Hematology Blood carries out many vital functions as it circulates through the body. It transports oxygen from the lungs to other body tissues and carries away carbon dioxide. It carries nutrients from the digestive system to the cells of the body, and carries away wastes for excretion by the kidneys. Blood helps our body fight off infectious agents and inactivates toxins, stops bleeding through its clotting ability, and regulates our body temperature. Doctors rely on many blood tests to diagnose and monitor diseases. Some tests measure the components of blood itself; others examine substances found in the blood to identify abnormal function of various organs.
a complete blood cell count (CBC) A CBC is one of the most commonly performed blood tests. It measures the red blood cells, white blood cells and platelets. Platelets are needed for blood to clot. Red blood cells carry oxygen from the lungs to the tissues and take carbon dioxide away. White blood cells help fight infections. In addition to determining the number of blood cells and platelets, the percentage of each type of white blood cell, and the content of hemoglobin (an oxygen-carrying protein in red blood cells), the CBC usually assesses the size and shape of red blood cells. Normal red blood cell counts vary with your age and gender: Men: 4.7 to 5.4 million red blood cells per microliter of blood Women: 4.2 to 5.4 million red blood cells per microliter of blood Hemoglobin concentration correlates closely with the red blood cell count. Normal white blood cell counts range from 4,000 to 10,000 white blood cells per cubic millimeter of whole blood. In an adult, a normal count is about 150,000 to 450,000 platelets per microliter (x 10–6/Liter) of blood.
A low red blood cell or hemoglobin count indicates anemia, or severe bleeding. An elevated red cell or hemoglobin count may indicate polycythemia, a rare blood disorder. Abnormally shaped red blood cells can also signal problems: sickle-shaped cells are characteristic of sickle cell disease, small red blood cells may indicate iron deficiency, and large oval red blood cells suggest folic acid or vitamins B12 deficiency (pernicious anemia). The number of white blood cells may increase or decrease significantly in certain diseases. An elevated white blood cell count often indicates infection, such as an abscess, meningitis, pneumonia, appendicitis or tonsillitis. A high count may also be caused by leukemia or by dead tissue from burns, heart attack or gangrene. A low white blood cell count may mean bone marrow problems. A number of disorders can lead to a low platelet count that increases the risk of bleeding.
A device used for determining the number of cells per unit volume of a suspension is called a counting chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts. To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid. The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the V-shaped wells with a pasteur or other type of pipet. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power.
It is essential to be extremely careful with higher power objectives, since the counting chamber is much thicker than a conventional slide. The chamber or an objective lens may be damaged if the user is not not careful. One entire grid on standard hemacytometers with Neubauer rulings can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed.
Cell suspensions should be dilute enough so that the cells do not overlap each other on the grid, and should be uniformly distributed. To perform the count, determine the magnification needed to recognize the desired cell type. Now systematically count the cells in selected squares so that the total count is 100 cells or so (number of cells needed for a statistically significant count). For large cells this may mean counting the four large corner squares and the middle one. For a dense suspension of small cells you may wish to count the cells in the four 1/25 sq. mm corners plus the middle square in the central square. Always decide on a specific counting patter to avoid bias. For cells that overlap a ruling, count a cell as "in" if it overlaps the top or right ruling, and "out" if it overlaps the bottom or left ruling. Here is how to determine a cell count using a standard hemocytometer. To get the final count in cells/ml, first divide the total count by 0.1 (chamber depth) then divide the result by the total surface area counted. For example suppose you counted 125 cells (total) in the four large corner squares plus the middle combined. Divide 125 by 0.1, then divide the result by 5 mm-squared, which is the total area counted (each large square is 1 mm-squared). You shoud get 125/ 0.1 = 1250. 1250/5 = 250 cells/mm-cubed. There are 1000 mm-cubed per ml, so you calculate 250,000 cells/ml. Sometimes you will need to dilute a cell suspension to
get the cell density low enough for counting. In that case you will need to multiply your final count by the dilution factor. For example, suppose that for counting we had to dilute a suspension of Chlamydomonas 10 fold. Suppose we obtained a final count of 250,000 cells/ml as above. Then the count in the original (undiluted) suspension is 10 x 250,000 which is 2,500,000 cells/ml.
