NUMERICAL ABNORMALITIES OF LEUKOCYTES

Published in Hemotology
Wednesday, 26 July 2017 16:28
For meaningful interpretation, absolute count of leukocytes should be reported. These are obtained as follows:
 
Absolute Leukocyte Count = Leukocyte% × Total Leukocyte Count/ml
 
 
Neutrophilia:
 
An absolute neutrophil count greater than 7500/μl is termed as neutrophilia or neutrophilic leukocytosis.
 
Causes
 
  1. Acute bacterial infections: Abscess, pneumonia, meningitis, septicemia, acute rheumatic fever, urinary tract infection.
  2. Tissue necrosis: Burns, injury, myocardial infarction.
  3. Acute blood loss
  4. Acute hemorrhage
  5. Myeloproliferative disorders
  6. Metabolic disorders: Uremia, acidosis, gout
  7. Poisoning
  8. Malignant tumors
  9. Physiologic causes: Exercise, labor, pregnancy, emotional stress.
 
Leukemoid reaction: This refers to the presence of markedly increased total leukocyte count (>50,000/cmm) with immature cells in peripheral blood resembling leukaemia but occurring in non-leukemic disorders (see Figure 801.2). Its causes are:
 
  • Severe bacterial infections, e.g. septicemia, pneumonia
  • Severe hemorrhage
  • Severe acute hemolysis
  • Poisoning
  • Burns
  • Carcinoma metastatic to bone marrow Leukemoid reaction should be differentiated from chronic myeloid leukemia (Table 801.1).
 
Table 801.1 Differences between leukemoid reaction and leukemia
Table 801.1 Differences between leukemoid reaction and leukemia
 
Figure 801.2 Leukemoid reaction in blood smear
Figure 801.2 Leukemoid reaction in blood smear
 
 
Absolute neutrophil count less than 2000/μl is neutropenia. It is graded as mild (2000-1000/μl), moderate (1000-500/μl), and severe (< 500/μl).
 
Causes
 
I. Decreased or ineffective production in bone marrow:
 
  1. Infections 
    (a) Bacterial: typhoid, paratyphoid, miliary tuberculosis, septicemia
    (b) Viral: influenza, measles, rubella, infectious mononucleosis, infective hepatitis.
    (c) Protozoal: malaria, kala azar
    (d) Overwhelming infection by any organism
  2. Hematologic disorders: megaloblastic anemia, aplastic anemia, aleukemic leukemia, myelophthisis.
  3. Drugs:
    (a) Idiosyncratic action: Analgesics, antibiotics, sulfonamides, phenothiazines, antithyroid drugs, anticonvulsants.
    (b) Dose-related: Anticancer drugs
  4. Ionizing radiation
  5. Congenital disorders: Kostman's syndrome, cyclic neutropenia, reticular dysgenesis.
 
II. Increased destruction in peripheral blood:
 
  1. Neonatal isoimmune neutropaenia
  2. Systemic lupus erythematosus
  3. Felty's syndrome
 
III. Increased sequestration in spleen:
 
  1. Hypersplenism
 
Eosinophilia:
 
This refers to absolute eosinophil count greater than 600/μl.
 
Causes
 
  1. Allergic diseases: Bronchial asthma, rhinitis, urticaria, drugs.
  2. Skin diseases: Eczema, pemphigus, dermatitis herpetiformis.
  3. Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis.
  4. Hematologic disorders: Chronic Myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma.
  5. Carcinoma with necrosis.
  6. Radiation therapy.
  7. Lung diseases: Loeffler's syndrome, tropical eosinophilia
  8. Hypereosinophilic syndrome.
 
Basophilia:
 
Increased numbers of basophils in blood (>100/μl) occurs in chronic myeloid leukemia, polycythemia vera, idiopathic myelofibrosis, basophilic leukemia, myxedema, and hypersensitivity to food or drugs.
 
Monocytosis:
 
This is an increase in the absolute monocyte count above 1000/μl.
 
Causes
 
  1. Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar.
  2. Recovery from neutropenia.
  3. Autoimmune disorders.
  4. Hematologic diseases: Myeloproliferative disorders, monocytic leukemia, Hodgkin's disease.
  5. Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis.
 
