CBC, Principle of Cell Counter, Interpretation of Histogram & Scattergram - Dr Rashmi.pptx

CBC, Principle of Cell Counter, Interpretation of Histogram & Scattergram

1. Complete Blood Count Principle of Cell Counter, Interpretation of Histogram and Scatter gram Presented by- Dr.Rashmi Soni Moderator- Dr.Yogesh Gupta sir

2. The Complete Blood Count is a series of tests used to evaluate the composition and concentration of the various cellular component of blood it consist: RBC count, WBC count and Platelets count. Measurement of Haemoglobin and calculation of haematocrit and Red blood cell indices. WBC total and differential count. Platelet count , Mean platelet volume, Plateletcrit, PDW. Histograms of RBC , WBC and Platelets .

3. RBC PARAMETER MEN WOMEN NEWBORN CHILD RBC count 4.5-6x10⁶/mm³ 4.2-5x10⁶/mm³ 5-7x10⁶/mm³ 4.5-5x10⁶/mm³ Haemoglobin 13.3-16.2g/dl 12-15.2g/dl 15.4-24.5g/dl 12.5-14g/dl Haematocrit 38.8-46.4% 35.4-44.4% 48-72% 33-39% Mean cell volume 79-98fl same 100-120fl 75-86fl Mean cell haemoglobin 26.7-31.9pg same 31-38pg 24-30pg Mean cell haemoglobin concentration 32-36% same 33-36% 31-37g/L RDW-SD RDW-CV 35-45fl 11.5-14.5% same

4. WBC PARAMETER MEN and WOMEN NEWBORN CHILD Total WBC count 4-11x10³/µL 4-40x10³/µl 5-15x10³/µl Differential white cells count Neutrophil (40-80%) 2-7 x10³/µl 4-7x10³/µl 2-8x10³/µl Lymphocytes (20-40%) 1-3 x10³/µl 2-4x10³/µl 6-9x10³/µl Monocytes (2-10%) 0.2-1x10³/µl 0.5-2x10³/µl 0.2-1x10³/µl Eosinophils (1-6%) 0.02-0.5x10³/µl 0.1-1x10³/µl 0.1-1x10³/µl Basophils (<1%) 0.02-o.1x10³/ ul 0.02-0.1x10³/µl 0.02-0.1x10³/µl

5. Platelet count 1.5 -4.5 lack/µl same 2-4.5 lack 1.8-4lack/µl MPV 7.4-10.4fl PDW 9-13fl plateletcrit 0.110-0.228 other Retic count 0.5 -2.5 %

6. Automated Hematology Analyzers There are two type of analysers: Semi Automated analysers: these require some step to be carried out manually like making a dilution of the blood sample. Fully Automated analysers: sample handling Is not required. Equipment is capable of piercing caps of sample tube, collecting the sample and processing it.

7. 3 p art differential 5 part differential 7 part differential Usually count Neutrophil It includes 5 part plus Granulocyte or large cells Eosinophil Large immature cells ( immature granulocyte) Lymphocytes or small cells Basophiles Atypical lymphocytes(blast cells) Mono nuclear cells Lymphocytes LUC Monocytes NRBC Atypical lymphocyte Other abnormal cell

8. Hematology Automation Two General Principles – Electronic resistance or impedance ( COULTER PRINCIPLE ) Light scattering Fluorescence and flow cyto metry Peroxidaes staining

9. In 1953, Wallace Coulter patented the Coulter principle in which particles are counted in fluid which passed through a hole. The incredulous attorneys who had told him “You can't patent a hole” were proven wrong. Automated Hematology Cell Counter—Basic Principles Most hematology instruments operate under several basic principles:

10. CELL COUNTING Process- Dilution Vacuum and pressure Electrical impedance Reagent systems

11. Using this technology, cells are sized and counted by detecting and measuring changes in the electrical resistance when a particle passes through a small aperture. This is called the electrical impedance principle of counting cells. A blood sample is diluted in saline (a good conductor of electrical current) and the cells are pulled through an aperture by creating a vacuum. Two electrodes establish an electrical current. The external electrode is located in the blood cell suspension. The second electrode is the internal electrode and is located in the glass hollow tube which contains the aperture. Low-frequency DC current is applied to both the electrodes. Electrical resistance or impedance occurs as the cells pass through the aperture causing a change in voltage.

