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A blood smear is examined for:
 
 
A peripheral blood smear has three parts: Head, body, and tail. Also read: PREPARATION OF BLOOD SMEAR BY WEDGE METHOD.
 
A blood smear should be examined in an orderly manner. Initially, blood smear should be observed under low power objective (10×) to assess whether the film is properly spread and stained, to assess cell distribution, and to select an area for examination of blood cells. Best morphologic details are seen in the area where red cells are just touching one another. Low power view is also helpful for the identification of Rouleaux formation, autoagglutination of red cells, and microfilaria. High power objective (45×) is suitable for examination of red cell morphology and for differential leukocyte count. A rough estimate of total leukocyte count can be obtained which also serves to crosscheck the total leukocyte count done by manual counting or automated method. Oil-immersion objective (100×) is used for more detailed examination of any abnormal cells.
 
Further Reading:
 
Box 802.1 Role of blood smear in thrombocytopeniaPlatelets are small, 1-3 μm in diameter, purple structures with tiny irregular projections on surface. In blood films prepared from non-anticoagulated blood (i.e. direct fingerstick), they occur in clumps. If platelet count is done on automated blood cell counters using EDTA-anticoagulated blood sample, about 1% of persons show falsely low count due to the presence in them of EDTA dependent antiplatelet antibody. Examination of a parallel blood film is useful in avoiding the false diagnosis of thrombocytopenia in such cases. Occasionally, platelets show rosetting around neutrophils (platelet satellitism) (see Figure 802.1). This is seen in patients with platelet antibodies and in apparently normal persons. Blood smear examination can be helpful in determining underlying cause of thrombocytopenia such as leukemia, lymphoma, or microangiopathic hemolytic anemia (Box 802.1).
 
Also Read:
 
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.
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:
 
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:
 

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

(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.

Compound Microscope

(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.

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:

COLLECTION OF BLOOD

It is necessary to follow a standard procedure for specimen collection to get the most accurate and trustworthy results of the laboratory test. The blood sample can be collected from the venipuncture or skin puncture for the hematological investigations.

SKIN PUNCTURE

This method is most common and mostly used in infants and small children and if the small amount of blood is required. This method is suitable for the estimation of hemoglobin, cell counts, determination of hematocrit (HCT) or packed cell volume (PCV) by micro method and preparation of blood films. Blood obtained by this method is also called as capillary blood. However, it is the mixture of blood from arterioles, venules, and capillaries. It also contains small amount of tissue fluid. In infants, blood is collected from the heel (the medial or lateral aspect of plantar surface or great toe). In adults, it is collected from the side of a middle or ring finger (distal digit) or from the earlobe. (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 the 70% ethanol or another suitable disinfectant. After drying, a puncture is made with a sterile, dry, disposable lancet, in deep to allow free flow of blood. The first drop of blood is wiped away with the dry and sterile cotton as it contains tissue fluid. After wiping the first drop of blood, next few drops of blood are collected. Excessive pressing should be avoided, as it may dilute the blood with the tissue fluid. After collection of blood, a piece of dry and sterile cotton is pressed over the puncture site till the bleeding ends. Hemoglobin, red cell count and hematocrit (HCT) or packed cell volume (PCV) are moderately higher in the blood collected from skin puncture, as compared to the venous blood. The reason behind this scenario is that platelets adhere to the puncture site and cause the lower count of platelet, and due to small sample size, instant repeat testing is not possible if the result is abnormal or unusual.

Avoid collecting blood from cold, cyanosed skin since the false elevation of values of red blood cells, white blood cells and hemoglobin will be obtained.

VENOUS BLOOD COLLECTION

Venous blood is obtained when the larger quantity of blood is needed to perform multiple tests. Different test tubes are filled with blood as per requirement of anticoagulant and blood ratio for the test. Anticoagulant is not required for the test performed by the serum.

Method

  1. Common sites of venepuncture in antecubital fossaThe best site for obtaining blood is the veins of antecubital fossa. A rubber tourniquet is applied to the upper arm (see Figure; Common sites of venepuncture in antecubital fossa (red circles)). It should not be too much tight and should not remain in a place for more than 120 seconds. To get veins more palpable and prominent, the patient is asked to make a fist.
  2. The puncture site is cleansed with the 70% ethanol or other suitable disinfectant and allowed to dry.
  3. The preferred vein is anchored by squeezing and pulling the soft tissues below the prick site with the left hand.
  4. Sterile, dry, disposable needles and syringes should be used for the collection of blood. Needle size should be 23-gauge in children and 19- to 21-gauge in adults. Venepuncture is made along with the direction of the vein and with the bevel of the needle up. Blood is withdrawn slowly. Pulling the piston quickly can cause hemolysis and collapse the vein. The tourniquet should be released as soon as the blood begins to flow into the syringe.
  5. When the required blood is collected, the patient is asked to open his/her fist. The needle is removed from the vein. A sterile alcohol swab is pressed over the puncture site. The patient is asked to press the alcohol swab over the site till the bleeding ends.
  6. The needle is removed from the syringe and the required amount of blood is carefully transferred into the test tube containing anticoagulant as per requirement of the laboratory test. If the blood is forced through the syringe without removing the needle, hemolysis can occur. Containers may be glass bottles or disposable plastic tubes with corks 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 strenuously as it can cause hemolysis and fizzing.
  8. Check whether the patient is dizzy and bleeding has stopped. Cover the site of puncture with a sticky bandage strip. Recapping the needle by hand can cause needle-prick injury. After the usage of disposable syringe, needles are crashed by the syringe needle destroyer and the syringe is disposed into the biohazard box. The blood container is labeled properly with the patient’s name, age, gender and the time of collection. The sample should be sent without delay to the laboratory with accompanying properly filled laboratory requisition form.

