Thick Blood Smear and Thin Blood Smear

Blood smears are one of the most common and useful laboratory techniques in hematology and parasitology. They involve spreading a thin layer of blood on a glass slide and staining it with a dye that highlights the different types of blood cells and parasites. Blood smears can provide valuable information about the health status of an individual, such as the number, shape, size, and distribution of red blood cells, white blood cells, platelets, and any abnormal or infectious agents in the blood.

A well-prepared blood smear is essential for obtaining accurate and reliable results from the analysis. A poorly prepared blood smear can lead to errors in interpretation, misdiagnosis, or missed diagnosis of various blood disorders and infections. Some of the factors that can affect the quality of a blood smear include:

• The type and volume of blood sample used
• The method and speed of spreading the blood on the slide
• The time and temperature of drying the smear
• The type and duration of staining the smear
• The quality and cleanliness of the glass slide and other equipment

Therefore, it is important to follow standard procedures and guidelines for preparing blood smears, as well as to check the quality of the smear before staining and examining it under a microscope. A good blood smear should have the following characteristics:

• A uniform thickness and distribution of blood cells across the slide
• A smooth transition from a thick to a thin area at the end of the smear
• A clear and well-defined edge without feathering or tailing
• No clumps, gaps, or artifacts in the smear
• No contamination or damage to the smear

A well-prepared blood smear can facilitate the identification and differentiation of various types of blood cells and parasites, as well as reveal any abnormalities or changes in their morphology, number, or function. This can help in diagnosing and monitoring various hematological and parasitic diseases, such as anemia, leukemia, malaria, babesiosis, ehrlichiosis, etc. Blood smears can also be used for other purposes, such as evaluating the response to treatment, assessing the prognosis, or screening for potential donors or recipients of blood transfusion.

In summary, blood smears are an important tool for hematology and parasitology that require careful preparation and quality control. A well-prepared blood smear can provide valuable information about the health status of an individual and help in detecting and managing various blood disorders and infections.

Giemsa stain is a type of Romanowsky stain named after Gustav Giemsa, a German chemist who created a dye solution. It is specific for the phosphate groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding.

Giemsa stain is used to stain chromosomes and blood cells, especially for the detection of blood parasites such as malaria, trypanosomes, and chlamydia.

Giemsa stain is a differential stain that contains a mixture of azure, methylene blue, and eosin dye. Azure and eosin are acidic dyes that variably stain the basic components of the cells like the cytoplasm, granules, etc. Methylene blue acts as the basic dye, which stains the acidic components, especially the nucleus of the cell. Methanol acts as a fixative as well as a cellular stain.

Depending on the objective of the smear, two types of smear can be prepared:

• Thin blood smear – for demonstration and differentiation of leukocytes.
• Thick blood smear – for diagnosis of blood protozoan parasites and blood abnormalities.

The procedures for preparing thin and thick smears for Giemsa staining technique are as follows:

Thin blood smear preparation

• Using a sterile pricking needle, make a prick on the index finger
• Apply some pressure on the finger and put two drops of blood at the edge, leaving a margin on a sterile microscopic slide.
• Place the edge of another sterile microscopic slide over the drops of blood, at an angle of 30-45 degrees, and make two streaks rapidly but smoothly forward from the blood sample and spread it. This will leave a thin film of blood resembling a tongue-shape.
• Allow the slide to air dry and fix it with methanol for 30 seconds.
• Stain with Giemsa working solution (1 part Giemsa stock solution + 9 parts buffered water) for 20-30 minutes.
• Rinse with buffered water and air dry.

Thick blood smear preparation

• Collect blood sample by venipuncture and put in a clean test tube
• Using a capillary tube collect blood from the tube and put two large drops at the center of a sterile microscopic slide.
• Holding the slide between your thumb and index finger, gently

Blood smears are one of the most common and informative diagnostic tests in hematology and parasitology. They provide valuable information on the number, shape, size, and structure of blood cells and the presence of parasites or microorganisms in the blood. Blood smears can help to diagnose various blood disorders, infections, and immune-mediated inflammatory diseases.

