Friday, November 9, 2012

complete blood count or picture


Hemogram

A hemogram consists of a white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), red blood cell indices, and a platelet count. A complete blood count (CBC) consists of a hemogram plus a differential WBC.


Complete Blood Count (CBC) 

The CBC is a basic screening test and is one of the most frequently ordered laboratory procedures. The findings in the CBC give valuable diagnostic information about the hematologic and other body systems, prognosis, response to treatment, and recovery. The CBC consists of a series of tests that determine number, variety, percentage, concentrations, and quality of blood cells:


White blood cell count (WBC): leukocytes fight infection

Differential white blood cell count (Diff):specific patterns of WBC

Red blood cell count (RBC): red blood cells carry O 2 from lungs to blood tissues and CO 2 from tissue to lungs

Hematocrit (Hct): measures RBC mass

Hemoglobin (Hb): main component of RBCs and transports O 2 and CO 2

Red blood cell indices: calculated values of size and Hb content of RBCs; important in anemia evaluations
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin concentration (MCHC)
- Mean corpuscular hemoglobin (MCH)
Stained red cell examination (film or peripheral blood smear)

Platelet count (often included in CBC):thrombocytes are necessary for clotting and control of bleeding

Red blood cell distribution width (RDW):indicates degree variability and abnormal cell size.

Mean platelet volume (MPV): index of platelet production


WBC Test

White Blood Cell Count (WBC; Leukocyte Count)

White blood cells (or leukocytes) are divided into two main groups: granulocytes and agranulocytes. The granulocytes receive their name from the distinctive granules that are present in the cytoplasm of neutrophils, basophils, and eosinophils. However, each of these cells also contains a multilobed nucleus, which accounts for their also being called polymorphonuclear leukocytes. In laboratory terminology, they are often called "polys" or PMNs. The nongranulocytes, which consist of the lymphocytes and monocytes, do not contain distinctive granules and have nonlobular nuclei that are not necessarily spherical. The term mononuclear leukocytes is applied to these cells.


Differential White Blood Cell Count (Diff; Differential Leukocyte Count)

The total count of circulating white blood cells is differentiated according to the five types of leukocytes, each of which performs a specific function.


Function of Circulating WBCs According to Leukocyte Type
  • Cell - These Cells Function to Combat
  • Neutrophils - Pyogenic infections (bacterial)
  • Eosinophils - Allergic disorders and parasitic infestations
  • Basophils - Parasitic infections, some allergic disorders
  • Lymphocytes - Viral infections (measles, rubella, chickenpox, infectious mononucleosis)
  • Monocytes - Severe infections, by phagocytosis

Segmented Neutrophils (Polymorphonuclear Neutrophils, PMNs, Segs, Polys)

Neutrophils, the most numerous and important type of leukocytes in the body's reaction to inflammation, constitute a primary defense against microbial invasion through the process of phagocytosis. These cells can also cause some body tissue damage by their release of enzymes and endogenous pyogenes. In their immature stage of development, neutrophils are referred to as "stab" or "band" cells. The term band stems from the appearance of the nucleus, which has not yet assumed the lobed shape of the mature cell.


Eosinophils

Eosinophils, capable of phagocytosis, ingest antigen-antibody complexes and become active in the later stages of inflammation. Eosinophils respond to allergic and parasitic diseases. Eosinophilic granules contain histamine (one third of all the histamine in the body).
This test is used to diagnose allergic infections, assess severity of infestations with worms and other large parasites, and monitor response to treatment.


Basophils

Basophils, which constitute a small percentage of the total leukocyte count, are considered phagocytic. The basophilic granules contain heparin, histamines, and serotonin. Tissue basophils are called mast cells and are similar to blood basophils. Normally, mast cells are not found in peripheral blood and are rarely seen in healthy bone marrow. Basophil counts are used to study chronic inflammation. There is a positive correlation between high basophil counts and high concentrations of blood histamines, although this correlation does not imply cause and effect. It is extremely difficult to diagnose basopenia because a 1000–10,000 count differential would have to be done to get an absolute count.


Monocytes (Monomorphonuclear Monocytes)

These agranulocytes, the largest cells of normal blood, are the body's second line of defense against infection. Histiocytes, which are large macrophagic phagocytes, are classified as monocytes in a differential leukocyte count. Histiocytes and monocytes are capable of reversible transformation from one to the other.