Lymphocytosis:
 
Box 801.1 Differential diagnosis of LymphocytosisThis is an increase in absolute lymphocyte count above upper limit of normal for age (4000/μl in adults, >7200/μl in adolescents, >9000/μl in children and infants) (Box 801.1).
 
Causes
 
  1. Infections: 
    (a) Viral: Acute infectious lymphocytosis, infective hepatitis, cytomegalovirus, mumps, rubella, varicella
    (b) Bacterial: Pertussis, tuberculosis
    (c) Protozoal: Toxoplasmosis
  2. Hematological disorders: Acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma.
  3. Other: Serum sickness, post-vaccination, drug reactions.

WHITE BLOOD CELLS MORPHOLOGY

Published in Hemotology
Wednesday, 26 July 2017 13:33
Approximate idea about total leukocyte count can be gained from the examination of the smear under high power objective (40× or 45×). A differential leukocyte count should be carried out. Abnormal appearing white cells are evaluated under oil-immersion objective.
 
Morphology of normal leukocytes (see Figure 800.1):
 
  1. Polymorphonuclear neutrophil: Neutrophil measures 14-15 μm in size. Its cytoplasm is colorless or lightly eosinophilic and contains multiple, small, fine, mauve granules. Nucleus has 2-5 lobes that are connected by fine chromatin strands. Nuclear chromatin is condensed and stains deep purple in color. A segmented neutrophil has at least 2 lobes connected by a chromatin strand. A band neutrophil shows non-segmented U-shaped nucleus of even width. Normally band neutrophils comprise less than 3% of all leukocytes. Majority of neutrophils have 3 lobes, while less than 5% have 5 lobes. In females, 2-3% of neutrophils show a small projection (called drumstick) on the nuclear lobe. It represents one inactivated X chromosome.
  2. Eosinophil: Eosinophils are slightly larger than neutrophils (15-16 μm). The nucleus is often bilobed and the cytoplasm is packed with numerous, large, bright orange-red granules. On blood smears, some of the eosinophils are often ruptured.
  3. Basophils: Basophils are seen rarely on normal smears. They are small (9-12 μm), round to oval cells, which contain very large, coarse, deep purple granules. It is difficult to make out the nucleus since granules cover it.
  4. Monocytes: Monocyte is the largest of the leukocytes (15-20 μm). It is irregular in shape, with oval or clefted (kidney-shaped) nucleus and fine, delicate chromatin. Cytoplasm is abundant, bluegray with ground glass appearance and often contains fine azurophil granules and vacuoles. After migration to the tissues from blood, they are called as macrophages.
  5. Lymphocytes: On peripheral blood smear, two types of lymphocytes are distinguished: small and large. The majority of lymphocytes are small (7-8 μm). These cells have a high nuclearcytoplasmic ratio with a thin rim of deep blue cytoplasm. The nucleus is round or slightly clefted with coarsely clumped chromatin. Large lymphocytes (10-15 μm) have a more abundant, pale blue cytoplasm, which may contain a few azurophil granules. Nucleus is oval or round and often placed on one side of the cell.
 
Figure 800.1 Normal mature white blood cells in peripheral blood
Figure 800.1 Normal mature white blood cells in peripheral blood
 
Morphology of abnormal leukocytes:
 
