Differential count of leucocytes by field’s method.

 Introduction

The function of leucocyte is to fight infection, defend the body by phagocytosis against invasion by foreign organisms, and to produce , or at least transport and distribute, antibodies in the immune response. Behaving as separate yet related system, the various types of leucocytes serve different functions.

The life span of leucocytes varies from 13 to20 days, after which the cells are destroyed in the lymphatic system; many are excreted from the body in the faecal matter. Differential count is also known as qualitative leucocyte count which aims to determine the proportion of different kinds of leucocytes' was first done in spinal fluid films by Einhorn in 1884 followed by Kraig in 1899. It was also known as Ehrlich’s count and study leucocytes. Schilling in 1913 called it `differential count’ of leucocytes.

Differential count is expressed as a percentage of the total number of white cells-relative number The absolute number of each type obtained mathematically by multiplying the percentile  value of one type of leucocyte by the total leucocyte count.

Absolute value[WBC/mm3]  =  Relative value[%]  *Total WBC count  [cells/mm3]

Principle:

Three mechanism are involve in producing the characteristics staining of Blood cells

1.      Coulombic attraction between dye and tissue substrate.

2.     Mechachromasia of Azure-B

3.     Dye-dye interactions betwwwn Azure- B and Eosine Y.

The coulombic attraction are between molecules carrying opposite electrostatic charge,  thus a cationic dye is attracted to an acid substrate and anionic dye to basic groups. Metachromasia  depends on the property of certain dyes to form polymers which result in a color change of the polymer. Azure- A and Azure-B are metachrometic stain and Azure B in particular plays an important part in providing the reddish-purple staing effect . This seems to be the result of specific intraction between two molecules of Azure- B and One of Eosine-Y

Romanowasky type stains are combination of methylene blue,. Azure-B, Azure-A , Azure-C and  Eosine-Y. When dissolved in water, all components that form positively charge basic dye ion, combined with negatively charges, nucleic acid.  Azure –A and Azuer- B are metachrometic stain and Azure-B particular plays and important part in providing the reddish purple colour staining effect in leucocytes in nuclei. At the same time methylene blue reacts well with RNA giving blur colour the cytoplasm of lymphocytes and monocytes. Eosin impart pink colour to the granules of  Eosinophills and Cytoplasm of RBC. Granules

 

Composition of field ’stain-A

Methylene blue            0.8g

Azure-I                        0.5g

Na₂HPO₄                    5.0g

KH₂PO₄                      6.25g

Distilled water             500ml

Composition of Field’s stain-B

Na₂HPO₄                        5.0g

KH₂PO₄                          6.25g

Eosin                               1.0g

Distilled water               500ml

 

 

present in the cells stains according to their chemical nature. The full Romanowasky’s  effect is optained through the combined action the components dyes.

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The characteristics appearance of romanowasky stained structure in blood are: Pink erythrocytes,purple chromatin, blue leucocyte cytoplasm, purple black basophil granules, red pink eosinophil granules, fawn to ilac neutrophil granules, purple platelet granules and purplish blue nucleoli, with reticulocytes staining their characteristics polychromatic shades.

 

Requirment:

Field’s stain A, Field’s stain B, Methanol, Distilled water, 70% alcohol, Sterile Lancet, Clean glass slide Spreader slide Cotton swab

Procedure

The process of differential count  of leucocyte can be divided into three separate stages:

A.     Preparation of blood film

B.     Fixation and staing of blood film

C.     Identification classification and counting of relative number of different cells.

A.    Preparation of Blood Film-Push wedge or two-slide method

1.      The tip of 3^rd or 4^thfingers cleaned with 70 % alcohol  and is dry it

2.      Puncture the fingertip quickly using sterile lancet or picker

3.     Wipeout the first drop of blood with  cotton swab and  allow the second drop to form on the finger tip.

4.     Gently put the blood drop on to the clean & dry slide surface.

5.     With help of the another slide edge.  Spread the blood and make the smeared allow it to air dry

B.     Fixation and staing of blood film

6.     Prepare a thin blood film, allow the film to dry in air and fix with menthol for ½ to 1 minute.

7.     Dip slide in Field’s stain B for 7-8 seconds.

8.     Rinse in a beaker of clean tap water and then drain off excess water onto absorbent paper.

9.     Dip the slide in Field’s stain A for 7-8 seconds.

10.   Rinse thoroughly in a beaker of clean tap water and allow to dry in a vertical position. Do not blot. When dry examine under high power and oil immersion lens.

11.    Count the 100 cells in non overlapping manner(Longitude or Battlement method) and record the types of cells in observation table.