I. Complete Blood count

1. PURPOSE OF EXAMINATION

The examination in whole blood is used mainly for diagnosis and management of complete blood count.

2. PRINCIPLE OF EXAMINATION

Blood sample is aspirated, measured to a predetermined volume, diluted at the specified ratio,and then fed into each transducer. The transducer chamber has a minute hole called the aperture. On both side of the aperture, there are the electrodes between which flows direct current. Blood cells suspended in the diluted sample pass through the aperture, causing direct current resistance to change between the electrodes. As direct current resistance changes, the blood cell size is detected as electrical pulses. All counts are derived by Electrical Impedance method and Hemoglobin by Non cyanide hemoglobin analysis method

Blood cell count is calculated by counting the pulses, and a histogram of blood cell sizes is plotted by determining the pulses sizes. Also, analyzing a histogram makes it possible to obtain various analysis data.

Differential WBC count done by differentiating lysing action followed by generation of electronic impulses.

PRINCIPLE OF HORIBA YUMIZEN H500& PENTRA XLR

Multi distribution sampling system[MDSS]

Specimen distribution in the chamber is carried out in a tangential flow of reagent which allows perfect mixing of the dilution and avoids any viscosity problems.

LMNE Matrix count: differential count in the flowcell is based on three essential principles:

1. The double hydrodynamic sleeving System “DHSS” which allows a linear flow of the cells through the light path.

2. The cell volume which is measured by electrical current.

3. The measurement of transmitted light at a 0ºangle which allows a measured response according to the internal structure of each cell and its absorbance, as unabsorbed light passes through the spaces in the nuclear material of each cell. This is known as diffused light.

4. 25 µl of whole blood is delivered to the LMNE chamber in a flow of ABX Eosinofix. This reagent lyses the RBC, stabilizes the WBC in their native forms and stains the eosinophil nuclei with a specific coloration.

5. The solution is then stabilized with ABX diluents and transferred to the flow cell. Each cell is measured both in absorbance (cytochemistry) and resistivity (volume).

Parameters RBC, WBCs and platelates are counted by impedence variation measures. Hb is measured by spectrophotometry. Parameters of the WBC differential count obtained by impedence and absorbance.

3. PERFORMANCE SPECIFICATIONS:

4. SAMPLE TYPE REQUIRED

Whole blood.

Stored at 2ºC-8ºC after analysis for 24 hrs

5. PRESERVATIVES NEEDED

Samples are collected in EDTA vaccutainer.

6. REAGENTS REQUIRED

• For MICROS 60 cell counter:

Lyser, cleaner and Diluent of MICROS 60

• For Yumizen H500 cell counter:

1. ABX Minoclair- 100 ml
2. Whitediff -1000 ml
3. ABX Cleaner-1000 ml
4. ABX Diluent-20,000 ml

• For PENTRA XLR part cell counter

1. ABX Lysebio-1000ml
2. ABX Basolyse-1000ml
3. ABX Eosinofix-1000ml
4. ABX Cleaner-1000ml
5. ABX Fluocyte-500ml
6. ABX Diluent-20,000ml

7. CALIBRATION:

Calibration is done by manufacture annually and calibration report is received in the laboratory.

Calibration reports are filed in HI: C\ Records\File\21\Calibration

Records

8. DETAILED WORK DESK INSTRUCTIONS / PROGRAMMING STEPS:

1] For MICROS 60 cell counter:

START:

1. Switch on main power supply
2. Switch on UPS
3. Switch on the cell counter by pressing the button at the rear of the instrument.
4. Press ID keys on front panel to enter the sample ID.
5. Press enter key.
6. Well mixed the sample.
7. Place the sample beneath the sample probe.
8. Press the manual sample bar.

SHUTDOWN:-

1. Press the stand by key
2. Switches to ‘stand by mode’
3. Switch off the instrument
4. Switch off the UPS
5. Switch off main power supply.