Precautions

  1. The tourniquet should not be too tight and should not be applied for more than 120 seconds as it will cause hemoconcentration and variation of test results.
  2. The tourniquet should be released before removing the needle from the vein to prevent the formation of a hematoma.
  3. Blood is never collected from the arm being used for the intravenous line since it will dilute the blood sample.
  4. Blood is never collected from an area with hematoma and from a sclerosed vein.
  5. A small bore needle should not be used, blood is withdrawn gradually and the needle is removed from the syringe before transferring blood into the container to avoid hemolysis.
  6. Proper precautions should be noticed while collecting blood either from a skin or a vein puncture since all blood samples are considered as infectious.
  7. The anticoagulated blood sample should be tested within 1-2 hours of collection. If this is not possible, the sample can be stored for 24 hours in a refrigerator at 4-6° C. After the sample is taken out of the refrigerator, it should be allowed to return to room temperature, mixed properly, and then laboratory test is performed.

Complications

  1. Failure to obtain blood: This is very common and usually painful for the patient. This happens if the vein is missed, or excessive pull is applied to the piston causing collapse of the vein.
  2. Formation of hematoma, abscess, thrombosis, thrombophlebitis, or bleeding.
  3. Transmission of infection like human immunodeficiency virus (HIV) or hepatitis B virus (HBV) if reusable syringes and needles, which are not properly sterilized, are used.

Further Reading:

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:
 
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 (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.
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.
Anticoagulants used for hematological investigations are ethylene diamine tetra-acetic acid (EDTA), heparin, double oxalate, and trisodium citrate (Table 791.1).
 
Table 791.1 Salient features of three main anticoagulants used in the hematology laboratory
Salient features of three main anticoagulants used in the hematology laboratory
 
Ethylene Diamine Tetra-acetic Acid (EDTA)
 
Changes occurring due to prolonged storage of blood in EDTAThis is also called as Sequestrene or Versene. This is the recommended anticoagulant for routine hematological investigations. However, it cannot be used for coagulation studies. Disodium and dipotassium salts of EDTA are in common use. International Committee for Standardization in Hematology recommends dipotassium EDTA since it is more soluble. It is used in a concentration of 1.5 mg/ml of blood. Dried form of anticoagulant is used as it avoids dilution of sample. Its mechanism of action is chelation of calcium. Proportion of anticoagulant to blood should be maintained. EDTA in excess of 2mg/ml causes shrinkage of and degenerative changes in red and white blood cells, decrease in hematocrit, and increase in mean corpuscular hemoglobin concentration. Excess EDTA also causess welling and fragmentation of platelets, which leads to erroneously high platelet counts. Prolonged storage of blood in EDTA anticoagulant leads to alterations as shown in Figure 791.1 and Box 791.1. EDTA is used for estimation of hemoglobin, hematocrit, cell counts, making blood films, sickling test, reticulocyte count, and hemoglobin electrophoresis.
 
Preparation
 
Dipotassium EDTA 20 gm
Distilled water 200 ml
 
Mix to dissolve. Place 0.04 ml of this solution in a bottle for 2.5 ml of blood. Anticoagulant should be dried on a warm bench or in an incubator at 37°C before use. For routine hematological investigations, 2-3 ml of EDTA blood is required.
 
Changes in blood cell morphology crenation of red cells separation of nuclear lobes of neutrophil vacuoles in cytoplasm and irregular lobulation of monocyte and lymphocyte nuclei due to storage of blood in EDTA anti
Figure 791.1 Changes in blood cell morphology (crenation of red cells, separation of nuclear lobes of neutrophil, vacuoles in cytoplasm, and irregular lobulation of monocyte and lymphocyte nuclei) due to storage of blood in EDTA anticoagulant for prolonged time
 
Heparin
 
Heparin prevents coagulation by enhancing the activity of anti-thrombin III (AT III). AT III inhibits thrombin and some other coagulation factors. It is used in the proportion of 15-20 IU/ ml of blood. Sodium, lithium, or ammonium salt of heparin is used. Heparin should not be used for total leukocyte count (since it causes leukocyte clumping) and for making of blood films (since it imparts a blue background). It is used for osmotic fragility test (since it does not alter the size of cells) and for immunophenotyping.
 
Double Oxalate (Wintrobe Mixture)
 
This consists of ammonium oxalate and potassium oxalate in 3:2 proportion. This combination is used to balance the swelling of red cells caused by ammonium oxalate and shrinkage caused by potassium oxalate. Mechanism of anticoagulant action is removal of calcium. It is used for routine hematological tests and for estimation of erythrocyte sedimentation rate by Wintrobe method. As it causes crenation of red cells and morphologic alteration in white blood cells, it cannot be used for making of blood films.
 
Preparation
 
Ammonium oxalate 1.2 gm
Potassium oxalate 0.8 gm
Distilled water upto 100 ml
 
Place 0.5 ml of this solution in a bottle for 5 ml of blood. Anticoagulant should be dried in an incubator at 37°C or on a warm bench before use.
 
Trisodium Citrate (3.2%)
 
This is the anticoagulant of choice for coagulation studies and for estimation of erythrocyte sedimentation rate by Westergren method.
 
Preparation
 
Trisodium citrate 3.2 gm
Distilled water upto 100 ml
 
Mix well to dissolve. Store in a refrigerator at 2-8°C.
 
Use 1:9 (anticoagulant: blood) proportion for coagulation studies; for ESR, 1:4 proportion is recommended.
 
ESR should be measured within 4 hours of collection of blood, while coagulation studies should be performed within 2 hours.
 
Further Reading:
 

ABO Grouping

There are two methods for ABO grouping:

  • Cell grouping (forward grouping): Red cells are tested for the presence of A and B antigens employing known specific anti-A and anti-B (and sometimes anti-A, B) sera.
  • Serum grouping (reverse grouping): Serum is tested for the presence of anti-A and anti-B antibodies by employing known group A and group B reagent red cells.