Some of the important and common applications of blood smears are:

• Differential leukocyte count (DLC): This is a quantitative analysis of the five types of white blood cells (WBCs) in the blood: neutrophils, lymphocytes, monocytes, eosinophils, and basophils. DLC can reveal the relative proportions of each WBC type and indicate the presence of abnormal or immature cells. DLC can help to diagnose infections, inflammations, allergies, leukemias, and other hematological conditions.
• Red blood cell (RBC) morphology: This is a qualitative analysis of the shape, size, color, and structure of RBCs in the blood. RBC morphology can reveal various abnormalities such as anisocytosis (variation in size), poikilocytosis (variation in shape), hypochromia (reduced hemoglobin content), polychromasia (presence of immature RBCs), and inclusion bodies (such as Howell-Jolly bodies or malaria parasites). RBC morphology can help to diagnose anemias, hemoglobinopathies, hemolytic disorders, and parasitic infections.
• Platelet count and morphology: This is a quantitative and qualitative analysis of the number and appearance of platelets in the blood. Platelets are small cell fragments that play a vital role in blood clotting and wound healing. Platelet count and morphology can reveal various abnormalities such as thrombocytopenia (low platelet count), thrombocytosis (high platelet count), giant platelets, platelet clumps, or platelet satellitism. Platelet count and morphology can help to diagnose bleeding disorders, thrombotic disorders, infections, inflammations, and bone marrow diseases.
• Blood parasite detection: This is a qualitative analysis of the presence and identification of parasites or microorganisms in the blood. Blood parasites are organisms that live in the blood of their hosts and cause various diseases. Some of the common blood parasites are Plasmodium spp. (causing malaria), Babesia spp. (causing babesiosis), Trypanosoma spp. (causing sleeping sickness or Chagas disease), Leishmania spp. (causing leishmaniasis), and Bartonella spp. (causing cat scratch disease or trench fever). Blood parasite detection can help to diagnose parasitic infections and monitor their treatment.

Blood smears are simple, rapid, and inexpensive tests that can provide a wealth of information on the health status of an individual. They are essential tools for clinical diagnosis and research in hematology and parasitology.

Leukocytes, or white blood cells, are the immune cells of the blood that protect the body from infections and foreign substances. There are five types of leukocytes: neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Each type has a different function and appearance under the microscope.

A thin blood smear is a simple and effective way to demonstrate and differentiate the leukocytes in a blood sample. A thin blood smear is prepared by spreading a small drop of blood on a glass slide, creating a thin film of blood cells that can be stained and examined under high magnification.

The most commonly used stain for blood smears is the Giemsa stain, which is a mixture of dyes that selectively binds to different components of the cells, such as DNA, RNA, proteins, and lipids. The Giemsa stain produces characteristic colors for each type of leukocyte, as well as for erythrocytes (red blood cells) and platelets.

The Giemsa stain can also reveal the presence of parasites, such as malaria or other blood-borne protozoa, that may infect the blood cells.

The thin blood smear allows the observer to count and classify the leukocytes based on their size, shape, nucleus, cytoplasm, and granules. The normal range and appearance of each type of leukocyte are as follows:

• Neutrophils: These are the most abundant leukocytes in the blood, accounting for 50-70% of the total leukocyte count. They are phagocytic cells that ingest and destroy bacteria and other microorganisms. They have a multilobed nucleus (usually 2-5 lobes) and a pale pink cytoplasm with fine lilac granules. They are also called polymorphonuclear leukocytes (PMNs) or segs.
• Lymphocytes: These are the second most common leukocytes in the blood, accounting for 20-40% of the total leukocyte count. They are involved in specific immune responses against foreign antigens. They have a large round nucleus that occupies most of the cell and a thin rim of sky blue cytoplasm. They are divided into two main types: B lymphocytes (which produce antibodies) and T lymphocytes (which regulate cell-mediated immunity).
• Monocytes: These are the largest leukocytes in the blood, accounting for 2-10% of the total leukocyte count. They are precursors of macrophages, which are phagocytic cells that migrate to tissues and organs to remove dead cells and debris. They have a kidney-shaped or horseshoe-shaped nucleus and a gray-blue cytoplasm with fine azurophilic granules. They are also called mononuclear phagocytes.
• Eosinophils: These are rare leukocytes in the blood, accounting for 1-6% of the total leukocyte count. They are involved in allergic reactions and parasitic infections. They have a bilobed nucleus and a bright orange-red cytoplasm with coarse granules that contain histamine and other inflammatory mediators.
• Basophils: These are the least common leukocytes in the blood, accounting for 0-1% of the total leukocyte count. They are also involved in allergic reactions and parasitic infections. They have a lobed nucleus that is often obscured by large dark purple granules that fill the cytoplasm. The granules contain histamine, heparin, and other vasoactive substances.

