Automatic Electronic Blood Cell Counting Prepared by lecturer Dr Adnan Anwer M.B.Ch.B — F.I.B.M.S

Advantages of electronic cell counter Rapid & shortens the time of tests and complete a large number of tests quickly Reduce the cost of the tests Reduce the testing equipments (instruments) Maintain and improve accuracy of tests

Complete blood count (CBC) A complete blood count (CBC) is a series of tests used to evaluate the composition and concentration of the cellular components of blood. It consists of the following tests: 1. Red blood cell (RBC) count, 2 . White blood cell (WBC) count, & differentiation. 3 . Platelet count (Plts), 4 . Measurement of hemoglobin (HgB) & hematocrit (HCT or PCV) 5 . Red blood cell indices (MCV, MCH,MCHC,RDW)

18 Parameter electronic cell counter WBC: White Blood Cells RBC: Red Blood Cells HgB: Hemoglobin Hct: Hematocrit MCV: Mean Cell Volume MCH: Mean Cell Hemoglobin MCHC: Mean Cell Hemoglobin Concentration RDW: Red Blood Cell Distribution Width

18 Parameter Cell Counter 8 parameters plus 9. LYM#: Lymphocytes total count 10. MID#: Mid Cells total count 11. GRN#: Granulocytes total count 12. LYM%: Lymphocytes Percentage 13. MID%: Mid Cells Percentage 14. GRN%: Granulocytes Percentage 15. Pct: Plateletcrit 16. PDW: Platelet Distribution Width 17. WBC histogram 18. Plt Histogram

Electronic blood cell counter Two General Principles Electronic resistance ( impedance) Light scattering Counts blood cells by electronic impedance (resistance). Discrimination & differentiation of blood cells based on cell size. Estimation of hemoglobin by Cyanomethemoglobin method using optical way. Reagents and diluent solution included.

The Coulter (Impedance) Principle of hematology analyzer Aperture...   A small opening (orifice) in a device by which blood cells may be drawn into an analyzer for counting. Principle. Electrical impedance..   Sizing and counting cells by detecting and measuring changes in electrical resistance when a cell passes through a small aperture. Tens of thousands of cells are counted, providing low statistical deviations with an accuracy of measurement estimated by Coulter to be within 1% of the true count

A small sample of the well mixed blood is aspirated and diluted with a balanced isotonic saline solution. The diluted blood sample is split into two parts, one for counting RBCs and platelets and the other for counting WBCs. The portion of the WBC bath and a detergent (lysing agent) is added to destroy (hemolyze) the red blood cells. The lysing agent contains potassium cyanide that reacts with the hemoglobin to form cyanmethemoglobin & the hemoglobin concentration is measured optically. The results are printed out or sent to a computer for review.

Light scattering (The second principle of electronic cell counting) Light scatter...  Cells counted as passed through focused beam of light( LASER) . Optical scatter... A cell counting principle :  The cell is identified and differentiated on the basis of light scatter and effects when it strikes the cell.

Optical Principle Outlet Aperture Inlet Photodiode Darkfield stop disk Light Source Beam Aperture Photodiode

Red blood cell (RBC) indices The three main RBC indices are used to determine the average size and hemoglobin content of the RBCs and they help determine the cause of anemia. The three indices are : Mean cell volume (MCV) -the average size of the red blood cells expressed in femtoliters fL(10-15 Liter). Mean cell hemoglobin (MCH) - the average amount of hemoglobin inside an RBC expressed in picograms pg(10-12 gm) . Mean cell hemoglobin concentration (MCHC) -the average concentration of hemoglobin in the RBCs expressed in g/dL or percent %.

RBC indices MCV - mean cell volume Indicates the average size (volume) of the red cells Calculation: PCV x 10 RBC If 80-100 fL, normal range, RBCs considered normocytic If < 80 fL are microcytic If > 100 fL are macrocytic

RBC indices MCH - mean cell hemoglobin weight Normal: 27-31 pg A measurement of the hemoglobin content in RBC’s Calculation: Hgb x 10 RBC If MCH is normal, cell described as normochromic If MCH is less than normal, cell described as hypochromic There are no hyperchromic RBCs

RBC indices MCHC - mean cell hemoglobin concentration Normal: 32-36 % A measure of the concentration of hemoglobin in the average RBC Calculation: Hgb x 100 Hct

RBC count Normal level of RBC Men 4.5 – 5.5 x 1012/L Women 3.8 – 4.8 x 1012/L RBC is reduced in anemia when there is reduction of RBC production in marrow, or destruction in peripheral vessels . RBC production increased in some conditions lead to increased Hb & PCV.

Normochromic Normocytic Hypochromic Microcytic

RBC indices Classification of anemia by RBC indices Normal size and color: normocytic/normochromic Causes: bone marrow failure, hemolytic anemia. Increased size, normal color: macrocytic/normochromic Causes: Folate or B12 deficiency, liver disease. Decreased size, decreased color: microcytic/hypochromic Most common anemia Causes: iron deficiency,thalassemia, chronic diseases

RBC indices RBC indices are readily available from the automated hematology counting devices RBC, Hbg & MCV is measured directly; MCH and MCHC are both calculated

Hematocrit (Hct or PCV) Automated cell counters calculate the hematocrit by multiplying the RBC count by the mean red cell volume. HCT = RBC count X MCV expressed as (%) A decrease in the number or size of red cells also decreases the amount of space they occupy, resulting in a lower hematocrit. Conversely, an increase in the number or size of red cells increases the amount of space they occupy, resulting in a higher hematocrit.

13 DESCRIBE THE VOLUME Histograms HISTOGRAM...   A visualization graph that formed based upon cell size and/or cell number and indicate frequency of distribution. Volume histograms performed for erythrocytes, leukocytes, and/or platelets. These types of histograms will provide an approximate number of cells on the Y-axis and the cell size on the X-axis. Y.Cell No X.Cell size fL

Histogram for erythrocytes The instrument counts those cells with volume sizes between 36 fL and 360 fL as erythrocytes. Deviations in the shape and position of the RBC histogram becomes an indicator of changes that are occurring in the size and/or shape of the erythrocytes. If the RBC’s are larger than normal, the curve will shift toward the right. If the RBC’s are smaller than normal the curve will shift to the left. Y.Cell No X.Cell size fL

13 DESCRIBE THE VOLUME Hisogram for WBC There are three analysis groups of leukocyte histogram : Lymphocytes, Granulocytes which include neutrophils, basophils, and eosinophils; and Mononuclear cells comprised of monocytes. Y.Cell No X.Cell size fL

Histogram for platelet Platelet histogram are obtained from volume sizes of 2 to 20 fL.   Y.Cell No X.Cell size fL

X-Axis = cell size in femtoliters (fL) Y.axis X. axis Y.axis X. axis Y.axis X-Axis = cell size in femtoliters (fL) Y-Axis =no of cells X. axis Histograms of RBC, Platelets & WBC

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