Both cell and serum grouping should be done since each test acts as a check on the other.

There are three methods for blood grouping: slide, tube and microplate. Tube and microplate methods are better and are employed in blood banks.

Further Reading:

  1. Autoagglutination: Presence of IgM autoantibodies reactive at room temperature in patient’s serum can lead to autoagglutination. If autocontrol is not used, blood group in such a case will be wrongly typed as AB. Therefore, for correct result, if autocontrol is also showing agglutination, cell grouping should be repeated after washing red cells with warm saline, and serum grouping should be repeated at 37°C.
  2. Rouleaux formation: Rouleux formation refers to red cells adhering to each other like a stack of coins and can be mistaken for agglutination. Rouleaux formation is caused by high levels of fibrinogen, immunoglobulins, or intravenous administration of a plasma expander such as dextran. Rouleaux formation (but not agglutination) can be dispersed by addition of normal saline during serum grouping.
  3. False-negative result due to inactivated antisera: For preservation of potency of antisera, they should be kept stored at 4°-6°C. If kept at room temperature for long, antisera are inactivated and will give false-negative result.
  4. Age: Infants start producing ABO antibodies by 3-6 months of age and serum grouping done before this age will yield false-negative result. Elderly individuals also have low antibody levels.
D antigen is the most immunogenic after ABO antigens and therefore red cells are routinely tested for D. Individuals are called as Rh-positive or Rh-negative depending on presence or absence of D antigen on their red cells. Following transfusion of Rhpositive blood to Rh-negative persons, 70% of them will develop anti Rh-D antibodies. This is of particular importance in women of childbearing age as anti-D antibodies can crosss the placenta during pregnancy and destroy Dpositive fetal red cells and cause hemolytic disease of newborn. In other sensitized individuals, reexposure to D antigen can cause hemolytic transfusion reaction.
 
In Rh D grouping, patient’s red cells are mixed with anti-D reagent. Serum or reverse grouping is not carried out because most Rhnegative persons do not have anti-D antibodies; anti-D develops in Rh-negative individuals only following exposure to Rh-positive red cells.
 
Rh typing is done at the same time as ABO grouping. Method of Rh D grouping is similar in principle to ABO grouping. Since serum or reverse grouping is not possible, each sample is tested in duplicate. Dosage effect (stronger antigenantibody reaction in homozygous cells i.e. stronger reaction with DD) is observed with antigens of the Rh system. Autocontrol (patient’s red cell + patient’s serum) and positive and negative controls are included in every test run. Monoclonal IgM anti-D antiserum should be used for cell grouping, which allows Rh grouping to be caried out at the same time as ABO grouping at room temperature. With monoclonal antisera, most weak and variant forms of D antigen are detected and further testing for weak forms of D antigen (Du) is not required. Differences between ABO and Rh grouping are shown in Table 788.1.
 
Table 788.1 Comparison of ABO grouping and Rh typing
Comparison of ABO grouping and Rh typing
Microplate is a polystyrene plate consisting of 96 micro wells of either U- or V-shape. Grouping is carried out in micro wells. This method is sensitive and ideal for large number of samples (see Figure 787.1).
 
Further reading: Rh D GROUPING METHOD
Principle
 
Red cells from the specimen are reacted with reagent antisera (anti-A and anti-B). Agglutination of red cells indicates presence of corresponding antigen (agglutinogen) on red cells.
 
Specimen
 
Capillary blood from finger prick, or venous blood collected in EDTA anticoagulant.
 
Reagents
 
ABO antisera: See box 786.1 and Figure 786.1.
 
BOX ABO antisera
Box 786.1: ABO antisera
 
Anti A and anti B sera used for cell grouping
 Figure 786.1 Anti-A and anti-B sera used for cell grouping
 
Method
 
  1. A clean and dry glass slide is divided into two sections with a glass marking pencil. The sections are labeled as anti-A and anti-B to identify the antisera (see Figure 786.2).
  2. Place one drop of anti-A serum and one drop of anti-B serum in the center of the corresponding section of the slide. Antiserum must be taken first to ensure that no reagents are missed.
  3. Add one drop of blood sample to be tested to each drop of antiserum.
  4. Mix antiserum and blood by using a separate stick or a separate corner of a slide for each section over an area about 1 inch in diameter.
  5. By tilting the slide backwards and forwards, examine for agglutination after exactly two minutes.
  6. Result:
    Positive (+): Little clumps of red cells are seen floating in a clear liquid.
    Negative (–): Red cells are floating homogeneously in a uniform suspension.
  7. Interpretation: Interpret the result as shown in the Table 786.1 and Figure 786.2.
 
Table 786.1 Interpretation of cell grouping (forward grouping) by slide test
Anti-A Anti-B Blood Group
+ - A
- + B
+ + AB
- - O
 
Cell grouping by slide method
Figure 786.2 Cell grouping by slide method
 
Slide test is quick and needs only simple equipment. It can be used in blood donation camps and in case of an emergency. However, it is not recommended as a routine test in blood banks since weakly reactive antigens on cells on forward grouping and low titer anti-A and anti-B on reverse grouping may be missed. Also, drying of the reaction mixture at the edges causes aggregation that may be mistaken for agglutination. Results of slide test should always be confirmed by cell and serum grouping by tube method.
Test tube method is more reliable than slide test, but takes longer time and more equipment. For cell grouping, patient’s saline-washed red cells are mixed with known antiserum in a test tube; the mixture is incubated at room temperature, and centrifuged. For serum grouping, patient’s serum is mixed with reagent red cells of known group (available commercially or prepared in the laboratory), incubated at room temperature, and centrifuged (See Table). Following centrifugation, a red cell button (sediment) will be seen at the bottom of the tube. Cell button is dislodged by gently tapping the base of the tube and examined for agglutination.
 
