.
Leave capillary tube inside. Press actuator.
I 49 I
4.11 Volume
Calibration allows getting an accurate ratio between peristaltic pump turns and aspirated volume expressed in microliters.
Press 1 and follow instructions written in the screen.
Measure volume with a graduated flask and introduce it. Remember that it must be done in microliters.
The Air gap function allows the emptying of the cell after the reading. This parameter can take values from 0 to 1500uL. With 0, the emptying of the cell is suppressed after the reading.
5- Semi-Automatic mode
When an external ke yboard is detected during instrument startup, instrument is automatically set to the semi-automatic mode and operation is controlled by the use of external keyboard:
Enter key is used to confirm or select an entry and to move the cursor to the next input field.
Backspace key is used to delete the numerical character to the left of the cursor.
Up/Down arrow key moves the cursor to the next option on a menu or entry during an edition.
Delete key Erases a patient.
Pg Up/Pg Dn key navigates on a list of results.
Left/right arrow keys navigates on a list of methods.
Esc key Cancels a menu or option.
When instrument is running a worklist or when it is on settings – utilities menu, additional control is achieved by the built-in keyboard as follows:
Pump actuator reads water/blank/standard or sample as required by the instrument.
Selection keys chooses between different available options shown on instrument display.
SIP / RCVR selection key Load the cell without reading or recovers sample.
Enter confirms factor or data.
Blank key recalibrates blank (at any time) Standard key recalibrates standard (at any time)
I 51 I
5.1. Main menu overview
MAIN MENU Ver 5.xxx
Instrument main menu is divided into two main screens,
MENU 1
screen options include the loading and edition of patients, printing of results by method or patients, definition and review of control results, definition/running of methods and profiles, and start of the run for the loaded patients.
MENU 2
screen option includes the access to absorbance reading mode, instrument internal settings (printer status, keybaord settings and calibration utilities), and calibration of peristaltic pump volume.
5.2. Methods settings and operation
METHODS
Method screen allows definition, run, priorities setting and printing of methods.
Select “Set method” option to edit a new method.
5.2.1. Create / Edit a method
This option defines a new method or modifies an existing one. The arrow keys navigate through the available me thods.
Enter key selects the method to be modified.
Once the values are changed, the instrument will ask to confirm the changes.
For details on method types and preparation, please see section 4.3. – Method preparation.
5.2.2. Modifying method priority settings
During sample batch processing, it can be necessary to run some methods earlier than others to ensure that fast reactions will be measured properly (e.g. kinetics reactions are stable for few minutes while color reactions are stable for about half an hour).
Priority setting table defines the order for processing each chemistry.
Methods
Use arrow keys and spacebar to select two methods to be swapped on the priority list. Repeat the operation to obtain desired ordering and press enter key when finished.
5.2.3. Run method (single method operation)
This option allows running a method outside from the batch (e.g. stat mode operation).
Details are provided on section 4.4 – Measuring.
5.2.4. Print Methods