Module 10 The Differential Cell Count Acknowledgments Ministry of Health Guyana Centers for Disease Control and Prevention CDC Global AIDS Program GAP Guyana Centers for Disease Control and Prevention CDC Atlanta American Society for Clinical Pathology ASCP Learning Objectives After completion of this unit of instruction and laboratory exercise the student will be able to 1 Discuss the three 3 general features that are evaluated during the examination of a stained peripheral blood smear 2 Define differential cell count 3 Differentiate a relative cell count from an absolute cell count Objectives Continued 4 Accurately perform a 100 cell differential cell count according to criteria established by the instructor 5 Perform a WBC and platelet estimate on a stained peripheral blood smear according to criteria provided by the instructor 6 Interpret results of a differential cell count Objectives Continued 7 Identify mature and immature leukocytes using images immature are not supposed to be in peripheral blood 8 Given appropriate information calculate an absolute cell count 9 Identify toxic granulation Dohle bodies and vacuoles in the neutrophils including associated clinical conditions 10 Identify reactive lymphocytes including clinical situations associated with their presence The Differential Cell Count Used to determine the relative numbers of each type of leukocyte A smear is examined and each time a leukocyte is seen it is both tabulated and classified on a special counter The counting and classifying continues until 100 cells have been observed WBC Diff Counter manual method The Differential Cell Count Since 100 cells are counted each different type of WBC represents a percentage of the total number of cells tabulated for the differential cell count The differential cell count also includes an evaluation of RBC morphology platelet morphology and numbers and general WBC morphology Preparation and Staining of Smears Remember that a quality differential cell count can only be performed on a peripheral blood smear that is well made and properly stained Evaluating Staining Properties Locate a cell that can be positively identified such as a polysegmented neutrophil Note the staining characteristics of the nucleus and cytoplasmic granules Relate these features to any unidentified cells that may be present Evaluating Staining Properties Remember that cells don t always appear picture perfect The actual staining color of cells may vary from what is seen in textbooks or even in images Normal Percentages of Leukocytes Cell Neutrophils Bands Eosinophils Lymphocytes Basophils Monocytes Percentage 40 75 2 6 0 7 20 45 0 2 2 10 Variations in Normal Cell Percentages Occur in different age groups Seen in various disease conditions Likewise morphologically abnormal cells can also be seen in pathologic or reactive conditions Performing the Differential Cell Count Performing a WBC Estimate Under 40x High Dry Look for estimate area which has 200 RBCs that are partially overlapping with many single red cells Determine a WBC estimate using the following conversion information hpf high power field 2 4 cells hpf 4 0 7 0x109 L 4 6 cells hpf 7 0 10 0x109 L 6 10 cells hpf 10 0 13 0x109 L 10 20 cells hpf 13 0 18 0x109 L Remember Remember estimates estimatesdo do not notget get reported reported Performing the Differential Cell Count Also note if any platelet clumps are present Note other unusual or irregular characteristics of cells Locate a thin area of the smear in which the RBCs are evenly distributed only slightly overlap and the central pallor or whites of their eyes is seen Place a drop of oil on the slide and switch to the oil immersion objective Platelet Clump When platelets clump clotting has started inadequate specimen do another one Blood was not properly mixed with EDTA Will give false readings Evaluating a Thin Blood Smear 100x Oil Immersion Perform a normal WBC differential Count 100 consecutive cells using the battlement pattern The battlement pattern starts in a good counting area at the edge of the slide and goes up to the other edge and then moves slightly over and repeats the pattern until 100 cells are counted Platelets are estimated by counting 10 fields taking the average in 10 fields and multiplying by 20 000 The normal platelet count is 150 400 x 109 L Example of a Battlement Pattern The battlement pattern refers to the direction in which a technologist examines a peripheral smear in a good counting area 200 red cells not touching Nucleated RBCs If present must be counted separately from WBCs Not included in the 100 cell differential Must be reported and used to correct the total WBC can cause false reading in WBCs What About Red Blood Cells Red blood cell morphology is an important aspect of completing a WBC differential Red blood cells are assessed for variations in Size Shape Color refers to hemoglobin Inclusions Distribution Grading RBC Morphology Red blood cell morphology is assessed in a well distributed counting field 200 RBCs Use this scale to grade RBC morphology If less than 5 of cells show variation 0 or normal If 5 10 of cells show variation very slight If 10 25 of cells show variation 1 or slight If 25 50 of cells show variation 2 or moderate If 50 75 of cells show variation 3 4 or many or marked One Additional Note Absolute vs Relative Count Absolute count Derived by multiplying the percentage of the identified cell times the white count If 40 lymphs are counted and the white cell count is 5 0x109 L then the absolute lymphocyte count is 5 0x109 L x 40 or 2 0x109 L This number is then compared to the healthy reference range Relative count Strictly the percent counted in the WBC differential The relative number is then compared to the reference range for normal white cell differentials So it is possible for an individual to have a relative increase an absolute increase and a relative and absolute increase of a particular cell The absolute cell count is indicative of a true increase of a specific cell and gives important information to assist the clinician in making a diagnosis Individuals with HIV will show a decreased absolute neutrophil count and will risk infection Immunocompetent individuals will show white cells with an intact phagocytic function which is the ability of segmented neutrophils to recognise and kill a pathogenic organism through the function of its granules and its ability to make hydrogen peroxide CD 4 and CD 8 are lymphocyte antigen markers CD 4