HSBTE DMLT IInd Semester. Differential leucocyte counting (DLC): Preparation and staining of blood film, Performance of DLC, Normal values and significance of DLC.

Differential leucocyte counting (DLC)

3.1 Preparation and staining of blood film

3.2 Performance of DLC

3.3 Normal values and significance of DLC.

Blood film is also known as blood smear, peripheral blood smear, or peripheral blood film. A stained thin film of blood is used to examine blood cells' appearance, their number, and their shape. It is also helpful to examine parasites present in the blood specimen. Capillary blood or venous blood containing EDTA is used to prepare a thin blood film. EDTA is used as anticoagulant. Anticoagulants maintain the morphology of blood cells, and parasite cells and prevent coagulation of blood.

Differential leucocyte count measures the relative percentage of white blood cells (WBCs) in peripheral blood circulation. It is expressed in the percentage or absolute value. The percentage indicates a relative number of each type of white blood cells (WBCs). Absolute value indicates multiple of percentage and total WBCs (Percentage X Total WBCs). There are five types of white blood cells (WBCs). Absolute value is more commonly used than the relative value of WBC in the differential count of WBC. These are Eosinophils, Basophils, Neutrophils Lymphocytes, and Monocytes.

Differential leukocyte counts:

Differential leukocyte count is carried out on 100 leukocytes. Select the area of the blood smear with the optimal spread of leukocytes. Turn to the higher resolution of the microscope (50X or 100X oil immersion objective). Start differential count of leukocyte and their morphology assessment. High dry objective 40X is not recommended because this magnification is too low. Cell features can’t be assessed in this resolution. Start leukocyte count by blood smear slide. Back and forth. Avoid repeated covering of the same area of the blood smear. This can be carried out by moving sideways or lengthways across the blood smear slide sideways movement is preferred. Identify each leukocyte till 100 leukocytes have been counted.

Human blood has five types of leukocytes: Eosinophils, basophils, neutrophils lymphocytes, and monocytes. Also, count immature neutrophils. It is considered under the category of bad neutrophils. If other leukocytes such as mast cells, histocytes, and leukemic blast are present. They are also noted as a part of the WBC morphological assessment. If their numbers are low they are counted separately. If their numbers are large they are counted as “other cells”.

PREPARATION AND STAINING OF BLOOD FILM:

Microscope slide with frosted end, spreader slide, capillary tubes (microhematocrit tube), pencil or pen to label microscope slide, EDTA tube, and sterile distilled water are used in the preparation of blood film.

A. Preparation of Blood Film/Smear:

Blood film preparation: There are several techniques to make blood film on a microscope slide. The most commonly used technique to make a film on a microscope slide is the wedge or push technique. It forms a uniform blood film that gets progressively thinner. The following steps are followed to make uniform blood film by wedge or push technique.

Step 1: Select a premium quality, pre-cleaned microscope slide with a frosted end and one pre-cleaned spreader slide. These slides should not have glass dirt or dust.

Always use well-mixed blood, clot-free blood, and EDTA anticoagulant blood at room temperature to make blood film on a microscope slide.

Step 2: Fill blood into the micro-hematocrit tube by blood capillary action. Blood should cover at least a quarter of the full length of the microhaematocrit tube. This will prevent the blood from coming out of the tube.

Step 3: Place one drop of blood onto a microscope slide approximately 0.5 cm away from the frosted end. The diameter of the droplet should be approximately 4mm. If there is not enough blood in the microhaematocrit tube, tap the tube on a microscope slide.

Step 4: Hold the spreader slide at 30 to 40 ○angle in front of the blood droplet on a microscope slide. Ensure even contact of the spreader slide with a microscope slide. Hold the microscope slide to block its movement during blood filmmaking.

Step 5: Maintain even contact and fix the angle of the spreader slide on a microscope slide. Move the spreader slide on the microscope slide in the backward direction towards the blood drop. Blood drops will spread evenly along the edge of the spreader slide in contact with the microscope slide. This movement of blood drop is due to capillary action along the edge of the spreader slide on a microscope slide.

Step 6: Maintain even contact and fix the angle of the spreader slide on a microscope slide. Push spreader slide forward in smooth and moderately fast motion. Sliding of the spreader slide on the microscope slide should take only one second. Put only normal pressure on the spreader slide during this movement. Excess pressure shall push too much blood forward. It will not form good blood film on a microscope slide. If the blood is very thin, then increase the angle of spreader slide to make blood film.