Positive (+) Test
 
Clumps of red cells suspended in a clear fluid. Agglutination in tube test is graded from 1+ to 4+ and read macroscopically (See Figure). 
 
Grading of ABO tube test
Grading of ABO tube test. Negative: Uniform suspension of red cells; Grade 1 (1+): Many small clumps of red cells (fine granular appearance); Grade 2 (2+): Many large clumps with many free red cells; Grade 3 (3+): Three or four individual clumps with few free red cells; and Grade 4 (4+): One solid clump of red cells with no free red cells
 
Negative (–) Test
 
Uniform suspension of red cells.

Separate tubes of auto-control, positive control, and negative control should always be setup along with the test sample tube. Auto-control tube consists of mixture of patient’s red cells and patient’s own serum. This is required to rule out false-positive result due to auto antibodies in patient’s serum causing auto agglutination of patient’s own red cells. Auto-control test is particularly essential when ABO grouping is being done only by forward method and blood group is typed as AB. If there are auto antibodies in recipient’s serum, ABO grouping, Rh typing, antibody screening, and cross matching all will show positive result.
 
In two positive control tubes, anti-A serum is mixed with group. A red cells and anti-B is mixed with group B red cells respectively. In two negative control tubes, anti-A serum is mixed with group B red cells and anti-B serum is mixed with group. A red cells respectively. These controls are necessary to confirm that reagents are working properly.
 
Interpretation of forward (cell) and reverse (serum) grouping
Interpretation of forward cell and reverse serum grouping
 
Why test tube method of blood grouping is more reliable than slide method?
 
Test tube method of blood grouping is more reliable than slide method. This is because centrifugation enhances the reaction by bringing antigen and antibodies closer together and allows detection of weaker antigen antibody reactions; in addition drying is avoided and smaller amounts of reagent are required.
 
If forward grouping, reverse grouping, and autocontrol tests are all positive, then these results are probably indicative of a cold-reactive autoantibody. Before performing forward typing, red cells should be washed with normal saline to elute the antibody. Before performing reverse grouping, autoantibody should be adsorbed by washed cells till autocontrol is negative.
A WBC differential count gives us information regarding the proportion and numbers of individual leukocytes in the patient’s sample, including significant morphological changes. This can provide useful diagnostic information in cases of inflammation, infection, and antigenic responses.

METHOD
 
Equipment
 
Stained PBS, microscope with 100×objective lens and cell counter.
 
Procedure
 
It is important that examinationand counts be performed withinthe monolayer area of your slide
 
  1. Scan the slide in a methodical grid pattern, in order not to cover the same area twice. Counts can be completed quickly under 400×magnification, but if you are also evaluating morphology, 1000×magnification should be used.
  2. Count a minimum of 100 WBCs.
 
(If the total WBC Count is increased, 200 cells should be counted to maintain accuracy.)
 
Calculations
 
Relative count:
 
No. of Cell Type Seen = ___%
100
 
Absolute count:
 
Relative (%) x WBC Count (10³/ L) = ___ x 10³/μL
100
 
Note: Check your math:
 
• Relative counts of each cell type should add up to equal 100
• Absolute counts of each cell type should add up to equal your WBC count.

Erythrocyte (Gr. erythros, red; kytos, cell) or red blood corpuscles are circular, anucleated, highly flexible, biconcave disc-shaped cells with high edges. The sixe of each cell averages 7.2 micrometer in diameter and 2.1 micrometer in thickness. It is 1.0 micrometer thick in the center. A complex membrane surrounds it, which is a bimolecular layer of protein. There is an inner most structure, called stroma, which is composed of lipids and proteins in the form of a fibrous protein. The cell contents are 90% hemoglobin. There are two methods for estimation of erythrocyte count:

  • Manual or microscopic method
  • Automated method

MANUAL METHOD

Equipment

Hemocytometer with cover glass, compound microscope.

Reagent

Hayem’s diluting solution is prepared as follows:

  • HgCl2 0.05 gm
  • NaSO4 2.5 gm
  • NaCl 0.5 gm
  • Distilled water 100 ml

Specimen

EDTA anticoagulated venous blood or blood obtained by skin puncture is used.

Method

  1. Wipe finger with cotton soaked with alcohol, with a sterile lancet do small prick on the finger tip. Use pipette. Aspirate blood to 0.5.
  2. Aspirate diluting Hayem’s solution to the 101 mark. It will give 1:200 dilution of the blood.
  3. Hold the pipette horizontally and role it with both hands between finger and thumb.
  4. Place the counting chamber, absolutely free from dust and grease, on the table and lay the cover glass in place over the ruled area.
  5. Discard the first two or three drops from the pipette. Charge the counting chamber by holding the pipette in an inclined position. Allow 3 minutes for the cells to settle.
  6. Locate the central square, which is divided into 25 medium sized squares. Each of the medium sized squares is further divided into 16 smallest squares.
  7. Count the erythrocytes in medium sized squares (80 smallest squares) using high power objective.
  8. In order to avoid confusion in counting, count all cells wihich touch the upper and left outer double line of the group of 16 squares as if they were inside the square. Neglect all those cells, which touch the lower and right inner line.

Calculation

You may calculate total number of erythrocytes per cu mm of the blood as shown in the following.

