CellCounter_DAE_V0.0.pptx

Cell counter for hematology

1. 5 Part automated cell counter Presented By, Dr. Divya Donepudi Moderator, Dr. Shailaja prabhala

2. Introduction Cell counting is a method used to determine the number of cells in a sample

3. Manual Counting Pros Low cost : Done with basic equipment like a microscope and a hemocytometer, making it a cost-effective method. Flexibility : Counting cells from various samples and use different types of stains to visualize cells. Control : Control over the counting process, making it easier to identify specific cell types or distinguish between living and dead cells. Cons Time-consuming : Labor-intensive process taking hours or even days depending on the sample size. Subjectivity : Prone to human error, and different researchers may count cells differently, leading to variations in results. Limited accuracy : Might be inaccurate due to variations in the size and shape of cells, as well as cell clumping.

4. Automated Counting Pros Speed : Much faster than manual counting, with results obtained in a matter of minutes. Accuracy : Less prone to human error, resulting in more accurate and consistent results across different experiments and researchers. Multi-parameter analysis : Cell count, viability, morphology, size, and granularity, providing more detailed data than manual counting. Cons High cost : Requires specialized equipment, which can be expensive and may not be affordable for all labs. Sample limitations : Automated counters may not be suitable for all types of samples, as certain samples may require manual processing or specialized equipment. Complexity : Automated counting can be complex, requiring training and expertise to operate and interpret the results.

5. Features Three-Part Automated Cell Counter Five-Part Automated Cell Counter Number of cell types counted Red blood cells, white blood cells (Neutrophils, monocytes and lymphocytes) and platelets Red blood cells, Platelets W hite blood cells (neutrophils, lymphocytes, monocytes, eosinophils, basophils) Accuracy and precision Lower due to limited differentiation capabilities Higher due to wider range of cell type differentiation Cost Less expensive due to simpler technology and reagents Expensive due to more complex technology and reagents required Sample volume Requires a larger sample volume for accurate results Requires a smaller sample volume for accurate results

6. Coulter & Flow Cytometry

7. Coulter cell counter In 1953, Wallace Coulter patented the Coulter principle in which particles were counted in fluid which passed through an orifice. Coulter Corporation introduced the first commercial cell counter, which used the principle of impedance to count cells. Following years : Various types of automated cell counters were developed, including those that used light scattering, fluorescence, and image analysis.

8. The Coulter Principle Works on Ohm’s law V = I *R As a cell passes through the aperture, electrolytic solution is displaced which is equal to size of cell, increasing impedance of electrolytic solution . Current is constant, therefore in the formula the voltage V will increase. The bigger the cell, the more the resistance, the greater the voltage. Each voltage spike is directly proportional to the size of the cell.

9. There are two chambers one measures Hb + WBCs and second chamber counts RBCs + PLTs The aspirated whole blood specimen is divided into two aliquots and mixed with an isotonic diluent The first dilution is delivered to the RBC aperture bath, and the second is delivered to the WBC aperture bath. In the RBC chamber, both the RBCs and the platelets are counted and discriminated by electrical impedance. Particles between 2 and 20 fL are counted as platelets (4.5 – 12.3 fL ) , and those greater than 36 fL are counted as RBCs (70-120 fL ) . A reagent to lyse RBCs and release hemoglobin is added to the WBC dilution before the WBCs are counted by impedance in the WBC bath. The electrical pulses obtained in the counting cycles are sent to the analyzer for editing and digital conversion. Working of cell counter

10. Raw data from Oscilloscope X- Axis : Time Y- Axis : cell size Data from analyzer is organized in an ascending order X-Axis : Occurrence Y- Axis : Cell size

11. In the electrical impedance system, raw data is generated and the analyzer’s computer classifies the raw data which is sorted and histograms are then smoothed (smooth curve) into distribution curve. In the above graph, X- Axis : Cell Size in fL Y- Axis : Frequency of occurrence

12. Flow cytometry Derived from the Greek words " cyto " (cell) and " metry " (measurement) by using “Flow”- movement of cells This technology enabled the use of fluorescent markers to label specific cellular components Allowing sorting and analysis of different cell populations based on their fluorescence properties.

13. Each cell will pass through a detection device called a flow cell. This flow cell will have a laser device focused on it and as the cell passes through the laser light path, it will scatter light in several directions. The unit has one detector that captures the forward scatter light (FSL) and a second detector that captures the light that is scattered (side scattered or SS) at 90° Light scattering & Flow cytometry

14. Principle Light Scattering Principle Interaction of light with cells Coulter Principle (Electrical Impedance) Change in electrical impedance Detection Photodetectors capture scattered light Electrodes measure impedance change Advantages Information about cell size, shape, granularity, and internal complexity Non-invasive Suitable for live cell analysis Highly accurate cell counts and size measurements Less affected by debris or cell aggregates High resolution for small particles and cells Limitations Affected by sample quality, debris, or aggregates Limited resolution for very small cells or particles Requires conductive liquid media and specific calibration Limited information about cell shape, internal complexity, and granularity Invasive method, may affect cell viability Applications Cell counting Cell size and shape analysis Internal complexity and granularity analysis Cell counting Cell volume measurements Counting small particles or cells Comparison between light scattering & Coulter