MAINTENANCE:-

1. Concentration cleaning-daily
2. Start up pass-daily
3. Backflush-Weekly
4. Drain chambers-Weekly
5. Prime-Weekly
6. Auto cleaning-Weekly

DAILY MAINTENANCE

1. Startup and standby cycles
   1. Press the “startup” key of the front panel at the beginning of each day.
   2. Press the “standby” key of the front panel at the end of each day.
2. Automatic cleaning
   1. When an automatic cleaning cycle begins a message will appear on the screen.
   2. A manual cleaning done by press the “auto clean” in the main menu of the screen.
3. General cleaning
   1. External cleaning
      1. Slightly wet a sponge with disinfectant product and wipe the dirty surface.
         • Never spill liquids on the instrument
         • Never use disinfectant that contains alcohol.
         • Never use solvent or abrasive materials.
         • Wipe of any trace of blood spillage as soon as possible.
   2. Internal cleaning
      1. Concentrated cleaning
         • Go in the main menu, then in service, and in concentrated cleaning.
         • Follow the steps that appear on the displays.
      2. Sampling probe
         • Prepare a solution of sodium hypochlorite to 100ml/l.
         • Fill a 5ml tube with this solution.
         • Run 5 analyses on bleach.
      3. Backflush

Go in the main menu, then in service, then in backflush.

2] Horiba Yumizen H500 cell counter:

START:-

1. Switch on main power supply
2. Switch on UPS
3. Switchthe instrument on by pressing the button at the rear of the instrument.
4. >Wait during initialization.
5. Switch on the printer.
6. After that login to application , to login into application select username and enter password .
7. Press OK
8. After that click on start up icon for start up pass
9. Wait until the cycle is over. cycle takes approx 1 minute
10. Blank cycles (cycles without any blood specimen) are performed during the startup cycle.
11. After start up pass, instrument is “ready” for sample and control run.*

SAMPLE PROCESSING:-

If start up is passed we can run the sample.

To Run the Specimen in STAT Mode

1. Press STAT Mode to open the tube holder door.

2. Enter information about the sample.

3.Press Validate in the contextual toolbar.

4. Press Icon (Next analysis) on function toolbar for sampling needle to come out.

5. Gently and thoroughly mix the sample.

6.open the tube and place it below the sampling needle, lift it so that the needle can sample its content.

7. press the manual sampling bar, or press “validate”

8. Remove the tube and put the cap back on once needle has moved up.

SHUTDOWN:- A shutdown cycle has to be from every 24 hrs.

It is highly recommended to perform a shutdown cycle before switching off the instrument.

1. Press shutdown icon to perform a Shutdown cycle manually.
2. Wait while the instrument shuts down. Shut down takes about 10 min.
3. Press Software exit Icon on contextual toolbar
4. Switch off the instrument
5. Switch off the printer

MAINTENANCE:-

A. Concentration cleaning- to clean the counting chambers and hydraulic parts.

1. Press Concentrated Cleaning.

2. A window informing you to connect Minoclair and press

validate

3. Disconnect the “ABX cleaner luer connected'

4. Connect ABX minoclair luer connector instead

5. Press validate in the contextual toolbar

6. Wait until the cycle is over

- A concentration cleaning cycle takes approx. 10 min.

7. When a window informing you to reconnect a reagent and press

“validate”, disconnect the ABX minoclair luer connecter and

connect ABY Cleaner luerconnector instead

8. Press validate in the contextual toolbar.

9. Startup cycle and an nalysis on a control bood sample

B. Auto clean Cycle

• Give the counting chambers a quick cleaning.

1. Press Autoclean.

2. Wait until the cycle is over

An Autoclean cycle takes about 3 MINUTES

C. Backflush

Clean the counting chamber apertures in case of blockage.

1. Press “ BackFlush RBC/PLT” to backflush the RBC/PLT aperture

2. Wait for the instrument to complete the backflush cycle which takes

about 35 sec.

3. Press ” backflush LMNEB“ to backflush the flowcell

4. Wait for the instrument to complete the backflush cyde, which takes

about 1 min.

D. To prime a reagent

1. Select the reagent you want to prime in Priming/Unpriming area

2. Press prime

3. Wait Until the cycle is over.

E. To Unprime a reagent

1. Disconnect and remove the bottle of reagents You want to unprime.

2. Select the reagents you want to unprime in the Priming /unpriming area

3. Press Unprime

4. Wait until the cycle is over

F. Rinse the system

LMNE flow cells can sometimes be defective due to airbubbles and/or

blockage, this allows to fix both these issues.

1. Press the rinse in the cleaning area

2. Wait for the instrument to complete the rinsing cycle,

which takes approx. 40 sec.