CHARACTERISTICS OF GOOD BLOOD SMEAR:

• 1. Blood smear should cover three-quarters of a microscope slide.

• 2. Blood film is thick at its origin point called the head of blood film. It becomes gradually thin called a tail or feather edge. The tail has no clear border area.

• 3. Blood film should blend smoothly to form the monolayer area.

• 4. Blood film edges should have a fine feathery appearance.

• 5. Appearance of a rainbow just behind the feather edge.

• 6. A thick line at the end of the blood smear indicates the formation of a poor blood smear

• 7. There should be a monolayer area just behind the feather edge.

• 8. Blood smear has a tongue-shaped appearance.

Reason for poor blood smear: Excessive pressure, slow spread motion or wobbling of spreader slide on the surface of microscope slide develop poor quality blood smear. Slow spreading motion develops a long thin smear that does not have a dense body.

The end edge of the blood smear may have a line of blood. The presence of a horizontal linear line at the edge is called a hesitation mark. Excessive pressure develops this hesitation mark on a blood smear. The too-slow motion shall cause poor leucocyte distribution. It will push larger cells toward the end of the blood smear. It also develops vertical streaks. The too-quick motion of the spreader slide shall develop a short blood smear with poor separation.

B. Staining of blood film:

Theory: Blood film is stained as soon as possible. Romanowsky stains are used to stain blood film. It consists of water-soluble eosin, methylene blue, acetone-free methanol, and eosin. The most suitable pH to stain blood film is 6.8 to 7. At this pH, both acidic and basic components of blood cells accept the color of stains. Methylene blue is a basic stain and eosin is an acidic stain. Acidic components of blood cells accept the color of basic stain that is methylene blue such as nucleus and nucleic acids (Cytoplasmic RNA and nuclear DNA). They are bluish-purple after accepting the color of methylene blue. The basic component of blood cells accepts the color of an acidic stain which is eosin. Granules in the cytoplasm accept the color of eosin to become orange to pink color. Neutral components of blood cells accept the color of both stains. Examples of Romanowsky stains are

• 1. Wright stain,

• 2. Leishman stain and

• 3. Giemsa stain. (MGG Stain or May-Grunwald Geimsa Stain).

They are also called polychromic staining solutions.

Acetone is a strong decolorizing agent and a strong dehydrating agent. Thus acetone-free methanol (Absolute methanol) is used as an ingredient in the Romanowsky stain. This acetone will destroy the cell membrane. Methanol acts as a fixative. Fixative maintains the morphology of blood cells and adheres them with the glass slides.

Romanowsky stain imparts different color to cellular structure.

GIEMSA STAIN ((MGG Stain or May-Grunwald Geimsa Stain).

It belongs to a group of Romanowsky stains. This is a neutral stain made of a mixture of methylene blue, azure, and eosin dye. It is used in the differentiation of cells, pathological examination of blood, bone marrow film and to demonstrate the parasites in blood.

Preparation of Giemsa stain solution (stock solution):

Giemsa stain consists of Giemsa powder, acetone-free absolute methanol, glycerine, and buffer solution. It is prepared by dissolving 0.3 gm of Giemsa staining powder in glycerol. Heat mixture of 0.3 gm Giemsa stains with glycerol in a water bath at temp 56 to 60 degrees C for 2 hrs. Add 25 ml acetone-free methanol to this mixture. Keep this Giemsa stain solution at room temperature for one week with occasional stirring. Filter the solution and store it in a tightly closed amber-colored bottle at room temperature.

Preparation of working solution: Dilute it with buffer solution in a ratio of 1:10 before use. Normally, a buffer solution pH 7 is used to dilute the Giemsa stain solution.

Preparation of buffer solution pH 7 for 100 ml: Formulation

a. M/15 Disodium hydrogen phosphate (9.47gm/Ltr): 61.1ml

b. M/15 Potassium dihydrogen phosphate (9.08/Ltr): 38.9ml

Mix both solutions to prepare a 100 ml buffer solution with pH 7.0

Principle: It is composed of the acidic dye eosin and the basic dye polychromed methylene blue (a mixture of methylene blue, azure, and other dyes). Basic dyes bind with acidic nuclei to produce a blue-purple color. Acidic dye eosin combines with alkaline cytoplasm and cytoplasm granules to develop red coloration.

Giemsa stain can develop several colors inside the cellular structure. This helps to differentiate various cellular components. The ability of stain to develop various colorations is called the Romanowsky effect or metachromasia.

