DILUTING PIPETS a.

The blood cell diluting pipet (see figure 2-1) consists of a graduated capillary tube having an arbitrary volume of one unit and marked in increments of that unit, each designated as 0.1; note that this unit is not a standard measurement but merely an arbitrarily selected unit. Above the capillary tube is a mixing bulb containing a color-coded glass bead, and above the bulb another shorter capillary tube with an engraved mark. The pipet for performing the white blood cell count has a white bead, the mixing bulb is smaller than that of the red count pipet, and the marking above the bulb reads "11." The pipet for performing the red blood cell count has a red bead in the mixing bulb and the marking above the bulb is "101." b. These pipets are used to take the specimen directly from a capillary puncture or, after careful mixing, from a vial of fluid or of blood treated with an anticoagulant, such as EDTA. The blood or fluid is drawn into the pipet to a predetermined point and diluted to the correct mark with diluting fluid. After proper mixing, the diluted substance is placed in the counting chamber and the cells are counted. c. Usually, the technique for diluting the blood specimen with a pipet calls for whole blood to be drawn exactly to the 0.5 mark and diluted only to the "11" or "101" mark with appropriate diluting fluid dependent upon the type of cell count. Since the volume of fluid in the stem does not enter into the dilution, the dilution is calculated on the volume in the bulb, thus with the white blood cell count: Whole blood = 0.5 = 1 Dilution = Volume in bulb 10 20 and in the RBC count Whole blood = 0.5 = 1

Dilution = Volume in bulb 100 200 The dilution of the blood sample in the white blood count is 1 to 20 and the dilution factor is 20. In the red blood count the blood sample is diluted 1 to 200 and the dilution factor is 200. The permissible error of the red cell pipet is +5 percent and the white cell pipet has a permissible error of +3.5 percent. d. Blood cell diluting pipets are delicate pieces of equipment and should have careful treatment. It is also important that clean, dry diluting pipets be used to prevent dilution errors and hemolysis of cells. e. If an aspirator is available, pipets can be easily washed by drawing cold tap water (distilled water is preferred) through the pipet. If a deposit remains, it can be dissolved by drawing dilute sodium hypochlorite (household bleach) through the pipet followed by several washings with distilled water. If pipets become plugged through neglect, the hole may be opened with a fine steel wire, and the above procedures may then be carried out. When necessary, pipets may be dried rapidly by drawing a small

amount of alcohol through then and then ether or acetone followed by a stream of air until the glass bead removes freely and no moisture remains in the pipet. f. All pipets should be inspected each time before use so as to prevent the use of equipment that is dirty or that has chipped ends. http://www.free-ed.net/sweethaven/MedTech/Hematology/lessonMain.asp?iNum=0204 http://www.oiml.org/publications/R/R078-e89.pdf To perform An erythrocyte sedimentation rate (ESR), also called a sedimentation rate or Westergren ESR, is the rate at which red blood cells sediment in a period of one hour. anticoagulated blood is placed in an upright tube, known as a Westergren tube, and the rate at which the red blood cells fall is measured and reported in mm/h. It is a common hematology test, It is a simple glass tube with marking on the side to measure the layers formed when blood separates. The hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine the concentration of cells in a liquid sample. It is frequently used to determine the concentration of blood cells (hence the name hemo-) but also the concentration of sperm cells in a sample. 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. http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber/ http://www.che.udel.edu/mranton/pdf/protocol_counting_cells.pdf Wintrobe method: The Wintrobe method is performed similarly except that the Wintrobe tube is smaller in diameter than the Westergren tube and only 100 mm long. EDTA anticoagulated blood without extra diluent is drawn into the tube, and the rate of fall of red blood cells is measured in millimeters after 1 hour. The shorter column makes this method less sensitive than the Westergren method because the maximal possible abnormal value is lower. However, this method is more practical for demonstration purposes. http://www.medicine.mcgill.ca/physio/vlab/bloodlab/esr.htm
Sedimentation markings on our Wintrobe tube are graduated from 0 minus 2 to 95 in 1mm intervals. Reverse markings are graduated from 5 to 102 for macrohematocrit determinations. Contrasting light blue markings are easily visible. The glass tube has a sealed bottom with an approximate 3mm bore and 115mm length.

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