By examining a thin blood smear stained with Giemsa stain, one can identify and quantify the different types of leukocytes in a blood sample and assess their morphology for any signs of abnormality or disease.

A thick blood smear is a type of blood smear that is used to detect and identify blood protozoan parasites and blood abnormalities such as anemia. A thick blood smear is prepared by spreading a large drop of blood in a small area of about 1 cm on a glass slide. The blood is not fixed with any chemical, but allowed to air-dry completely. The dried blood is then stained with a Romanowski stain, such as Giemsa stain, which differentially colors the nuclei and cytoplasm of the cells and parasites.

The advantage of a thick blood smear is that it concentrates the blood cells and parasites in a thin layer, making them easier to see under the microscope. A thick blood smear can detect low levels of parasitemia (the number of parasites in the blood) that may be missed by a thin blood smear. A thick blood smear can also provide an estimate of the parasite density by counting the number of parasites per 200 leukocytes or per 500 erythrocytes.

The disadvantage of a thick blood smear is that it distorts the morphology of the cells and parasites, making them difficult to identify and differentiate. A thick blood smear also requires more time and skill to prepare and examine than a thin blood smear. A thick blood smear may also be affected by factors such as anticoagulants, hemolysis, or clotting of the blood sample.

Some examples of blood protozoan parasites that can be detected by a thick blood smear are:

• Plasmodium spp., which cause malaria. The different species of Plasmodium have different morphological features and life cycles in the human host and the mosquito vector. The most common and dangerous species is Plasmodium falciparum, which can cause severe complications such as cerebral malaria, anemia, renal failure, and death.
• Trypanosoma spp., which cause trypanosomiasis or sleeping sickness. The two main types of trypanosomiasis are African trypanosomiasis, caused by Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense, and American trypanosomiasis, caused by Trypanosoma cruzi. The parasites are transmitted by tsetse flies or triatomine bugs, respectively. The symptoms of trypanosomiasis include fever, headache, lymphadenopathy, anemia, neurological disorders, and cardiac dysfunction.
• Leishmania spp., which cause leishmaniasis or kala-azar. The parasites are transmitted by sandflies and infect various tissues and organs such as the skin, mucous membranes, spleen, liver, bone marrow, and lymph nodes. The symptoms of leishmaniasis vary depending on the species and the site of infection, ranging from cutaneous ulcers to visceral organ failure.

Some examples of blood abnormalities that can be detected by a thick blood smear are:

• Anemia, which is a condition characterized by low levels of hemoglobin or red blood cells in the blood. Anemia can have various causes such as iron deficiency, vitamin B12 deficiency, hemolysis, bleeding, infection, or bone marrow disorders. Anemia can cause symptoms such as fatigue, weakness, pallor, shortness of breath, dizziness, and chest pain.
• Thalassemia, which is a genetic disorder that affects the production or function of hemoglobin. Hemoglobin is the protein that carries oxygen in the red blood cells. There are two main types of thalassemia: alpha thalassemia and beta thalassemia. Depending on the severity and type of thalassemia, the symptoms may include anemia, jaundice, splenomegaly (enlarged spleen), hepatomegaly (enlarged liver), bone deformities, growth retardation, and heart problems.
• Sickle cell disease (SCD), which is another genetic disorder that affects the shape and function of hemoglobin. Hemoglobin in SCD forms abnormal crescent-shaped or sickle-shaped red blood cells that are rigid and sticky. These cells can block the small blood vessels and cause pain crises (vaso-occlusive crises), organ damage (such as stroke or kidney failure), infection (such as pneumonia or meningitis), anemia (hemolytic anemia), and reduced life expectancy.

A thick blood smear is a useful diagnostic tool for detecting and identifying blood protozoan parasites and blood abnormalities. However, it should be complemented by other tests such as a thin blood smear, molecular tests (such as PCR or antigen detection), serological tests (such as antibody detection), or biochemical tests (such as hemoglobin level or liver function tests) to confirm the diagnosis and monitor the treatment outcome.

