Peripheral blood film or peripheral blood smear or Peripheral smear examination forms an important and basic lab investigation for diagnosing a number of hematological (blood) disorders.
With the advent of hematological automated machines, complete and highly accurate hematological data and parameters can be obtained within a few minutes. In spite of this, peripheral blood smear examination still remains a low-cost, valuable and indispensable investigation to assess the morphology of different blood cell types.
Constituents of Blood
Blood consists of following major cell types
Red Blood Cells (RBCs) or Erythrocytes
These are the heoglobine carrying cells and carry oxygen throughout our body.
White blood cells (WBCs) or Leucocytes
These are mainly concerned with fighting infections and other inflammatory diseases. They are of following sub-types
These cells protect the body from bacterial and fungal infections. They are first line of defense against microbial infections.
These cells protect the body against viruses and chronic infections.
They have a phagocytic function (consume dead or damaged cells) and live longer than neutrophils.
They have a role in allergic reactions and parasitic infections.
These cells release histamines during allergic reactions
Platelets or thrombocytes
They play a role in blood clotting.
Indications for Peripheral Blood Smear Examination
Peripheral blood smear examination is usually done on clinical request by the clinician due to suspicion of a blood disorder.
The test may also be initiated by the laboratory based on abnormal findings from an automated count.
Moreover, smear evaluation is a check on the values obtained from automated cell counters.
Common clinical indications for peripheral blood film analysis include
- Cytopenias (decreased counts of one or more cell types)
- Anemia: Decreased number of RBCs or decreased hemoglobin levels (nutritional, hemolytic or congenital anemias)
- lLeucopenia: Decreased white blood cell counts
- Thrombocytopenia: Decreased platelets
- Leukocytosis (increased number of white blood cells)
- Depending on the type of cell increased these could be further termed as neutrophilia, lymphocytosis, monocytosis, eosinophilia or basophilia
- Unexplained jaundice or haemolysis
- To rule out or confirm blood parasitic infections like malaria, filaria, etc.
- Severe bacterial sepsis
- Suspected organ disease such as renal disease, liver disease, alcoholism, thyroid disorders
- Suspected acute leukemias
- Suspected chronic myeloproliferative disorder
- Suspected chronic lymphoproliferative diseases such as chronic lymphocytic leukemia, lymphoma with leukemic spills, multiple myeloma, etc
- Hyperviscosity syndrome [as in paraproteinaemias, leukaemic hyperleucocytosis, polycythaemia]
- Non-hematological malignancies with suspicion of bone marrow involvement;
- Evaluation of therapeutic response in anemias, haemoglobinopathies, etc.
How to Make a Peripheral Blood Smear
Proper preparation of the blood smear is critical for correct interpretation and obtaining accurate results.
- Take one drop of venous or capillary blood. Blood can also be taken from an EDTA sample. This drop of blood is then placed on a clean glass slide about 1 cm from one end in the center line of the slide.
- Another slide (spreader) having a smooth edge is taken.
- Place the spreader at a 30° angle just in front of the drop of blood.
- Draw the spreader backwards until it just touches the drop of blood.
- Allow the drop of blood to spread in the angle between the slide and the spreader.
- The spreader is then rapidly brought forward across the stationary slide with one even stroke and pressure, avoiding any jerky movements.
- The smear is allowed to air-dry.
- Label the blood smear with patient’s name, date and identification number.
The aim is to get a region, called a monolayer, where the cells are spaced far enough apart to be counted and differentiated. The monolayer is found in the “feathered edge” created by the spreader slide as it draws the blood forward.
Precautions and sources of error
- The smear should be made as soon as possible. Delay in preparation of blood smear results in degeneration of the cells and formation of platelet aggregates.
- If the slide is greasy or dirty, the smear will have blank oval areas. Hence it is very important to use a clean glass slide.
- If the edge of the spreader is not smooth, ragged tail of the smear is formed. This is known as tailing of the smear. Such a smear shows irregular distribution of the leucocytes (with majority of neutrophils at the tail end and reduced number of neutrophils in the rest of the smear) resulting in a fallacious DLC. So it is important that the spreader has a smooth edge.
- Pressure exerted on the spreader should be directed across the stationary slide in the direction that the film is made rather than down on the stationary slide.
- Smear should ideally cover two-third of the length of the slide. The faster the spreader slide is moved, the longer and thinner the film will be. The slower the slide is moved, the shorter and thicker the slide will be. The angle will also affect the results. An angle greater than 30° makes the smear thicker; less than 30° degrees will produce a thinner smear. Speed, angle and drop size can be varied slightly to produce a good smear.
Common causes of a poor blood smear
- Drop of blood too large or too small.
- Spreader slide pushed across the slide in a jerky manner.
- Failure to keep the entire edge of the spreader slide against the slide while making the smear.
