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# Hemocytometer manual cell counting (1)

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Hemocytometer manual cell counting (1)

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### Hemocytometer manual cell counting (1)

1. 1. RAJESH MOHESS ,CLT.
2. 2.  The hemocytometer is a specimen slide which is used to determine the concentration of cells in a liquid sample.  It has a rectangular indentation that that creates a chamber  The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known.
3. 3.  Given the known parameters it is possible to count the number of cells or particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall  The hemocytometer is frequently used to determine the concentration of blood cells (hence the name “hemo-”)  However, it can also be used for other samples, such as sperm cells.
4. 4.  The cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height (usually 0.1mm).  Additionally, a grid is etched into the glass of the hemocytometer.  This grid, an arrangement of squares of different sizes, allows for an easy counting of cells.  This way it is possible to determine the number of cells in a specified volume.
5. 5.  The fluid containing the cells must be appropriately prepared before applying it to the hemocytometer. Proper mixing: The fluid should be a homogenous suspension. Cells that stick together in clumps are difficult to count and they are usually not evenly distributed.
6. 6. Appropriate concentration:  The concentration of the cells should neither be too high or too low.  If the concentration is too high, then the cells overlap and are difficult to count.  A low concentration of only a few cells per square results in a higher statistical error and it is then necessary to count more squares (which takes time).  Suspensions that have a too high concentration should be diluted 1:10, 1:100 and 1:1000.  A 1:10 dilution can be made by taking 1 part of the sample and mixing it with 9 parts normal saline  The dilution must later be considered when calculating the final concentration.
7. 7. Counting cells that are on a line:  Cells that are on the line of a grid require special attention.  Cells that touch the top and right lines of a square should not be counted  Cells on the bottom and left side should be counted.
8. 8. Number of squares to count:  The lower the concentration, the more squares should be counted.  Otherwise one introduces statistical errors.  Cells should be counted on both sides of the chamber.  If the final result is very different, then this can be an indication of sampling error.
9. 9.  When a liquid sample containing immobilized cells is placed on the chamber, it is covered with a cover glass, and capillary action completely fills the chamber with the sample.  Looking at the chamber through a microscope, the number of cells in the chamber can be determined by counting.  Different kinds of cells can be counted separately as long as they are visually distinguishable.  The concentration of the cells can be calculated from the cells counted from the mixture using simple formulas
10. 10.  Rulings cover 9 square millimeters.  Boundary lines of the Neubauer ruling are the center lines of the groups of three
11. 11.  The central square millimeter is ruled into 25 groups of 16 small squares  The ruled surface is 0.10mm below the cover glass  One (1) Milliliter = 1000 cubic millimeters (cu mm)  One (1) Microliter (ul) = One (1) cubic millimeter (cu mm)  The number of cells per cubic millimeter = Number of cells counted per square millimeter X dilution (eg. 100 for WBC count) X 10 (depth factor)
12. 12.  Depth: 0.1 mm
13. 13.  Red square = 1 x 1 mm = 1 mm2 (AREA) = 0.1 cubic millimeter (VOLUME - mm3)  Green square = 0.25 x 0.25 mm = 0.0625 mm2 = 0.00625 mm3
14. 14.  Yellow square = 0.2 x 0.2 mm = 0.04 mm2 = 0.004 mm3  Blue square = 0.05 x 0.05 mm = 0.0025 mm2 = 0.00025 mm3
15. 15.  There are different types of counting chambers available, with different grid sizes.  Know the grid size and height (read the instruction manual) otherwise you’ll make calculation errors.  The provided cover-glasses are thicker than the standard 0.15mm cover glasses.  They are therefore less flexible and the surface tension of the fluid will not deform them.  This way the height of the fluid is standardized.
16. 16.  Moving cells (such as sperm cells) are difficult to count.  These cells must first be immobilized.  The hemocytometer is much thicker than a regular slide.  Be careful that you do not crash the objective into the hemocytometer when focusing
17. 17.  The Unopette system is a system of prefilled blood dilution vials containing solutions that will preserve certain cell types while lysing others.  It utilizes a premeasured volume of diluent in a chamber into which a specified amount of blood is drawn
18. 18.  The Unopette test system consists of a self-filling capillary pipette  It consists of a straight, thin-wall, uniform-bore plastic capillary tube fitted into a plastic holder  Also has a plastic reservoir containing a premeasured volume of reagent for diluting
19. 19.  The reservoir is punctured to open access to the reagent  The dilution is determined by the type of capillary used since each type have different volumes  The diluted blood is added to a hemocytometer chamber and cells are counted in a specified area.
