How to Prepare & Interpret Peripheral Blood Smears

 

How to Prepare & Interpret Peripheral Blood Smears


Roger S. Riley, M.D., Ph.D.
G. Watson James, M.D.
Sandra Sommer, Ph.D., M.T., S.H. (ASCP)
Mary Jo Martin, M.D., M.T. (ASCP)
Medical College of Virginia
Virginia Commonwealth University
Richmond, VA
Table of Contents

Peripheral Blood Smear

Peripheral blood cell preparation
Peripheral blood smear examination
Disadvantages of the blood smear

Automated Hematologic Evaluation

RBC Counts and indices
WBC Counts and the leukocyte differential
Platelet counts

Morphologic Evaluation of Red Blood Cells

Clinical importance of RBC morphology
Classification of RBC morphologic abnormalities

Morphologic Evaluation of White Blood Cells

Clinical importance of RBC morphology
Classification of RBC morphologic abnormalities

Morphologic Evaluation of Platelets

Clinical importance of RBC morphology
Classification of RBC morphologic abnormalities

References and Additional Reading

Version #1 09/18/99

Watch for updates to this document. Photographs will be added in the near future. Images are also available in the Image Archives and Case Studies sections of this web site. See Other Resources for links to additional web sites with high quality images.

The Peripheral Blood Smear

A peripheral blood smear (peripheral blood film) is a glass microscope slide coated on one side with a thin layer of venous blood. The slide is stained with a dye, usually Wright’s stain, and examined under a microscope.

Microscopic examination of the peripheral blood is used to supplement the information provided by automated hematology analyzers ("blood cell counters"). Hematology analyzers provide accurate quantitative information about blood cells and can even identify specimens with abnormal cells. However, the precise classification of abnormal cells requires a trained microscopist, a well-made peripheral blood smear, and a light microscope with good optical characteristics. In practice, hematology analyzers of varying sophistication are used for cell counting in all but the smallest hematology laboratories. In addition to providing cell counts and graphical displays of the information recovered, these instruments also provide a warning ("flag") that atypical cells were found and provide a presumptive identification of the abnormality. The instrument operator reviews the information from each specimen and decides if smear preparation and light microscopy are necessary. If not, the information is released to the clinician.

Peripheral Blood Smear Preparation

The wedge slide ("push slide") technique developed by Maxwell Wintrobe remains the standard method for the preparation of peripheral blood smears (films). The following procedure (Fig. 1) is utilized to prepare a peripheral smear.

  • Place a 1" x 3" glass microscope slide with a frosted end on a flat surface (usually the counter top of a laboratory bench).
  • Attach a label on the slide or write the patient name, specimen identification number, and date of preparation on the frosted surface.
  • Place a 2 - 3 mm drop of blood approximately 1/4" from the frosted slide, using a wooden applicator stick or 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 at the frosted end.
  • 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 until it almost reaches the edges of the spreader slide.
  • Push the spreader slide forward at a 30o angle with a rapid, even motion. Let the weight of the slide do the work.


Table 1
Preparation of Peripheral Blood Smear
Step 1. Placing a small drop of venous blood on a glass microscope slide, using a glass capillary pipette. A wooden applicator stick can also be used for this purpose. Step 2. A spreader slide has been positioned at an angle and slowly drawn toward the drop of blood. Step 3. The spreader slide has been brought in contact with the drop of blood and is being drawn away. Note layer of blood at the edge of the spreader slide. Step 4. The spreader slide is further pulled out, leaving a thin layer of blood behind. Step 5. The blood smear is nearly complete. Step 6. End result. A glass slide with a well-formed blood film. After drying for about 10 minutes, the slide can be stained manually or placed on an automated slide stainer.


Fig. 1. Wedge slide technique for preparation of a peripheral blood smear.

A well-made peripheral smear is thick at the frosted end and becomes progressively thinner toward the opposite end. The "zone of morphology" (area of optimal thickness for light microscopic examination) should be at least 2 cm in length. The smear should occupy the central area of the slide and be margin-free at the edges (Fig 2).



Fig. 2. Photograph of the peripheral blood smear prepared above. The arrow points to the zone of morphology.

Peripheral Blood Smear Examination

Peripheral smear examination requires a systematic approach in order to gather all possible information. In addition, all specimens must be evaluated in the same manner, to assure that consistent information is obtained. The following approach is recommended:

  • An examination at low power (10X ocular, 10x objective) is first performed to evaluate the quality of the smear, ascertain the approximate number of white blood cells and platelets, and to detect rouleaux formation, platelet clumps, and leukocyte clumps and other abnormalities visible at low magnification. An optimal area for evaluation at higher magnification is also chosen. This should be an intact portion of the smear free of preparation artifact where the red blood cells are separated by 1/3 to 1/2 of a cell diameter. The red blood cells should stain a pink color, while neutrophils show "crisp" features, with deep blue-purple nuclear material and lilac to pinkish to violet cytoplasmic granules. Optimal preparation and staining of the peripheral blood smear is critical for morphologic examination; an inadequate smear should not be examined.

