Blood Film

A blood film was once prepared on nearly everyone who had a full blood count (FBC). With the automated blood cell counting instruments currently used, an automated  differential is also provided. However, if the presence of abnormal WBCs, RBCs or platelets is suspected, a blood film, examined by a trained eye, is still the best method for identifying immature and abnormal cells.

There are many diseases, disorders and deficiencies that can have an effect on the number and type of blood cells produced, their function and their lifespan. Although usually only normal mature cells are released into the bloodstream, circumstances can force the bone marrow to release immature and/or malformed cells into the blood. When a significant number of abnormal cells are present, they can indicate disease and prompt the doctor to do further testing.

The blood film is primarily used when a FBC with differential, performed with an automated blood cell counter, shows the presence of abnormal or immature cells. It may also be used when a doctor suspects a deficiency, disease or disorder that is affecting blood cell production, such as an anaemia, decreased or abnormal production of cells in the bone marrow, or increased cell destruction. A blood film may also be requested when a patient is being treated or monitored for a blood cell-related disease.

Findings from the blood film test do not always give a diagnosis but can provide information indicating the presence of an underlying condition and its severity and the need for further diagnostic testing. Blood film findings may include:

RBC (Red blood cells)
Normal, mature red blood cells are uniform in size (7 µm). Unlike most other cells, they do not have a nucleus. They are round and flattened like a doughnut but with a depression in the middle instead of a hole (biconcave). With routine staining, due to the haemoglobin inside the RBCs, they appear pink to red in colour with a pale centre. While not every RBC will be perfect, the presence of many cells that are different in shape or size may indicate a more severe problem. There may be one or more irregularities present and may include:

• Anisocytosis - variable sizes of red cells. The presence of smaller RBCs (<7 µm) is referred to as microcytosis and RBCs larger than 7 µm is macrocytosis.
• Poikilocytosis - various shapes of red cells. These may include echinocytes, acanthocytes, elliptocytes, keratocytes, sickle cells, target cells, teardrop cells (dacrocytes), smear cells (also known as smudge or basket cells), and schistocytes.

See the section below for Details on Red Blood Cell Irregularities.

WBC (White Blood Cells)
White blood cells have a nucleus surrounded by cytoplasm. All WBCs come from bone marrow stem cells. In the marrow, they change into two groups: myeloid and lymphoid cells. They then mature into five different types of WBCs.

• Granulocytes – the three types of cell making up this group are distinguished by granules of characteristic size and colour in their cytoplasm.
  • Neutrophils - these cells have nuclei with multiple lobes and have pink or purple granules in their cytoplasm. They compose the majority of WBCs in a healthy adult. They increase in episodes of inflammation, such as with bacterial infections or in rheumatoid arthritis.
  • Eosinophils  - are easily recognised in stained smears with their large, red-orange granules and nuclei usually with 2 lobes (bi-lobed). They are normally present in relatively low numbers (1-3%) but increase in number with allergies and parasitic infections.
  • Basophils - all have large, dark purple-black granules and are the least often seen type of WBC (1%). Like eosinophils they usually have bi-lobed nuclei. Increased numbers of basophils are not often encountered but may be found with certain haematological diseases most notably some leukaemias and myeloproliferative disorders. Elevated numbers of basophils can also be found associated with chicken pox, ulcerative colitis , or after an immunisation.
  • Monocytes - are usually the largest of the WBCs (12-20 µm) and are often referred to as scavenger cells (phagocytes). They can ingest particles such as cellular debris, bacteria, or other insoluble particles.
  • Lymphoid cells - lymphocytes are smaller in size (10-12 µm) and many have a smooth, round nucleus which is not lobated. Normally most lymphocytes seen on a smear don’t have visible granules or have just a few granules in their cytoplasm. Lymphocytes are responsible for the production of antibodies (immunoglobulins) or have complicated roles in organising the responses of other white blood cells or in recognising and attacking germs or even cancer cells

See the section below for more Details on White Blood Cells.

Platelets
These are cell fragments that come from large bone marrow cells called megakaryocytes. Upon release from the bone marrow, they appear as fragments in the peripheral blood. When there is blood vessel injury or other bleeding, the platelets become activated and begin to clump together to form aggregates. This is the first step in making a blood clot. You must have a sufficient number of platelets to control bleeding. If there are too few, the ability to form a clot is impaired and can be life-threatening. In some people, too many platelets may be produced, which interferes with the flow of blood and increases a person's risk of developing a blood clot. These same people may also experience bleeding because many of the extra platelets may be dysfunctional even though they appear normal.

Details of platelet number and size is usually part of a FBC. An abnormally low number or high number of platelets may be further evaluated by preparing a peripheral blood film to visualise any anomalies in shape or size directly.