Before applying any staining technique to the blood smears, it is essential to ensure that they are completely dry. This is because wet or moist smears can cause several problems that can affect the quality and accuracy of the results. Some of these problems are:

• Wet smears can cause the blood cells to lyse or burst, resulting in distorted cell morphology and loss of cellular details.
• Wet smears can cause the stain to run or diffuse unevenly, resulting in poor contrast and visibility of the cells and parasites.
• Wet smears can cause the stain to precipitate or form crystals on the surface of the smear, resulting in artifacts and interference with the identification of the cells and parasites.
• Wet smears can cause the smear to stick to the cover slip or the microscope slide, resulting in damage or loss of the smear during examination.

Therefore, it is important to air-dry the smears for 20-30 minutes or until they are completely dry before applying any stain. This will ensure that the blood cells and parasites are well-preserved and clearly visible under the microscope. Drying can be done at room temperature or by using a fan or a hair dryer. However, avoid using heat sources such as ovens or hot plates as they can cause overheating and denaturation of the cells and parasites.

Blood smears are simple procedures to perform aiming at demonstrating and acquiring information on blood cells, qualitatively and quantitatively. Quantitative importance enables the numbering of blood cells while the qualitative function is to demonstrate and identify the cell morphologies, including types of leukocytes, erythrocytes, monocytes, and platelets.

Quantitative analysis of blood smears involves counting the number of different types of blood cells per unit volume of blood. This can be done manually using a hemocytometer or an automated cell counter. The results can provide useful information on the hematological status of the patient, such as the presence of anemia, leukocytosis, leukopenia, thrombocytosis, or thrombocytopenia. The quantitative analysis can also help to calculate indices such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW).

Qualitative analysis of blood smears involves examining the shape, size, color, and arrangement of blood cells under a microscope. This can reveal abnormalities in the morphology of blood cells, such as poikilocytosis, anisocytosis, hypochromia, polychromasia, rouleaux formation, agglutination, or parasitism. The qualitative analysis can also help to identify the specific types of leukocytes based on their nuclear and cytoplasmic features, such as neutrophils, eosinophils, basophils, lymphocytes, or monocytes. The qualitative analysis can also help to detect immature or abnormal cells in the blood, such as blasts, metamyelocytes, myelocytes, promyelocytes, nucleated red blood cells (nRBCs), or sickle cells.

Both quantitative and qualitative analyses of blood smears are important for diagnosing and monitoring various hematological disorders and infections. They can also provide clues to the underlying causes of these conditions and guide appropriate treatment and management. Therefore, blood smears are essential tools in clinical hematology and parasitology.

The main objectives for preparing thin and thick blood smears are:

• To observe the morphology and number of blood cells, such as erythrocytes, leukocytes, and platelets.
• To identify and differentiate the types of leukocytes, such as neutrophils, eosinophils, basophils, lymphocytes, and monocytes.
• To detect the presence and species of blood parasites, such as malaria, babesiosis, trypanosomiasis, leishmaniasis, and filariasis.
• To diagnose various hematological disorders and infections, such as anemia, leukemia, lymphoma, thrombocytopenia, hemolytic disease, and sepsis.

By preparing thin and thick blood smears, one can obtain valuable information about the health status of the patient and the possible causes of their symptoms. Thin blood smears allow for a detailed examination of the shape, size, color, and arrangement of blood cells under a microscope. Thick blood smears concentrate the blood cells in a smaller area and increase the chances of finding rare or low-density parasites. Both types of smears require proper staining techniques to enhance the visibility and contrast of the cellular structures. Giemsa stain is one of the most commonly used stains for blood smears as it can differentiate between various types of cells and parasites based on their affinity to the stain.

Therefore, preparing thin and thick blood smears is an essential skill for laboratory technicians and clinicians who work with blood samples. By following the correct procedures and using quality materials, one can ensure accurate and reliable results that can help in diagnosis and treatment.