  • Following low power examination of a peripheral blood smear, the 50X or 100X objective of the microscope is selected (500X or 1000X total magnification when using a 10x ocular) and the area of morphology is examined in a consistent scanning pattern (Fig 3) to avoid counting the same cell(s) twice. A differential count of at least 100 white blood cells (200, 500, or 1000 is even better) is performed, and any abnormal morphology of RBCs, WBCs, and platelets observed during the differential count is recorded. Each morphologic abnormality observed should be quantitated ("graded") separately as to severity ("slight to marked" or "1+ to 4+"). Medical technologists are well trained in the reproducible quantitation of morphologic abnormalities; details are available in medical technology textbooks.

  • A fairly accurate estimate of the white blood cell count (cells/mL) can be obtained by counting the total number of leukocytes in ten 500X microscopic fields, dividing the total by 10, and multiplying by 3000. These estimates should approximate that obtained by the cell analyzer. If the estimate does not match the automated cell count, obtain the original blood specimen, confirm patient identity, repeat the automated analysis, and prepare a new smear.



Fig. 3. Scanning technique for peripheral blood differential count and morphologic evaluation. (a) Ten microscopic fields are examined in a vertical direction from bottom to top (or top to bottom). (b) The slide is horizontally moved to the next field (c) Ten microscopic fields are counted vertically. (d) The procedure is repeated until 100 leukocytes have been counted (for a 100-cell count).

A peripheral smear must be interpreted in the context of the clinical situation. That is, only limited information can be obtained unless the following information is available with the peripheral smear.

  • The age and sex of the patient must be known, since absolute cell numbers and the significance of some findings vary with age. For example, relative lymphocytosis with NRBCs and atypical lymphocytes would be unusual and pathologic in an adult, but appear in any infant under stress.

  • The red blood cell count (RBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), and red cell distribution width (RDW) cannot be accurately determined by manual smear examination and should be available. The white blood cell count (WBC) and platelet count can be approximated manually, but an automated ("machine") count is helpful.

  • Graphical information provided by the hematology analyzer is helpful but not essential for peripheral blood smear interpretation. Hematology analyzers utilize light scatter, electrical impedance, and other physical parameters to count cells, determine cell size and differentiate different types of blood cells. For example, many modern hematology analyzers measure electrical impedance, light scatter, cell viability, and other parameters during the evaluation process. Light scatter at 0 o roughly corresponds to cell size, 10 o light scatter to cellular internal "complexity," 90 o light scatter to nuclear lobularity, and 90 o depolarized light scatter to cytoplasmic granularity. An example of a light scatter histogram produced by a modern hematology analyzer is shown in Fig 4.

Disadvantages of the Peripheral Blood Smear

Peripheral blood smear examination provides information that cannot be obtained from automated cell counting. However, peripheral smear evaluation has some limitations and special considerations. These include:

  • Experience is required to make technically adequate smears.
  • There is a non-uniform distribution of white blood cells over the smear, with larger leukocytes concentrated near the edges and lymphocytes scattered throughout.
  • There is a non-uniform distribution of red blood cells over the smear, with small crowded red blood cells at the thick edge and large flat red blood cells without central pallor at the feathered edge.

Automated Hematological Evaluation

The total red cell count (RBC), RBC size (mean corpuscular volume, MCV), and red cell distribution width (RDW) are determined from analysis of electrical impedance and/or light scattering data by the hematology analyzer. These measurements are used to calculate the hematocrit, MCH, and MCHC.

In unusual circumstances the automated hematology analyzer produces cell counts which are falsely increased or decreased. Fortunately, in almost all cases the instruments "flag" the specimen as abnormal so that the operator can verify the results manually or perform necessary corrections. Causes of spurious red blood cell counts include:

  • Very small red blood cells (microcytosis) may be counted as large platelets and result in a falsely decreased RBC.
  • Autoagglutination or cryoglobulins lead to RBC clumping, which may falsely increase the RBC.
  • Spurious elevations in the RBC may also occur in patients with very high WBCs (> 100 x109).

Fig. 4. A modern hematology analyzer (Cell Dyn 4000, Abbott Laboratories, Chicago, IL). Sample analysis is performed in the

RBC Evaluation

Importance of the MCV and RDW

The MCV is the median value of the histogram distribution obtained when red blood cell size is plotted against the number of cells ("red cell histogram")(Fig. 5). The MCV, measured in femtoliters (fL, or 10-15 L), is the most important of the red cell indices. It is used to classify anemias as normocytic (normal MCV), microcytic (decreased MCV), or macrocytic (increased MCV). However, the MCV may be falsely elevated in patients with red blood cell agglutination, since the hematology analyzer may identify some of the cell clumps as single cells. In patients with severe hyperglycemia (glucose > 600 mg/dL), osmotic swelling of the red blood cells may also spuriously elevate the MCV.