  1. Box 800.1 Role of blood smear in leukemiaToxic granules: These are darkly staining, bluepurple, coarse granules in the cytoplasm of neutrophils. They are commonly seen in severe bacterial infections.
  2. Döhle inclusion bodies: These are small, oval, pale blue cytoplasmic inclusions in the periphery of neutrophils. They represent remnants of ribosomes and rough endoplasmic reticulum. They are often associated with toxic granules and are seen in bacterial infections.
  3. Cytoplasmic vacuoles: Vacuoles in neutrophils are indicative of phagocytosis and are seen in bacterial infections.
  4. Shift to left of neutrophils: This refers to presence of immature cells of neutrophil series (band forms and metamyelocytes) in peripheral blood and occurs in infections and inflammatory disorders.
  5. Hypersegmented neutrophils: Hypersegmentation of neutrophils is said to be present when >5% of neutrophils have 5 or more lobes. They are large in size and are also called as macropolycytes. They are seen in folate or vitamin B12 deficiency and represent one of the earliest signs.
  6. Pelger-Huet cells: In Pelger-Huet anomaly (a benign autosomal dominant condition), there is failure of nuclear segmentation of granulocytes so that nuclei are rod-like, round, or have two segments. Such granulocytes are also observed in myeloproliferative disorders (pseudo-Pelger-Huet cells).
  7. Atypical lymphocytes: These are seen in viral infections, especially infectious mononucleosis. Atypical lymphocytes are large, irregularly shaped lymphocytes with abundant cytoplasm and irregular nuclei. Cytoplasm shows deep basophilia at the edges and scalloping of borders. Nuclear chromatin is less dense and occasional nucleolus may be present.
  8. Blast cells: These are most premature of the leukocytes. They are large (15-25 μm), round to oval cells, with high nuclear cytoplasmic ratio. Nucleus shows one or more nucleoli and nuclear chromatin is immature. These cells are seen in severe infections, infiltrative disorders, and leukemia. In leukemia and lymphoma, blood smear suggests the diagnosis or differential diagnosis and helps in ordering further tests (see Figure 800.2 and Box 800.1).
 
Figure 800.2 Morphological abnormalities of white blood cells
Figure 800.2 Morphological abnormalities of white blood cells: (A) Toxic granules; (B) Döhle inclusion body; (C) Shift to left in neutrophil series; (D) Hypersegmented neutrophil in megaloblastic anemia; (E) Atypical lymphocyte in infectious mononucleosis; (F) Blast cell in acute leukemia
 
Further Reading:
 

RED CELLS MORPHOLOGY

Published in Hemotology
Tuesday, 25 July 2017 18:19
Role of blood smear in anemiasRed cells are best examined in an area where they are just touching one another (towards the tail of the film). Normal red cells are 7-8 μm in size, round with smooth contours, and stain deep pink at the periphery and paler in the center. Area of central pallor is about 1/3rd the diameter of the red cell. Size of a normal red cell corresponds roughly with the size of the nucleus of a small lymphocyte. Normal red cells are described as normocytic (of normal size) and normochromic (with normal staining intensity i.e. hemoglobin content).
 
Morphologic abnormalities of red cells in peripheral blood smear can be grouped as follows:
 
  • Red cells with abnormal size (see Figure 799.1)
  • Red cells with abnormal staining
  • Red cells with abnormal shape (see Figure 799.1)
  • Red cell inclusions (see Figure 799.2)
  • Immature red cells (see Figure799.3)
  • Abnormal red cell arrangement(see Figure 799.4).
 
Red cells with abnormal size:
 
Mild variation in red cell size is normal. Increased variation in red cell size is called as anisocytosis. This is a feature of most anemias and is non-specific. Anisocytosis is due to the presence of microcytes, macrocytes, or both in addition to red cells of normal size.
 
Microcytes are red cells smaller in size than normal. They are seen when hemoglobin synthesis is defective i.e. in iron deficiency anemia, thalassemias, anemia of chronic disease, and sideroblastic anemia.

Macrocytes are red cells larger in size than normal. Oval macrocytes (macro-ovalocytes) are seen in megaloblastic anemia, myelodysplastic syndrome, and in patients being treated with cancer chemotherapy. Round macrocytes are seen in liver disease, alcoholism, and hypothyroidism.
 
Red cells with abnormal staining (hemoglobin content):

Staining intensity of red cells depends on hemoglobin content. Red cells with increased area of central pallor (i.e. containing less hemoglobin) are called as hypochromic. They are seen when hemoglobin synthesis is defective, i.e. in iron deficiency, thalassemias, anaemia of chronic disease, and sideroblastic anemia.
 
In dimorphic anemia, there are two distinct populations of red cells in the same smear. An example is presence of both normochromic and hypochromic red cells seen in sideroblastic anemia, iron deficiency anemia responding to treatment, and following blood transfusion in a patient of hypochromic anemia. In myelodysplastic syndrome, dimorphic picture results from admixture of microcytic hypochromic cells and macrocytes.
 
Red cells with abnormal shape:
 
Increased variation in red cell shape is called as poikilocytosis and is a feature of many anemias. A red cell that is abnormal in shape is called as a poikilocyte.
 
