Dr Nikhil Dr Nikhil BansalBansal

J.N.M.C.,WardhJ.N.M.C.,Wardhaa

DLC BY PERIPHERAL SMEAR DLC BY PERIPHERAL SMEAR COMPARISION WITH AUTOMATED COMPARISION WITH AUTOMATED

DIFFERENTIAL COUNTDIFFERENTIAL COUNT

ACKNOWLEGMENTACKNOWLEGMENTSkill based project is an art to enhance the creativity

of a student and to increase his/her interest in clinical basis.

As per student manner I firstly would like to thank “Dr. S. Vagha Ma’am”“Dr. S. Vagha Ma’am” for arranging such project. I also thank “Dr Samarth Shukla Sir”“Dr Samarth Shukla Sir” for helping us whenever required. My pay my heartful gratitude to “Dr.Vijay Kumar Dembra Sir”“Dr.Vijay Kumar Dembra Sir” for guiding us throughout the project.

Last but not the least I thank all my “group mates” group mates” for co-operating with me and lending a helpful hand when required.

INTRODUCTIONINTRODUCTIONWBC or leukocyte is the colourless and

nucleated formed element of blood. Leukocyte play very important role in defense mechanism of body.

Leukocyte are classified into 2 types namely –

1.Granulocyte-with granules2.Agranulocyte- without granulesThe granulocyte are-Neutrophills Eosinophills BasophillsThe agranulocyte are-Monocytes Lymphocytes

MORPHOLOGYMORPHOLOGYNeutrophils-neutrophils have fine or small

granules in the cytoplasm granules appear violet in colour. The nucleus is multilobed.nucleus has 4-5 lobes.

diameter – 10-12 microns Eosinophils-eosinophils have larger granules in

the cytoplasm which stain bright red colour.the nucleus is bilobed.

diameter-10 and 14 micronsBasophils-basophils also have coarse granules

in the cytoplasm.the granules stain purple blue with basic dyes like methylene blue.

diameter-8 to 10 microns

Monocytes-monocytes are largest leucocytes with diameter of 14 to 18 microns.the cytoplasm is clear without granules.the nucleus is kidney,round,oval,horseshoe shaped.

Lymphocytes-the lymphocyte also have clear cytoplasm without granules.the nucleus is ovel shaped occupying the whole of the cytoplasm.depending upon the size the lymphocyte are divided into two group as:

large lymphocytes-the younger cell with a diameter of 10 to 12 microns.

Small lymphocytes-the older cell with a diameter of 7 to 10 microns

Depending upon the function,the lymphocytes are divided into 2 types as:

T lymphocytes-concerned with cellular immunity B lymphocytes-concerned with humoral immunity

NORMAL COUNT OF WBCNORMAL COUNT OF WBCTotal WBC count – 4,000 to 11,000/cu.mm of

blood.Differential WBC count:LEUCKOCYTE PERCENTAGE ABSOLUTE VALUENeutrophils 50 to 70 % 3000-6000Lymphocytes 20 to 30 % 1500-

2700 Eosinophils 2 to 4 % 150-450Monocytes 2 to 6 % 200-600Basophils 0 to 1 % 0-100

CONDITION IN WHICH CONDITION IN WHICH ALTERATION IN DLCALTERATION IN DLC

Neutrophilia-increased in neutrophil count called neutrophilic leukocytosis.this occurs in the following condition:

1)Acute infections2)Metabolic disorders 3)Injection of vaccines4)After acute hemorrhage

Lymphocytosis - increasd in

lymphocyte count is called lymphocytosis and this occurs in

1)Diptheria2) Infectious hepatitis3)Mumps4)Malnutrition5)Rickets6)Syphilis

Eosinophilia-increasd in eosinophil count is called eosinophilia and this occurs in

1)Allergic condition2)Asthma3)Blood parasitism4)Scarlet fever

Monocytosis-increasd in monocytes count is known as monocytosis and occurs in

1)Tuberculosis 2)Syphilis3)Malaria4)Kala azar

Basophilia-increased in basophill count is called basophilia and it occur in

1)Small pox 2)Chicken pox 3)Polycythemia vera

AIM & OBJECTIVESAIM & OBJECTIVES

“ To determine the Differential leukocyte count of the given sample in peripheral smear and compare it with the automated cell counter ’’

To compare differential leukocyte count in peripheral smear with automated cell counter.

