- 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).
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.
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.
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.
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.
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.
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.
- Plasma contains fibrinogen as well as all the other proteins, while serum does not contain fibrinogen.
- 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.
- Amount of sample is greater with plasma than with serum for a given amount of blood.
- Use of anticoagulant may alter the concentration of some constituents if they are to be measured like sodium, potassium, lithium, etc.
|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.
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.
- 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.