- 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 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).
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.
- The 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.
- The puncture site is cleansed with the 70% ethanol or other suitable disinfectant and allowed to dry.
- The preferred vein is anchored by squeezing and pulling the soft tissues below the prick site with the left hand.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- The tourniquet should be released before removing the needle from the vein to prevent the formation of a hematoma.
- Blood is never collected from the arm being used for the intravenous line since it will dilute the blood sample.
- Blood is never collected from an area with hematoma and from a sclerosed vein.
- 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.
- Proper precautions should be noticed while collecting blood either from a skin or a vein puncture since all blood samples are considered as infectious.
- 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.
- 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.
- Formation of hematoma, abscess, thrombosis, thrombophlebitis, or bleeding.
- 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.
- 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.