Evaluation of the Peripheral Blood Smear

A peripheral blood smear is a key clinical tool used in the assessment of patients in hematology. It is an inexpensive and readily available test in most medical centers and laboratories. Smears can provide rapid information that can help guide our management decisions. Automated machines can provide relevant information usually reported in the differential of a complete blood count (CBC). However, machines may not note subtle findings and do not provide an interpretation if a certain finding is significant. Medical professionals particularly hematologists must become familiar with performing and interpreting peripheral blood smears.

How to Obtain a Peripheral Smear

Peripheral smears are predominantly obtained from peripheral veins. However, any blood source can be analyzed and may include central lines, arterial lines, and other methods. The blood sample is collected in an EDTA purple-top tube and then used for the preparation of the slides. It is crucial to use clean slides before obtaining the sample.

Equipment and Preparation

In general, a drop of non-clotted blood is used and placed on a slide for analysis. Having an appropriate technique will help obtain a good-quality smear and provide accurate information. Even though in most medical centers, laboratory personnel perform the preparation of a peripheral smear for review, medical professionals need to be aware of the necessary equipment and preparation of a smear for review. Here is a summary of the equipment and preparation of a peripheral blood smear.

1. Gathering Materials: Materials can include clean glass slides, disposable gloves, alcohol swabs, lancets, and microscopic slides. A blood sample obtained from our patient will be used to be spread across a slide for analysis.
2. Smear Preparation: Place a drop of blood close to one end (about 1 cm from the edge) with a pipette or capillary tube. Use another clean glass slide at a 30-45 degree angle to the first slide, touching the edge of the blood drop. Using a smooth and steady movement, spread the blood across the surface of the second slide to create a thin and even smear. Blood should extend to approximately 2/3 of the slide’s length. Please, watch the following video by Robert Bounds.
3. Drying and Fixation: Allow the smear to air dry at room temperature to help avoid any possible artifacts that could form.
4. Staining: Several stains can be used during the visualization of a peripheral smear such as a Wright-Giemsa or May-Grünwald-Giemsa. An optional step is to fix the smear by using a solution such as methanol for 1-2 minutes. Fixation helps preserve cell morphology and prevents cellular distortion.
5. Mounting and Cover Slipping: After staining the smear, one could rinse the slide with distilled water to remove excess stain. A mounting medium could be applied to enhance the clarity of the cells and preserve them.
6. Labeling and Storage: Make sure to appropriately label the prepared slide with the patient’s identification information, specimen source, and any pertinent clinical data. Store the slides in a slide box or rack to protect them from damage and contamination. Most of the time, clinicians interpreting a peripheral smear would get a slide that has been already prepared by laboratory personnel).

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Interpretation and Analysis of a Peripheral Blood Smear

Once a smear is produced, it can be visualized under a microscope or uploaded into a digital microscope for virtual analysis. There are many software applications available at certain medical centers in which accessing a peripheral smear remotely is possible and can be quite convenient. However, it is important to become familiar with these applications and learn the limitations that they may have. For example, certain applications may not allow visualization of the entire peripheral smear and could lead to missing important findings. However, they are excellent tools that are convenient and helpful in patient care.

Systematic Evaluation

Begin by systematically scanning the entire smear at low magnification to assess the overall cellularity, distribution, and quality of the smear. A recommendation is to choose a particular method to review a smear and use it when interpreting any blood smear. For example, one can choose to scan a peripheral smear from one corner (e.g. top left) and move the slide down to the bottom (e.g. bottom left) vertically. Once that vertical stripe has been reviewed, move horizontally to the next one (e.g. to the right) and repeat the process until the entire slide has been analyzed. Some large cells or elements in a smear could accumulate mostly at the borders of a slide. For example, a blast may be seen close to the border or thick area and not in the thin area of a smear. Any systemic evaluation technique could be considered, understanding that a standard technique will help us not to miss findings in particular areas of a slide.

