- Acute bacterial infections: Abscess, pneumonia, meningitis, septicemia, acute rheumatic fever, urinary tract infection.
- Tissue necrosis: Burns, injury, myocardial infarction.
- Acute blood loss
- Acute hemorrhage
- Myeloproliferative disorders
- Metabolic disorders: Uremia, acidosis, gout
- Malignant tumors
- Physiologic causes: Exercise, labor, pregnancy, emotional stress.
- Severe bacterial infections, e.g. septicemia, pneumonia
- Severe hemorrhage
- Severe acute hemolysis
- Carcinoma metastatic to bone marrow Leukemoid reaction should be differentiated from chronic myeloid leukemia (Table 801.1).
(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
- Hematologic disorders: megaloblastic anemia, aplastic anemia, aleukemic leukemia, myelophthisis.
(a) Idiosyncratic action: Analgesics, antibiotics, sulfonamides, phenothiazines, antithyroid drugs, anticonvulsants.
(b) Dose-related: Anticancer drugs
- Ionizing radiation
- Congenital disorders: Kostman's syndrome, cyclic neutropenia, reticular dysgenesis.
- Neonatal isoimmune neutropaenia
- Systemic lupus erythematosus
- Felty's syndrome
- Allergic diseases: Bronchial asthma, rhinitis, urticaria, drugs.
- Skin diseases: Eczema, pemphigus, dermatitis herpetiformis.
- Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis.
- Hematologic disorders: Chronic Myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma.
- Carcinoma with necrosis.
- Radiation therapy.
- Lung diseases: Loeffler's syndrome, tropical eosinophilia
- Hypereosinophilic syndrome.
- Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar.
- Recovery from neutropenia.
- Autoimmune disorders.
- Hematologic diseases: Myeloproliferative disorders, monocytic leukemia, Hodgkin's disease.
- Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis.
(a) Viral: Acute infectious lymphocytosis, infective hepatitis, cytomegalovirus, mumps, rubella, varicella
(b) Bacterial: Pertussis, tuberculosis
(c) Protozoal: Toxoplasmosis
- Hematological disorders: Acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma.
- Other: Serum sickness, post-vaccination, drug reactions.
- 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.
- 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.
- 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.
- 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.
- 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.
- Toxic granules: These are darkly staining, bluepurple, coarse granules in the cytoplasm of neutrophils. They are commonly seen in severe bacterial infections.
- 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.
- Cytoplasmic vacuoles: Vacuoles in neutrophils are indicative of phagocytosis and are seen in bacterial infections.
- 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.
- 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.
- 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).
- 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.
- 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).
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
- Specimen slide (will need tofocus under 10× power)
- Compound microscope.
- Mount the specimen slide onthe stage and focus under 10×.
- Close the iris diaphragm completely.
- If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
- 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).
- Open the iris diaphragm until the entire field is illuminated.
- During regular microscope maintenance
- After the microscope is moved/transported
- Whenever you suspect objects do not appear as sharp as they could be.
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).
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.
- Due 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.
- Venepuncture site is cleansed with 70% ethanol and allowed to dry.
- The selected vein is anchored by compressing and pulling the soft tissues below the puncture site with the left hand.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Puncture site should be allowed to dry completely after cleaning with alcohol (before performing the venepuncture).
- Tourniquet should be released before removing the needle from the vein (to prevent hematoma formation).
- 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.
- 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.
- Failure to obtain blood: This happens if vein is missed, or excessive pull is applied to the plunger causing collapse of the vein.
- Occurrence of hematoma, thrombosis, thrombophlebitis, abscess, or bleeding.
- Transmission of infections like hepatitis B or human immuno-deficiency virus if reusable needles and syringes, which are not properly sterilised, are used.
- First tube: Blood culture.
- Second tube: Plain tube (serum).
- Third tube: Tube containing anticoagulant (EDTA, citrate, or heparin).
- Fourth tube: Tube containing additional stabilizing agent like fluoride.
|Dipotassium EDTA||20 gm|
|Distilled water||200 ml|
|Ammonium oxalate||1.2 gm|
|Potassium oxalate||0.8 gm|
|Distilled water||upto 100 ml|
|Trisodium citrate||3.2 gm|
|Distilled water||upto 100 ml|
Use 1:9 (anticoagulant: blood) proportion for coagulation studies; for ESR, 1:4 proportion is recommended.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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.
- Add one drop of blood sample to be tested to each drop of antiserum.
- 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.
- By tilting the slide backwards and forwards, examine for agglutination after exactly two minutes.
Positive (+): Little clumps of red cells are seen floating in a clear liquid.
Negative (–): Red cells are floating homogeneously in a uniform suspension.
- Interpretation: Interpret the result as shown in the Table 786.1 and Figure 786.2.