- Failure to keep the spreader slide at a 30° angle with the slide.
- Failure to push the spreader slide completely across the slide.
Fixation and staining of the smear
The air dried smears are first fixed by giving 4-6 dips in methanol or any other fixative. Then they are stained using one of the Romanowsky stains. The commonly used stains are Leishman stain, Giemsa stain, Wright’s stain, Jenner’s stain and Jenner-giemsa stain.
- The smear is flooded with stain for about 2-3 minutes.
- Then it is double diluted with distilled water and allowed to stand for about 5–10 minutes.
- The slide is thoroughly rinsed under running water.
- The underside of the slide is wiped with tissue paper to remove excess stain.
- The slide is then made to dry by placing it on a rack with the tail end upwards.
The intensity of the staining varies with the duration of stain contact time and concentration of the stain. Contact time needed for appropriate staining will vary and needs to be determined with every new batch of stain used.
Stain artifact such as debris and precipitates may be caused by over-staining and inadequate washing under running water.
Automated slide stainers are available these days. This is especially useful in centers where large number of smears are to be made.
Sometimes smears are made from buffy layer (white area between the plasma and red cell layer, rich in leucocytes and platelets formed after centrifugation of EDTA blood). Since buffy coat is a concentrated leucocyte suspension, it is useful in case of leucopenic patients or do detect parasites like microfilaria, etc when present in low concentration.
Evaluation and Interpretation of a Peripheral Smear
A very thin layer of oil is applied over the smear. It is then examined under low power objective to choose the most suitable area for morphological assessment. The area between tail and body of the smear where red cells are evenly distributed and there is slight overlap of RBCs is chosen.
Evaluation of cellular morphology including differential leucocyte count is then carried out at 40 X magnification. For estimating platelet counts, oil immersion power is used. Parasites, platelet clumps and large cells like monocytes and blasts, being heavier than the RBCs, are carried to the tail by the spreader. So their detection is best carried out by examining the tail and edges of the smear.
A step wise analysis is carried out and following parameters are noted.
Average size of RBC is roughly equal to the size of the nucleus of a small lymphocyte with a mean diameter of about 7.5 μm.
Based on this, RBCs are classified as
- macrocytic (larger than normal)
- microcytic (smaller than normal)
- normocytic (normal sized).
This morphological assessment is very useful in classifying the major types of anemias. Iron deficiency anemia, thalessemia, anemia of chronic disorders and sideroblastic anemia show microcytic RBCs.
Anemia due to Vitamin B-12 and folic acid deficiency, liver disease or alcoholism is of macrocytic type.
Dimorphic anemia is a type of anemia in which dual population of macrocytic and microcytic cells is seen. It can be due to combined deficiency of iron, vitamin B12 and folic acid or is seen in sideroblastic anemia.
Hemolytic anemias are usually of normocytic type.
Variation in size of RBCs is known as anisocytosis.
Color and Pallor
Normal RBCs are disc shaped and have an area of central pallor that occupies approximately one-third of the cell’s diameter. These normal erythrocytes are known as normochromic RBCs. RBCs that have an area of pallor that is larger than the normal are called hypochromic. This is seen in iron deficiency anemia, anemia of chronic diseases, hemoglobinopathies, sideroblastic anemia, etc.
Normal RBCs are biconcave discs. A variety of diseases can result in abnormal shapes of RBCs; identification of which provides important clues to the underlying disorder. Variation in the shape of RBC is called poikilocytosis. Some of the abnormal RBC shapes are given in the table
|Abnormal shape||Morphology||Associated diseases|
|Spherocytes||RBCs become more spherical, no central pallor is present and there is increased hemoglobin content.||Hereditary spherocytosis, autoimmune hemolytic anemia and post transfusion reaction.|
|Ovalocytes||Oval shaped RBCs||Thalassemia major, hereditary ovalocytosis, sickle cell anemia.|
|Elliptocytes||RBCs are elliptical in shape with long axis being twice the short axis||Hereditary elliptocytosis, megaloblastic anemia, iron deficiency anemia, thalassemia|
|Target cells||RBCs have a centrally located disk of hemoglobin surrounded by an area of pallor with an outer rim of hemoglobin adjacent to the cell membrane giving the cell the appearance of a target.||Iron deficiency anemia, thalassemia, post splenectomy, obstructive liver disease|
|Teardrop cells||Tear or pear shaped RBCs||Myelofibrosis, megaloblastic anemia, iron deficiency anemia, thalassemia|
|Fragmented, helmet or triangular shaped RBCs.||Thalassemia, microangiopathic hemolytic anemia, mechanical hemolytic anemia, uremia, artificial heart valves|
|Acanthocytes||RBCs with irregularly spaced projections.||Liver diseases, post splenectomy, anorexia nervosa and starvation, alcoholism, vitamin C deficiency|
|Stomatocytes||Red cells with a central linear slit or stoma. Seen as mouth shaped form in smears.||Hereditary stomatocytosis, alcoholic liver disease|
|Keratocytes||Half-moon shaped RBCs with two or more spicules.||G6PD deficiency, pulmonary embolism, disseminated intravascular coagulation|
|Burr cells||RBCs with uniformly spaced pointed projections on their surface.||Hemolytic anemia, uremia, megaloblastic anemia|
|Sickle cells||RBCs become sickle shaped.||Sickle cell anemia|
|Red cell agglutinate||RBCs are present in irregular clumps||Cold agglutination, warm autoimmune hemolysis|
|Rouleaux Formation||RBCs resembling a stack of coins||Multiple myeloma, hyperglobulinemia, hyperfibrinogenaemia|
The following tale describes the type of inclusion bodies can be seen in red blood cells.