These phagocytic cells of varying size and mobility remove injured and dead cells, microorganisms, and insoluble particles from the circulating blood. Monocytes escaping from the upper and lower respiratory tracts and the gastrointestinal and genitourinary organs perform a scavenger function, clearing the body of debris. These phagocytic cells produce the antiviral agent called interferon.

This test counts monocytes, which circulate in certain specific conditions such as tuberculosis, subacute bacterial endocarditis, and the recovery phase of acute infections.


Lymphocytes (Monomorphonuclear Lymphocytes); CD4, CD8 Count; Plasma Cells

Lymphocytes are small, mononuclear cells without specific granules. These agranulocytes are motile cells that migrate to areas of inflammation in both early and late stages of the process. These cells are the source of serum immunoglobulins and of cellular immune response and play an important role in immunologic reactions. All lymphocytes are manufactured in the bone marrow. 

B lymphocytes mature in the bone marrow, and T lymphocytes mature in the thymus gland. B cells control the antigen-antibody response that is specific to the offending antigen and is said to have "memory." The T cells, the master immune cells, include CD4 + helper T cells, killer cells, cytotoxic cells, and CD8 + suppressor T cells. Plasma cells (fully differentiated B cells) are similar in appearance to lymphocytes. They have abundant blue cytoplasm and an eccentric, round nucleus. Plasma cells are not normally present in blood. This test measures the number of lymphocytes in the peripheral blood. Lymphocytosis is present in various diseases and is especially prominent in viral disorders. Lymphocytes and their derivatives, the plasma cells, operate in the immune defenses of the body.


Lymphocyte Immunophenotyping (T and B Cells)

Lymphocytes are divided into two categories, T and B cells, according to their primary function within the immune system. In the body, T and B cells work together to help provide protection against infections, oncogenic agents, and foreign tissue, and they play a vital role in regulating self-destruction or autoimmunity.

Most circulating lymphocytes are T cells with a life span of months to years. The life span of B cells is measured in days. B cells (antibody) are considered "bursa or bone marrow dependent" and are responsible for humoral immunity (in which antibodies are present in the serum). 

T cells (cellular) are thymus derived and are responsible for cellular immunity. T cells are further divided into helper T (CD3 +, CD4 +) cells and suppressor T (CD3 +, CD8 +) cells. Evaluation of lymp hocytes in the clinical laboratory is performed by quantitation of the lymphocytes and their subpopulations and by assessment of their function activity. These laboratory analyses have become an essential component of the clinical assessment of two major disease states: lymphoproliferative states (eg, leukemia, lymphoma), in which characterization of the malignant cell in terms of lineage and stage of differentiation provides valuable information to the oncologist to guide prognosis and appropriate therapy; and immunodeficient states (eg, HIV infection, organ transplantation), in which the alterations in the immune system that occur secondary to infection are evaluated.


Tests of red blood cells

Many tests look at the red blood cells: their number and size, amount of Hb, rate of production, and percent composition of the blood. The red blood cell count (RBC), hematocrit (Hct), and hemoglobin (Hb) are closely related but different ways to look at the adequacy of erythrocyte production. The same conditions cause an increase (or decrease) in each of these indicators.


Red Blood Cell Count (RBC; Erythrocyte Count)

The main function of the red blood cell (RBC or erythrocyte) is to carry oxygen from the lungs to the body tissues and to transfer carbon dioxide from the tissues to the lungs. This process is achieved by means of the Hb in the RBCs, which combines easily with oxygen and carbon dioxide and gives arterial blood a bright red appearance. To enable use of the maximal amount of Hb, the RBC is shaped like a biconcave disk; this affords more surface area for the Hb to combine with oxygen. The cell is also able to change its shape when necessary to allow for passage through the smaller capillaries.

The RBC test, an important measurement in the evaluation of anemia or polycythemia, determines the total number of erythrocytes in a microliter (cubic millimeter) of blood.


Hematocrit (Hct); Packed Cell Volume (PCV)

The word hematocrit means "to separate blood," which underscores the mechanism of the test because the plasma and blood cells are separated by centrifugation.

The Hct test is part of the CBC. This test indirectly measures the RBC mass. The results are expressed as the percentage by volume of packed RBCs in whole blood (PCV). It is an important measurement in the determination of anemia or polycythemia.