20. 20.  Unopette System discontinued  The BMP LeukoChek is used to measure and dilute whole blood for manual counting of leukocytes (WBC) and platelets  It replaces the Unopette system  Tested to CLIA guidelines  Clinical Laboratory Improvement Amendments (CLIA) – establish quality standards for all laboratory testing to ensure the accuracy, reliability and timeliness of patient test results regardless of where the test was performed
21. 21.  For this procedure, whole blood is added to ammonium oxalate (diluent), which lyses the red cells while preserving platelets, leukocytes
22. 22. PRINCIPLE  Whole blood is added to the diluent, which lyses red cells but preserves platelets, leukocytes  When erythrocytes are completely lysed, the solution will be clear red and counting can proceed.  The diluted blood is placed in a hemocytometer according to accepted technique.  Cells are allowed to settle for 10-15 minutes before leukocytes and platelets are counted.  Under 100X magnification (x10 objective) using bright-light microscopy, leukocytes appear refractile (can be seen as dark dots)  Under 400X magnification (x40 objective) using bright-light microscopy, platelets appear oval or round and frequently have one or more dendritic processes.
23. 23.  BMP LeukoChek containing ammonium oxalate Check expiration dates and do not use expired test kits. Protect from sunlight.  BMP LeukoChek capillary pipette, 20 μL.  Hemocytometer : improved Neubauer ruling  Hemocytometer coverslips  Petri dish lined with filter paper that has been moistened and two applicator sticks to hold the hemocytometer  Microscope  Hand counter  EDTA whole blood
24. 24. DILUTION RATIO  Sample to total volume.......................1:100  That is 1.98 ml of diluent to 20μl of sample
25. 25.  1. Specimen should be well mixed and left on a rocker for at least 5 minutes before using.  2. Check BMP LeukoChek for clarity and contents. If the BMP LeukoChek chambers appear cloudy or the amount of reagent looks questionable, do not use.
26. 26.  3. With the reservoir on a flat surface, puncture the diaphragm of the reservoir using the protective shield of the capillary pipette.  A. Using a twist action, remove protective shield from the pipette assembly.  B. Holding the pipette and the tube of blood almost horizontally, touch the tip of the pipette to the blood (fill with 20μl of blood).  The pipette will fill by capillary action and will stop automatically when the blood reaches the end of the capillary bore in the neck of the pipette.
27. 27.  C. Wipe the excess blood from the outside of the capillary pipette. Be careful not to touch the tip of the capillary when wiping off excess blood.  D. Before entering the reservoir, it is necessary to force some air out of the reservoir by squeezing it. Do not expel any liquid and maintain pressure on reservoir.  E. Place an index finger over opening of overflow chamber and position pipette into reservoir neck.   F. Release pressure on reservoir and then remove finger. The negative pressure will draw blood into pipette.
28. 28.  G. Rinse the capillary pipette with the diluents by squeezing the reservoir gently two or three times. This forces diluent up into, but not out of, the overflow chamber and releases pressure each time to ensure the mixture returns to the reservoir.  H. Return protective shield over upper opening and gently invert several times to mix blood adequately.   I. Allow the BMP LeukoChek to stand for 10 minutes to allow RBCs to hemolyze. Leukocyte counts should be performed within 3 hours.
29. 29.  4. Charge hemocytometer  A. Mix the dilution by inversion and convert the BMP LeukoChek to the dropper assembly.   B. Gently squeeze BMP LeukoChek and discard first 3 or 4 drops. This allows proper mixing, with no excess diluent in the tip of the capillary.  C. Carefully charge hemocytometer with the diluted blood, gently squeezing the reservoir to release contents until chamber is properly filled. Be sure to charge both sides and not to overfill chambers.
30. 30.  5. Place the hemocytometer in the pre-moistened Petri dish and leave for 15 minutes. This allows the sample to settle evenly.  6. Cell count can now be performed
31. 31.  A WBC count is performed with a Neubauer hemocytometer.  Using the X10 microscope magnification, count WBC using the four outer large squares on the outer sections of the counting chamber  Count both sides of the chamber and average the count.
