Blood film

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Blood film
Peripheral blood smear - stained and unstained.jpg
Two push-type peripheral blood smears suitable for characterization of cellular blood elements. Left smear is unstained, right smear is stained with Wright-Giemsa stain.
ICD-9-CM 90.5
MedlinePlus 003665

A blood film—or peripheral blood smear—is a thin layer of blood smeared on a glass microscope slide and then stained in such a way as to allow the various blood cells to be examined microscopically. Blood films are examined in the investigation of hematological (blood) disorders and are routinely employed to look for blood parasites, such as those of malaria and filariasis.

Blood specialized bodily fluid in animals

Blood is a body fluid in humans and other animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells.

Microscope slide

A microscope slide is a thin flat piece of glass, typically 75 by 26 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is mounted (secured) on the slide, and then both are inserted together in the microscope for viewing. This arrangement allows several slide-mounted objects to be quickly inserted and removed from the microscope, labeled, transported, and stored in appropriate slide cases or folders etc.

Hematology, also spelled haematology, is the branch of medicine concerned with the study of the cause, prognosis, treatment, and prevention of diseases related to blood. It involves treating diseases that affect the production of blood and its components, such as blood cells, hemoglobin, blood proteins, bone marrow, platelets, blood vessels, spleen, and the mechanism of coagulation. Such diseases might include hemophilia, blood clots, other bleeding disorders and blood cancers such as leukemia, multiple myeloma, and lymphoma. The laboratory work that goes into the study of blood is frequently performed by a medical technologist or medical laboratory scientist.

Contents

Preparation

Blood films are made by placing a drop of blood on one end of a slide, and using a spreader slide to disperse the blood over the slide's length. 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.

Unstained peripheral blood smear closeup.jpg
Unstained
Stained peripheral blood smear closeup.jpg
Wright-Giemsa stained
Closeups of the feathered edge of blood smears. The pale middle band of the gradient is the monolayer.

The slide is left to air dry, after which the blood is fixed to the slide by immersing it briefly in methanol. The fixative is essential for good staining and presentation of cellular detail. After fixation, the slide is stained to distinguish the cells from each other.

In the field of anatomy, fixation is the preservation of biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability. Tissue fixation is a critical step in the preparation of histological sections, its broad objective being to preserve cells and tissue components and to do this in such a way as to allow for the preparation of thin, stained sections. This allows the investigation of the tissues' structure, which is determined by the shapes and sizes of such macromolecules as proteins and nucleic acids.

Methanol, also known as methyl alcohol amongst other names, is a chemical with the formula CH3OH (a methyl group linked to a hydroxyl group, often abbreviated MeOH). Methanol acquired the name wood alcohol because it was once produced chiefly by the destructive distillation of wood. Today, methanol is mainly produced industrially by hydrogenation of carbon monoxide.

Routine analysis of blood in medical laboratories is usually performed on blood films stained with Romanowsky stains such as Wright's stain, Giemsa stain, or Diff-Quik. Wright-Giemsa combination stain is also a popular choice. These stains allow for the detection of white blood cell, red blood cell, and platelet abnormalities. Hematopathologists often use other specialized stains to aid in the differential diagnosis of blood disorders.

Romanowsky stain Family of related stains for examination of blood including the detection of parasites

Romanowsky staining, also known as Romanowsky–Giemsa staining, is a prototypical staining technique that was the forerunner of several distinct but similar stains widely used in hematology and cytopathology. Romanowsky-type stains are used to differentiate cells for microscopic examination in pathologic specimens, especially blood and bone marrow films, and to detect parasites such as malaria within the blood. Stains that are related to or derived from the Romanowsky-type stains include Giemsa, Jenner, Wright, Field, May–Grünwald stain and Leishman stains. The staining technique is named after the Russian physician Dmitri Leonidovich Romanowsky (1861–1921), who was one of the first to recognize its potential for use as a blood stain.