To prepare blood smears, you will need the following items:

• Sterile syringe and needle: These are used to collect venous blood samples from the patient. Make sure to use a new syringe and needle for each patient to avoid cross-contamination and infection.
• EDTA vials: These are tubes that contain ethylenediaminetetraacetic acid (EDTA), an anticoagulant that prevents blood from clotting. You will need to transfer the venous blood samples into these vials and mix them well by gently inverting them several times.
• Tourniquet: This is a device that applies pressure to a limb to restrict blood flow and make the veins more visible and accessible for venipuncture. You will need to wrap it around the patient`s arm above the elbow and tighten it until you feel a pulse.
• 70% isopropyl alcohol: This is a disinfectant that you will use to clean the skin of the patient before and after venipuncture. You will need to soak a cotton ball or swab with alcohol and rub it on the puncture site in a circular motion.
• Sterile lancet: This is a sharp instrument that you will use to make a small prick on the finger of the patient to obtain peripheral blood samples. You will need to use a new lancet for each patient and dispose of it in a sharps container after use.
• Microscopic glass slides: These are thin pieces of glass that you will use to spread the blood samples into thin and thick smears. You will need to use clean and dry slides that are free of dust, grease, or scratches. You will also need to label each slide with the patient`s name, date, and type of smear (thin or thick).
• Spreader slide: This is another glass slide that you will use to make thin blood smears. You will need to hold it at an angle of 30-45 degrees and slide it across the drop of blood on the first slide, creating a thin film of blood.
• Romanowski stain: This is a mixture of dyes that you will use to stain the blood smears and make the cells and parasites more visible under the microscope. There are different types of Romanowski stains, such as Giemsa, Wright, or Leishman stains. You will need to follow the manufacturer`s instructions on how to prepare and apply the stain.

###Specimen: Venous blood sample

###Principle: Thick blood smears require larger volumes of blood than the thin blood smears. This allows them to be used for the detection of blood parasites in the blood samples. A thick blood smear is made by spreading a large blood drop in a small area of about 1 cm which provides a better opportunity to detect various parasitic forms against a more transparent background .

###Procedure:

• Collect blood sample by venipuncture and put in a clean test tube
• Using a capillary tube collects blood from the tube and put two large drops at the center of a sterile microscopic slide.
• Holding the slide between your thumb and index finger, gently shake the slide to spread the blood about 10mm in diameter.
• A thick smear of proper density is one which, if placed (wet) over newsprint, allows you to barely read the words.
• Lay the slides flat and allow the smears to dry thoroughly (protect from dust and insects!). Insufficiently dried smears (and/or smears that are too thick) can detach from the slides during staining. The risk is increased in smears made with anticoagulated blood. At room temperature, drying can take several hours; 30 minutes is the minimum; in the latter case, handle the smear very delicately during staining. You can accelerate the drying by using a fan or hair dryer (use cool setting). Protect thick smears from hot environments to prevent heat-fixing the smear.
• Do not fix thick smears with methanol or heat. If there will be a delay in staining smears, dip the thick smear briefly in water to hemolyse the RBCs.
• Apply the appropriate Romanowski stain, such as Giemsa stain, following the manufacturer`s instructions.

Specimen: Peripheral Blood sample

Principle: The Thin Blood smear is prepared by making a drop of well-mixed venous blood, 2mm in diameter at the center of a sterilized microscopic glass slide. Some borders are left around the smear for easy counting and differentiating of the cells. A second glass slide is used as a spreader, streaking the blood into a thin film across the glass slide. This preparation is allowed to dry and then fixed with an appropriate Romanowski stain, depending on your objective.

Procedure:

1. Using a sterile pricking needle, make a prick on the index finger
2. Apply some pressure on the finger and put two drops of blood at the edge, leaving a margin on a sterile Microscopic slide.
3. Place the edge of the sterile microscopic slide over the drops of blood, at an angle of 30-45°, and make two streaks rapidly but smoothly forward from the blood sample and spread it. This will leave a thin film of blood resembling a tongue-shape.
4. Allow the slide to air dry and stain with an appropriate staining technique.