Some examples of situations or conditions that may affect or invalidate results of a blood film include:

• The patient has received a recent blood transfusion
• The patient has increased levels of protein
• The blood specimen has a blood clot in it
• Blood collected in the wrong specimen tube
• Not enough blood collected into the specimen tube
• Specimen tube not kept at the correct temperature
• Blood film not prepared or stained correctly
• Sample too old for preparation of film

Automated blood cell counters are used for routine testing of FBCs. These machines provide information based on the shape, size, and electrical or photometric properties RBCs, WBCs and platelets. A variety of physiological and external stimuli can lead to some variation in the numbers of cells the body produces and the proportions of the different cell. Automated instruments can often identify the presence of abnormal cells but lack the sophistication to subclassify them definitively. Cell fragments and platelet clumps, particularly if they are large in size, can be mistakenly counted as WBCs, thus falsely elevating a white cell count. Healthcare scientists and haematologists have been trained to identify and classify these abnormalities by examining a blood film.

• Anisocytosis: this is abnormal variation in size of RBCs
• Macrocytosis: large RBCs that may be due to a vitamin B12 or folate deficiency (megaloblastic anaemia). They are seen in pernicious anaemia, liver disease, in patients with underactive thyroid glands, in some bone marrow diseases, and sometimes in pregnancy.
• Microcytosis: this is the presence of small RBCs that may be due to iron deficiency anaemia, long-standing inflammation due to infections or conditions such as rheumatoid arthritis, or to an inherited disorder such as thalassaemia.

• Poikilocytosis is a variation in the shape of an RBC and may include several different abnormalities at the same time.
• Acanthocytes (spur, thorn or spiculated cells): irregular shaped cells with 5-10 spicules. May be present with liver or thyroid conditions, or in post-splenectomy patients.
• Echinocytes (burr, crenated or berry cells): may have 10-30 spiny projections and often seen in patients with renal failure or malnutrition May be an artefact - something caused during sample preparation.
• Elliptocytes elliptical-shaped RBC seen in hereditary elliptocytosis and various anaemias.
• Keratocyte (horn cell): A half-moon or spindle shaped RBC that may be seen in patients with disseminated intravascular coagulation (DIC) or a vascular prosthesis.
• Rouleaux: RBCs that appear as a stack of coins and seen in patients with inflammatory illnesses, anaemia, multiple myeloma or macroglobulinaemia.
• Sickle cells: crescent-shaped RBCs characteristic of sickle cell anaemia.
• Target cells (leptocytes or codocytes): RBCs that resemble a bull's-eye. Commonly seen in patients with liver disease, iron deficiency, haemoglobinopathies (abnormal inherited forms of haemoglobin), thalassaemia.
• Teardrop cells (dacrocytes): RBCs that resemble a teardrop. Often seen in patients with myelofibrosis.
• Schistocytes: fragments or broken pieces of RBCs. This may be due to a disorder that is causing the red blood cells to be especially fragile or due to mechanical haemolysis as seen sometimes with devices such as artificial heart valves, or in severely burned patients.
• Spherocytosis: sphere-shaped RBCs found in hereditary spherocytosis or in autoimmune haemolytic anaemia.

• Hypochromasia: this may be seen in a variety of disorders including thalassaemia and iron deficiency. The RBC is pale in colour due to insufficient haemoglobin and contains a large, hollow middle (central pallor) of the cell.
• Polychromasia: blue-staining RBCs, indicating that they are immature due to early release from the bone marrow.

• Nucleated RBCs (normoblasts): a very immature form of RBCs seen when there is a severe demand for RBCs to be released by the bone marrow. May be seen in abrupt blood loss, severe anaemia, myelofibrosis, thalassaemia, miliary tuberculosis and in cancers that involve the bone marrow. nucleated RBCs can be normal in infants for a short time after birth.
• Reticulocytes: these are immature RBCs that are usually polychromatic in colour. A few of these young red blood cells are normal in the circulation. Elevated numbers may be seen with acute blood loss, hypoxia, RBC destruction (“haemolysis”, such as in sickle cell disease, glucose-6-phosphate dehydrogenase (G6PD) deficiency and autoimmune haemolytic anaemia).
• Siderocyte, sideroblast, ringed sideroblast: When RBCs are stained with Prussian blue dye, iron granules may be seen. Sideroblasts are immature siderocytes. Ring sideroblasts are particularly found in some forms of hereditary anaemia and in myelodysplastic syndrome.
• Basophilic stippling (dark blue dots inside the RBC): due to the precipitation of nuclear material (ribosomes) and may be present in heavy metal poisoning (such as lead), nutritional deficiencies, or myelofibrosis.
• Heinz bodies: large inclusion bodies (granules) in the RBCs when stained with crystal violet. May be due to an enzyme (G6PD) deficiency, unstable haemoglobin variant, thalassaemia, and autoimmune haemolytic anaemia.
• Howell-Jolly bodies (small round remnants of nuclear DNA inside cell): present in sickle cell anaemia, haemolytic or megaloblastic anaemias, and may be seen after a splenectomy.
• Cabot's Rings: threadlike inclusions that form a ring within the RBC. May be seen in a variety of anaemias.
• Malarial parasites: patients with malaria may have these parasites living inside RBCs breaking them apart as they leave. This is not a routine finding; it would normally only be detected on a blood film when an investigating doctor suspects that a patient has an active case of malaria.