Giemsa stain solution is mixed with buffer in a 1:10 ratio just before its use. Buffer solution with pH 6.8 or 7 precipitates the dyes from the Giemsa stain solution. These precipitated dyes bind with cellular components. Acetone-free methanol acts as a fixative. This prevents perforation. Use eosin-Y in place of eosin. pH 6.8 is suitable for studying the morphology of blood cells. pH 7 is suitable for studying parasites in the blood.

Giemsa Stain procedure (Staining Technique): Mix one 1 part of Giemsa stain solution and nine parts of buffer solution. Flood this mixture on a slide having dry, fixed thin blood film. Set aside for 10 to 15 minutes. Drain out the mixture from slide. Allow slide to dry in air and examine it under microscope.

LEISHMAN STAIN:

Leishman stain belongs to a group of Romanowsky stains. It is a differential stain that is used to stain various cellular components, especially blood cells. Leishman stain is the best stain for routine blood staining. Leishman stain is widely used to stain thin blood smears for the identification of toxic granules, Blood cell morphology, anemia type, etc.

Preparation of Leishman stain (Stock solution)

a. Leishman stain powder: 0.15gm.

b. Acetone-free absolute methanol q.s: 100ml.

Grind 0.15gm Leishman stain powder in a glass mortar. Put it into the bottle by using a funnel. Add 20 ml acetone-free methanol into the bottle through a funnel. Ensure to wash out all powder from the funnel into the bottle. Recap the bottle and shake it in a circular motion for 2 to 3 minutes. To dissolve Leishman stain crystals warm it in a water bath at temp 50○C for 15 minutes with occasional shaking. Add sufficient acetone-free methanol into the bottle through the funnel to make a volume of 100 ml. Close the bottle and shake it for a few minutes. Set aside for a week. Filter the solution after one week. It is the stock solution. This solution remains stable for 3 months.

Preparation of working solution: Dilute the Leishman stain solution with buffer solution in ratio 1:2.

Principal: (Same as Geimsa stain) Eosin is a fluorescent red dye. It is an artificial derivative of fluorescein. It consists of eosin y and eosin b. Eosin y is a more commonly used eosin dye. It is slightly yellowish and called eosin y.

Leishman Stain procedure (Staining Technique): Put 10 to 15 drops of Leishman stain solution on a slide having dry, fixed thin blood film. Set aside for 1 to 2 minutes. Put 10 to 15 drops of buffer solution to dilute the Leishman stain solution present on a thin blood film. Set aside for 10 minutes. Drain out the mixture from the slide. Wash the slide with buffer solutionor tao water. Allow slide to dry in air and examine it under a microscope.

PERFORMANCE OF DLC

It is the microscopic examination of stained blood film. The following steps are followed to examine dry stained blood film.

A. Use low power objective (10X objective): Examine stained blood film under a 10X objective. The main aims of using the 10 X objective are

1. Evaluation of blood film quality

2. Rough estimation of Red Blood Cells (RBCs) and White Blood Cells (WBCs) in blood film.

Select the area just before tail end of the stained blood film.

B. Use of oil immersion objective (100X): Put immersion oil over stained blood film. Select oil immersion objective. Open the iris of the diaphragm of the microscope. This will increase the amount of light on stained blood film. Move the stained blood film over the stage of the microscope systematically. Examine stained blood film during this movement. Avoid repetition of the same area. Record the types and number of white blood cells. Count at least 100 white blood cells. Counting 500 white blood cells will give more accuracy in the result.

Identification of WBCs (Leucocytes) after staining blood film:

There are five types of WBC. These are:

A. Granulocytes

1. Neutrophils: They have lobular nuclei. Each lobe is connected through a thin strand. It accepts the color of eosin to develop a deep reddish-purple color. The cytoplasm of neutrophils has a large number of granules that develop a light pink color. The average diameter of neutrophils is 10 to 12 µm.

2. Eosinophils: They are similar or slightly larger than neutrophils. They have a bilobed nucleus. Cytoplasm has a large number of granules that accept the color of eosinophil to develop orange-red. Number of eosinophils varies from 4% to 6% of total WBCs.

3. Basophils: They are slightly smaller than neutrophils. Cytoplasm develops a purple or black color after staining the blood film. The nucleus of basophils has two or more lobes. The nucleus is not visible due to the presence of large, round deep purple-coloured granules. The average diameter of basophils is 8 to 10 µm.