Sickle cells are narrow and elongated red cells with one or both ends pointed. Sickle form is assumed when a red cell containing hemoglobin S is deprived of oxygen. Sickle cells are seen in sickle cell disorders, particularly sickle cell anemia. Sickle cells are not seen on blood smear in neonates with sickle cell disease because high percentage of fetal hemoglobin in red cells prevents sickling.
 
Spherocytes are red cells, which are slightly smaller in size than normal, round, stain intensely, and do not have central area of pallor. The surface area of spherocytes is less as compared to the volume. They are seen in hereditary spherocytosis, autoimmune hemolytic anemia (warm antibody type), and ABO hemolytic disease of newborn.
 
Schistocytes are fragmented red cells, which take various forms like helmet, crescent, triangle, etc. and usually have surface projections or spicules. They are seen in microangiopathic hemolytic anemia, cardiac valve prosthesis, and severe burns.
 
Target cells are red cells with bull's eye appearance. These red cells show a central stained area and a peripheral stained rim with unstained cytoplasm in between. They are seen in hemoglobinopathies (e.g. thalassemias, hemoglobin disease, sickle cell disease), obstructive jaundice, and following splenectomy.disease, sickle cell disease), obstructive jaundice, and following splenectomy.
 
Burr cells or echinocytes are small red cells with regularly placed small projections on surface. They are seen in uremia.
 
Acanthocytes are red cells with irregularly spaced sharp projections of variable length on surface. They are seen in spur cell anemia of liver disease, McLeod phenotype, and following splenectomy.
 
Teardrop cells or dacryocytes have a tapering droplike shape. Numerous teardrop red cells are seen in myelofibrosis and myelophthisic anemia.
 
Blister cells or hemi ghost cells are irregularly contracted cells in which hemoglobin is contracted and condensed away from the cell membrane. This is seen in glucose-6-phosphate dehydrogenase defici-ency during acute hemolytic episode.
 
Bite cells result from removal of Heinz bodies by the pitting action of the spleen (i.e. a part of red cell is bitten off by the splenic macrophages). They are seen in glucose-6-phosphate dehydrogena-se deficiency and unstable hemoglobin disease.
 
Red cell inclusions:
 
Those inclusions that can be visualized on Romanowsky-stained smears are basophilic stippling, Howell-Jolly bodies, Pappenheimer bodies, and Cabot's rings.

Basophilic stippling or punctate basophilia refers to the presence of numerous, irregular basophilic (purple-blue) granules which are uniformly distributed in the red cell. These granules represent aggregates of ribosomes. Their presence is indicative of impaired erythropoiesis and they are seen in thalassemias, megaloblastic anemia, heavy metal poisoning (e.g. lead), and liver disease.cell. These granules represent aggregates of ribosomes. Their presence is indicative of impaired erythropoiesis and they are seen in thalassemias, megaloblastic anemia, heavy metal poisoning (e.g. lead), and liver disease.
 
Red cell inclusions
Figure 799.2 Red cell inclusions: (A) Basophilic stippling; (B) Howell-Jolly bodies; (C) Pappenheimer bodies; (D) Cabot’s ring
 
Howell-Jolly bodies are small, round, purple-staining nuclear remnants located peripherally in red cells. They are seen in megaloblastic anemia, thalasse-mias, hemolytic anemia, and following splenectomy.

Pappenheimer bodies are basophilic, small, ironcontaining granules in red cells. They give positive Perl's Prussian blue reaction. Unlike basophilic stippling, Pappenheimer bodies are few in number and are not distributed throughout the red cell. They are seen following splenectomy and in thalassemias and sideroblastic anemia.

Cabot's rings are fine, reddish-purple or red, ring-like structures. They appear like loops or figure of eight structures. They indicate impaired erythropoiesis and are seen in megaloblastic anemia and lead poisoning.
 