Neutrophils (also called segmented neutrophils, polymorphonuclear cells, polys or PMNs) are about 12 microns in diameter and their function is to engulf and destroy invading organisms. They make up about 50 to 70% of the total WBC count in the peripheral blood and may have two to five nuclear lobes connected by a thin strand of nuclear material. This WBC may be seen in greater numbers during infections, malignancies or extreme stress situations. Using a standard Romanowsky dye, the blood slides are stained to visualise the various cell types better. The cytoplasm of neutrophils is pale and often contains small pink to purple granules. These granules (specific granules and azurophilic granules) contain enzymes and proteins that neutralise or destroy microorganisms.

Anomalies of neutrophils include:

• Toxic granulation: large dark blue granules in the cytoplasm, associated with severe infection, chemical poisoning, alcohol abuse.
• Vacuolisation: vacuoles appear as holes in the cytoplasm and are frequently found in association with toxic granulation.
• Döhle bodies: irregular greyish or greenish inclusions in the peripheral cytoplasm of neutrophils. They are nuclear remnants that are often seen in association with toxic granules and vacuoles. They may be present in association with burns, trauma, acute or systemic infections, and may be present with exposure to cytotoxic agents (ie, chemotherapy). They may also be seen during a normal pregnancy.
• Auer Bodies (Auer Rods): unique, pink or red rod-shaped inclusions that are seen in very immature granulocytes ("blasts") in patients with acute myeloid leukaemia (AML).
• Bands: immature neutrophils with a non-segmented but elongated nucleus. Bands are normal in the circulation in small numbers but if there is a percentage increase of them, there is said to be a “left shift.” This may happen when an acute infection stimulates increased neutrophil production causing the bone marrow to prematurely release some WBCs before they have matured to the neutrophil stage.
• Hypersegmentation: neutrophils with 6 or more nuclear segments. This is mainly associated with B12 and folate deficiency, less often with iron deficiency and rarely with infections.
• Pelger-Huët: hereditary anomaly where neutrophils appear with fewer than two lobes. The nucleus is often in the shape of a peanut or dumbbell, or may consist of two lobes connected with an obvious filament. They may also appear in myelodysplastic syndromes where they are referred to as "pseudo- Pelger-Huët" or pelgeroid cells.
• Alder-Reilly granules: large, dark leucocyte granules that stain purple. They are indicative of mucopolysaccharidosis (an inherited enzyme deficiency disorder, e.g. Hurler's and Hunter's syndromes).
• Chédiak-Higashi syndrome: an inherited anomaly characterised by the presence of big red, blue, or greenish granules of variable size that are peroxidase positive and indicate a lethal metabolic disorder, they may be found in granulocytes, lymphocytes, and monocytes. Patients with this syndrome may exhibit neurological problems as well as a compromised immune system and photophobia. Death often ensues.

Eosinophils have 2 or 3 lobes to their nucleus and contain characteristic reddish/orange granules in their cytoplasm. They are most often involved in allergic responses and parasitic infections. About 1 - 3% of WBCs are eosinophils.

Basophils have a multi-lobed nucleus and have many dark blue granules (which contain histamine) in their cytoplasm. Only about 1% of WBCs are basophils. A slight elevation in number may be seen during an allergic response, ulcerative colitis, chronic sinusitis, chicken pox and immunisations. A significant increase is not uncommon in certain myeloid leukaemias.

Lymphocytes are often relatively small (7 - 10 µm) and round in shape. The nucleus is generally large in relation to the amount of cytoplasm. The cytoplasm is pale blue and generally does not contain any granules. The nucleus of most lymphocytes is smooth in appearance and is dark blue. There are two major types of lymphocytes, B cell and T-cell, but they cannot be distinguished when viewed under the microscope using standard staining techniques. B cells can be differentiated from T cells using specific fluorescent-labelled antibody stains in conjunction with a special instrument called a flow cytometer. B cells create specific antibodies while T-cells can activate B cells as well as recognise and destroy invading organisms. Lymphocytes make up about 20% to 40% of the total WBC count.

• Reactive lymphocyte (atypical, activated, Downey cells): these cells are large lymphocytes that contain a greater amount of cytoplasm and can vary in size and shape. Often a characteristic bluish tinge of cytoplasm is seen where the cell abuts with surrounding RBCs. Increase numbers of atypical lymphocytes are found in viral illnesses such as infectious mononucleosis.
• Hairy cells: these lymphocytes have tiny projections that make them appear hairy under the microscope. They are found in hairy cell leukaemia.

Monocytes are the largest of the WBCs and comprise less than 6% in normal blood. They are characterised by their abundant blue-grey cytoplasm that is irregular in shape and have a folded nucleus. The main function of monocytes is to ingest microorganisms and respond to infection and inflammation by releasing certain proteins (monokines) that can inactivate bacteria. When stimulated by cytokines, monocytes can move out of the bloodstream and become tissue macrophages.