Wrights stain Histologic stain that facilitates the differentiation of blood cell types

Wright's stain is a histologic stain that facilitates the differentiation of blood cell types. It is classically a mixture of eosin (red) and methylene blue dyes. It is used primarily to stain peripheral blood smears, urine samples, and bone marrow aspirates which are examined under a light microscope. In cytogenetics, it is used to stain chromosomes to facilitate diagnosis of syndromes and diseases.

Giemsa stain

Giemsa stain, named after German chemist and bacteriologist Gustav Giemsa, is used in cytogenetics and for the histopathological diagnosis of malaria and other parasites.

After staining, the monolayer is viewed under a microscope using magnification up to 1000x. Individual cells are examined and their morphology is characterized and recorded. [1] [2]

Clinical significance

Normal Adult Blood Smear.JPG
Chronic Myeloid Leukemia smear 2009-04-09.JPG
The left image shows a microscopic view of a normal adult blood film, while the right image shows a blood film from a patient with chronic myeloid leukemia.

Blood smear examination is usually performed in conjunction with a complete blood count in order to investigate abnormal results or confirm results that the automated analyzer has flagged as unreliable. [3]

Complete blood count medical laboratory test

A complete blood count (CBC) is a blood panel requested by a doctor or other medical professional that gives information about the cells in a patient's blood, such as the cell count for each blood cell type and the concentrations of hemoglobin. A scientist or lab technician performs the requested testing and provides the requesting medical professional with the results of the CBC.

Microscopic examination of the shape, size, and coloration of red blood cells is useful for determining the cause of anemia. Disorders such as iron deficiency anemia, sickle cell anemia, megaloblastic anemia and microangiopathic hemolytic anemia result in characteristic abnormalities on the blood film. [2]

Anemia Decrease in the total number of red blood cells or amount of hemoglobin in the blood, or lowered ability of the blood to carry oxygen

Anemia is a decrease in the total amount of red blood cells (RBCs) or hemoglobin in the blood, or a lowered ability of the blood to carry oxygen. When anemia comes on slowly, the symptoms are often vague and may include feeling tired, weakness, shortness of breath, and a poor ability to exercise. When the anemia comes on quickly, symptoms may include confusion, feeling like one is going to pass out, loss of consciousness, and increased thirst. Anemia must be significant before a person becomes noticeably pale. Additional symptoms may occur depending on the underlying cause.

Megaloblastic anemia macrocytic anemia that is characterized by inhibition of DNA synthesis during red blood cell production

Megaloblastic anemia is an anemia that results from inhibition of DNA synthesis during red blood cell production. When DNA synthesis is impaired, the cell cycle cannot progress from the G2 growth stage to the mitosis (M) stage. This leads to continuing cell growth without division, which presents as macrocytosis. Megaloblastic anemia has a rather slow onset, especially when compared to that of other anemias. The defect in red cell DNA synthesis is most often due to hypovitaminosis, specifically vitamin B12 deficiency or folate deficiency. Loss of micronutrients may also be a cause. Copper deficiency resulting from an excess of zinc from unusually high oral consumption of zinc-containing denture-fixation creams has been found to be a cause.

Microangiopathic hemolytic anemia (MAHA) is a microangiopathic subgroup of hemolytic anemia caused by factors in the small blood vessels. It is identified by the finding of anemia and schistocytes on microscopy of the blood film.

The proportions of different types of white blood cells can be determined from the blood smear. This is known as a manual white blood cell differential. The white blood cell differential can reveal abnormalities in the proportions of white blood cell types, such as neutrophilia and eosinophilia, as well as the presence of abnormal cells such as the circulating blast cells seen in acute leukemia. [4] Qualitative abnormalities of white blood cells, like toxic granulation, are also visible on the blood smear. Modern complete blood count analyzers can provide an automated white blood cell differential, but they have a limited ability to differentiate immature and abnormal cells, so manual examination of the blood smear is frequently indicated. [5] [6]

Blood smear examination is the preferred diagnostic method for certain parasitic infections, such as malaria and babesiosis. [7] Rarely, bacteria may be visible on the blood smear in patients with severe sepsis. [8]

Malaria

Blood films showing various developmental stages of the malaria parasite Plasmodium falciparum, stained with Wright stain and Giemsa stain. Synchronsed P falciparum cultures.jpg
Blood films showing various developmental stages of the malaria parasite Plasmodium falciparum , stained with Wright stain and Giemsa stain.

