Clin Path Lab 3

CLINICAL PATHOLOGY LABORATORY 3 USTMED ’07 Sec C - AsM Differential Leucocyte Count Preparation of Blood Smears for Differential Count Procedure: 1. Make a finger puncture and wipe of the first drop of blood 2. Touch the slide to the second drop, transferring a small drop of blood to the slide about ½ inch from one end or if venipuncture was done, place a drop of oxalated blood onto a slide 3. Lay the slide on a flat surface and place the end of the second (spreader) slide on it, held by the fingertips at an angle no greater than 30 degrees, in front of the drop of blood 4. Pull the spreader slide back into the drop of blood. When the blood has spread along the two thirds of the width, push the spread along the two thirds of the width, push the spreader slide forward with a steady even motion. The weight of the slid is the only pressure applied

Purpose: to establish the percentage distribution of the different leukocytes Equipment: Microscope with oil immersion objective Stained slide Procedure: 1. Under LPO, slect area where cells are evenly distributed 2. Place a drop of cedar wood oil and examine under oil immersion lens. 3. Count 100 leukocytes in the smear. Suitable and unsuitable regions for observation of stained smear

left – (unsuitable) erythrocytes are scattered but their three-dimensional structure is difficult to observe middle – (suitable) erythrocytes are uniformly distributed and their three-dimensional structure is well observed (the central part is bright) right – (unsuitable) erythrocytes are stacking

Pathway for differential count

Normal values (adult) Myelocytes Juvenile Stabs Segmenters Lymphocytes Monocytes Eosinophils Basophils

% 0% 0 - 1% 0–5% 50 – 70% 20 – 40 % 0 – 7% 0 – 5% 0 – 1%

SI 0 - 0.01 0 – 0.05 0.50 – 0.70 0.20 – 0.40 0 – 0.07 0 – 0.05 0 – 0.01

Tally counter – used for differential leukocyte count (peripheral smears)

Nucleated RBC under the Microscope Corrected WBC Count Formula: WBC count Corrected WBC count

x

100

= 100 + number of NRBC counted

Microscopic view of 1 large square (Area for counting WBCs under LPO)

Note: if nucleated RBCs are present, the leukocyte count can be corrected by using the following formula Total WBC count x 100 Corrected WBC

= 100 + No. of nucleated RBC

Computation: Cells counted in 4 large squares WBC/cu mm = of blood no of squares x depth of counting x dilution*** counted* chamber** Examples of white blood cells counted in a representative area.

*no. of squares counted = 4 **depth of counting chamber = 1/10 or 0.1mm ***dilution = 1/20

Leukocyte Count A. Leukocyte count using oxalated blood

Normal values S.I. 4.5 – 10.0 x 109 / L (5,000-10,000 /mm3)

Principle: blood is diluted with a fluid that lyses the nonnucleated RBC and not the nucleated WBC

Complete Blood Count A complete blood count (CBC), a routine hematology screening, includes the following determinations: • total white blood cell count • total red blood cell count • hematocrit • hemoglobin • differential white cell count With an accurate determination of these values, approximately 70% to 80% of the hematologic diagnosis can be made as well as significant amount of information gathered for the purpose of _____________ the stages of a particular disease or of diagnosing some disease entities not directly related to the hematopoetic system.

Equipment: WBC pipette Counting chamber Sucking tube WBC Diluting fluid o Glacial acetic acid 1% with tinge of 1% gentian violet

Small lymphocyte - small lymphocytes are slightly larger than erythrocytes (about 11 um in diameter and circular in shape. Their cytoplasm is scanty and clear blue, having no granules. The nucleus stains deeply due to condensation of chromatin. In healthy adults, 30-45% of leukocytes are classed as lymphocytes. Large Lymphocyte - large lymphocytes are round and twice as large as the erythrocyte (about 15 um in diameter). The nucleus is large and the cytoplasm plentiful. The nuclear chromatin structure and the coloring of the cytoplasm are similar to those of small lymphocytes, except that a few azurophilic granules are present in the cytoplasm.

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Fill a WBC pipette with blood to the 0.5 line Dilute with diluting fluid to the 11 line above the bulb so that the blood is diluted 1:20

shake, discharge 3 drops, then fill the counting chamber count (10x objective) the cells of the 4 large corner squares (1mm each) calculate the leukocyte count on the basis of cells counted, area counted and the dilution

Large Lymphocyte (ulit) The cell shown here has larger azurophilic granules scattered in the cytoplasm. This is called a large granular lymphocyte (LGL) and is considered, by some, to be a natural killer cell. To classify lymphocytes into T or B forms or into subclasses, surface antigen exploration with monoclonal antibodies is required. Polymorphonuclear neutrophil - the size, cytoplasm and nuclear chromatin structure of polymorphonuclear neutrophils are similar to those of neutrophilic band granulocytes. However, the nucleus is segmented into three lobes by 2 filaments. In healthy adults,a bout 50% of leukocytes are polymorphonuclear neutrophils. Most polymorphonuclear

neutrophils have tow or three lobes. Eosinophil - eosinophils are slightly larger than neutrophils. The cytoplasm contains large orange-red granules. The nuclear chromatin structure is similar to that of neutrophils but the nucleus is only segmented into two swollen oval lobes of equal size by a filiform strand. In the normal blood, about 3% of leukocytes are eosinophils. The level increases in cases of parasitic diseases, such as ancylostomiasis, allergic reactions, such as bronchial asthma, and chronic granulocytic leukemia. Neutrophil band granulocyte (neutrophil band form) Neutrophil band granulocytes have diameters about twice that of erythrocytes (about 15 um). The cytoplasm contains fine granules staining lightly orange-purple. The nuclear chromatin is condensed, forming coarse, dark purplishred clumps. The nucleus is like a band of varying widths but the narrow portions are not filiform as in the mature neutrophil. In normal blood, 5-10% of leukocytes are neutropil band granulocytes. - fin [email protected] [email protected]