3] ForPentra XLR cell counter:

START:

1. Switch on main power supply
2. Switch on UPS
3. Switchthe instrument on by pressing the button at the rear of the instrument.
4. Wait during initialization.
5. Switch on the printer.
6. After that login to application , to login into application select username and enter password .
7. (Optional) Select Disable Auto Loader if you want to work in STAT mode (runs a manual sampling) only. (Start Rack: runs an automatic sampling).
8. Select the following options: ■ Erase Worklist ■ Reset AutoNumbering ■ Archive Reports .(This step can only be performed when you start the instrument for the first time of the day. The first two options are optional, depending on your laboratory's rate of analysis. It is highly recommended to select the third option at the first startup of each day).
9. Press OK
10. After that click on start upicon forstart up pass .
11. Select which cycle you want to perform.(startup/retic blank cycle)
12. Wait until the cycle is over. cycle takes about 3 minutes 30 seconds. Blank cycles (cycles without any blood specimen) are performed during the startup cycle.
13. After start up pass, instrument is ok for sample and control run.

SAMPLE PROCESSING:

If start up is passed we can run the sample.

To Run the Specimen in STAT Mode

1. Press STAT Mode to open the tube holder door.

2. Enter information about the sample.

3. Select the test to perform by pressing CBC/DIFF/RET.

4. Press CDR if you need a post-dilution on the sample.

5. Select the appropriate dilution ratio for WBC/LMNE and for RBC/PLT/HGB.6. Press Validate in the contextual toolbar.

7. Gently mix the blood specimen.

8. Put the tube in the tube holder and close the door to start sampling.

9. When the door opens, remove the tube from the tube holder and recap it if needed.

To Run the Specimen in Rack Mode

1. Prepare your worklist.

2. If you work with tubes with barcode labels, place the tube on any rack, at any position. Make sure that the barcode label is visible for the internal barcode reader.

3. If you work with tubes with no barcode labels, place the tube on the rack and at the position specified in the worklist.

4. Place the rack on the rack loader.

5. Press Start Rack.

SHUTDOWN:-

1. It is highly recommended to perform a shutdown cycle before switching off the instrument.
2. Press Quit in the contextual toolbar.
3. Press shutdown icon*to perform a Shutdown cycle manually.
4. Wait while the instrument shuts down. When the instrument is ready to be switched off, the following message appears on your screen: “It's now safe to turn off your computer.
5. Switch off the instrument.

SOP TO REPLACE REAGENT:-

1.When a reagent “Low Level” alarm is displayed, press Check.

2. Press the icon of the reagent you need to change

3.Press Edit in the contextual toolbar.

4. Enter the lot number in Lot Nb using the external barcode reader. All the fields are updated and the reagent level is set to an automatic default level. 5. Open the front door and remove the empty bottle from the reagent compartment.

6.Uncap a new reagent bottle. 7. Insert the stopper assembly tube into the new bottle and tighten the stopper assembly to ensure an adequate seal

8.Install the new reagent bottle into the reagent compartment and close the door.

9.Press Validate in the contextual toolbar. The instrument automatically starts priming the reagent.

MAINTENANCE:-

Miniclean: launches a short rinsing cycle of the counting chambers.

Concentrated Cleaning: starts a concentrated cleaning procedure using ABX Minoclair.

AutoClean: launches a cleaning cycle using ABX Cleaner.

Super User Menu: gives access to customer maintenance.

Technician Menu: (reserved to technicians) gives access to advanced maintenance

(Super User is made of three additional menus: ■ Mechanical System which allows the management of mechanical cycles. ■ Hydraulic System which allows the management of hydraulic cycles. ■ Others which facilitates certain maintenance procedures, and allows to force calibration coefficient values and RET parameters)

MAINTENANCE:-

1.Concentration cleaning- to clean the counting chambers and hydraulic parts.

■ Remove the right front cover and the right-hand side panel.

• Press Concentrated Cleaning.
• Press OK. A popup window informing you to pour ABX Minoclair in each chamber is displayed.
• Use the syringe to pour 3 mL of ABX Minoclair (or diluted bleach) in each chamber.
• Press OK. A popup window is displayed.
• Use the syringe to pour 3 mL of ABX Minoclair (or diluted bleach) in the WBC/BASO chamber (last chamber on the right).
• Press OK.
• Wait for the instrument to complete the cleaning cycle.
• A concentrated cleaning takes about five minutes.

2. Miniclean Cycle

• Press Miniclean.
• Wait for the instrument to complete the cleaning cycle.

A Miniclean cycle takes about 35 seconds.

3. Autoclean Cycle

• Give the counting chambers a quick cleaning.

• Press Autoclean.
• Wait for the instrument to complete the cleaning cycle.

An Autoclean cycle takes about 90 seconds.

4. Backflush

Clean the counting chamber apertures in case of blockage.

Only a super user can perform this action.