Supose number of erythrocytes counted in 5 intermediate squares

= E
 
Area of each of the five squares in which cells are counted
 
= 1/25 sq mm
 
Therefore, total area counted
 
= 1/25 sq mm x 5
= 1/5 sq mm
 
Depth of chamber = 1/10 mm
 
Therefore, the volume in which cells are counted
 
= Area x Depth
= 1/5 sqmm x 1/10 mm
= 1/50 cu mm
 
Now, in 1/50 cu mm of diluted blood, the number of erythrocyte counted = E
 
Number of erythrocyte in one cu mm in diluted blood = E x 50
 
Since the dilution of the blood is 1 in 200, the number of erythrocytes in one cu mm of undiluted blood
 
= E x 50 x 200
 

GENERAL NOTES


(1) Increased in numbers of RBC called polycythemia it is due to
 
Congenital heart disease
• Cor pulmonale
Dehydration
• Pulmonary fibrosis
• Polycythemia vera
 
(2) Decreased in numbers of RBC is due to
 
• Anemia
Bone marrow failure
• Erythropoietin deficiency (2ndry to kidney disease)
Hemolysis (RBC destruction) from transfusion reaction
Hemorrhage
• Leukemia
• Multiple myloma
• Nutritional deficiencies of (Iron, Copper, Folate, Vit B12, B6)
 

REFERENCE RANGES

  • Newborns 4.8-7.2 millions
  • Children 3.8-5.5 millions
  • Adult (Male) 4.6-6.0 millions
  • Adult (Female) 4.2-5.0 millions
  • Pregnancy slightly lower than normal

Principle

Anticoagulated whole blood is centrifuged in a capillary tube of uniform bore to pack the red cells. Centrifugation is done in a special microhematocrit centrifuge till packing of red cells is as complete as possible. The reading (length of packed red cells and total length of the column) is taken using a microhematocrit reader, a ruler, or arithmetic graph paper.

Equipment

  1. Microhematocrit centrifuge: It should provide relative centrifugal force of 12000 g for 5 minutes.
  2. Capillary hematocrit tubes: These are disposable glass tubes 75 mm in length and 1 mm in internal diameter. They are of two types: plain (containing no anticoagulant) and heparinised (coated with a dried film of 2 units of heparin). For plain tubes, anticoagulated venous blood is needed. Heparinised tubes are used for blood obtained from skin puncture.
  3. Tube sealant like plastic sealant or modeling clay; if not available, a spirit lamp for heat sealing.
  4. Microhematocrit reader; if not available, a ruler or arithmetic graph paper.

Specimen

Venous blood collected in EDTA (dipotassium salt) for plain tubes or blood from skin puncture collected directly in heparinised tubes. Venous blood should be collected with minimal stasis to avoid hemoconcentration and false rise in PCV.

Method

  1. Fill the capillary tube by applying its tip to the blood (either from skin puncture or anticoagulated venous blood, depending on the type of tube used). About 2/3rds to 3/4ths length of the capillary tube should be filled with blood.
  2. Seal the other end of the capillary tube (which was not in contact with blood) with a plastic sealant. If it is not available, heatseal the tube using a spirit lamp.
  3. The filled tubes are placed in the radial grooves of the centrifuge with the sealed ends toward the outer rim gasket. Counterbalance by placing the tubes in the grooves opposite to each other.
  4. Centrifuge at relative centrifu-gal force 12000 g for 5 minutes to completely pack the red cells.
  5. Immediately remove the tubes from the centrifuge and stand them upright. The tube will show three layers from top to bottom: column of plasma, thin layer of buffy coat, and column of red cells.
  6. With the microhematocrit reader, hematocrit is directly read from the scale. If hematocrit reader is not available, the tube is held against a ruler and the hematocrit is obtained by the following formula:
Length of red cell column in mm
-------------------------------------------------------
Length of total column in mm

To obtain PCV, the above result is multiplied by 100.

GENERAL NOTES

  1. Prolonged application of tourniquet during venepuncture causes hemoconcentration and rise in hematocrit.
  2. Excess squeezing of the finger during skin puncture dilutes the sample with tissue fluid and lowers the hematocrit.
  3. Correct proportion of blood with anticoagulant should be used. Excess EDTA causes shrinkage of red cells and falsely lowers the hematocrit.
  4. Inadequate mixing of blood with anticoagulant, and inadequate mixing of blood before testing can cause false results.
  5. Low hematocrit can result if there are clots in the sample.
  6. Centrifugation at lower speed and for less time falsely increases PCV.
  7. A small amount of plasma is trapped in the lower part of the red cell column which is usually insignificant. Increased amount of plasma is trapped in microcytosis, macrocytosis, spherocytosis, and sickle cell anemia, which cause an artifactual rise in hematocrit. Larger volume of plasma is trapped in Wintrobe tube than in capillary tube.
  8. As PCV requires whole blood sample, it is affected by plasma volume (e.g. PCV is higher in dehydration, and lower in fluid overload).
  9. Expression of PCV: Occasionally, PCV is expressed as a percentage. In SI units, PCV is expressed as a volume fraction. Conversion factor from conventional to SI units is 0.1 and from SI to conventional units is 100.
  10. Rules of 3 and 9: These rules of thumb are commonly used to check the accuracy of results and are applicable only if red cells are of normal size and shape.
    Hemoglobin (gm/dl) × 3 = PCV
    Red cell count (million/cmm) × 9 = PCV
  11. Automated hematocrit: In automated hematology analyzers, hematocrit is obtained by multiplying red cell count (in millions/cmm) by mean cell volume (in femtoliters).

REFERENCE RANGES

  • Adult males: 40-50%
  • Adult females (nonpregnant): 38 45%
  • Adult females (pregnant): 36-42%
  • Children 6 to 12 years: 37-46%
  • Children 6 months to 6 years: 36 42%
  • Infants 2 to 6 months: 32-42%
  • Newborns: 44-60%

CRITICAL VALUES

  • Packed cell volume: < 20% or > 60%

Principle

Anticoagulated whole blood is centrifuged in a Wintrobe tube to completely pack the red cells. The volume of packed red cells is read directly from the tube. An advantage with this method is that before performing PCV, test for erythrocyte sedimentation rate can be set up.

Equipment

  1. Wintrobe tube: This tube is about 110 mm in length and has 100 markings, each at the interval of 1 mm. Internal diameter is 3 mm. It can hold about 3 ml of blood.
  2. Pasteur pipette with a rubber bulb and a sufficient length of capillary to reach the bottom of the Wintrobe tube.
  3. Centrifuge with a speed of 2300 g.