Note: The quality of the thin blood smear depends on several factors, such as:

• The size and shape of the blood drop
• The angle and speed of the spreader slide
• The cleanliness and smoothness of the slides
• The humidity and temperature of the environment
• The proper drying and fixation of the smear

To ensure a good thin blood smear, follow these tips:

• Use clean slides to avoid the formation of grease spots (holes in the smear).
• Use fresh blood samples or well-mixed anticoagulated blood samples.
• Make sure the blood drop is not too large or too small (about 2mm in diameter).
• Hold the spreader slide at a 30-45° angle and move it quickly and evenly across the slide.
• Avoid making multiple passes over the same area or changing direction while spreading.
• Avoid touching the edge of the spreader slide to the slide before or after spreading.
• Rapidly air dry the smear to preserve cell morphologies.
• Fix the smear with methanol or other suitable fixative before staining.

Blood smears are useful tools for diagnosing and monitoring various blood-related conditions, such as anemia, leukemia, malaria, and mononucleosis. By examining the number, shape, and appearance of the different types of blood cells under a microscope, a blood smear can provide valuable information about the health and function of the blood and the bone marrow.

Some of the applications of blood smears are:

• Classification of blood disorders: Blood smears can help identify the type and severity of anemia, which is a condition where the red blood cells are low in number or abnormal in shape or size. Anemia can have many causes, such as iron deficiency, vitamin B12 deficiency, hemolysis (destruction of red blood cells), or inherited disorders like sickle cell anemia or thalassemia. Blood smears can also help detect bleeding disorders, such as thrombocytopenia (low platelet count) or hemophilia (impaired clotting factor), which can affect the ability of the blood to stop bleeding.

• Characterization of blood-related disorders: Blood smears can help diagnose and classify leukemias, which are cancers of the white blood cells that originate from the bone marrow. Leukemias can be classified into acute or chronic, and lymphoid or myeloid, based on the type and maturity of the abnormal white blood cells. Blood smears can also help detect other blood cancers, such as lymphomas (cancers of the lymph nodes) or multiple myeloma (cancer of the plasma cells).

• Detection of immune-mediated inflammatory disorders and infections: Blood smears can help identify infections caused by bacteria, viruses, or parasites that affect the blood cells. For example, malaria is a parasitic infection that invades and destroys the red blood cells, causing fever, chills, and anemia. Blood smears can show the presence and stage of the malaria parasite inside the red blood cells. Similarly, mononucleosis is a viral infection that causes an increase in the number and size of lymphocytes (a type of white blood cell) and atypical lymphocytes (abnormal-looking lymphocytes). Blood smears can help confirm the diagnosis of mononucleosis by showing these changes in the white blood cells. Blood smears can also help detect immune-mediated inflammatory disorders, such as lupus or rheumatoid arthritis, which cause inflammation and damage to various organs and tissues. Blood smears can show signs of inflammation, such as increased or decreased white blood cells, or changes in their shape or function.

Blood smears are simple and quick tests that can provide a lot of information about the health and function of the blood and the bone marrow. They can help diagnose and monitor various hematological disorders and infections that affect the blood cells. However, blood smears are not always conclusive or definitive, and they may need to be supplemented by other tests, such as bone marrow biopsy, chemistry panel, or thyroid tests, to confirm or rule out certain conditions.

Blood smears are widely used in clinical and laboratory settings to examine the morphology and number of blood cells and to detect blood parasites or other abnormalities. However, like any other diagnostic method, blood smears have some advantages and disadvantages that should be considered.

Some of the advantages of using blood smears are:

• It is a simple and rapid technique that requires basic equipment and skills.
• It can be performed with very small volumes of blood, which is convenient for patients and animals.
• It provides qualitative and quantitative information on blood cells, such as their types, shapes, sizes, and abnormalities.
• It can help diagnose various blood disorders, such as anemia, leukemia, infections, and parasitic diseases.
• It can help monitor the response to treatment and the prognosis of some blood conditions.

Some of the disadvantages of using blood smears are:

• The quality and accuracy of the results depend on several factors, such as the preparation of the smear, the staining technique, the microscope, and the expertise of the examiner.
• The results may vary depending on the time of collection, the anticoagulant used, and the storage conditions of the blood sample.
• The results may not be conclusive or specific for some blood disorders or infections, and may require further confirmation by other tests.
• The technique may not detect low levels of parasites or abnormal cells that are present in a small proportion of the blood sample.
• The technique may cause discomfort or bleeding at the site of blood collection, especially for patients with bleeding disorders or those taking anticoagulants.

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