• Press BackFlush.
• Wait for the instrument to complete the backflush cycle.

A backflush cycle takes about 35 seconds.

5. Rinse the Cytometer

Only a super user can perform this action.

The LMNE flow cell can sometimes be defective due to air bubbles and/or blockage. Rinsing the cytometer allows to fix both these issues. To do so, you need to:

• Press Rinse Cytometer.
• Wait for the instrument to complete the cytometer rinsing cycle.

A cytometer rinsing cycle takes about 90 seconds.

Cleaning Frequency

QUALITY CONTROL PROCEDURE

• External Quality Assessment:

Participate in external quality assessment program ISHTM – AIIMS EQAS programme.

EQAS 3 monthly for Micros 60 (EQ.28 and EQ- 49), Horiba Yumizen H500(EQ- 50) and Pentra XLR (EQ-08)are being done.

Data is maintained inC\Records\File\2\Results of EQA and interlaboratory comparison

• Internal Quality Control:-

Quality control samples are run on the cell counters Micros 60[EQ.28,EQ.49] , Horiba Yumizen H500[EQ. 50] and Pentra XLR[EQ.08].

1. Daily all three level QC sample run, depending on the availability.
2. All the records are kept in file C\Records\File\9\Internal Quality Control Records.
3. In case of unavailability of QC sample for any of the cell counter, inter-instrument comparison is done with cell counter on which QC sample is being run.

4.Inter Instrument comparison :

• Inter instrument comparison between Micros 60 [EQ-HI-49] and Micros 60 [EQ- HI-01],
• Inter instrument comparison between Micros 60 [EQ-HI-28] and PENTRA XLR [EQ- HI-008],
• Inter instrument comparison between Micros 60 [EQ-HI-49] andHoriba Yumizen H500 [EQ- HI-50].
• Inter instrument comparison between Micros 60 [EQ-HI-49] and Micros 60 [EQ- HI(S)-02].

Records of comparative data are maintained in C\Records\File\9\Inter-instrument comparison.

5.Daily one blank run is checked before running the sample for checking the background counts and the data are entered in separate register. C\Records\File\9\Internal Quality Control Records.

• For any nonconformance,Procedure for identification, control, correction and prevention of nonconformities is activated and the report of which is filed in C\Records\File\12\Records of Nonconformities

J. INTERFERENCES:

1. Coincidence(i.e. by two cells passing through an orifice simultaneously and being counted as one cellor by a pulse being generated during the electronic dead time of the circuit).

2. By recirculation of cells that have already been counted.

3. By red cell agglutination (which causes a clump of cells to be counted as one cell).

4. By the counting of bubbles, lipid droplets, microorganisms, or extraneous particles as cells.

5. Faulty maintenance may lead to variation in the volume aspirated or the flow rate.

6.pH, temperature, and rate of ionization have to be standardized and remain constant because changes alter the electric field and may lead to artifactual alterations in the size, shape, and stability of the blood cells in the diluent.

7. Diluent must be free of particles and give a background count of less than 50 particles in the measured volume.

11. CALCULATION OF RESULTS AND UNCERTAINTY:

Not Applicable

12. BIOLOGICAL REFERENCE INTERVAL

13. LINEARITY:

1] For MICROS 60

2] For Horiba Yumizen H500

3] For Pentra XLR

14. REPORTABLE INTERVAL FOR EXAMINATION RESULTS:

1]FOR MICROS 60

2] For Horiba Yumizen H500

3] For Pentra ES XLR

15. CRITICAL VALUES:

16. INTERPRETATION BY THE LABORATORY:

WBC count (approximate), Differential count and platelet must be counterchecked with P/S examination.

17. POTENTIAL SOURCES OF VARIABILITY:

1. Preanalytical sources of variability: Delayed sample receipt, Leucocytosis, Leukemia, Septicemia, short draw in vacuum tube.

2. Coincidence (i.e. by two cells passing through an orifice simultaneously and being counted as one cellor by a pulse being generated during the electronic dead time of the circuit).

3. By recirculation of cells that have already been counted.

4. by red cell agglutination (which causes a clump of cells to be counted as one cell).

5. By the counting of bubbles, lipid droplets, microorganisms, or extraneous particles as cells.

6. Faulty maintenance may lead to variation in the volume aspirated or the flow rate.

7. pH, temperature, and rate of ionization of have to be standardized and remain constant because changes alter the electric field and may lead to artefactual alterations in the size, shape, and stability of the blood cells in the diluents.