Hematocrit The hematocrit (HCT), or packed cell volume (PCV) represents the proportion of blood composed of red blood cells, expressed as % (vol/vol). It is the quickest and most accurate measure of the red cell component of blood. Traditionally, it is determined by measuring the height of the red cell column in a microhematocrit tube following centrifugation (see fig. at right). Automated analyzers (such as the Advia) calculate the HCT by multiplying the red cell count and the mean red cell volume, both of which are measured directly by the machine. Examination of the "crit tube" can also provide subjective information about the color and clarity of the plasma (icterus, hemolysis, lipemia), and the size of the "buffy coat" (which contains WBC and platelets). Additionally, one can score and break the tube as desired to remove the plasma for refractometric protein estimation, or to extrude the buffy coat for smear-making. The "buffy coat smear" has the advantage of providing a concentrated preparation of nucleated cells, which can be useful if looking for low-incidence cell-types of potential significance (e.g., mast cells).
Hemoglobin Hemoglobin concentration (Hb) is reported as grams of hemoglobin per deciliter of blood (g/dL). Since red cells are approximately 33% hemoglobin, the hemoglobin concentration of whole blood normally is about one third of the HCT The Hb molecule is a tetramer composed of 2 alpha and 2 beta chains. (i.e., the MCHC is 33%). Mean Cell Volume (MCV) The mean cell volume indicates the volume of the "average" red cell in a sample. It is expressed in femtoliters (fl; 10-15 liters). Traditionally, MCV was a calculated parameter, derived by using the following formula: MCV = (PCV ÷ RBC) x 10 Mean Cell Hemoglobin MHC is the mean cell hemoglobin. This represents the absolute amount of hemoglobin in the average red cell in a sample. Its units are picograms (pg) per cell. The MCH is calculated from the [Hb] and the RBC using the following equation: MCH (pg) = (Hb x 10) ÷ RBC
Mean Cell Hemoglobin Concentration (MCHC) MCHC is the mean cell hemoglobin concentration, expressed in g/dL. It can be calculated from the [Hb] and the PCV using the following formula: MCHC = (Hb ÷ PCV) x 100
Blood groups The differences in human blood are due to the presence or absence of certain protein molecules called antigens and antibodies. The antigens are located on the surface of the red blood cells and the antibodies are in the blood plasma. Individuals have different types and combinations of these molecules. The blood group you belong to depends on what you have inherited from your parents. There are more than 20 genetically determined blood group systems known today, but the AB0 and Rh systems are the most important ones used for blood transfusions. Not all blood groups are compatible with each other. Mixing incompatible blood groups leads to blood clumping or agglutination, which is dangerous for individuals.
Blood group A If you belong to the blood group A, you have A antigens on the surface of your red blood cells and B antibodies in your blood plasma Blood group B If you belong to the blood group B, you have B antigens on the surface of your red blood cells and A antibodies in your blood plasma. Blood group AB If you belong to the blood group AB, you have both A and B antigens on the surface of your red blood cells and no A or B antibodies at all in your blood plasma. Blood group 0 If you belong to the blood group 0 (null), you have neither A or B antigens on the surface of your red blood cells but you have both A and B antibodies in your blood plasma.
Rh factor blood grouping system Many people also have a so called Rh factor on the red blood cell's surface. This is also an antigen and those who have it are called Rh+. Those who haven't are called Rh-. A person with Rh- blood does not have Rh antibodies naturally in the blood plasma (as one can have A or B antibodies, for instance). But a person with Rh- blood can develop Rh antibodies in the blood plasma if he or she receives blood from a person with Rh+ blood, whose Rh antigens can trigger the production of Rh antibodies. A person with Rh+ blood can receive blood from a person with Rh- blood without any problems.
Blood typing – how do you find out to which blood group someone belongs? A person with A+ blood receives B+ blood. The B antibodies (yellow) in the A+ blood attack the foreign red blood cells by binding to them. The B antibodies in the A+ blood bind the antigens in the B+ blood and agglutination occurs. This is dangerous because the agglutinated red blood cells break after a while and their contents leak out and become toxic. 1. You mix the blood with three different reagents including either of the three different antibodies, A, B or Rh antibodies. 2. Then you take a look at what has happened. In which mixtures has agglutination occurred? The agglutination indicates that the blood has reacted with a certain antibody and therefore is not compatible with blood containing that kind of antibody. If the blood does not agglutinate, it indicates that the blood does not have the antigens binding the special antibody in the reagent. 3. If you know which antigens are in the person's blood, it's easy to figure out which blood group he or she belongs to!