|Howell-Jolly bodies||Small round cytoplasmic red cell inclusion with same staining characteristics as nuclei. These are fragments of DNA.||Post splenectomy, megaloblastic anemia, hemolytic anemia|
|Heinz bodies||These represent denatured hemoglobin (methemoglobin) within a cell. With a supravital stain like crystal violet, these appear as round blue precipitates.||G6PD deficiency, splenectomy|
|Pappenheimer bodies||These represent iron deposits which are seen as dense blue, irregular granules.||Hemolytic anemia, splenectomy, sideroblastic anemia, thalassemia|
|Basophilic stippling||Small basophilic inclusions in red cells which represent precipitated RNA.||Thalassemia, megaloblastic anemia, hemolytic anemia, liver damage, heavy metal poisoning|
|Cabbot’s ring||Reddish, purple, thread-like rings in RBCs. These are the remnants of nuclear membrane.
Parasites causing malaria, filaria, trypanosomiasis, babesiosis, etc can be identified by their special morphological characteristics. Both thick and thin smears are employed for this purpose.
White Blood Cells
The first thing to be assessed about WBCs in blood smears is the total leucocyte count. Although exact count is possible only through automated analysers or manual Neubauer chamber, blood smears can give a fair assessment about the count and whether it is abnormally low or high. Normally, about 2-5 WBCs per high power field (HPF) are present.
This assessment is to be done in the area between tail and body of the smear where red cells are evenly distributed and there is slight overlap of RBCs. An average count of less than 2 WBCs/ HPF is indicative of leucopenia while an average count of more than 5 WBCs/HPF suggests leucocytosis.
Large number of fields need to be assessed to get an accurate estimate. It is mandatory to examine a peripheral blood smear if automated counts show flagging (alerts) due to excessively high or low counts. Falsely elevated WBC count may be given by the automated or manual counts due to circulating nucleated red cells. Nucleated RBCs are seen in neonates (physiological), hypoxia, hemolytic anemias, bone marrow invasion by leukemias, lymphomas, tuberculosis, etc. The correcting formula is given as:
Corrected WBC=[estimated WBC/(100 + Number of nucleated RBC among 100 WBC)] x 100 %.
100 consecutive WBCs are counted and classified according to their morphology to obtain a differential leucocyte count.
Following table would give an idea about counts in different conditions
|DLC||%age||Morphology||Increase in count||Decrease in count|
|Neutrophils (Polymorphonuclear cells, segmented neutrophils)||40-75 %||2.0 – 7.5 µm in diameter. Segmented nucleus with 2-5 lobes, pink to colorless cytoplasm with azurophilic granules.||Neutrophilia
– Acute bacterial infections
-Acute inflammatory conditions- acute appendicitis
– Severe overwhelming infection
– Infections like typhoid, influenza, rocky mountain spotted fever
– Aplastic anemia
– Bone marrow damage (e.g., chemotherapy, radiation therapy
|Lymphocytes||20-40 %||Small lymphocytes are round with high N: C ratio and scanty pale dark blue cytoplasm. Large lymphocytes have lower N: C ratio with less condensed chromatin. When large lymphocytes have cytoplasmic granules, there are termed large granular lymphocytes.||Lymphocytosis
– Aplastic anemia
-Infections like HIV, viral hepatitis
-Bone marrow damage in case of chemotherapy, radiation therapy
|Monocytes||2-8 %||They are the largest cells with blue-grey ground glass cytoplasm. Its nucleus is large having various shapes but often horse shoe shaped||Monocytosis
-Infections like malaria, Kala-azar, tuberculosis
|Not of much significance|
|Eosinophils||1-6 %||They are slightly larger than neutrophils. The nucleus is bilobed. Orange-red granules are present in the cytoplasm||Eosinophilia
-Allergic conditions / asthma
-Some cancers, Hodgkin’s lymphoma, leukemias
|Not of much significance|
|Basophils||<1 %||They are slightly smaller than neutrophils. They have large deeply basophilic (bluish) granules that may even totally obscure the nucleus.||Basophilia
– Chronic myeloid leukemia
– Urticaria pigmentosa
|Not of much significance|
Normal physiological lymphocytosis is seen in children less than 6-7 years of age.
