Dayyal Dg.

Dayyal Dg.

Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger

Tuesday, 25 July 2017 13:19

RED CELLS MORPHOLOGY

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.

Tuesday, 25 July 2017 08:43

KOHLER ILLUMINATION

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.
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:
 
Friday, 21 July 2017 06:48

ABO GROUPING AND Rh D GROUPING

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:

Friday, 21 July 2017 06:19

FALSE REACTION IN ABO GROUPING

  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.
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