B. Agranulocytes

1. Monocytes: They have kidney-shaped nuclei. Its cytoplasm develops a pale blue color. Cytoplasm has numerous pink blue fine granules. The average diameter of monocytes is 16 to 22µm.

2. Lymphocytes: There are two types of lymphocytes. These are large lymphocytes and small lymphocytes.

• i. Large lymphocytes: 12 to 15 µm in diameter. Pale blue cytoplasm. Large round nucleus.

• ii. Small lymphocytes: 10 to 12 µm in diameter. Blue cytoplasm and dark round nucleus.

Normal values of white blood cells (Lymphocytes) in males and females:

• 1. Neutrophils: 40 to 75% 1800 to 7800 per µL

• 2. Eosinophils: 1 to 4% 50 to 450 per µL

• 3. Basophils: 0 to 1% 0 to 200 per µL

• 4. Monocytes: 2 to 8% 100 to 800 per µL.

• 5. Lymphocytes: 20 to 45% 1000 to 4500 per µL

Clinical significance of DLC (Differential Leucocyte Count):

DLC helps to find out changes in normal values of WBCs in blood. It gives an idea regarding the severity of several diseases. Such as

• 1. Leucocytosis: An increase in leucocyte number above its normal value is called leucocytosis. It occurs in certain infections such as bacteria, fungi, viruses, or pathogens.

• 2. Neutrophilia: An increase in several neutrophils above their normal values in blood is called neutrophilia. It may be due to bacterial infection.

• 3. Neutropenia: A decrease in several neutrophils below their normal values in blood is called neutropenia. It happens in typhoid fever, viral fever, anemia, etc.

• 4. Lymphocytosis: An increase in several lymphocytes above its normal value is called lymphocytosis or absolute lymphocytosis. It occurs in certain infections such as influenza, cough, mumps, tuberculosis, etc.

• 5. Lymphopenia: A decrease in several lymphocytes below its normal value is called lymphopenia. It is observed during acute infection, irradiation, etc.

• 6. Eosinophilia: An increase in several eosinophils above its normal value is called eosinophilia. It happens during a hypersensitivity reaction.

• 7. Monocytosis: An increase in number of monocytes is called monocytosis. It happens during malaria, tuberculosis, typhoid, etc.

• 8. Basophilia: An increase in several basophils above its normal value is called basophilia.

BLOOD CELL MORPHOLOGY IN HEALTH AND DISEASE (PERIPHERAL BLOOD FILM)

Stained blood film on the microscopic slide is used to examine the morphology of blood cells in normal health conditions and diseased conditions.

Blood cell morphology in a healthy human body

 Blood has three types of blood cells. These are Erythrocytes or RBCs (Red blood cells), Leucocytes or WBCs (White blood cells), and Thrombocytes or Platelets

1. Erythrocytes (Red Blood Cells): Erythrocytes are non-nucleated, circular, biconcave disc-shaped cells. Single RBC appearance is pale buff color (pale yellow color). In the group, it appears red color due to the presence of hemoglobin in RBCs. Haemoglobin occupies maximum space inside RBC.

2. Leucocytes (White Blood Corpuscles): Size of WBCs is large than RBCs. WBCs are amoeboid in shape and have the ability of amoeboidal movement. WBCs consist of cytoplasm and nucleus.

There are two types of WBCs: Granulocytes or Polymorphonuclear leucocytes and Agranulocytes or Mononuclear leucocytes.

i. Granulocytes:

Granulocytes have granules in the cytoplasm. They have irregularly subdivided or lobulated nuclei. They are also called polymorphonuclear leucocytes due to the presence of lobbed nuclei. There are three types of granulocytes depending on their staining properties. These are Eosinophils, Basophils, and Neutrophils.

• Eosinophils: The Cytoplasm of eosinophils has granules that accept the color of acidic stains like eosin. Cytoplasm has a bilobed nucleus, a narrow strand connecting both lobes. In allergic conditions and worm infections, there is an increase in several eosinophils.

• Basophils: Basophils have a lesser number of granules than eosinophils. These granules quickly accept the color of basic stains like methylene blue. There are three lobes in the nucleus of basophils, they form an “S” shaped configuration.

