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RED CELLS MORPHOLOGY

Published in Hemotology
Tuesday, 25 July 2017 13:19
Role of blood smear in anemiasRed cells are best examined in an area where they are just touching one another (towards the tail of the film). Normal red cells are 7-8 μm in size, round with smooth contours, and stain deep pink at the periphery and paler in the center. Area of central pallor is about 1/3rd the diameter of the red cell. Size of a normal red cell corresponds roughly with the size of the nucleus of a small lymphocyte. Normal red cells are described as normocytic (of normal size) and normochromic (with normal staining intensity i.e. hemoglobin content).
 
Morphologic abnormalities of red cells in peripheral blood smear can be grouped as follows:
 
  • Red cells with abnormal size (see Figure 799.1)
  • Red cells with abnormal staining
  • Red cells with abnormal shape (see Figure 799.1)
  • Red cell inclusions (see Figure 799.2)
  • Immature red cells (see Figure799.3)
  • Abnormal red cell arrangement(see Figure 799.4).
 
Red cells with abnormal size:
 
Mild variation in red cell size is normal. Increased variation in red cell size is called as anisocytosis. This is a feature of most anemias and is non-specific. Anisocytosis is due to the presence of microcytes, macrocytes, or both in addition to red cells of normal size.
 
Microcytes are red cells smaller in size than normal. They are seen when hemoglobin synthesis is defective i.e. in iron deficiency anemia, thalassemias, anemia of chronic disease, and sideroblastic anemia.

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

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

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

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

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

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

PROCEDURES FOR THE COLLECTION OF BLOOD FOR HEMOTOLOGICAL INVESTIGATIONS

Published in Hemotology
Monday, 24 July 2017 05:21
COLLECTION OF BLOOD
 
For reliable and accurate results of laboratory tests, it is essential to follow a standard procedure for specimen collection. For hematological investigations, blood sample can be obtained from the skin puncture or venepuncture.
 
SKIN PUNCTURE

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

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

VENOUS BLOOD COLLECTION

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

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

SEQUENCE OF FILLING OF TUBES FOR HEMOTOLOGICAL INVESTIGATIONS

Published in Hemotology
Saturday, 22 July 2017 07:14
SEQUENCE OF FILLING OF TUBES
 
Following order of filling of tubes should be followed after withdrawal of blood from the patient if multiple investigations are ordered:
 
  1. First tube: Blood culture.
  2. Second tube: Plain tube (serum).
  3. Third tube: Tube containing anticoagulant (EDTA, citrate, or heparin).
  4. Fourth tube: Tube containing additional stabilizing agent like fluoride.
 
Further Reading:
 

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

Published in Hemotology
Saturday, 22 July 2017 06:59
Plasma is the supernatant liquid obtained after centrifugation of anticoagulated whole blood.
 
Serum is the liquid obtained after clotting of whole blood sample collected in a plain tube.
 
Some of the differences between the two are as follows:
 
  1. Plasma contains fibrinogen as well as all the other proteins, while serum does not contain fibrinogen.
  2. Plasma can be obtained immediately after sample collection by centrifugation, while minimum of 30 minutes are required for separation of serum from the clotted blood.
  3. Amount of sample is greater with plasma than with serum for a given amount of blood.
  4. Use of anticoagulant may alter the concentration of some constituents if they are to be measured like sodium, potassium, lithium, etc.

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

Published in Hemotology
Saturday, 22 July 2017 06:43
Plain tubes (i.e. without any anticoagulant) are used for chemistry studies after separation of serum: liver function tests (total proteins, albumin, aspartate aminotransferase, alanine aminotransferase, bilirubin), renal function tests (blood urea nitrogen, creatinine), calcium, lipid profile, electrolytes, hormones, and serum osmolality. Fluoride bulb is used for collection of whole blood for estimation of blood glucose. Addition of sodium fluoride (2.5 mg/ml of blood) maintains stable glucose level by inhibiting glycolysis. Sodium fluoride is commonly used along with an anticoagulant such as potassium oxalate or EDTA.

International Council for Standardization in Haematology (ICSH)

Published in Hemotology
Saturday, 22 July 2017 06:23
The International Council for Standardization in Haematology (ICSH) was initiated as a standardization committee by the European Society of Haematology (ESH) in 1963 and officially constituted by the International Society of Hematology (ISH) and the ESH in Stockholm in 1964. The ICSH is recognised as a Non-Governmental Organisation with official relations to the World Health Organisation (WHO).
 
The ICSH is a not-for-profit organisation that aims to achieve reliable and reproducible results in laboratory analysis in the field of diagnostic haematology.
 
The ICSH coordinates Working Groups of experts to examine laboratory methods and instruments for haematological analyses, to deliberate on issues of standardization and to stimulate and coordinate scientific work as necessary towards the development of international standardization materials and guidelines.

USES OF ANTICOAGULANTS FOR HEMOTOLOGICAL INVESTIGATIONS

Published in Hemotology
Saturday, 22 July 2017 05:04
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 AND Rh D GROUPING

Published in Hemotology
Friday, 21 July 2017 06:48
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:
 

FALSE REACTION IN ABO GROUPING

Published in Hemotology
Friday, 21 July 2017 06:19
  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.

Rh D GROUPING METHOD

Published in Hemotology
Friday, 21 July 2017 05:47
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
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