Hemoglobin (Hb)

Hb, the main component of erythrocytes, serves as the vehicle for the transportation of oxygen and carbon dioxide. It is composed of amino acids that form a single protein called globin, and a compound called heme, which contains iron atoms and the red pigment porphyrin. It is the iron pigment that combines readily with oxygen and gives blood its characteristic red color. Each gram of Hb can carry 1.34 mL of oxygen per 100 mL of blood. The oxygen-combining capacity of the blood is directly proportional to the Hb concentration rather than to the RBC because some RBCs contain more Hb than others. This is why Hb determinations are important in the evaluation of anemia.

The Hb determination is part of a CBC. It is used to screen for disease associated with anemia, to determine the severity of anemia, to monitor the response to treatment for anemia, and to evaluate polycythemia.

Hb also serves as an important buffer in the extracellular fluid. In tissue, the oxygen concentration is lower, and the carbon dioxide level and hydrogen ion concentration are higher. At a lower pH, more oxygen dissociates from Hb. The unoxygenated Hb binds to hydrogen ion, thereby raising the pH. 

As carbon dioxide diffuses into the RBC, carbonic anhydrase converts carbon dioxide to bicarbonate and protons. As the protons are bound to Hb, the bicarbonate ions leave the cell. For every bicarbonate ion leaving the cell, a chloride ion enters. The efficiency of this buffer system depends on the ability of the lungs and kidneys to eliminate, respectively, carbon dioxide and bicarbonate.


Red Blood Cell Indices

The red cell indices define the size and Hb content of the RBC and consist of the mean corpuscular volume (MCV), the mean corpuscular hemoglobin concentration (MCHC), and the mean corpuscular hemoglobin (MCH).

The RBC indices are used in differentiating anemias. When they are used together with an examination of the erythrocytes on the stained smear, a clear picture of RBC morphology may be ascertained. On the basis of the RBC indices, the erythrocytes can be characterized as normal in every respect or as abnormal in volume or Hb content. In deficient states, the anemias can be classified by cell size as macrocytic, normocytic, or microcytic, or by cell size and color as microcytic hypochromic.


Mean Corpuscular Hemoglobin Concentration (MCHC)

The MCHC measures the average concentration of Hb in the RBCs. The MCHC is most valuable in monitoring therapy for anemia because the two most accurate hematologic determinations (Hb and Hct) are used in its calculation.


Mean Corpuscular Hemoglobin (MCH)

The MCH is a measure of the average weight of Hb per RBC. This index is of value in diagnosing severely anemic patients.


Red Cell Size Distribution Width (RDW)

This automated method of measurement is helpful in the investigation of some hematologic disorders and in monitoring response to therapy. The RDW is essentially an indication of the degree of anisocytosis (abnormal variation in size of RBCs). Normal RBCs have a slight degree of variation.


Stained Red Cell Examination (Film; Stained Erythrocyte Examination)

The stained film examination determines variations and abnormalities in erythrocyte size, shape, structure, Hb content, and staining properties. It is useful in diagnosing blood disorders such as anemia, thalassemia, and other hemoglobinopathies. This examination also serves as a guide to therapy and as an indicator of harmful effects of chemotherapy and radiation therapy. The leukocytes are also examined at this time.


Reticulocyte Count

A reticulocyte—young, immature, nonnucleated RBC—contains reticular material (RNA) that stains gray-blue. Reticulum is present in newly released blood cells for 1 to 2 days before the cell reaches its full mature state. Normally, a small number of these cells are found in circulating blood. For the reticulocyte count to be meaningful, it must be viewed in relation to the total number of erythrocytes (absolute reticulocyte count = % reticulocytes × erythrocyte count).

The reticulocyte count is used to differentiate anemias caused by bone marrow failure from those caused by hemorrhage or hemolysis (destruction of RBCs), to check the effectiveness of treatment in pernicious anemia and folate and iron deficiency, to assess the recovery of bone marrow function in aplastic anemia, and to determine the effects of radioactive substances on exposed workers.


Sedimentation Rate (Sed Rate); Erythrocyte Sedimentation Rate (ESR)

Sedimentation occurs when the erythrocytes clump or aggregate together in a column-like manner (rouleaux formation). These changes are related to alterations in the plasma proteins. Normally, erythrocytes settle slowly because normal RBCs do not form rouleaux.
The ESR is the rate at which erythrocytes settle out of anticoagulated blood in 1 hour. This test is based on the fact that inflammatory and necrotic processes cause an alteration in blood proteins, resulting in aggregation of RBCs, which makes them heavier and more likely to fall rapidly when placed in a special vertical test tube.