A related parameter, the red cell distribution width (RDW) is the coefficient of variation of the red blood cell distribution histogram. As a quantitative measure of variation in red blood cell size (anisocytosis), the RDW is elevated in iron deficiency anemia, myelodysplastic syndromes, macrocytic anemia secondary to vitamin B12 or folate deficiency, and some malignancies. In contrast, the RDW is usually normal or only mildly elevated in the microcytic anemia of thalassemia.


Fig. 5. Red cell distribution histograms. In these histograms, RBC volume (x-axis) is plotted vs. the cell count (number of events counted (y-axis). The mean corpuscular volume (MCV) is the median value of the histogram distribution. The red cell distribution width (RDW) is the coefficient of variation of the curve. Microcytic red cells (a) fall to the left portion of the curve, while macrocytic red cells fall to the right (c). The histogram in the center is from a normocytic, normochromic specimen with an MCV of 88 fL.
Measurement of Hematocrit

The hematocrit (Hct, "crit") is the ratio of the volume of red blood cells to the volume of whole blood. In the past, the hematocrit was determined by centrifugation of whole blood in a narrow glass tube (capillary blood tube) sealed at one end ("spun hematocrit"). The spun hematocrit is spuriously elevated if plasma becomes trapped in the red cell layer. This phenomenon occurs in patients with polycythemia, macrocytosis, spherocytosis, hypochromic anemias, and RBC fragment syndromes. Improper mixing of the specimen or the addition of excessive anticoagulant can also lead to false Hct values. Since "crit" tubes are also fragile and dangerous to use, spun hematocrits are rarely used today.

The automated hematology analyzer calculates the Hct from the RBC and MCV by the following formula:

Hct (L/L, %) = RBC (cells/L) x MCV (L/cell)

Since the Hct is a calculated value, it is less accurate than either the RBC or Hb, and is affected by errors in either or both of these measurements.

The MCV, mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are the red blood cell indices. The MCH is the hemoglobin concentration per cell (hemoglobin mass/red blood cell), expressed in picograms per cell (pg, 10-12 g). The MCH is calculated from the hemoglobin and RBC by the following formula:


The MCH is decreased in patients with anemia caused by impaired hemoglobin synthesis. The MCH may be falsely elevated in blood specimens with turbid plasma (usually caused by hyperlipidemia) or severe leukocytosis.

The MCHC is the average hemoglobin concentration per total red blood cell volume (ratio of hemoglobin mass to RBC volume), as determined from the following equation:

The MCHC is decreased in microcytic anemias where the decrease in hemoglobin mass exceeds the decrease in the size of the red blood cell. It is increased in hereditary spherocytosis and in patients with hemoglobin variants, such as sickle cell disease and hemoglobin C disease).

Measurement of Hemoglobin

Nearly all automated hematology analyzers utilize the cyanomethemoglobin method to measure the hemoglobin content of the red blood cell. In this method, hemoglobin is converted to cyanated methemoglobin (cyanmethemoglobin) by the addition of a solution (Drabkin solution) containing potassium ferricyanide and potassium cyanide. Cyanated methemoglobin maximally absorbs light at 540 nm, and the total amount of hemoglobin is determined by spectrophotometry. The hemoglobin concentration is measured in grams per deciliter (g/dL) of whole blood.

Hyperlipidemia, fat droplets (from hyperalimentation), hypergammaglobinemia, cryoglobulinemia, and leukocytosis (> 50 x 109/mL) can result in spurious elevations of hemoglobin concentration. Spurious hemoconcentration or hemodilution, occurring with improper specimen collection, may falsely elevate or decrease hemoglobin concentrations. In addition, the age, sex, and race of the patient must also be considered in the interpretation of hemoglobin levels. Hemoglobin levels fall during the first month of life and remain relatively low until after puberty. The mean male hemoglobin level is 1 - 2 g/dL higher than the mean female level. Blacks of both sexes and all ages have hemoglobin levels which are 0.5 - 1.0 g/dL lower than whites of the same age and sex.

White Blood Cell Evaluation

The total white blood cell count (WBC, leukocyte count) includes all circulating nucleated hematopoietic cells with the exception of nucleated red blood cells (NRBCs). The WBC is of great importance in the diagnosis and management of patients with hematologic and infectious diseases. It is also used to monitor patients receiving cytotoxic drugs, radiation therapy, and some antimicrobial drugs.