Left shift is a term used to describe an abnormal rise in the proportion of immature leucocytes. This is seen in leukemias or rarely infections
Myeloblasts are the earliest recognizable granulocyte precursors. They are large with round to oval nucleus, basophilic cytoplasm, 2 to 5 nucleoli and fine reticular chromatin pattern. Promyelocytes are slightly larger with primary granules, faint nucleoli and slightly more condensed chromatin pattern. Myelocytes are smaller with oval or round nucleus, no nucleoli, condensed chromatin and more cytoplasm. Metamyelocytes are similar to myelocytes but have indented nucleus. The band cells are neutrophils having unsegmented nuclear morphology.
These cells are not normally present in peripheral blood but may spill over into the blood in situations that cause marked left shift.
presence of large number of immature WBCs precursors- myeloblasts or lymphoblasts usually indicates leukemia or lymphoma.
A severe neutrophilia usually accompanied with left shift. It is seen after, in severe infection, burns, following an acute haemolysis or prolonged hypoxia.
These are coarse granules seen in neutrophils in cases of severe infection.
It is characterised by the presence of hypersegmented neutrophils (>5 lobed nucleus). It is characteristically seen in megaloblastic anaemia.
Leukoerythroblastic blood picture
This is the presence of immature cells (immature leucocytes-myeloblasts and immature RBCs-normoblasts) in the peripheral film. It is seen in cases of marrow infiltration – eg., in metastatic tumours, malignancy, thalassemia, prolonged hypoxia or severe infection.
These are lymphocytes having less condensed chromatin and abundant, pale blue cytoplasm that may appear to “hug” adjacent red blood cells. They are seen in viral infections like infectious mononucleosis, etc.
These are lymphoid cells having fine, irregular hair like extensions. They are seen only in hairy cell leukemia.
Rod like structures seen within myeloblasts and are characteristic of acute myeloid leukemia.
Presence of bilobed neutrophils.It can be hereditary (in which case the neutrophils are functionally normal) or acquired – eg, myelodysplastic syndrome.
These are lymphocytes whose cell membranes have ruptured during preparation of the blood film. It is an artifact seen in chronic lymphocytic leukemia.
Platelets are approximately 2-4 by 0.5 microns in size (which is about a third of a normal sized red cell) with coarse cytoplasmic granules. In a normal individual approximately 7– 15 platelets are seen per oil immersion field (x100 objective). Single platelet/oil immersion field is roughly equivalent to 15,000- 20,000 platelets in circulation. For estimating platelet counts on smear, large number of fields should be counted so as to reduce sample bias. Also the fields chosen should have even distribution with slight overlap of RBCs.
A low platelet count (thrombocytopenia) can occur in certain infections like dengue fever, aplastic anemia, megaloblastic anemia, hypersplenism, idiopathic thrombocytopenic purpura or leukemias.
A high platelet count (thrombocytosis) is seen in iron deficiency anemia, post-haemorrhage, idiopathic thrombocythemia or chronic myeloid leukemia.
Qualitative abnormalities of platelets are termed thrombasthenia and require platelet functional studies to identify them.
Giant platelets These are large platelets about the size of a normal RBC or more which are caused by hyperactivity of megakaryocytes. Any stimulus which causes increased demand of platelets stimulates the bone marrow to produce platelets at an increased rate. As a result, younger and larger plalelets are thrown into circulation. They are seen in inherited conditions like Bernard Soulier syndrome, May-Haggelin anomaly or Wiskott Aldrich syndrome and acquired states like myeloproliferative disorders, idiopathic thrombocytopenic purpura, etc.
Abnormalities of Platelets
It describes platelets encircling a neutrophil. It occurs when a patient has a serum factor that reacts to the anticoagulant EDTA.
It is an in vitro artefact which is seen in some cases and results in false low counts (spurious or pseudo-thrombocytopenia) on automated cell counters. Large clumps of platelets are seen on the smear towards the tail end and towards the edges of the smears. Thus all cases of thrombocytopenia reported by automatic counters need to be cross-checked and confirmed by peripheral blood smear examination.
Thorough examination and proper interpretation of a good quality smear in the light of proper clinical context should be done by the hemato-pathologist. Clinicians should be aware of the clinical utility and proper application of the reports for appropriate management of patients.
Despite the major advances in genetic and molecular techniques in diagnosis of blood disorders, the examination of blood smear morphology remains a basic, valuable and indispensable tool in hematology.