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Complete Blood Count – Automated Haematology Analyser

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Published by bismetro, 2015-11-18 02:47:24

Complete Blood Count – Automated Haematology Analyser

Complete Blood Count – Automated Haematology Analyser

Keywords: Complete Blood Count

Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

1.Purpose: Quantitative enumeration of different formed elements (cells) of blood and red

blood cell indices in whole blood by Sysmex xs-800i, an automated haematology analyzer

equipped with 5 part differential functionality (NEUT + LYMPH + MONO + EO + BASO).

Sysmex xs-800i enumerates the following parameters:

 Total RBC count.

 Total WBC count.

 Differential WBC Count – both absolute and percentage.

 Haemoglobin.

 Packed Cell Volume (PCV).

 Mean Corpuscular Volume (MCV).

 Mean Corpuscular Haemoglobin (MCH).

 Mean Corpuscular Haemoglobin Concentration (MCHC).

 Platelet count.

 Mean platelet Volume (MPV).

 Platelet haematocrit (PCT).

 RBC Distribution Width (RDW).

The various parameters generated from the automated haematology analyzer are of

clinical importance to differentiate between diseases of haematological abnormalities.

Reference values vary across the life cycle and between sexes and differ in different age

groups and in various disease conditions.

2. Principle:

The RBC’s and platelets are analyzed by the RBC detector using the Hydro Dynamic

Focusing (HDF) method. In HDF, diluents provides a sheath, aligning the cells to pass

through centre of aperture thereby eliminating recirculation, non-axial flow and

coincidences obtained during cell count by impedance method. Automatic

discriminators separate the two cell populations based on complex algorithms. The

intensity of the electronic pulse from each analyzed RBC is proportional to the cell

volume. The haematocrit (HCT) is directly determined based on the count and volume

detection of each individual RBC.

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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

Fig: Illustration of Hydrodynamic Focusing System

Analysis of WBC and differential is performed with an optical detector block based
on the flow cytometry method, using a semiconductor laser. A surfactant Stromatolyser 4
DL lyses and dissolves RBCs and PLTs and perforates cell membrane of WBC. Later, a
fluorescence stainer, polymethine dye enters perforated WBCs and stains their nucleic
acids (RNA/DNA). When the laser light beam strikes a fluorescent stained cell, the light
of various intensity is scattered with respect to the physical characteristics of the cell and
the following characteristics are determined; cell size from forward scatter, internal
structure and granulation from side scatter and nucleic acid contents from side
fluorescence.

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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

Fig: Illustration of laser flow cytometry

Haemoglobin (Hgb) is analyzed by the HGB detector based on the SLS haemoglobin
detection method. The Sysmex sodium lauryl sulfate (SLS) method for haemoglobin analysis

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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

is a non-cyanide method. Haemoglobin is determined in a separate channel, minimizing

interference from high leukocyte concentrations.

2.1. MCV: calculated from RBC and Hct.

2.2. MCH: Calculated from RBC and Hb.

2.3. MCHC: calculated from Hb and Hct.

2.4. RDW: Calculated from deviation of RBC Distribution Width.

3.Linearity and measurement range (Performance characteristics):

WBC : 0 – 440 X 103/µl.

RBC : 0 – 8 x 106/µl.

HGB : 0 – 25 g/dl.

HCT : 0 – 60%

PLT : 0 – 5000 x 103/µl.

4.Type of samples:

4.1. Use whole blood as sample collected in EDTA.

4.2. Collect 2 ml blood in lavender top vacutainer tube (Containing 5.4 mg EDTA) or in

tubes containing 40 μl of 10 % EDTA per 2 ml of blood).

4.3. Sample rejection criteria: Inadequate sample volume.

Exception: Upon clinicians request regarding the samples of neonates, pediatric and

critical case, low volume samples (1 – 2 ml) are accepted. Result is released with a

comment stating “Inadequate sample volume. Report released after clinician’s

request”.

4.4. Mix the specimen thoroughly but gently in a hemomixer for 3 – 5 minutes

immediately prior to aspiration of the sample by the machine / by 3- 5 gentle

inversion and rotation between the palms of hands.