The preferred and most reliable diagnosis of malaria is microscopic examination of blood films, because each of the four major parasite species has distinguishing characteristics. Two sorts of blood film are traditionally used.

From the thick film, an experienced microscopist can detect all parasites they encounter. Microscopic diagnosis can be difficult because the early trophozoites ("ring form") of all four species look identical and it is never possible to diagnose species on the basis of a single ring form; species identification is always based on several trophozoites.

The biggest pitfall in most laboratories in developed countries is leaving too great a delay between taking the blood sample and making the blood films. As blood cools to room temperature, male gametocytes will divide and release microgametes: these are long sinuous filamentous structures that can be mistaken for organisms such as Borrelia. If the blood is kept at warmer temperatures, schizonts will rupture and merozoites invading erythrocytes will mistakenly give the appearance of the accolé form of P. falciparum. If P. vivax or P. ovale is left for several hours in EDTA, the buildup of acid in the sample will cause the parasitised erythrocytes to shrink and the parasite will roll up, simulating the appearance of P. malariae. This problem is made worse if anticoagulants such as heparin or citrate are used. The anticoagulant that causes the least problems is EDTA. Romanowsky stain or a variant stain is usually used. Some laboratories mistakenly use the same staining pH as they do for routine haematology blood films (pH 6.8): malaria blood films must be stained at pH 7.2, or Schüffner's dots and James's dots will not be seen.

Immunochromatographic capture procedures (rapid diagnostic tests such as the malaria antigen detection tests) are nonmicroscopic diagnostic options for the laboratory that may not have appropriate microscopy expertise available. [10]

Related Research Articles

Babesiosis malaria-like parasitic disease caused by infection with Babesia, a genus of Apicomplexa

Babesiosis is a malaria-like parasitic disease caused by infection with Babesia, a type of Apicomplexa. Human babesiosis transmission via tick bite is most common in the Northeastern and Midwestern United States and parts of Europe, and sporadic throughout the rest of the world. It occurs in warm weather. People can get infected with Babesia parasites by the bite of an infected tick, by getting a blood transfusion from an infected donor of blood products, or by congenital transmission . Ticks transmit the human strain of babesiosis, so it often presents with other tick-borne illnesses such as Lyme disease. After trypanosomes, Babesia is thought to be the second-most common blood parasite of mammals, and they can have a major impact on health of domestic animals in areas without severe winters. In cattle the disease is known as Texas cattle fever, redwater, or piroplasmosis.

Hemolytic anemia is a form of anemia due to hemolysis, the abnormal breakdown of red blood cells (RBCs), either in the blood vessels or elsewhere in the human body. It has numerous possible consequences, ranging from relatively harmless to life-threatening. The general classification of hemolytic anemia is either inherited or acquired. Treatment depends on the cause and nature of the breakdown.

Sideroblastic anemia aplastic anemia where the bone marrow produces ringed sideroblasts rather than healthy red blood cells (erythrocytes)

Sideroblastic anemia, or sideroachrestic anemia, is a form of anemia in which the bone marrow produces ringed sideroblasts rather than healthy red blood cells (erythrocytes). In sideroblastic anemia, the body has iron available but cannot incorporate it into hemoglobin, which red blood cells need in order to transport oxygen efficiently. The disorder may be caused either by a genetic disorder or indirectly as part of myelodysplastic syndrome, which can develop into hematological malignancies.

