A peripheral blood smear or peripheral blood film is a glass slide covered on one side with a thin layer of venous blood. The slide is the stained and viewed under a microscope.The examination of a stained peripheral blood film is a hematology test and is an integral part of laboratory evaluation of patients. This procedure provides information on leucocyte, red cells and platelets and is used to supplement the information provided by automated hematology analyzers.It also helps to identify any parasite if present on the smear and the detection of abnormal cells in patients with hematological malignancies such as blood cancer like lymphoma or lymphoma cancer,multiple myeloma,leukemia,acute myeloid leukemia,chronic lymphocytic leukemia,myeloid leukemia and other hematologic cancer.
Staining of peripheral blood film or smear
peripheral blood smear test can be made from fresh venous or capillary blood to which no anticoagulant or with a common anticoagulants such as EDTA anticoagulant added to the blood.
In order to make blood films, clean glass slides should be used and each smears slide should be labeled with the patient number or other relevant identification code depending on each laboratory.
Peripheral blood smears are routinely stained using Romanowsky stains which provides very good results. These stains have the property to of staining different organelles with distinct colors. Romanowsky stains also stain granules differentially.
The most commonly used Romanowski stains are giemsa stain using giemsa dye, wright stain,may grunwald stain and Jenner’s stain but of all these stains,Leishman and Wright are widely used in routine staining. However, the results are inferior as compared to Giemsa and Jenner stain.
The International Council of Standardization in Hematology (ICSH) advocates a combination of pure Azure B and Eosin Y in the stain to be used.This is because Azure B is a basic dye which binds to anionic molecules such as the phosphate group of DNA and proteins of cell nuclei meanwhile Eosin Y is an acid dye which binds to the basic molecules such as hemoglobin and cationic sites on proteins.This difference in the binding of the dye to different structures of the cell leads to the staining of this cell component with certain dyes whereby the red cells turn pink, the leukocyte cytoplasm is light pink, the nuclei are stained purple black and the granules of the different leukocytes stain with different colours.
The advantages of using this combination are that the procedure can be standardized and produces consistent results from batch to batch while the disadvantage is the fact the these stains are expensive
How to Make peripheral blood smear
As earlier mentioned,the examination of a stained peripheral smear is an integral part of laboratory evaluation of patients and this provides information on red cells, leukocytes and platelets and is used to supplement the information provided by automated hematology analyzers . All these is possible only if the peripheral smear is well prepared as follows
- Start by placing a 1″ × 3″ glass microscope slide which is preferable than plastic microscope slides on a countertop of a laboratory bench. If frosted slides are used, the frosted end of the slide should face upward.
- Then write the laboratory number of the patient on the slide.
- Place a 2 – 3 mm drop of blood approximately 1/4″ from the edge, using a glass capillary tube.
- Hold the slide by the narrow side between the thumb and forefinger of one hand at the end farthest from the frosted end
- Grasp a second slide (“spreader slide”) between the thumb and forefinger of the other hand
- Place the edge of the spreader slide on the lower slide in front of the drop of blood (side farthest from the frosted end).
- Pull the spreader slide toward the frosted end until it touches the drop of blood. Permit the blood to spread by capillary motion or action until it almost reaches the edges of the spreader slide.
- Push the spreader slide forward at an angle of 30 °C with a rapid, even motion.
This is illustrated below :
- A : Place a drop of blood on the slide
- B : Hold the spreader as shown
- C : Pull the spreader over the drop of blood so that it spreads
- D : Push the spreader with a firm motion
- E : This is how the prepared film appears
Note : Cleaning microscope slides can be done with Xylene and kept in microscope slide holder while those microscope slides with specimens can be washed or discard depending on the laboratory waste management policy
Preparation of Hematological staining solutions
Preparation of Giemsa stain
- Add 1g of Giemsa powder in a conical flask.
- Add 100 ml of methanol and warm the mixture to a temperature of 50°C.
- Allow to cool for 15 min.
- Shake occasionally then filter and keep for few hours before using.