Specimen

Venous blood collected in EDTA (1.5 mg EDTA for 1 ml of blood) or in double oxalate. Test should be performed within 6 hours of collection.

Method

  1. Mix the anticoagulated blood sample thoroughly.
  2. Draw the blood sample in a Pasteur pipette and introduce the pipette up to the bottom of the Wintrobe tube. Fill the tube from the bottom exactly up to the 100 mark. During filling, tip of the pipette is raised, but should remain under the rising meniscus to avoid foaming.
  3. Centrifuge the sample at 2300 g for 30 min (To counterbalance a second Wintrobe tube filled with blood from another patient or water should be placed in the centrifuge).
  4. Take the reading of the length of the column of red cells.

Hematocrit can be expressed either as a percentage or as a fraction of the total volume of blood sample.

Significance

In anemia, PCV is below the lower level of normal range. PCV is raised in dehydration, shock, burns, and polycythemia.

After centrifugation of anticoagulated whole blood, three zones can be distinguished in the Wintrobe tube from above downwards-plasma, buffy coat layer (a small greyish layer of white cells and platelets, about 1 mm thick), and packed red cells. Normal plasma is straw-colored. It is colorless in iron deficiency anemia, pink in the presence of hemolysis or hemoglobinemia, and yellow if serum bilirubin is raised (jaundice). In hypertriglyceridemia, plasma appears milky. Increased thickness of buffy coat layer occur if white cells or platelets are increased in number (e.g. in leukocytosis, thrombocytosis, or leukemia). Smears can be made from the buffy coat layer for demonstration of lupus erythematosus (LE) cells, malaria parasites, or immature cells.

Packed cell volume (PCV) is the volume occupied by the red cells when a sample of anticoagulated blood is centrifuged. It indicates relative proportion of red cells to plasma. PCV is also called as hematocrit or erythrocyte volume fraction. It is expressed either as a percentage of original volume of blood or as a decimal fraction.

USES OF PCV

  • Detection of presence or absence of anemia or polycythemia
  • Estimation of red cell indices (mean cell volume and mean corpuscular hemoglobin concentration)
  • Checking accuracy of hemoglobin value (Hemoglobin in grams/dl × 3 = PCV).

There are two methods for estimation of PCV: macro method (Wintrobe method) and micro method (microhematocrit method). Micro method is preferred because it is rapid, convenient, requires only a small amount of blood, capillary blood from skin puncture can be used, and a large number of samples can be tested at one time.

This method is also more accurate as plasma trapping in red cell column is less.

By this method, the approximate value of hemoglobin is estimated. This method is simple and rapid. This method is most common in the blood bank for the selection of blood donors.

In this method, a drop of the blood sample is allowed to fall in the solution of copper sulfate having specific gravity 1.053 from the altitude of 1 cm. The hemoglobin concentration of 12.5 g/dl is equivalent to the specific gravity of 1.053. The drop of blood gets covered with copper proteinate and remains separate and distinct for 15-20 seconds. If the drop of blood sample sinks within 15-20 seconds, the specific gravity of copper sulfate solution is lower than the specific gravity of blood sample and the approximate value of hemoglobin is more than 12.5 grams/dl and hemoglobin level is acceptable for the donation of blood. If the drop of blood sample floats, hemoglobin value is less than 12.5 grams/dl and unacceptable for blood donation. However, the concentration of plasma proteins and total leukocyte count also influence the specific gravity of whole blood which may lead false-positive result. In the existence of hypergammaglobulinemia (e.g. multiple myeloma) or leukocytosis (e.g. myeloid or lymphoid reaction, chronic myeloid or lymphocytic leukemia), hemoglobin level will be misleadingly high.

For the estimation of hemoglobin by oxyhemoglobin method, blood sample is mixed with a weak ammonia solution and then absorbance of this solution is deliberated in a photometer using a yellow-green filter or measured in a spectrophotometer at 540 nanometer. Absorbance of the test sample is corresponded with that of the standard solution.For the estimation of hemoglobin by oxyhemoglobin method, blood sample is mixed with a weak ammonia solution and then absorbance of this solution is deliberated in a photometer using a yellow-green filter or measured in a spectrophotometer at 540 nanometer. Absorbance of the test sample is corresponded with that of the standard solution.

This method is much similar to cyanmethemoglobin (hemoglobin-cyanide) method.

This method is very simple and rapid but this method is not much reliable as compared to cyanmethemoglobin method because there is no stable standard solution is available, derivatives of hemoglobin except oxyhemoglobin are not measured, and color of oxyhemoglobin solution swiftly dims.

The major cells of the immune system are lymphocytes. Lymphocytes that are critical for immune reactions are of two types namely B-cells and T-cells. Both cells develop from stem cells located in the liver of the foetus and in bone marrow cells of adults.
 
The lymphocytes which are differentiated in the bone marrow are B-cells. The lymphocytes that migrate to thymus and differentiate under its influence are called T-cells. The young lymphocytes migrate to lymphoid tissues such as spleen, lymph nodes and tonsils where they undergo final maturation. Matured lymphocytes circulate in the body fluids. T-cells are responsible for cellular immunity and B-cells produce antibodies about 20 trillion per day.
 
Both components require antigens to trigger them into action but they respond differently.
 
Antigens
 
An antigen is a substance when introduce into an individual, stimulates the production of an antibody with which it reacts. Antigens are large molecules of proteins or polysaccharides. Some of the antigens are the parts of microorganisms others include pollen, egg white, certain fruits, vegetables, chicken, feathers etc.