The WBC is determined on EDTA-anticoagulated blood. RBCs are removed by lysis, and the total WBC is measured by electrical impedance or light scatter techniques. Unlysed red blood cells, nucleated red blood cells, platelet clumps, large platelets, and cryoglobulins may result in spurious WBC results. If these conditions are detected by the hematology analyzer, the specimen is "flagged" for a manual peripheral smear evaluation. Occasionally, a manual WBC, using a hemacytometer, may be necessary to verify the accuracy of an automated WBC. If NRBCs are present, a relative estimate of their number is obtained by light microscopy and expressed as # NRBCs/100 WBC. Under these circumstances, the total WBC must be corrected by use of the following formula:

Differential Leukocyte Count

The differential leukocyte count (leukocyte differential, white blood cell differential) is probably the least understood and overutilized of all hematologic assays. Until the 1980’s, the relative number (%) of each type of white blood cell was determined by manual examination of the peripheral blood smear and multiplied by the white cell count to obtain the absolute leukocyte count (cells/mL). Unfortunately, the manual differential is labor intensive, subjective, statistically unreliable (only 100-200 cells are counted), and inaccurate because of nonrandom distribution of cells on the smear (monocytes at the edge, lymphocytes in the middle).

Hematology analyzers are more accurate than the manual count for leukocyte elaboration under normal circumstances. For example, the hematology analyzers used in the Hematology Laboratories at WVU Hospitals generates a five-part differential based on electronic impedance, conductivity, and light scatter measurements (Fig. 6). Cell counting with these instruments is rapid, objective, statistically significant (8000 or more cells are counted), and not subject to the distributional bias of the manual count. In addition, the precision of the automated differential makes the absolute leukocyte count reliable and reproducible.

Hematology analyzers cannot yet correctly identify all abnormal white blood cells, and manual examination of the peripheral smear is still needed under some circumstances (blasts and immature cells, atypical lymphocytes, leukopenia, etc.). Specimens that meet one or more of abnormal criteria are flagged for manual examination. Depending on the patient population, a manual smear examination is required in only approximately 25% of peripheral smears.


Fig. 6. Normal leukocyte differential histogram from a modern hematology analyzer (Cell-Dyn 4000, Abbott Diagnostics). Scatterplot obtained from laser light scatter analysis of white blood cells. y-axis represents data obtained by light scatter at 0o (measure of cell size), while x-axis represents laser light scatter at 7o (cell internal complexity). Each "dot" represents data from a single cell. Clusters of cells represent neutrophils (66.6%), monocytes (8.63%), lymphocytes (22.2%), eosinophils (2.23%), and basophils (0.35%) present in the specimen. The total white blood count was 6.89 x 109/L.


Absolute vs. Relative Leukocyte Counts

The absolute leukocyte count provides clinical information of much greater value than the relative differential count. In fact, the relative count can be misleading, and the sole use of this parameter can conceal the diagnosis of certain cytopenias or obscure clinically significant trends that are occurring. The absolute neutrophil count, (ANC) and not the relative count, is helpful in monitoring chemotherapy patients, and the absolute neutrophil count is a superior indicator of infection and inflammation.

The report of an abnormal blood count is often the first clue to an abnormality of the white cell series; less commonly, the patient presents with an infection or other clinical problem. However, the peripheral blood leukocyte count is only one measure of white cell activity, and several factors must be considered in data interpretation. For example, a patient with a total white blood cell count of 2000/mL and 100 neutrophils/mL has a different problem from a patient with a white blood count of 2000/mL and 100 lymphocytes/mL. Absolute leukocyte numbers must be always be reviewed. In addition, the peripheral blood is only a conduit for leukocytes, and only a small percentage of the total white blood cells in the body are present in the peripheral blood at any one time. Therefore, the total white blood count and absolute leukocyte count must be interpreted in light of the physical findings and other laboratory data. Common causes are summarized in Table I.

Table I
Common Causes of Altered Leukocyte Counts
  Decreased Increased Neutrophil

Congenital
Hereditary neutropenia

Acquired
Bone marrow disease
Immune reactions
Drugs
Gram-negative septicemia
Myeloproliferative disorders

Acquired
Infections
Tissue destruction
Corticosteroids, lithium
Neoplastic growth
Leukemoid reaction
Lymphocyte Congenital
Congenital immunodeficiency disease

Acquired
Severe infection
Drugs (Corticosteroids, alkylating)
GI disease
Immunodeficiency
Acquired
Viral infection (EBV, hepatitis, etc.)
Some fungal, parasitic infections
Rare bacterial infection (Pertussis)
Allergic reactions/drug sensitivities
Immunologic disease
Monocyte Acquired
Hairy cell leukemia
Corticosteroids
Acquired
Mycobacterial infection
Tuberculosis, syphilis
Subacute bacterial endocarditis
Inflammatory responses
Recovery phase of neutropenia
Myeloproliferative disorders
Eosinophil Acquired
Bacterial infection
ACTH administration

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