"ractical o , 3#

WHITE BLOOD CELL COUNT & DIFFERENTIAL COUNT

Objectives :At the end of the practical the student should be able to, 1. Identify the apparatus used in counting WBCs. 2. Determine the total white cell count using the eubauer counting chamber. !. "repare a peripheral blood film #. Identify the different types of white blood cells. $. Describe how to obtain a differential white cell count. %. &ist the normal 'alues of, i. (otal white cell count ii. eutrophils iii. )osinophils i'. Basophils '. &ymphocytes * 'i. +onocytes And the conditions where these 'alues are abnormal. Calculatio o! t"e Total W"ite Cell Cou t Apparatus and materials ,(he haemacytometer set which contains, eubauer counting chamber White cell pipette .uction apparatus Co'er glasses +icroscope .terile lancet. .urgical spirit and cotton swabs. Diluting fluid /lacial acetic acid 0212 1.$ ml C malachite green 3.$ ml /entian 'iolet a4ueous 3.$ ml Distilled water 56 ml 07or this, the blood is diluted with a fluid which lyses red blood cells, but spares white blood cells and nucleated red cells.2 -

3.$ ml Column of blood

1 ml

11 ml

White Bead

+ethod ,-

Wipe a 'olunteer8s finger with surgical spirit and allow to dry. .tab the pulp of the finger with a sterile lancet and wipe away the first drop of blood 0do not rub or s4uee9e finger to assist flow of blood2. When a big enough drop of blood forms, apply the tip of the pipette to the drop and suc: blood upto the #$%&l &a'(. Draw up the diluting fluid till the )) &a'( - till glass bubble is full. Dilution would be 1 , 23. ;old the pipette hori9ontally and rotate it 'igorously between finger and thumb to mi<. &et out the fluid contained within the stem of the pipette. (a:e the eubauer counting chamber, place it on the stage of the microscope and put the co'er glass o'er it. (ouch the pipette to the side of the counting chamber, where it pro=ects beyond the co'er glass. >nown as ?charging the chamber?. (he chamber will fill with the diluted blood. ow wait two minutes to allow for the cells to settle. @bser'e under low power. As WBCs are larger cells and are found in less amounts than ABCs, the larger s4uares of the counting chamber are used.

B
Depth B 1C13 mm

D
Calculation ,-

C
1mm

1mm

(he 'olume in one large s4uare 0eg. A2 (he 'olume in # large s4uares

o. of WBCs in one s4uare Appro<. o. of WBC in # s4uares concentration of WBC Blood was diluted 23 times o. of WBC in 1mm! of undiluted blood B B +'e,a'atio o! a ,e'i,"e'al bloo- !il& -

B B B B B B B

1 < 1 < 1C13 mm! 1C13 mm! # < 1C13 mm! #C13 mm! D #D B E E * #C13 13E < 23 C # $3E

Clean fingertip with surgical spirit and allow to dry .tab with a sterile lancet and wait for blood droplet to form. "lace the drop of blood on a clean slide.

(a:e another clean dry slide and place it 'ertically, touching the drop of blood. Drag this slide slightly bac:wards and then slide it smoothly forward to spread the drop of blood e'enly.

Allow the slide to air dry for around one minute

When dry, add 6 - 13 drops of &eishmann stain to the slide and allow to stain for 1 minute. 0stain is in alcohol medium2. e<t, add double the amount of drops of water to the slide and mi< slightly Allow to air dry.

Add a drop of oil and 'iew under oil immersion lens of the microscope

T"e W"ite Bloo- Cells

Neut'o,"ils
T"e Di!!e'e tial Leu(oc.te Cou t

Eosi o,"ils

- (he differential count is obtained by counting theBilobed number of white cells on the Coarse Aed &obulated Aed, "in:Cblue peripheral blood film /ranules nucleus 0!-$2 Body ucleus granules ;ead (ail
Abundant cytoplasm - (he is done using a chart as follows, this chart has 13 rows.

field
Absolute Count , "ercentage Description , ,

&

1 2 2333 - F$33 ! $3 # - F3 1 $ umerous, small, purple % granules F 6 &arge, round well defined cell 5 13

.i9e , )4ual to eutrophils Absolute Count, #3 G #33 CIl "ercentage , Description 1G#1 ,

&arge, round cell blood film A field is chosen the microscope, in the 8body8 area of the &obulesunder are lin:ed by Characteristic, bilobed nucleus to the 13 white cells are counted, the abo'e chart according chromatin strands in a field, while filling different numbers of white cells contained in the total 13 cells Abundant cytoplasm eg. % neutrophils, ! lymphocytes, 1 monocyte Increased in ,

&obulated lobules2

ucleus 0! G $

umerous, closely pac:ed, orange red granules

"arasitic Infections Increased , - in A similar count is done in 5 more fields, bringing the total of cells - number Amoebiasis Bacterial infections counted, to 133. - as(ropical pulmonary - (he&eu:aemias total number of each type of white cell is then ta:en the percentage. eosinophilia .e'ere inflammatory conditions - 7ilariasis - (o obtain thenecrosis absolute count, these percentages are multiplied by the total white and tissue - ;oo: worm infection cell count Allergic Disorders - Bronchial Asthma Decreased in , - ;ay fe'er Hiral infections "ancytopaenia (yphoid fe'er and on reco'ery from acute infection

Baso,"ils
ucleus with a constriction in the center /ranules 0Dar:2 Contains ;istamine , ;eparin

L.&,"oc.tes

&arge ucleus

&ess cytoplasm

.i9e

Absolute Count, "ercentage , Description ,

much smaller than eutrophils 23 - 133 CIl 3.# 1 Hery large, deep purple granules in the cytoplasm Densely arranged ucleus is :idney shaped, co'ered in granules and is difficult to see.

.i9e

much smaller than eutrophils 1$33 - #333 CIl 23 - #3 1 Bone +arrow &ymphoid tissue (onsils .pleen (hymus &ymph nodes

Absolute Count, "ercentage +ade in , ,

Increased in

, Asthma Anaphyla<is Chronic +yelocytic &eu:aemia Decreased in , "ancytopaenia )lderly

Description

Hery scanty cytoplasm 0hardly seen2 ucleus is large and occupies most of the cell

Increased in

Chronic infections 0(uberculosis C .yphilis2 Hiral infections 0;IH C ;epatitis2 Decreased in , "ancytopaenia )lderly

/o oc.tes
>idney shaped nucleus Cytoplasm 0blue2 G ground glass appearance

.i9e , Absolute Count, "ercentage , 7ormed in , Description ,

e4ual to lymphocytes 233 - 633 CIl 2-61 Bone +arrow +ore cytoplasm than in lymphocytes Contains 'ery fine, dar: granules ucleus is :idney shaped

Increased in , (uberculosis +alaria (yphoid ;as phagocytic properties