To check the accuracy of automated cell counter.

MATERIAL & METHODMATERIAL & METHOD

MATERIAL:- Cell counter Microscope Cotton Glass slide Leishmen’s stain Distilled water Needle Staining track

METHODS:

1) Slide method

-place a drop of blood in the centre of a clean glass slide 1 to 2 cm from one end.

-place another slide with smooth edge at an angle of 30-45 0 near the drop of blood.

-move the spreader backward so that it makes contact with drop of blood.

-then move the spreader forward rapidly over the slide.

-a thin peripheral blood film is thus prepared.

-Dry it and stain.

Qualities of a good blood film-1)It should not cover the entire surface of

slide.2)It should have smooth and even

appearance.3)It should be free from waves and holes.4)It should not have irregular tail.

Parts of a thin blood film-consists of 3 parts

1)Head-i.e. the portion of blood film near the drop of blood.

2)Body-i.e. the main part of blood film.3)Tail-i.e. the tapering end of the blood

film.

2) cover glass method--Take a no.1 (22 mm square)clean cover slide.-Touch it on to the drop of blood.-place it on another similar cover glass in cross

wise direction with slide containing drop of blood facing down.

-pull the cover glass quickly.-Dry it and stain.

3) spine method-This is an automated method-place a drop of blood in the center of a glass slide.-spin at a high speed in a special centrifuge

cytospin.-blood spreads uniformly.-dry it and stain it.

PROCEDURE FOR STAINING

-pour leishman’s stain drop wise on the slide and wait for 2 min.this allows fixation of the PBF in methyl alcohol.

-add double the quantity of buffered water drop wise over the slide.

-mix by rocking for 8 mints.

-wash it water for 1 to 2 mints.

-dry in air and examine under oil immersion lens of the microscope.

COMPONENT OF LEISHMEN’S COMPONENT OF LEISHMEN’S STAINSTAIN

-preparation dissolve 0.2g of powdered leishmens dye in 100 ml of acetone free methyl alcohol in a conical flask.

-warm it to 50˚c for half an hour with occasional shaking.

-cool it and filter it.

OBSERVATIONOBSERVATIONNAME A/S MRD

NO.

LYMPH

GRA MID P L E M B

1 SAINA 4/F 987808

21.0 74.7 4.3 75 22 02 01 00

2 KAVITA 22/F 907818

40.0 54.6 5.4 55 40 03 02 00

3 SUMAN 60/F 789149

21.6 74.3 4.1 76 20 02 02 00

4 SINDHUBAI

52/F 906676

46.4 47.4 06 48 47 03 02 00

5 NOOTAN 6/F 906739

27.4 67.9 4.7 70 27 02 01 00

CELL COUNTER(%) PEREPHERAL SMEAR (%)

NAME A/S MRDNO.

LYMPH

GRA MID P L E M B

6 RAVI 7/M 908240

44.7 48.7 6.6 49 47 05 01 00

7 DEVASHIS

5/M 908241

50.0 42.4 7.6 47 50 02 01 00

8 SHALU 27/F 908285

31.6 63.4 5.0 65 32 02 01 00

9 MOTIRANM

72/M 908277

27.7 66.9 5.4 66 30 03 01 00

10

SUNITA 20/F 908266

31.0 61.5 7.5 65 32 02 01 00

CELL COUNTER(%) PEREPHERAL SMEAR (%)

RESULTRESULT

- As per the above observation we can see that there isn’t much of a difference between cell counter reading and the reading by peripheral smear.

- Hence after the comparative study

between the two we can conclude that cell counter reading are equally efficient and automat zed reading scan be preferred over the manual to save time.

DISCUSSIONDISCUSSIONCauses of alteration in DLC count-1)Errors in calculation2)Increased erythropoietin in a blood sample.3)Improper admixture of anticoagulant in a blood

sample.4)If blood sample is clotted.5)If sample collected in a plain blub.6)Overstaining.7)Suboptimal staining. As per the causes mentioned above before

going to analysis or observe for differential leukocyte count one must nn of all those things to avoid errors in differential leukocyte count.Accordingly some of the precautions to be taken by examining the peripheral smear or analyzed by automated cell counter