• Neutrophils: Neutrophils in a non-motile state are round. In a motile state, they are flat and amoeboidal. Their cytoplasm has fine granules. But compared to eosinophils and basophils, cytoplasm has coarse granules. However, neutrophil cytoplasmic granules have two sizes – small and large granules. Larger granules are called primary granules. Smaller granules are of various shapes and are called secondary granules. Primary granules accept the color of neutral red dyes. Secondary granules accept the color of Giemsa and Leishmen’s stain. Cytoplasm has a multilobed nucleus. These lobes are attached by narrow strands, and the lobes are arranged in horseshoe-shaped.

ii. Agranulocytes

The cytoplasm of these leucocytes does not have granules. There are two types of agranulocytes: Lymphocytes and Monocytes.

• Lymphocytes: They have a large round nucleus with a small amount of cytoplasm. Lymphocytes have two sizes: large and small. Large lymphocytes are young lymphocytes while small lymphocytes are mature lymphocytes. The nucleus accepts basic dye color more than the cytoplasm.

• Monocytes: A Monocyte nucleus is a bean-shaped nucleus. They are the largest leucocytes and have an amoeboidal shape.

Thrombocytes (Platelets):.

They are colorless, round, oval, biconvex or irregular, non-nucleated.

Blood cell morphology in the healthy human body: Morphology and number of abnormal Red Blood Cells, White Blood Cells, and platelets are examined in stained blood film under oil immersion objective of microscope.

Dr Pramila Singh

Blood cell morphology in a healthy human body:

Morphology and several abnormal Red Blood Cells, White Blood Cells, and platelets are examined in stained blood film under the oil immersion objective of a microscope.

A. Abnormal Erythrocytes (Red Blood Cells):

• 1. Microcytes: Size of RBCs lower than normal size RBCs called microcytes. It is related to microcytic anemia

• 2. Macrocytes: The size of RBC is bigger than the normal size of RBCs called aicrocyte. It is related to macrocytic anemia.

• 3. Ovalocytes: Oval-shaped or egg-shaped It is related to megaloblastic anaemia.RBC is called ovalocytes.

• 4. Anisocytosis: A variation in the size of RBCs is called anisocytosis.

• 5. Spherocytes: RBC spherical in shape without biconcave disc structure is called spherocyte. Its size is smaller than the normal size of RBC. It is related to hemolytic anemia

• 6. Hypochromia: It has less color intensity than normal RBC. It is due to the low concentration of hemoglobin in RBC.

• 7. Stomatocytes: Cup-shaped or bean-shaped RBC is called stomatocytes. It is related to hemolytic anemia. The faster death rate of RBC than the synthesis rate of RBC is called hemolytic anemia.

• 8. Sickle cells: Crescent-shaped or sickle-shaped RBC is called sickle cells. Its one end is pointed-shaped. It is related to sickle cell anemia.

• 9. Schistocytes: Fragmented RBC is called schistocyte.

• 10. Target cells: It has a centrally stained area.

• 11. Burr cells: RBCs with a pointed structure on their surface are called burr cells.

• 12. Crenated cells: RBCs with irregular outer surfaces are called crenated cells. It is mainly due to faulty drying of blood film.

• 13. Acanthocytes: RBC with a sharp thorn-like structure on its outer surface is called an acanthocyte.

• 14. Howell-Jolly body: Nuclear fragments round and purple in blood film is called Howell-Jolly's body.

• 15. Cabot ring: RBC containing thread-like filament in ring shape is called Cabot ring.

• 16. Basophilic stippling: RBC containing purple granules is called basophilic stippling.

B. Abnormal Lymphocytes (White Blood Cells):

All are already discussed under DLC. Some additional abnormalities are following.

• 1. Toxic granules: Neutrophils with prominent granules are called toxic granules. It is related to severe bacterial infection

• 2. Hypersegmentation: The presence of more than four lobes in the nucleus of a neutrophil is called hypersegmentation.

• 3. Hypo segmentation: The presence of fewer than three lobes in the nucleus of neutrophils is called hypopigmentation.

• 4. Double bodies: The presence of light blue round or oval stained bodies in the cytoplasm is called double bodies

• 5. Auer bodies: The presence of needle or rod-shaped bodies in the cytoplasm is called Auer bodies.

C. Thrombocytes (Platelets): A. Either increase or decrease in number of platelets during abnormal thrombocytes.

1. Thrombocytosis: An abnormally high number of thrombocytes than normal thrombocytes number in blood is called thrombocytosis. This increases the risk of blood clotting in blood vessels. This leads to stroke or heart attack.

2. Thrombocytopenia: An abnormally low number of thrombocytes than normal thrombocytes number is called thrombocytopenia. This leads to excessive bleeding even on minor injuries.