12. This change in voltage generates a pulse . This weak pulse is amplified and measured. The number of pulses is proportional to the number of cells counted as cell count, and is displayed on a screen. The size of the voltage pulse is also directly proportional to the volume or size of the cell. This information can be transmitted to a microprocessor of a computer and is used to provide an accurate cell count and identification of the types of cells.

13. LIGHT SCATTERING Diluted cell suspension pass through an aperture. Cell pass in a single file in front of a light source, light scattered by the cells. The scattered light detected by a photomultiplier, which convert it into the electrical impulse. That accumulated and counted. Amount of light scattered proportional to surface area and volume of cell.

14. Variable measured using Variable measured using electronic impedance RBC count MCV MCH MCHC RDW Haematocrit Size distribution histogram Total WBC count and 3 part differential count Platelet count and platelet histogram Optical scatter 5 part differential count (fluorescence flowcytometry) 7 part differential count ( peroxidase staining) Scattogram of RBC and platelet in siemens

15. HEMATOCRIT MEASUREMENT After getting HCT and Hb values, MCV, MCH and MCHC values are derived.

16. RDW is an important parameter for measurement of degree of variation in red cell size or anisocytosis. The two parameter available on the instrument are RDW-CV and RDW-SD. RDW-CV is calculated by SD/MCV × 100 and is equivalent to 68.26% of the distribution curve. and normally ranges between 11.5 and 14.5%. RDW-SD is the width of RBC volume histogram measured by drawing an arbitrary line at a height of 20% on the y-axis in femtoliters is termed as RDW-SD. Its normal value is in the range of 35 to 45 fL.

17. HISTOGRAMS

18. • RBC detection: Between 36-360fl. Platelets: between 2 to30fl • Distribution curves are separated by flexible discriminators: RL and RU. • The histogram curve should start and end at the baseline within the discriminators.

19. RBC Histogram The volume histogram reflects the size of erythrocytes or any other particle in the erythrocyte size range. The instrument counts the cells as erythrocyte with volume sizes between 36 fL and 360 fL. RBC histogram is a symmetrical bell-shaped curve LD=25-75fl UD=200-250fl

20. ABNORMAL RBC HISTOGRAMS AND FLAGS 1. Abnormal height at lower discriminator RL flag- This flag is seen when the LD exceeds the preset height by greater than 10%.

21. 2. Abnormal height at upper discriminator RU flag- This flag is seen when the UD exceeds the preset height by greater than 5%.

22. 3. RBC anisocytosis-Multiple peaks (MP)

23. IDA

24. PLATELET HISTOGRAM Platelet derived histograms are obtained from volume sizes of 2 to 30 fL . . Small particles, such as bubbles or dust , can overlap at the lower end of the histogram; microcytic erythrocytes can interfere at the upper end.. UD=12-30fl LD=2-6fl

25. PLATELET HISTOGRAM

26. 1. PL Flag This occurs when the lower discriminator exceeds the preset height by 10% the platelet count.

27. 2. PU Flag This occurs when the upper discriminator exceeds the preset height by more than 40%.

28. 3. MP Flag (Multipeaks in PLT Histogram)

29. The Volume/Hemoglobin Concentration (V/HC) cytogram is a linear version of the RBC map that appears on the RBC cytogram. On the V/HC cytogram, hemoglobin concentration is plotted along the x axis and cell volume is plotted along the y axis. Only red blood cells appear on this cytogram. 1. 60 fL volume marker 2. 120 fL volume marker 3. 28 g/ dL HC marker 4. 41 g/ dL HC marker The RBC Method

30. (2A) RBC cytogram from a normal individual. Most RBC plots fall in the central zone (normocytic normochromic). (2B) Iron deficiency anemia most of the RBC plots lie in the microcytic hypochromic zone. (2C) Beta thalassaemia trait. Note the comma-shaped curve in the microcytic hypochromic region of the cytogram. (2D) Beta thalassaemia major. The cytogram is much wider due to heterogenity of population .

31. (3A) RBC cytogram pattern in a patient with non- megaloblastic macrocytosis. Note the close clustering of the RBC plots in the macrocytic zone (arrow). (3B) Megaloblastic anemia. The RBC plot shows a wider spread (arrows) in the macrocytic zone as compared to that seen in non- megaloblastic macrocytosis. (3C) Dual deficiency anemia. Note the RBC plots in the macrocytic (arrow) as well as the microcytic (arrowhead) zones. (3D) recovery from IDA.