32. 32.  When counting, the cells that touch the extreme lower and the extreme left lines are included in the count. Those on top and right are not included. Count both sides of the chamber and average the numbers
33. 33.  Use the following formulas to calculate the WBC.  Cells/mm3 = Average No. of cells + 10% X depth factor (10) X dilution factor (100) divided by the Area (number of squares counted)  Depth factor is multiplied by 10 to convert area to volume in μl  Area of each large square = 1mm, so for the 4 large squares = 4mm
34. 34.  Example: If have 36 cells on one side and 44 cells on the other side Average = 80/2 = 40 cells + 10% (=4) = 44 cells  Cells/mm3 = (44 x 10 x 100)/ 4 (since 4 squares counted)  Cells/mm3 = 44,000/ 4 = 11,000  Total WBC = 11,000 mm3
35. 35. Normal Value:  Adult: 4,000 – 10,000  Newborn: 10,000 – 30,000
36. 36.  Platelet counts are performed with a Neubauer hemocytometer  Counting is done using x40 dry phase contrast objective. Platelets will have a faint halo.  The middle square of the hemocytometer chamber is counted.  It contains 25 small squares.
37. 37.  Count the 25 squares in the middle of the counting chamber No. of platelets/mm3 = Multiply No. of platelets (+ 10%) X 1000 OR  Count 5 of the 25 squares  Take the average of both sides add 10%  Multiply No. of platelets x 5000 = No. of platelets/mm3
38. 38.  Example: If have 36 cells on one side in 5 squares and 44 cells on the other side in 5 squares Average = 80/2 = 40 cells + 10% = 44  Cells/mm3 = 44 x 5000 (since 5 squares counted)  Cells/mm3 = 220,000  Total Platelets = 220,000 mm3
39. 39.  Normal Value: Platelets: 150,000 to 400,000 mm3
40. 40. LIMITATIONS  1. Specimen should be properly mixed and have sufficient volume of blood so there is no dilution of anticoagulant.  2. The capillary tube must be filled completely and be free of any air bubbles.  3. After the hemocytometer is charged, it should be placed in a pre-moistened Petri dish to prevent evaporation while the cells are settling out.  4. The light adjustment is critical. It is important for both WBCs and especially platelets. If the condenser is not in the correct position, it will fade out platelets.  5. Debris and bacteria can be mistaken for platelets.
41. 41. LIMITATIONS  6. Clumped platelets cannot be counted properly; the specimen must be recollected. The anticoagulant of choice is EDTA for preventing platelet clumping.  7. Avoiding overloading of hemocytometer chamber.  8. A highly elevated leukocyte or platelet count may make accurate counting difficult. In either instance, a secondary dilution should be made. When calculating the total count, adjust the formulas to allow for secondary dilution.  9. All WBC and platelet counts are done in duplicate. WBC counts should agree +/- 15%. Platelet counts must agree +/- 25%. If they do not agree, repeat counts
42. 42.  Infections – most common is bacterial infections It also occur in viral (lymphocytosis)  Allergy and drug hypersensitivity  Parasitic infections  Inflammation: eg. Inflammatory bowel disease, RA, and vasculitis  Extremely low birth weight  Malignancy and myeloproliferative disorders: eg. Leukemias, lymphomas  Increased release of WBC from bone marrow:- This occurs in infection, stress, and hypoxia it also occurs due to endotoxin stimulation and steroid administration
43. 43.  Viral infections – eg. HIV  Medications – abx, diuretics  Chemotherapy/Radiation therapy  Hyperthyroidism  Malignancy  Leukemia  Lupus  Aplastic anemia
44. 44.  Acute blood loss/Hemolytic Anemia  Malignancy  Splenomegaly  Inflammatory conditions – RA, IBS, Celia disease, Connective tissue disorder  Pancreatitis  Kidney disease
45. 45.  Idiopathic thrombocytopenic purpura (ITP)  Thrombotic thrombocytopenic purpura (TTP)  Hemolytic uremic syndrome – heparin, sulfa drugs, quinidine  Bacteremia  Autoimmune diseases  Pregnancy  Trapping of platelets in the spleen  Reduced production of platelets  Increased breakdown of platelets
46. 46. THE END