Here is a summary of the main blood smear elements that can be analyzed:

Red Blood Cells (RBCs)

Red blood cells are the predominant cells seen in a peripheral smear in normal conditions. Here is a table that summarizes the main characteristics of a normal red blood cell:

Characteristic	Description
Shape	Biconcave and discoid
Size (Diameter)	6 to 8 micrometers (µm) Can compare to the size of the nucleus of a small normal lymphocyte
Color	Pale pink to red
Central Pallor	Present, lighter area in the center
Uniformity	Minimal variation in size, shape, and color
Flexibility	High flexibility and deformability
Lifespan	Approximately 120 days

Multiple RBC abnormalities could be observed in a peripheral smear and can point toward certain conditions that could be present. Here is a summary of the main RBC findings that could be detected:

Abnormality	Description	Associated Conditions
Anisocytosis	Variation in RBC size (when comparing one to another)	Nutritional anemias, hemolysis (due to larger erythrocytes)
Poikilocytosis	Variation in RBC shape (e.g., presence of spherocytes, schistocytes)	Hemolytic anemias, hereditary spherocytosis, thrombotic microangiopathies (TMA)
Hypochromia	Decreased hemoglobin concentration, resulting in increased central pallor	Iron deficiency anemia, thalassemia, sideroblastic anemia
Polychromasia	Increased central staining due to the presence of residual RNA	Reticulocytosis, hemolytic anemias, bone marrow recovery
Spherocytosis	Presence of spherical or hyperchromic RBCs	Hereditary spherocytosis, autoimmune hemolytic anemia
Elliptocytosis	Presence of elliptical or oval-shaped RBCs, indicative of membrane abnormalities	Hereditary elliptocytosis, hereditary ovalocytosis
Target Cells (Codocytes)	RBCs with a central area of hemoglobin surrounded by a ring of pallor	Liver disease, thalassemia, hemoglobinopathies
Stomatocytes	RBCs with a central slit or mouth-like appearance	Liver disease, hereditary stomatocytosis
Spur cells (Acanthocytes)	RBCs with irregularly spaced, blunt projections on the cell surface (spurs)	Abetalipoproteinemia, liver disease, abnormally high cholesterol levels
Burr Cells (Echinocytes)	RBCs with evenly spaced, short, blunt projections (wheel-shaped)	Uremia, liver disease, pyruvate kinase deficiency
Tear-drop Cells (Dacrocytes)	RBCs with a teardrop-shaped morphology, often seen in myelofibrosis or bone marrow infiltration (think of cells squeezing out of the marrow)	Myelofibrosis, myelodysplastic syndromes, leukemias
Rouleaux Formation	Abnormal stacking of RBCs, resembling a stack of coins, indicative of increased plasma proteins	Multiple myeloma, Waldenström macroglobulinemia
Agglutination	Clumping of RBCs due to the presence of immunoglobulins or complement proteins	Autoimmune hemolytic anemia, cold agglutinins, transfusion reaction
Schistocytes (fragmented or helmet cells)	Presence of fragmented or fragmented RBCs, indicative of mechanical damage (sliced RBCs)	Disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS)
Heinz Bodies	Round or irregular inclusions within RBCs composed of denatured hemoglobin	G6PD deficiency, thalassemia, exposure to oxidizing agents
Howell-Jolly Bodies	Small, basophilic nuclear remnants within RBCs	Asplenia, functional hyposplenism, megaloblastic anemia

White Blood Cells (WBCs)

White blood cell abnormalities are important and can be diverse as well. Normal characteristics of white blood cells include:

White Blood Cell Type	Size (Diameter)	Nucleus	Cytoplasm	Cytoplasmic Granules
Neutrophils	10-12 µm	Multilobed, segmented nucleus (3-5)	Pale pink to light purple, fine granules	Present, primarily lysosomes
Lymphocytes	6-9 µm	Round or slightly indented nucleus	Scant, blue-gray cytoplasm	Absent
Monocytes	12-20 µm	Kidney-shaped or folded nucleus	Abundant, blue-gray cytoplasm	Present, fine, or vacuolated
Eosinophils	12-17 µm	Bilobed nucleus	Bright red to orange-red granules	Present, large specific granules
Basophils	10-14 µm	S-shaped or lobulated nucleus	Deep purple to black granules	Present, large specific granules

WBC abnormalities that can be detected in a peripheral smear include:

Abnormality	Description	Associated Conditions
Toxic Granulations	Increased cytoplasmic granules and staining intensity in neutrophils	Bacterial infections
Döhle Bodies	Blue-gray cytoplasmic inclusions in neutrophils	Infections, inflammatory conditions
Pelger-Huët Anomaly	Bilobed or hypolobulated neutrophil nuclei	Inherited abnormality, myelodysplastic syndromes
Hypersegmentation	Presence of neutrophils with more than five lobes	Megaloblastic anemia, vitamin B12, or folate deficiency
Atypical Lymphocytes	Enlarged lymphocytes with irregular nuclear contours	Viral infections, infectious mononucleosis
Smudge Cells (Basket Cells)	Fragile lymphocytes that rupture during smear preparation “smudged”	Chronic lymphocytic leukemia (CLL)
Dysmorphic PMNs	Usually irregularly shaped nuclei, cytoplasmic vacuolization, or nuclear hypo or hypersegmentation	Sepsis, severe infections, myelodysplastic syndromes
Clover Cells (flower cells)	Abnormal red blood cells with a tetra-lobed or cloverleaf-shaped appearance	Adult T-cell Leukemia/Lymphoma
Auer Rods	Cytoplasmic inclusions in myeloid blasts, rod-shaped structures	Acute promyelocytic leukemia (APML) and amyeloid leukemias (AML)
Reed-Sternberg Cells (owl’s eye)	Large, binucleated, or multinucleated cells with prominent nucleoli	Hodgkin lymphoma
Hairy Cells	Small lymphoid cells with “hairy” projections on their surface	Hairy cell leukemia
Plasma Cells (clock-faced cells)	Mature B lymphocytes with eccentric nuclei and abundant, deeply basophilic cytoplasm	Multiple myeloma, Waldenström macroglobulinemia
Blasts	Large cells with high nuclear-to-cytoplasmic ratio (big nuclei), prominent nucleoli, fine “open” chromatin (ready to proliferate)	Acute leukemias, myelodysplastic syndromes, myelofibrosis
Hypogranular Neutrophils	Neutrophils with decreased or absent cytoplasmic granules	Myelodysplastic syndromes (MDS), drug toxicities, congenital disorders affecting granulopoiesis
Hypoplastic Neutrophils	Neutrophils with reduced cytoplasmic volume or overall cell size	Aplastic anemia, chemotherapy-induced bone marrow suppression, viral infections

We will include images and cases of the different WBC morphology changes in future posts and the Q-Bank sections that will describe these findings in more detail.

Platelets

Platelets are small cell fragments that can also be evaluated in a peripheral smear. Here are the normal characteristics of platelets:

Characteristic	Description
Size	2-4 µm in diameter
Shape	Discoid or oval
Cytoplasm	Pale blue, containing granules
Granules	Alpha granules (containing proteins involved in clotting), dense granules (containing ADP and serotonin)
Distribution	Evenly distributed throughout the blood smear

One can observe several abnormalities in a peripheral smear that can help formulate a differential diagnosis or understand the overall clinical picture of a patient’s presentation including:

Abnormality	Description	Associated Conditions
Thrombocytopenia	Decreased platelet count (decreased overall quantity per field)	Many causes including inflammation, infection, idiopathic thrombocytopenic purpura (ITP), leukemias, medication-induced, and many others
Thrombocytosis	Increased platelet count	Essential thrombocythemia, reactive thrombocytosis (e.g., following surgery or trauma)
Giant Platelets	Abnormally large platelets (larger than surrounding RBCs)	Bernard-Soulier syndrome, May-Hegglin anomaly, ITP, and other conditions with increased platelet production
Platelet Clumps (pseudothrombocytopenia)	Aggregates of platelets leading to clumps or clusters	Artefactual phenomenon, often seen in EDTA-anticoagulated blood (resolves when testing in citrate tubes)
Platelet Satellitism	Platelets adhering to the surface of neutrophils or other cells	Artefactual phenomenon, often seen in EDTA-anticoagulated blood

Becoming familiar with the interpretation of peripheral smears is a great skill to develop and can help provide crucial information to guide our management decisions in hematology. It is challenging to learn all about peripheral smears in one post. However, we hope the current post serves as a starting point and a guide when evaluating smears in the future. Our website includes a peripheral smear section in the main menu area where you can find cases to review and continue practicing your skills. As with any other area in life, when it comes to peripheral smears, “practice makes perfect.” Maybe not perfect, but our best selves in the interest of our patients.

Although practice doesn’t ever make “perfect,” it almost always makes “better.”

Dale S. Wright