Antibodies
 
Antibodies are protein molecules called immunoglobulin (Ig). They are produced by lymphocytes. The antibodies inactivate antigens. An antibody  consists of four amino acid chains bounded together by disulphide bonds. Of the four chains two are long, heavy chains and two are short, light chains. All of them are arranged in the shape of the letter ‘Y’. The tail portion of antibody having two heavy chains is called constant fragment (Fc). On the tip of each short arm, an antigen- binding fragment (Fab) is present which specifically hold antigen.
 
Antibody immunity10
 
Based upon the five types of heavy chains, the immunoglobulin's are classified into five major types. Light chains are similar in all types of Immunoglobulin's.
 
TYPES OF IMMUNOGLOBULIN'S
 
lgG is the most important long acting antibody representing about 80% of the antibodies. The second important antibody is IgM. IgA is called secretory antibody, found in tears, saliva and colostrum, (the first milk secreted by mother). IgD serves as a receptor site at the surface of B cells to secrete other antibodies. IgE plays an important role in allergic reactions by sensitizing cells to certain antigens.
 
iimmunoglobulin types12
Lizard, bird and rabbit, all these three animals are included in group Amniota (Amniote). They excrete the unwanted nitrogenous waste products from their body, and the process is called excretion. If the process of excretion does not take place properly in the body, they become poisonous. In Vertebrates, main excretory organs are called as kidneys. Skin, gills, lungs, liver and intestine are also acts as accessory excretory organs.
 
Kidneys are located on the dorsal side of the coelom and they are made up with numerous uriniferous tubules.
 
Typical, uriniferous tubule is consist of three parts.
 
  1. Ciliated peritoneal funnel
  2. Malpighian body
  3. Ciliated convoluted tube
 
EXCRETORY SYSTEM - GARDEN LIZARD EXCRETORY SYSTEM - PIGEON EXCRETORY SYSTEM - RABBIT
1. Paired kidneys are dark red and irregular in shape. These are flattened organs. 1. Kidneys are dark red and somewhat rectangular and flattened organs. 1. Kidneys are dark red and bean-shaped organs.
2. Kidneys are located in the posterior region of the abdominal cavity and attached to the dorsal wall by a fold of peritoneum. 2. Kidneys are situated in the anterior part of the abdomen. 2. Kidneys are located in the posterior part of the abdominal cavity.
3. Right and left kidneys are opposite to each other. 3. Same as in calotes. 3. The two kidneys are distinct. The right kidney lies much ahead than the left kidney.
4. They are attached to the dorsal muscles. 4. They are fitted in the hollows of the pelvic girdle. 4. Same as in calotes.
5. They are very near to the median line kidneys are Metanephros type. 5. They are a little away from the median line. Kidneys are Meta nephros type. 5. They are well away from the median line. Kidneys are meta ne phros type.
6. Each kidney has two lobes Anterior lobe is broad and posterior lobe is broad Hilus is absent. 6. Each kidney has three lobes They are anterior, median and posterior lobes. Hilus is absent. 6. Each kidney is a single-lobed structure. Inner side of the kidney has a concave depression is known as the 'hilus'.
7. The two kidneys are united posteriorly forming a V-shaped structure. 7. The two kidneys are separate and do not fuse with each other. 7. The two kidneys are distinct.
8. The two ureters are narrow, thin-walled ducts extending behind from the kidneys to the cloaca, where these open into the urodaeum. 8. Same as in Calotes. 8. The ureters open into the urinary bladder. Ureters arise from the hilus of each kidney.
9. There is no pelvis. 9. There is no pelvis. 9. Each ureter is expanded in its kidney into a funnel like pelvis.
10. In males the ureters join at its posterior end with its corresponding vas deferens and both open by a common urino-genital aperture. 10. The ureters do not join with the vas deferens and both open separately into the cloaca. 10. Ureters open separately into the urinary bladder.
11. A thin walled urinary bladder opens on the ventral side of cloaca. 11. Urinary bladder is absent. 11. Urinary bladder is a large, median, pear, shaped, thin walled transparent sac.
12. Urinary bladder communicates with urodaeum thrumph its ventral wall. 12. __ 12. Urinary bladder opens into the urethra or unnogenital canal.
13. Calotes is uricotelic animal Urine consists n.ainly of uric acid. 13. Urine consists mainly of uric acid cotelic animal. 13. Urine consists mainly of urea - ureotelic animal.
14. Urine is excreted in a semi solid state. 14. Urine is excreted in a semisolid state (Bird droppinos). 14. Urine is passed out in a fluid state.
Kidneys are the major excretory organs in all vertebrates. Some other organs such as lungs, gills, liver, intestine and skin also remove certain waste materials besides their normal functions. These are also known as the accessory excretory organs. Both shark and frog are anamniotic animals.
 
The kidneys lie dorsal to the coelom and are composed of large number of renal or uriniferous tubules. A uriniferous tubule typically con­sists of three regions - a ciliated peritoneal funnel, a malpighian body and a ciliated convoluted tube. The malpighian body is a two layered cup, the 'Bowman's capsule' containing a mass of capillaries the 'glomerulus'. The convoluted tube opens into a Longitudinal duct which extends backwards and opens into the cloaca. The excretory organs remove the nitrogenous waste products formed during the metabolic activities from time to time. If these products are not removed from the body, they are changed to toxic substances.
 