Leishman stain

Leishman stain, also known as Leishman's stain, is used in microscopy for staining blood smears. It is generally used to differentiate between and identify white blood cells, malaria parasites, and trypanosomas. It is based on a methanolic mixture of "polychromed" methylene blue and eosin. The methanolic stock solution is stable and also serves the purpose of directly fixing the smear eliminating a prefixing step. If a working solution is made by dilution with an aqueous buffer, the resulting mixture is very unstable and cannot be used for long. Leishman stain is named after its inventor, the Scottish pathologist William Boog Leishman. It is a version of the Romanowsky stain, and is thus similar to and partially replaceable by Giemsa stain, Jenner's stain, and Wright's stain.

<i>Babesia</i> genus of protozoan parasites

Babesia, also called Nuttallia, is an Apicomplexan parasite that infects red blood cells, transmitted by ticks. Originally discovered by the Romanian bacteriologist Victor Babeș, over 100 species of Babesia have since been identified.

Gustav Giemsa German scientist

Gustav Giemsa was a German chemist and bacteriologist who was a native of Medar-Blechhammer. He is remembered for creating a dye solution commonly known as "Giemsa stain". This dye is used for the histopathological diagnosis of malaria and parasites such as Plasmodium, Trypanosoma, and Chlamydia.

Diff-Quik

Diff-Quik is a commercial Romanowsky stain variant used to rapidly stain and differentiate a variety of pathology specimens. It is most frequently used for blood films and cytopathological smears, including fine needle aspirates. The Diff-Quik procedure is based on a modification of the Wright-Giemsa stain pioneered by Harleco in the 1970s, and has advantages over the routine Wright-Giemsa staining technique in that it reduces the 4-minute process into a much shorter operation and allows for selective increased eosinophilic or basophilic staining depending upon the time the smear is left in the staining solutions.

Pappenheimer bodies

Pappenheimer bodies are abnormal basophilic granules of iron found inside red blood cells on routine blood stain. They are a type of inclusion body composed of ferritin aggregates, or mitochondria or phagosomes containing aggregated ferritin. They appear as dense, blue-purple granules within the red blood cell and there are usually only one or two, located in the cell periphery. They stain on a Romanowsky stain because clumps of ribosomes are co‐precipitated with the iron‐containing organelles.

The mainstay of malaria diagnosis has been the microscopic examination of blood, utilizing blood films. Although blood is the sample most frequently used to make a diagnosis, both saliva and urine have been investigated as alternative, less invasive specimens. More recently, modern techniques utilizing antigen tests or polymerase chain reaction have been discovered, though these are not widely implemented in malaria endemic regions. Areas that cannot afford laboratory diagnostic tests often use only a history of subjective fever as the indication to treat for malaria.

Leukocytes are one type of Blood cells, the other two being Red blood cells and Platelets. Until now, information given by most Hematology Automated analysers on Leukocytes used for the complete blood count has only been numerical, reporting the total amount of Leukocytes and the percentage and absolute number of the different types thereof: Neutrophils, Lymphocytes, Monocytes, Eosinophils and Basophils.

Dmitri Leonidovich Romanowsky

Dmitri Leonidovich Romanowsky was a Russian physician who is best known for his invention of an eponymous histological stain called Romanowsky stain. It paved the way for the discovery and diagnosis of microscopic pathogens, such as malarial parasites.

Jaswant Singh–Bhattacharji stain, commonly referred to as JSB stain, is a rapid staining method for detection of malaria. It is useful for the diagnosis of malaria in thick smear samples of blood. The JSB stain is commonly used throughout India, but rarely used in other countries.

Quartan fever

Quartan fever is one of the four types of malaria which can be contracted by humans.

White blood cell differential Blood test for types and amounts of white blood cells

A white blood cell differential is a medical laboratory test that provides information about the types and amounts of white blood cells in a person's blood. The test, which is usually ordered as part of a complete blood count, measures the amounts of the five white blood cell types in normal human blood – neutrophils, lymphocytes, monocytes, eosinophils and basophils – as well as abnormal cell types if they are present. These results are reported as percentages and absolute values. Changes in the amounts of white blood cells can aid in the diagnosis of many health conditions, including viral, bacterial, and parasitic infections and blood disorders such as leukemia.

References

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