4.5. Process the samples within 2 hours of collection.

4.6. Do not use samples which are clotted, containing fibrin micro-clots, decomposed or

contaminated sample.

4.7. Reject clotted samples and samples without ID.

5. Patient preparation:

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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

5.1. The patient is seated comfortably with his arm extended on the table and palm
facing upwards. Apply tourniquet just above the elbow.

5.2. The tourniquet makes the vein more prominent thus avoiding blind probing.
5.3. Select the vein for puncture and clean the site with spirit (70.0 % alcohol).
5.4. The needle is attached to the syringe. It is introduced into the vein with the bevel

facing upwards and at angle of 30 – 40 degree to the skin. Entrance in to the vein is
denoted by the appearance of blood in the hub of the needle or in the syringe.
5.5. Release the tourniquet and with draw the piston till the required amount of blood
fills the barrel. Place a cotton swab at the site of puncture and withdraw needle in a
single movement.
5.6. The blood is transferred to appropriate containers after removing the needle from
the syringe.

6.Type of container / Additive & preservative: Use a lavender color topped vacutainer tube

(containing 5.4 mg EDTA) or tubes containing 40 μ l of 10 % EDTA per 2 ml of blood).

7.Required equipment and Reagents :

7.1.Equipment :
(i) Sysmex xs-800i Automated Hematology Analyzer.
(ii) Hemomixer.

7.2.Reagents :
(i) Cell pack.
(ii) Sulfolyser.
(iii) Stromatolyser 4DS.
(iv) Stromatolyser 4DL.
(v) Transasia control (Level 1, 2,3).

8.Environmental and safety controls :

8.1. Handle all samples as potentially infectious.
8.2. Handle all reagents with care and avoid contact with eye mouth and skin.
8.3. Discard used reagents and sample as per disposal procedure.

9.Calibration procedures :

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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

9.1. The instrument (Sysmex xs 800i) is calibrated once in I year.

10. Procedure:

Before switching on the instrument:
10.1.Check the level of all reagents and its expiry.
10.2.Empty the waste container.
10.3.Switch on printer and ensure sufficient paper in printer.

Daily Startup:
10.4.Switch on the plug.
10.5.Switch on the CPU.
10.6.Please wait XS display appear.
10.7.Enter the username and password.
10.8.Switch on the instrument.
10.9.Wait till green glowing in the instrument.
10.10. When monitor shows “NEXT” the instrument is ready for analysis.
Running Quality Control:
10.11. Select QC files.
10.12. Press manual.
10.13. Select the control level.
10.14. Select lot number.
10.15. Press ok.
10.16. Aspirate the control.
10.17. Values will be displayed.
10.18. Check the values.
10.19. If displayed plain – within limits (press accept).
10.20. If displayed in yellow color – border line (press cancel, mix well and rerun).
10.21. If displayed in red color – out of limit (press cancel).
10.22. Take the corrective action.

Run sample in manual mode: Amendment No: 00
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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

10.23. Press manual.
10.24. Select test (CBC or CBC + DIFF).
10.25. Select mode (MANUAL or CAPILLARY).
10.26. Manual mode: sample is aspirated from open tube.
10.27. Capillary mode: analysis performed after manual dilution (1:7 ratio) (20µl blood +

120µl cell pack).
10.28. Enter sample ID or scan the barcode, name, age and sex.
10.29. Press ok.
10.30. Aspirate sample till green light off in the instrument.
10.31. Wait for 60 seconds.
10.32. The result will be displayed.

Maintenance:
10.33. Go to menu.
10.34. Go to controller.
10.35. Select maintenance.
10.36. Select monthly rinse.
10.37. Aspirate cell clean.
10.38. Wait till the process is complete.

Changing Reagents:
10.39. Go to menu.
10.40. Select reagents replacement.
10.41. Select the reagent to be changed.
10.42. Enter the lot number, expiry date and amount.
10.43. Press execute.
10.44. The priming starts automatically.
10.45. Wait till the process is complete.