Preparation of Wright stain
It is also available commercially in powder form. The preparation is done by
- Adding 2.5g of wright stain powder in a conical flask powder
- Add 2.5L of methanol to it then shake well.
- Keep the stain for 4-5 days for maturation
- Filter the stain prior to use.
How to stain peripheral blood smear
Peripheral blood smears are stained with Romanowsky dyes such as Giemsa and Wright stain.
|Buffer water pH 6.8|
The Buffer water pH 6.8 is made up of 9.1g/L Potassium dihydrogen phosphate (Solution A) and 9.5g/L Di sodium hydrogen phosphate (Solution B) which is prepared by mixing 50.8ml of solution A and 49.2ml of solution B.
Wright stain staining procedure
- Make a smear and air dry it.
- Place the smear on a staining rack then flood it with Wright stain and leave for 2 minutes. This is the time required for fixation(methanol acts as a fixative).
- Now,add twice the amount of buffered water, pH 6.8 from a plastic wash bottle.
- Leave for 10 minutes.
- Wash the stain with buffered water till the smear has a pinkish tinge.
- Wipe the back of the smear and stand upright to dry.
- Examine with 100x microscope magnification using immersion oil.
Some precaution when staining with wright stain
- Stain the or films or smear as soon as possible after they have been dried.
- Do not leave the smears unfixed for more than a few hours. If smears are left unfixed for a long time, there is distortion of cellular morphology. Also, dried plasma stains the background of the smear a pale blue. If a delay is expected always fix the smear.
- the staining rack should be leveled.
- do not let the stain solution dry over the smear.
Giemsa staining procedure
- Fix the slide in methanol.
- Then pour or flood the slide with Giemsa stain and allow to react for 20 minutes
- Wash off the smear with clean water
- Allow to air dry and examine with 100x microscope magnification using immersion oil.
How to interpret red morphology on peripheral blood smear
|Normal Red Cells||This appear well spread. The inner one third of each cell is devoid of hemoglobin and appears clear. There is very little variation in size or shape of normal red cells.|
|Anisocytosis||It means that the red cells are showing variation in size which can be due to larger red cells (macrocytic) or smaller red cells (microcytic).|
|Poikilocytosis||This refers to variation in shape of red cells. It can be seen in nutritional anemias, thalassemia and other hemolytic anemias.|
|Macrocytes||As the name suggests, these are red cells which are larger in size. They are found in megaloblastic anemia,aplastic anemia,myelodysplastic syndromes,chronic liver disease|
|Microcytes ||These are red cells which are smaller in size. They are seen in iron deficiency anemia,anemia of chronic disease,thalassemia,other hemoglobinopathies and sideroblastic anemia|
|Hypochromia||It refers to the presence of red cells in which the area of central pallor is increased. It is seen in iron deficiency anemia,anemia of chronic disease,thalassemia, other hemoglobinopathies and sideroblastic anemia|
|Polychromasia||This appearance is of reticulocytes which appear pale blue on smears fixed and stained subsequently. So in patients with reticulocytes an increased number of polychromatophils are seen in the smear. They can be seen in hemolytic anemias,nutritional anemias after treatment with hematinics|
|Target cells||These are cells in which there is a central round stained area and a peripheral rim of hemoglobin which are separated by non staining cytoplasm. They are found in chronic liver disease,iron deficiency anemia,thalassemia and other hemoglobinopathies|
|Spherocytes||These are spheroidal red cells with a regular outline. They are seen in hereditary spherocytosis,immune hemolytic anemia,hemolytic disease of the newborn and bacterial toxins|
|Basophilic stippling||This means the presence of numerous blue granules in the red cells. They are found in thalassemia,lead poisoning,unstable hemoglobins and megaloblastic anemia|
|Howell Jolly bodies||These are nuclear remnants and are present singly in a small number of red cells. They are basophilic and are seen in after splenectomy and pernicious anemia|
|Agglutination||This refers to clumping together of red cells as seen in autoimmune hemolytic anemia.|