15. Horiba Yumizen h550

16. General out line of processing of one sample

17. Post Analytic Pre-Analytic Analytic Factors affecting Quality

18. Interpretation

19. Normal RBC Histogram RL – Lower Range wrt detection of RBC RU – Upper Range wrt . detection of RBC Mean Corpuscular Volume (MCV) is calculated from the area under the peak. Red blood cell Distribution Width (RDW) is also calculated from the data used to calculate the MCV MCV * 100   MCH * 100   MCHC * 100  

20. RDW-CV (%) = 100 x s /µ RDW-CV : 11.5 – 14.5% 100 % 20 % RDW-SD is not a statistical SD, but measured by drawing an arbitrary line at a height of 20% on the y-axis in femtoliters RDW-SD : 35 - 45 fl The RBC distribution width gives a measure of anisocytosis. Red cell Distribution width (RDW) 100 % s s µ Points of inflection 68,26 % of all values - Standanrd Deviation µ - Mean

21. Abnormal RBC Histogram Abnormal height at lower discriminator RL (Lower Range) flag This flag is seen when the LD exceeds the preset height by greater than 10%

22. Abnormal RBC Histogram Abnormal height at upper discriminator RU flag This flag is seen when the UD exceeds the preset height by greater than 5%.

23. Abnormal RBC Histogram Abnormal distribution width (DW) Abnormal histogram distribution RDW – SD or RDW – CV is flagged .

24. RBC anisocytosis-Multiple peaks (MP) Abnormal RBC Histogram

25. Lower discriminator (LD) fluctuating between 30 and 60 fL Upper discriminator (UD) fixed at 300 fL. The number of cells between the UD and the LD is the WBC count. WBC histograms consist of two troughs, T1 between 78 and 114 fL and T2 < 150 fL. The peak between the LD and T1 represents small cells, i.e lymphocytes. The volume of the cells range from 35 to 90 fL. The peak that lies between T1 and T2 represents the mid cell count which includes the eosinophils, basophils, monocytes, blasts and promyelocytes. Volume of the cell ranges from 90 to 160 fL. The peak after T2 represents neutrophils. Volume ranging between 160 to 300 fL. Normal WBC Histogram

26. Abnormal WBC Histogram Abnormal Curve in Front of the Lower Discriminator

27. Abnormal WBC Histogram Abnormal Curve at the Lower Discriminator– Wl Flag This appears when height of LD is greater than the preset 20% of Y-axis. As a result of this the WBC count, W-SCR (small cell region), W-MCR (mid cell region) and W-LCR (large cell region) will show a Wl flag

28. Abnormal WBC Histogram Abnormal Curve at the Upper Discriminator–WU Flag This appears when the height of UD is greater than the preset 10% on the Y-axis. As a result the WBC count will show a WU flag. It occurs when there is insufficient WBC lysing.

29. Abnormal WBC Histogram Abnormal Curve at T1 Level—T1 Flag The T1 and T2 discriminators are flexible and will be set automatically according to the sample. In extreme pathological condition discrimination between 3 population is not possible. Flag T1 occurs when discrimination between lymphocytes and mid cell population is not done as in abnormal leukocytosis like chronic myeloid leukemia.

30. Abnormal WBC Histogram Abnormal Curve and Flagings —F1, F2 and F3 Flag F1 flag appears when the relative height of T1 exceeds preset limit of 40%. F1 flag means that the small cell and middle cell data may be inaccurate. It may occur in acute lymphoblastic leukemia. F2 flag appears when the relative heights exceed the preset of T1 (40%) or T2 (50%). F2 flag means that the middle cell data is inaccurate. This often occurs in eosinophilia, acute myeloid leukemia, monocytosis , etc. F3 flag appears when the T2 exceeds the preset limit of 50%. F3 Flag means that the large cell data is inaccurate.

31. Abnormal WBC Histogram Abnormal Curve at T2—T2 Flag Flag T2 appear when discrimination between mixed cell and neutrophil could not be done, as in chronic lymphocytic leukemia.

32. Normal Platelet Histogram Mean platelet volume (MPV) This is a mathematical calculation to determine the average size of the platelets. The average MPV range = 7.4 to 10.4 fL. MPV = PCtx1000/ Plt fl Platelet distribution width (PDW) The platelet distribution width (PDW) is the width of the curve of distribution of platelets related to the different sizes produced by these cells

33. Abnormal Platelet Histogram PL Flag This occurs when the lower discriminator exceeds the preset height by 10% the platelet count, MPV and P-LCR will show the PL flag.

34. Abnormal Platelet Histogram PU Flag This occurs when the upper discriminator exceeds the preset height by more than 40%.

35. Abnormal Platelet Histogram MP Flag ( Multipeaks in PLT Histogram)

36. WBC Differential Scattergram

37. Reference The ABC of CBC Interpretation of Complete Blood Count and Histograms, DP Lokwani . Flow Cytometry Animation GIF | Gfycat Flow Cytometry Animation GIF | Gfycat https://www.horiba.com/gbr/medical/products/detail/action/show/Product/yumizen-h550-1851 / https://www.semanticscholar.org/paper/Overview-of-Automated-Hematology-Analyzer-XE-2100-Inoue/bcda80fdb303611a9d1db457346128a252f447d5/figure/6