Immature red cells:
 
Polychromatic cells are young red cells containing remnants of ribonucleic acid. These cells are slightly larger than normal red cells and have a diffuse bluishgrey tint. (They represent reticulocytes when stained with a supravital stain like new methylene blue). Polychromasia is due to the uptake of acid stain by hemoglobin and basic stain by ribonucleic acid. Presence of polychromatic cells is indicative of active erythropoiesis and are increased in hemolytic anemia, acute blood loss, and following specific therapy for nutritional anemia.and are increased in hemolytic anemia, acute blood loss, and following specific therapy for nutritional anemia.
 
Nucleated red cells are red cell precursors (erythroblasts), which are released prematurely in peripheral blood from the bone marrow. They are a normal finding in cord blood of newborns. Large number of nucleated red cells in blood smear is seen in hemolytic disease of newborn, hemolytic anemia, leukemias, myelophthisic anemia, and myelofibrosis.
 
Immature red cells
Figure 799.3 Immature red cells: (A) Polychromatic red cell; (B) Nucleated red cell
 
Abnormal red cell arrangement:
 
Rouleaux formation refers to alignment of red cells on top of each other like a stack of coins. It occurs in multiple myeloma, Waldenström's macroglobulinemia, hypergammaglobulinemia, and hyper fibrinogenemia.
 
Abnormal red cell arrangement
Figure 799.4 Abnormal red cell arrangement: (A) Rouleaux formation; (B) Autoagglutination

Autoagglutination refers to the clumping of red cells in large, irregular groups on blood smear. It is seen in cold agglutinin disease. Role of blood smear in anemia is shown in Box 799.1 and Figures 799.5 to 799.7.
 
Figure 799.5 Differential diagnosis of macrocytic anemia on blood smear
Figure 799.5 Differential diagnosis of macrocytic anemia on blood smear: (A) Megaloblastic anemia; (B) Hemolytic anemia; (C) Liver disease; (D) Myelodysplastic syndrome
 
Figure 799.6 Differential diagnosis of microcytic anemia on blood smear
Figure 799.6 Differential diagnosis of microcytic anemia on blood smear: (A) Iron deficiency anemia; (B) Thalassemia minor; (C) Thalassemia major; (D) Sideroblastic anemia
 
Figure 799.7 Differential diagnosis of hemolytic anemia on blood smear
Figure 799.7 Differential diagnosis of hemolytic anemia on blood smear. (A) Microangiopathic hemolytic anemia showing fragmented red cells, (B) Hereditary spherocytosis showing spherocytes and a polychromatic red cell, and (C) Glucose-6-phosphate dehydrogenase deficiency showing a blister cell and a bite cell
 
Further Reading:
 

PARTS AND FUNCTIONS OF A COMPOUND MICROSCOPE

Published in Microbiology
Tuesday, 25 July 2017 15:42
The microscope is the most important piece of equipment in the clinic laboratory. The microscope is used to review fecal, urine, blood, and cytology samples on a daily basis (see Figure). Understanding how the microscope functions, how it operates, and how to care for it will improve the reliability of your results and prolong the life of this valuable piece of equipment.

Parts and functions of a compound microscope

Compound Microscope(A) Arm: Used to carry the microscope.
 
(B) Base: Supports the microscope and houses the light source.
 
(C) Oculars (or eyepieces): The lens of the microscope you look through. The ocular also magnifies the image. The total magnification can be calculated by multiplying the objective power by the ocular power. Oculars come in different magnifications, but 10× magnification is common.
 
(D) Diopter adjustment: The purpose of the diopter adjustment is to correct the differences in vision an individual may have between their left and right eyes.
 
(E) Interpupillary adjustment: This allows the oculars to move closer or further away from one another to match the width of an individual’s eyes. When looking through the microscope, one should see only a single field of view. When viewing a sample, always use both eyes. Using one eye can cause eye strain over a period of time.
 
(F) Nosepiece: The nosepiece holds the objective lenses. The objectives are mounted on a rotating turret so they can be moved into place as needed. Most nosepieces can hold up to five objectives.
 
(G) Objective lenses: The objective lens is the lens closest to the object being viewed, and its function is to magnify it. Objective lenses are available in many powers, but 4×, 10×, 40×, and 100× are standard. 4× objective is used mainly for scanning. 10× objective is considered “low power,” 40× is “high power” and 100× objective is referred to as “oil immersion.” Once magnified by the objective lens, the image is viewed through the oculars, which magnify it further. Total magnification can be calculated by multiplying the objective power by the ocular lens power.
 