EXCRETORY SYSTEM OF FISH EXCRETORY SYSTEM OF FROG
1. Paired kidneys are very long and ribbon like. 1. Paired kidneys are short and roughly oval in shape.
2. Each kidney is differentiated into a small non-renal part (genital part) and a long posterior renal part. The two parts exhibit morphological difference. 2. Each kidney possesses genital as well as renal region. But these are not morphologically differentiat­ed.
3. The kidneys are uriniferous 'Opisthonephros' but functional Mesonephros. 3. The kidneys are mesonephros.
4. Some uriniferous tubules retain peritoneal funnel. 4. The peritoneal funnels are absent.
5. The uriniferous tubules have a specialised urea - absorbing seg­ment. 5. The urea-absorbing segment is absent.
6. Uriniferous tubules lead into special tubes - the urinary ducts (ureters). These are distinct from wolffian ducts. 6. Uriniferous tubules lead into the wolffian ducts.
7. Ureters run back ward over the ventral surface of the kidneys. 7. Wolffian ducts leave outer border of kidneys and run backward.
8. Ureters are independent ducts to carry urine from the kidneys to the Urinogenital sinus'. 8. The ureters serve for the passage of genital elements as well as urine. So they are known as urino-genital ducts.Urino genital sinus is absent.
9. The urinary bladder is absent. 9. A large bilobed urinary bladder is present. It opens into the cloaca opposite the openings of the ureters.
10. The urine is hypotonic to blood. 10. The urine is hypertonic to blood.
11. Scoliodon is an ureotelic animal. The endproduct of nitrogen metabolism is urea. A large Quantity of urea is retained in the body as an adaptation to marine life.Excess of urea is excreted chiefly through its gills. 11. Frog is also ureotelic animal. It excretes urine from the cloaca in the form of urea.
Calotes is a cold blooded (poikilothermic) and terrestrial garden lizard. Pigeon is a ward blooded bird adapted for aerial mode of life. Rabbit is warm blooded and a herbivorous mammal which is also known as Oryctolagus. The circulation of blood in vertebrates is of closed type(circulation occurs is blood vessels. The blood vessels which collect blood from different parts of the body are called as veins. The walls of veins are thick and possess valves.Thier lumen is wide. They collect deoxygenated blood from different parts of the body and carry to the heart. The veins are formed by means of capillaries in the respective tissues or organs. The deoxygenated blood is received by the sinus venosus or the right auricle. The portal veins are having capillaries at their both ends. The pulmonary veins possess oxygenated blood.
 
VENOUS SYSTEM OF CALOTES (GARDEN LIZARD) VENOUS SYSTEM OF COLUMBA (PIGEON) VENOUS SYSTEM OF ORYCTOLAGUS (RABBIT)
1. The venous system consists of common pulmonary vein, two precaval and one post caval veins. These collect blood from the various parts of the body. 1. The venous system con­sists of three large veins-teeo precavak and one post caval along with four large pulmonary veins. 1. The venous system con­sists of four distinct divisions. i) System of venae carae ii) Hepatic portal system iii) Pulmonary system iv) Coronary system
2. The two precaval veins collect blood from the anterior part of the body. Each precaval is formed by the union of the internal and external jugular veins from head and the sub clavian vein from the arm. Transverse jugular vein is absent. Azygous vein is also absent. 2. The two precaval veins collect blood from the anterior part of the body. Each precaval vein is formed by the union of Jugular (head), brachial (arm) and pectoral (Pectoral muscfes) veins. Transverse jugular vessel is present in between the jugular veins. Azygous vein is absent. 2. The two precaval veins collect blood from the anterior part of the body. Each precaval vein is formed by the union of the external jugular vein (head) and subclavian vein (fore limb). The right precaval vein receives the azygous (unpaired) and intercostal veins (intercostal muscles and dorsal wall of theory). Left azygous vein is absent.
3. The post canal vein joins the posterior angle of the sinus venous. It forms by the union right and left efferent renal veins and brings blood from the posterior side. 3. The post caval vein is formed by the union of two large itac veins a tittle behind the liver. 3. The post caval vein is a large median vein. It stands at the cauda region (icaudal vein) and runs forward and receives blood in its course. The veins which join the posl caval vein are pairec ilio himbars, iliacs gonadial renal, anc hepatic.
4. The renal portal system collects blood from the posterior side of the body. Caudal vein bifurcates into two pelvic veins which . unite in front and form into the median anterior abdominal vein enters into the liver. Each pelvic vein joined by femoral, sciatic veins of that side. From the pelvic arise the renal portal veins which branch into capillaries in the substance of the kidneys coccygeo-mesenteric vein is absent. 4. Renal portal system is not well developed in pigeon caudal vein bifurcates into right and left renal portal veins (Hypo gastric veins) each of which enters the kidney. The hypogastric vein receives the Internal iliac vein abng with femoral & sciatic veins. At the bifurcation of the caudal vein into the two renal portal veins arise a median 'coccygeome-senteric vein'. It is characteristic of birds. The coccygeo- mesenteric vein joins the hepatic portal vein. 4. Renal portal system is completely absent in Rabbit.
5. The Hepatic portal vein collects blood from the alimentary canal and enters the liver and breaks upto capillaries. 5. The Hepatic portal vein collects bbod from the alimentary canal and emptied into the liver. From the Ever the blood is carried by the post caval vein through hepatic veins. 5. Same as in pigeon.
6. Epi gastric vein is absent. 6. Epi gastric vein returns the blood from the mesenteries and joins the hepatic veins. This vein corresponds to the abdominal vein of the frog. 6. Epi gastric vein is absent.
7. The right and left pulmonary veins bring pure blood from the right and left lungs and united into a common branch. Common pulmonary vein opens into the left auricle. 7. Four large pulmonary veins return blood from the posterior part of the left auricle. 7. A pair of pulmonary veins bring oxygenated blood from the lungs They unite by a common arch and open into the dorsal wall of the left auricle.
8. The right auricle receives deoxygenated blood through sinus venosus and left auricle possess oxygenated blood. In the partially divided ventricle the blood mixes to some extent. 8. The right side of the heart (right auricle & ventricle) receives de-oxygenated blood and left side folded with (left auricle & ventricle) oxygenated blood. 8. Same as in pigeon. Coronary veins collect deoxygenated blood from the wall of the heart. The coronary sinus opens into the right auricle through an aperture guarded by the Valve of The besius'. The opening is called as the 'formina of the The besius'.
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