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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

Shut Down:
10.46. Press shut down.
10.47. Wait for 3 minutes.
10.48. Switch off the instrument.
10.49. Close the XS software.
10.50. Shut down the system.
10.51. Please wait till monitor off.
10.52. Switch off the plug.

11. Interferences and cross reactions :

The following factors will affect cell counting in an automated haematology analyzer.
11.1. Visible clots, fibrin micro clots or cryoprecipitate in the specimen.
11.2. Haemolysis.
11.3. Inadequate dilution of specimen and inadequate calibration of the analyzer.
11.4. Non homogenous suspension of blood cells in the diluting fluid.
11.5. Deterioration of diluting solution.
11.6. Presence of dust particles in the diluents.
11.7. Should be free of dust particles especially for platelet counting.
11.8. Partial or total obstruction of the aperture.
11.9. Wrong threshold setting of the instrument.
11.10. Carry over from one to next measurement.
11.11. Fluctuation of electric current.

12. Biological reference intervals :

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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

Parameter Reference Range
WBC count
RBC count Adults 4500-11,000 cells/cu.mm
Male : 4.5 to 6.2 x 106cells/cu.mm
Haemoglobin Female: 4.0 to 5.5 x 10 6 cells/cu.mm
Platelet count Men 14 – 18g/100ml Women 12-16 g/ 100ml
Haematocrit 150,000 – 400,000/CU.MM
MCV Male: 40% – 54% Female 37% – 47%
MCH 82-98 microns
MCHC 26-34 pg
31.0-35.0 g/100ml

A. Any deviation is observed with reference to above ranges the samples are

submitted for repeated both in instrument as well as manual check for

pathologists.

B. The smear preparation for slide review is referred in BL/HM/13.

C. The criteria for repeat test and slide review are documented in BL/HM/T.

13. Reportable interval of examination results : 4 hours from the sample received.

14. Instructions for determining quantitative results when a result is not within the

measurement interval

15.1. The test can be repeated.

15.2. Cross check with the manual.

15.3. Repeat sample can be asked.

15. Critical / Alert Values:

PARAMETER BELOW ABOVE

Haemoglobin 7.0 g 20.0 g

Platelet 50,000 cells/cu.mm 600,000 cells/cu.mm

Total WBC Count 1900 cells/cu.mm 30,000 cells/cu.mm

Red Blood Cell count 2 million cells/cu.mm 7 million cells/cu.mm

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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

16. Laboratory clinical interpretation :

S.No Parameter Increased in Decreased in

01 Total WBC count Leucocytosis. Leucopenia
02 RBC count Erythrocytosis Anemia
03 Haemoglobin Polycythemia Anemia
04 PCV Polycythemia Anemia
05 Platelet count Thrombocytosis Thrombocytopenia.
06 Neutrophil Neutrophilia Neutropenia
07 Lymphocyte Lymphocytosis Lymphocytopenia

08 Monocyte Monocytosis Monocytopenia

09 Eosinophil Eosinophilia Eosinopenia

10 Basophil Basophilia -

17. Potential Sources of variability: The following factors will affect cell counting in an

automated haematology analyzer.

17.1. Visible clots, fibrin micro clots or cryoprecipitate in the specimen.

17.2. Haemolysis.

17.3. Inadequate dilution of specimen and inadequate calibration of the analyzer.

17.4. Non homogenous suspension of blood cells in the diluting fluid.

17.5. Deterioration of diluting solution.

17.6. Presence of dust particles in the diluents.

17.7. Should be free of dust particles especially for platelet counting.

17.8. Partial or total obstruction of the aperture.

17.9. Wrong threshold setting of the instrument.

17.10. Carry over from one to next measurement. Amendment No: 00
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Department of Haematology

Complete Blood Count –
Automated Haematology Analyser

17.11. Fluctuation of electric current.

18. Reference / Source Document:

18.1. User Manual of sysmex xs-800i automated haematology analyzer.
18.2. Dacie and Lewis Practical Haematology, 10th Edition, 2006, page 14, 51.
18.3. Methods and Interpretation – MLT, Ramnik Sood, 1999, 5th Edition, page 181 – 183.
18.4.Pathology practical book by Dr.Harsh mohan, Second edition, page 204.

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