For example: 100× objective lens with 10× oculars = 1000× total magnification.
 
(H) Stage: The platform on which the slide or object is placed for viewing.
 
(I) Stage brackets: Spring-loaded brackets, or clips, hold the slide or specimen in place on the stage.
 
(J) Stage control knobs: Located just below the stage are the stage control knobs. These knobs move the slide or specimen either horizontally (x-axis) or vertically (y-axis) when it is being viewed.
 
(K) Condenser: The condenser is located under the stage. As light travels from the illuminator, it passes through the condenser, where it is focused and directed at the specimen.
 
(L) Condenser control knob: Allows the condenser to be raised or lowered.
 
(M) Condenser centering screws: These crews center the condenser, and therefore the beam of light. Generally, they do not need much adjustment unless the microscope is moved or transported frequently.
 
(N) Iris diaphragm: This structure controls the amount of light that reaches the specimen. Opening and closing the iris diaphragm adjusts the diameter of the light beam.
 
(O) Coarse and fine focus adjustment knobs: These knobs bring the object into focus by raising and lowering the stage. Care should be taken when adjusting the stage height. When a higher power objective is in place (100× objective for example), there is a risk of raising the stage and slide and hitting the objective lens. This can break the slide and scratch the lens surface. Coarse adjustment is used for finding focus under low power and adjusting the stage height. Fine adjustment is used for more delicate, high power adjustment that would require fine tuning.
 
(P) Illuminator: The illuminator is the light source for the microscope, usually situated in the base. The brightness of the light from the illuminator can be adjusted to suit your preference and the object you are viewing.

KOHLER ILLUMINATION

Published in Microbiology
Tuesday, 25 July 2017 13:43
What is Kohler illumination?

Kohler illumination is a method of adjusting a microscope in order to provide optimal illumination by focusing the light on the specimen. When a microscope is in Kohler, specimens will appear clearer, and in more detail.

Process of setting Kohler
 
Materials required
 
  • Specimen slide (will need tofocus under 10× power)
  • Compound microscope.
 
Kohler illumination
 
  1. Mount the specimen slide onthe stage and focus under 10×.
  2. Close the iris diaphragm completely.
  3. If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
  4. Using the condenser adjustment knobs, raise or lower the condenser until the edges of the field becomes sharp (see Figure 797.1 and Figure 797.2).
  5. Open the iris diaphragm until the entire field is illuminated.
 
Note the blurry edges of the unfocused light
Figure 797.1 Note the blurry edges of the unfocused light
 
Adjusting the condenser height sharpens the edges of the ball of light
Figure 797.2 Adjusting the condenser height sharpens the edges of the “ball of light.”
 
When should you set/check Kohler?
 
  • During regular microscope maintenance
  • After the microscope is moved/transported
  • Whenever you suspect objects do not appear as sharp as they could be.
 
Further Reading:
 

PROCEDURES FOR THE COLLECTION OF BLOOD FOR HEMOTOLOGICAL INVESTIGATIONS

Published in Hemotology
Monday, 24 July 2017 10:21
COLLECTION OF BLOOD
 
For reliable and accurate results of laboratory tests, it is essential to follow a standard procedure for specimen collection. For hematological investigations, blood sample can be obtained from the skin puncture or venepuncture.
 
SKIN PUNCTURE

This method is commonly used in infants and small children and if the amount of blood required is small. It is suitable for cell counts, estimation of hemoglobin, determination of hematocrit by micro method, and preparation of blood films. Blood obtained by skin puncture is also called as capillary blood. However, it is a mixture of blood from capillaries, venules, and arterioles. It also contains some tissue fluid. In adults, blood is obtained from the side of a ring or middle finger (distal digit) or ear lobe. In infants, it is collected from the heel (lateral or medial aspect of plantar surface) or great toe (see Figure 796.1).
 
