White Blood Cell Count and Differential

Definition

A white blood cell (WBC) count determines the concentration of white blood cells in the patient's blood. A differential determines the percentage of each of the five types of mature white blood cells.

Purpose

This test is included in general health examinations and to help investigate a variety of illnesses. An elevated WBC count occurs in infection, allergy, systemic illness, inflammation, tissue injury, and leukemia. A low WBC count may occur in some viral infections, immunodeficiency states, and bone marrow failure. The WBC count provides clues about certain illnesses, and helps physicians monitor a patient's recovery from others. Abnormal counts that return to normal indicate that the condition is improving, while counts that become more abnormal indicate that the condition is worsening. The differential will reveal which WBC types are affected most. For example, an elevated WBC count with an absolute increase in lymphocytes having an atypical appearance is most often caused by infectious mononucleosis. The differential will also identify early WBCs which may be reactive (e.g. a response to acute infection) or the result of a leukemia.

Precautions

Many medications affect the WBC count. Both prescription and non-prescription drugs including herbal supplements should be noted. Normal values for both the WBC count and differential are age related.

Sources of error in manual WBC counting are largely due to variance in the dilution of the sample and the distribution of cells in the chamber, and the small number of WBCs that are counted. For electronic WBC counts and differentials, interference may be caused by small fibrin clots, nucleated RBCs, platelet clumping, and unlysed RBCs. Immature WBCs and nucleated RBCs may cause interference with the automated differential count. Automated cell counters may not be acceptable for counting white blood cells in other body fluids especially when the number of WBCs is less than 1000/μL or when other nucleated cell types are present.

Description

White cell counts are usually performed using an automated instrument, but may be done manually using a microscope and a counting chamber especially when counts are very low, or the person has a condition known to interfere with an automated WBC count. An electronic WBC count is based upon the principle of impedance. The red blood cells are lysed using a detergent in the counting diluent. As the cells move one at a time through a counting aperture, they displace electrolyte in the diluent causing a voltage pulse. The magnitude of the voltage pulse is dependent upon size, which allows the instrument to discriminate between different types of WBCs.

An automated differential may be performed by an electronic cell counter or by an image analysis instrument. The automated electronic cell counter uses a combination of impedence measurement and other means such as radio frequency conductance and angular light scattering to differentiate between closely related WBCs. Image analysis systems use morphometric and densitometric programs to distinguish the cells that are photographed from a stained slide by a digital color camera. When the electronic WBC count is abnormal or a cell population is flagged, meaning that one or more of the results is atypical, a manual differential is performed. The WBC differential is performed manually by microscopic examination of a blood sample that is spread in a thin film on a glass slide. The film is air-dried and stained with Wright stain, a polychromatic stain consisting of buffered solutions of methylene blue and eosin. Acidic structures such as DNA take up the basic methylene blue dye, while basic proteins, such as hemoglobin, take up the acidic eosin dye. White blood cells are identified by their size, the shape and texture of the nuclear chromatin, cytoplasmic and nuclear staining, and the presence and color of granules in the cytoplasm.

Causes for abnormalities in the white blood cell (WBC) differential count
Type of WBC and normal differential count	Elevated	Decreased
Source: Pagana, K.D. and T.J. Pagana. Mosby's Diagnostic and Laboratory Test Reference. 3rd ed. St. Louis: Mosby, 1997.
Neutrophils 55-70%	Neutrophilia	Neutrophilia
	Physical or emotional stress	Aplastic anemia
	Acute suppurative infection	Dietary deficiency
	Myelocytic leukemia	Overwhelming bacterial infection
	Trauma	Viral infections
	Cushing's syndrome	Radiation therapy
	Inflammatory disorders	Addison's disease
	Metabolic disorders	Drug therapy: myelotoxic drugs (as in chemotherapy)
Lymphocytes 20-40%	Lymphocytosis	Lymphocytopenia
	Chronic bacterial infection	Leukemia
	Viral infection	Sepsis
	Lymphocytic leukemia	Immunodeficiency diseases
	Multiple myeloma	Lupus erythematosus
	Infectious mononucleosis	Later stages of HIV infection
	Radiation	Drug therapy: adrenocorticosteroids, antineoplastics
	Infectious hepatitis	Radiation therapy
Monocytes 2-8%	Monocytosis	Monocytopenia
	Chronic inflammatory disorders	Drug therapy: prednisone
	Viral infections
	Tuberculosis
	Chronic ulcerative colitis
	Parasites
Eosinophils 1-4%	Eosinophilia	Eosinopenia
	Parasitic infections	Increased adrenosteroid production
	Allergic reactions
	Eczema
	Leukemia
	Autoimmune diseases
Basophils 0.5-1.0%	Basophilia	Basopenia
	Myeloproliferative disease (e.g., myelofibrosis, polycythemia rubra vera)	Acute allergic reactions
		Hyperthyroidism
	Leukemia	Stress reactions

The manual WBC differential involves a thorough evaluation of a stained blood film. In addition to determining the percentage of each mature white blood cell, the following tests are preformed as part of the differential:

Evaluation of RBC morphology is performed. This includes grading of the variation in RBC size (anisocytosis) and shape (poikioocytosis); reporting the type and number of any abnormal RBCs such as target cells, sickle cells, stippled cells, etc.; reporting the presence of immature RBCs (polychromasia); and counting the number of nucleated RBCs per 100 WBCs.
An estimate of the WBC count is made and compared to the automated or chamber WBC count. An estimate of the platelet count is made and compared to the automated or chamber platelet count. Abnormal platelets such as clumped platelets or excessively large platelets are noted on the report.
Any immature white blood cells are included in the differential count of 100 cells, and any inclusions or abnormalities of the WBCs are reported.