32. The Plt Method Integrated analysis is used to distinguish platelets, large platelets, red blood cells, RBC fragments, and RBC ghosts. This example illustrates how large platelets are counted: - Large platelets are identified on the PLT Scatter cytogram based on their refractive index values (1.35 to 1.40) and a volume greater than 20 fL. - Large platelets are also identified on the RBC Scatter cytogram (area 3 below) based on their refractive index values (1.35 to 1.40) and a volume less than 60 fL. 1 Area covered by the PLT Scatter Cytogram 2 RBC fragments 3 Large platelets 4 RBC s 5 RBC ghosts

33. The Plt Method 1 Platelets 2 Large platelets 3 Red blood cells 4 RBC fragments 5 Debris 6 RBC ghosts Using the Mie theory of light scattering for homogeneous spheres , the low- angle and high-angle light scatter signals for each cell are transformed into volume and refractive index (n) values.

34. WHITE BLOOD CELL TECHNOLOGY The sysmex haematology analyser utilize fluorescence flowcytometry The siemens haematology analyzer utilize peroxidase staining. Cell are stained by peroxidase reagent and analyzed for size and peroxidase stain intensity. Cell specific lysis reagent are used to separate basophils from other white cells

35. FLUORESCENCE FLOWCYTOMETRY Two event occur: Light scattering Emission of fluorescent light

36. FLUORESCENCE FLOWCYTOMETRY

37. WDF WNR This channel differentiate neutrophil,lympho,monocytes,eosinophils and Immature granulocyte. After reagent interaction,morphology of basophil well preserved compair to other WBC baso high SFL AND forward scatter. nRBC differentiated from WBC with low fluorescence. Basophils

38. Normal Scattergram Mono Neut Eo Ghost Baso Lymph

39. Neutrophilia < Normal > Manual : 89.00% XS : 86.9%

40. Lymphocytosis < Normal > Manual : 52.75% XS : 66.0%

41. Monocytosis < Normal > Manual : 15.25% XS : 15.6%

42. Basophilia < Normal > Manual : 4.75% XS : 3.7 %

43. Eosinophilia < Normal > Manual : 32.50 % XS : 30.5 %

44. The perox method The cells are stained by peroxidase reagent and analysed for size and peroxidase stain intensity.. H 2 O 2 + 4-chloro-1-naphthol + cellular peroxidase= dark precipitate within the cells Absorbance of white light from the tungsten light source is a measure of peroxidase reaction.

45. The Perox Method Absorption detector Filter Scatter detector Sample stream Tungsten lamp Dark stop Beam splitter

46. Scatter signal to measure the volume of the cells Absorption signal for peroxidase activity measurement Cells with medium peroxidase activity absorbs less light than cells with high peroxidase activity The Perox Method

47. The Perox Method Cytochemical classification according to peroxidase activity Cell type Peroxidase Myeloblasts -, sometimes ½+ (especially micromyeloblasts) Promyelocytes Myelocytes 3+ 3+ Metamyelocytes Band cells 3+ 2-3+ Neutrophils 2+ Eosinophils 4+ Basophils ½-1+ (stay unstained in the ADVIA 120) Lymphoblasts Prolymphocytes Lymphocytes Atypical lymphocytes Monoblasts - - - - - Promonocytes Monocytes Plasma cells Nucleated red blood cells ½-1+ 1+ - -

48. The Perox Method Absorbed light = Peroxidase Activity Light scatter = Cell Size 1 Noise 2 Nucleated Red Blood Cells 3 Platelet Clumps 4 Lymphocytes and Basophils 5 Large Unstained Cells 6 Monocytes 7 Neutrophils 8 Eosinophils

49. BLOOD BASOPHIL/ LOBULARITY METHOD In Baso chamber of siemens sample mix with Baso reagent. Baso reagent lyses the red cells , platelets and cytoplasm of all white cell types except basophils. Baso cytogram are ploted according the cell size and nuclear configuration of cell.

50. The Baso Method Cell Size Nuclear Configuration The BASO cytograms is representative of a patient specimen. 1 Noise 2 Blast cell nuclei 3 Mononuclear WBCs (Monocyte and Lymphocyte nuclei) 4 Basophils 5 Baso Suspect 6 Saturation 7 Polymorphonuclear WBCs (Neutrophil and Eosinophil nuclei)

51. The Baso Method Left Shift The following cytograms are from the sample of a patient with left shift

52. Acute leukaemia

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