A. Blood lancet and sites of B. finger puncture cross and C. heel puncture shaded areas
Figure 796.1 (A) Blood lancet and sites of (B) finger puncture (cross) and (C) heel puncture (shaded areas)

The puncture site is cleansed with 70% ethanol or other suitable disinfectant. After drying, a puncture, sufficiently deep to allow free flow of blood, is made with a sterile, dry, disposable lancet. The first drop of blood is wiped away with sterile, dry cotton as it contains tissue fluid. Next few drops of blood are collected. Excessive squeezing should be avoided, as it will dilute the blood with tissue fluid. After collection a piece of sterile cotton is pressed over the puncture site till bleeding ceases. As compared to the venous blood, hemoglobin, hematocrit, and red cell count are slightly higher in blood from skin puncture. As platelets adhere to the puncture site, platelet count is lower. Because of small sample size, immediate repeat testing is not possible if the result is abnormal. Blood should not be collected from cold, cyanosed skin since false elevation of values of hemoglobin and red/white cell counts will be obtained.

VENOUS BLOOD COLLECTION

When multiple tests are to be done and larger quantity of blood is needed, anticoagulated venous blood should be obtained.

Method
 
  1. Common sites of venepuncture in antecubital fossaDue to the ease of access, blood is best obtained from the veins of the antecubital fossa (see Figure; Common sites of venepuncture in antecubital fossa (red circles)). A rubber tourniquet (18 inches long × 3/4 or 1 inch in adults and 12 inches × 1/8 inch in children) is applied to the upper arm. It should not be too tight and should not remain in place for more than two minutes. Patient is asked to make a fist so that veins become more prominent and palpable.
  2. Venepuncture site is cleansed with 70% ethanol and allowed to dry.
  3. The selected vein is anchored by compressing and pulling the soft tissues below the puncture site with the left hand.
  4. Sterile, disposable needles and syringes should be used for venepuncture. Needle size should be 19- to 21-gauge in adults and 23-gauge in children. Venepuncture is performed with the bevel of the needle up and along the direction of the vein. Blood is withdrawn slowly. Pulling the plunger quickly can cause hemolysis and collapse of the vein. Tourniquet should be released as soon as the blood begins to flow into the syringe.
  5. When the required amount of blood is withdrawn, the patient is asked to open his/her fist. The needle is withdrawn from the vein. A sterile cotton gauze is pressed over the puncture site. Patient is asked to press the gauze over the site till bleeding stops.
  6. The needle is detached from the syringe and the required amount of blood is carefully delivered into the tube containing appropriate anticoagulant (see later). If the blood is forced through the needle without detaching it, hemolysis can occur. Containers may be glass bottles or disposable plastic tubes with caps and flat bottom.
  7. Blood is mixed with the anticoagulant in the container thoroughly by gently inverting the container several times. The container should not be shaken vigorously as it can cause frothing and hemolysis.
    Check whether the patient is feeling faint and bleeding has stopped. Cover the puncture site with an adhesive bandage strip. After use, disposable needles should be placed in a puncture-proof container for proper disposal. Recapping of needle by hand can cause needle-stick injury. The container is labeled. Time of collection should be noted on the label. Sample should be sent immediately to the laboratory with accompanying properly filled order form.
 
Precautions
 
  1. Blood is never collected from an intravenous line or from the arm being used for intravenous line (since it will dilute the blood sample). Blood is not collected from a sclerosed vein and from an area with hematoma.
  2. Tourniquet should not be too tight and should not be applied for more than 2 minutes as it will cause hemoconcentration and alteration of test results.
  3. Puncture site should be allowed to dry completely after cleaning with alcohol (before performing the venepuncture).
  4. Tourniquet should be released before removing the needle from the vein (to prevent hematoma formation).
  5. To avoid hemolysis, blood is withdrawn gradually, a small-bore needle should not be used, and the needle is detached from the syringe before dispensing blood into the container.
  6. All blood samples are considered as infectious and proper precautions should be observed while collecting blood either from a vein or a skin puncture. Anticoagulated blood sample should be tested within 1-2 hours of collection. If this is not possible, sample can be stored in a refrigerator at 4-6°C for maximum of 24 hours. After the sample is taken out of refrigerator, it should be allowed to return to room temperature, mixed properly, and then tested.
 