WBCs consist of two main subpopulations, the mononuclear cells and the granulocytic cells. Mononuclear cells include lymphocytes and monocytes. Granulocytes include neutropohils (also called polymorphonuclear leukocytes or segmented neutrophils), eosinophils, and basophils. Each cell type is described below:

Neutrophils are normally the most abundant WBCs. They measure 12-16 μm in diameter. The nucleus stains dark purple-blue, and is divided into several lobes (usually three to four) consisting of dense chromatin. A neutrophil just before the final stage of maturation will have an unsegmented nucleus in the shape of a band. These band neutrophils may be counted along with mature neutrophils or as a separate category. The cytoplasm of a neutrophil contains both primary (azurophilic) and secondary (specific) granules. The secondary granules are lilac in color and are more abundant almost covering the pink cytoplasm. Neutrophils are phagocytic cells and facilitate removal of bacteria and antibody-coated antigens. The neutrophilic granules are rich in peroxidase, and aid the cell in destroying bacteria and other ingested cells.
Eosinophils are 14-16 μm in diameter and contain a blue nucleus that is segmented into two distinct lobes. The cytoplasm is filled with large refractile orange-red granules. The granules contain peroxidase, hydrolases, and basic proteins which aid in the destruction of phagocytized cells. Eosinophils are increased in allergic reactions and parasitic infections.
Basophils, like eosinophils, are 14-16 μm in diameter and have a blue nucleus that is bilobed. The cytoplasm of the basophil is filled with large dark blue-black granules that may obscure the nucleus. These contain large amounts of histamine, heparin, and acid mucopolysaccharides. Basophils mediate the allergic response by releasing histamine.
Lymphocytes are the second most abundant WBCs. They may be small (7-9 μm in diameter) or large (12-16 μm in diameter). The nucleus is dark blue and is nearly round or slightly indented and the chromatin is clumped and very dense. The cytoplasm is medium blue and usually agranular. An occasional lymphocyte will have a few azurophilic granules in the cytoplasm. Lymphocytes originate in the lymphoid tissues and are not phagocytic. They are responsible for initiating and regulating the immune response by the production of antibodies and cytokines.
Monocytes are the largest WBCs measuring 14-20 mm in diameter. They have a large irregularly shaped and folded blue nucleus with chromatin that is less dense than other WBCs. The cytoplasm is grey-blue, and is filled with fine dust-like lilac colored granules. Monocytes are phagocytic cells that process and present antigens to lymphocytes, an event required for lymphocyte activation.

Preparation

This test requires a 3.5 mL sample of blood. Venipuncture is usually performed by a nurse or phlebotomist following standard precautions for the prevention of transmission of bloodborne pathogens. There is no restriction on diet or physical activity.

Aftercare

Discomfort or bruising may occur at the puncture site. Pressure to the puncture site until the bleeding stops reduces bruising; warm packs relieve discomfort. Some people feel dizzy or faint after blood has been drawn and should be treated accordingly.

Complications

Other than potential bruising at the puncture site, and/or dizziness, there are no complications associated with this test.

KEY TERMS

Band cell—An immature neutrophil at the stage just preceding a mature cell. The nucleus of a band cell is unsegmented.

Basophil—Segmented white blood cell with large dark blue-black granules that releases histamine in allergic reactions.

Differential—Blood test that determines the percentage of each type of white blood cell in a person's blood.

Eosinophil—Segmented white blood cell with large orange-red granules that increases in response to parasitic infections and allergic reactions.

Lymphocyte—Mononuclear white blood cell that is responsible for humoral (antibody mediated) and cell mediated immunity.

Monocyte—Mononuclear phagocytic white blood cell that removes debris and microorganisms by phagocytosis and processes antigens for recognition by immune lymphocytes.

Neutrophil—Segmented white blood cell normally comprising 50-70% of the total. The cytoplasm contains both primary and secondary granules that take up both acidic and basic dyes of the Wright stain. Neutrophils remove and kill bacteria by phagocytosis.

Phagocytosis—A process by which a white blood cell envelopes and digests debris and microorganisms to remove them from the blood.

Results

Normal values vary with age. White counts are highest in children under one year of age and then decrease somewhat until adulthood. The increase is largely in the lymphocyte population. Adult normal values are shown below.

WBC count: 4,500-11,000/μL
polymorphonuclear neutrophils: 1800-7800/μL; (50-70%)
band neutrophils: 0-700/μL; (0-10%)
lymphocytes: 1000-4800/μL; (15-45%)
monocytes: 0-800/μL; (0-10%)
eosinophils: 0-450/μL; (0-6%)
basophils: 0-200/μL; (0-2%)

Health care team roles

The WBC count and differential are ordered and interpreted by physicians. The samples may be collected by a nurse, physician assistant, phlebotomist, or technician. Testing is performed by a clinical laboratory scientist CLS(NCA)/medical technologist MT(ASCP) or by a clinical laboratory technician CLT(NCA)/medical laboratory technician MLT(ASCP).