Complications
 
  1. Failure to obtain blood: This happens if vein is missed, or excessive pull is applied to the plunger causing collapse of the vein.
  2. Occurrence of hematoma, thrombosis, thrombophlebitis, abscess, or bleeding.
  3. Transmission of infections like hepatitis B or human immuno-deficiency virus if reusable needles and syringes, which are not properly sterilised, are used.
 
Further Reading:
 

SEQUENCE OF FILLING OF TUBES FOR HEMOTOLOGICAL INVESTIGATIONS

Published in Hemotology
Saturday, 22 July 2017 12:14
SEQUENCE OF FILLING OF TUBES
 
Following order of filling of tubes should be followed after withdrawal of blood from the patient if multiple investigations are ordered:
 
  1. First tube: Blood culture.
  2. Second tube: Plain tube (serum).
  3. Third tube: Tube containing anticoagulant (EDTA, citrate, or heparin).
  4. Fourth tube: Tube containing additional stabilizing agent like fluoride.
 
Further Reading:
 

USE OF PLASMA VS. SERUM [DIFFERENCE BETWEEN PLASMA AND SERUM]

Published in Hemotology
Saturday, 22 July 2017 11:59
Plasma is the supernatant liquid obtained after centrifugation of anticoagulated whole blood.
 
Serum is the liquid obtained after clotting of whole blood sample collected in a plain tube.
 
Some of the differences between the two are as follows:
 
  1. Plasma contains fibrinogen as well as all the other proteins, while serum does not contain fibrinogen.
  2. Plasma can be obtained immediately after sample collection by centrifugation, while minimum of 30 minutes are required for separation of serum from the clotted blood.
  3. Amount of sample is greater with plasma than with serum for a given amount of blood.
  4. Use of anticoagulant may alter the concentration of some constituents if they are to be measured like sodium, potassium, lithium, etc.

PLAIN TUBES (Without any anticoagulant) AND FLUORIDE TUBES FOR COLLECTION OF BLOOD

Published in Hemotology
Saturday, 22 July 2017 11:43
Plain tubes (i.e. without any anticoagulant) are used for chemistry studies after separation of serum: liver function tests (total proteins, albumin, aspartate aminotransferase, alanine aminotransferase, bilirubin), renal function tests (blood urea nitrogen, creatinine), calcium, lipid profile, electrolytes, hormones, and serum osmolality. Fluoride bulb is used for collection of whole blood for estimation of blood glucose. Addition of sodium fluoride (2.5 mg/ml of blood) maintains stable glucose level by inhibiting glycolysis. Sodium fluoride is commonly used along with an anticoagulant such as potassium oxalate or EDTA.

International Council for Standardization in Haematology (ICSH)

Published in Hemotology
Saturday, 22 July 2017 11:23
The International Council for Standardization in Haematology (ICSH) was initiated as a standardization committee by the European Society of Haematology (ESH) in 1963 and officially constituted by the International Society of Hematology (ISH) and the ESH in Stockholm in 1964. The ICSH is recognised as a Non-Governmental Organisation with official relations to the World Health Organisation (WHO).
 
The ICSH is a not-for-profit organisation that aims to achieve reliable and reproducible results in laboratory analysis in the field of diagnostic haematology.
 
The ICSH coordinates Working Groups of experts to examine laboratory methods and instruments for haematological analyses, to deliberate on issues of standardization and to stimulate and coordinate scientific work as necessary towards the development of international standardization materials and guidelines.
Advertisement

Useful Sites

  • NCBI

    National Center for Biotechnology Information
  • LTO

    Lab Tests Online® by AACC
  • ASCP

    American Society for Clinical Pathology
  • ASM

    American Society for Microbiology
  • The Medical Library®

    Project of BioScience.pk
Advertisement

Connect With Us

Contact Us

All comments and suggestions about this web site are very welcome and a valuable source of information for us. Thanks!

Tel: +(92) 302 970 8985-6

Email: This email address is being protected from spambots. You need JavaScript enabled to view it.

Website: https://www.bioscience.pk



This website is certified by Health On the Net Foundation. Click to verify. This site complies with the HONcode standard for trustworthy health information:
verify here.

Our Sponsors

InsightGadgets.comPathLabStudyTheMedicalLibrary.orgThe Physio ClubB2BPakistan.com

By using BioScience.pk you agree to our use of cookies to enhance your experience on this website.