The faster the settling of cells, the higher the ESR. The ESR should not be used to screen asymptomatic patients for disease. It is most useful for diagnosis of temporal arteritis, rheumatoid arthritis, and polymyalgia rheumatica. The sedimentation rate is not diagnostic of any particular disease but rather is an indication that a disease process is ongoing and must be investigated. It is also useful in monitoring the progression of inflammatory diseases; if the patient is being treated with steroids, the ESR will decrease with clinical improvement.


Tests for porphyria

Porphyrins are chemical intermediates in the synthesis of Hb, myoglobin, and other respiratory pigments called cytochromes. They also form part of the peroxidase and catalase enzymes, which contribute to the efficiency of internal respiration. Iron is chelated within porphyrins to form heme. Heme is then incorporated into proteins to become biologically functional hemoproteins.


Erythropoietic Porphyrins; Free Erythrocyte Protoporphyrin (FEP)
Normally, there is a small amount of excess porphyrin at the completion of heme synthesis. This excess is cell-free erythrocyte protoporphyrin (FEP). The amount of FEP in the erythrocyte is elevated when the iron supply is diminished. This test is useful in screening RBC disorders such as iron deficiency and lead exposure, especially in children 6 months to 5 years of age. This is the test of choice to diagnose erythopoietic protoporphyria. 


Porphyrins; Fractionation of Erythrocytes and of Plasma
The primary porphyrins of erythrocytes are protoporphyrin, uroporphyrin, and coproporphyrin. Fractionation of erythrocytes is used to differentiate congenital erythropoietic coproporphyria from erythropoietic protoporphyria and to confirm a diagnosis of protoporphyria. This test establishes a specific type of porphyria by naming the specific porphyrin in plasma. In persons with renal failure, plasma fractionation can help to determine whether the porphyria is caused by a deficiency of uroporphyrinogenic decarboxylase or by failure of the renal system to excrete porphyrinogens.


Tests for hemolytic anemia

Several RBC enzyme and fragility tests can be done to screen, detect, and confirm the cause of chronic hemolytic anemia. Many persons with hemolytic anemia have no clinical signs or symptoms. Abnormal test outcomes are associated with inherited deficiencies, abnormal hemoglobins, and exposure to chemicals and drugs. Definitive test results indicate some type of injury to the RBC, oxidated activity that interferes with normal Hb function, and/or increased RBC fragility.


Pyruvate Kinase (PK)

PK deficiency is a genetic disorder characterized by a lowered concentration of adenosine triphosphate in the RBC and consequential membrane defect. The result is a nonspherocytic, chronic hemolytic anemia. PK deficiency is the most common and most important form of hemolytic anemia resulting from a deficiency of glycolytic enzymes in the RBC.


Erythrocyte Fragility (Osmotic Fragility and Autohemolysis)

Spherocytes of any origin (including conditions other than hereditary spherocytosis) are more susceptible than normal RBCs to hemolysis in dilute (hypotonic) saline and show increased osmotic fragility. Generally, fully expanded cells (spheroidal cells or spherocytes) have increased osmotic fragility, whereas cells with higher surface area-to-volume ratios (eg, thin cells, hypochromic cells, tart cells) have decreased osmotic fragility.

In hereditary spherocytosis, the osmotic fragility test may be normal initially. There-fore, the test is incubated at 37°C for 24 hours, at which time the test is positive for hereditary spherocytosis.


Glucose-6-Phosphate Dehydrogenase (G6PD)

G6PD is a sex-linked disorder. The major variants occur in specific ethnic groups. In a large group of African American men, the incidence of type A G6PD deficiency was found to be 11%. Approximately 20% of African American women are heterozygous. With some variants, there is chronic lifelong hemolysis, but more commonly, the condition is asymptomatic and results only in susceptibility to acute hemolytic episodes, which may be triggered by certain drugs, ingestion of fava beans, or viral or bacterial infection. G6PD hemolysis is associated with formation of Heinz bodies in peripheral RBCs.

The other two most common types are Mediterranean, which is common in Iraqis, Kurds, Sephardic Jews, and Lebanese and less common in Greeks, Italians, Turks, and North Africans, and the MAHIDOL variant, which is common in Southeast Asians (22% males).


Heinz Bodies; Heinz Stain; Glutathione Instability

Heinz bodies are insoluble intracellular inclusions of Hb attached to RBC membrane. Heinz bodies are uncommon except with G6PD deficiency immediately after hemolysis and in patients with unstable Hb variants. Oxidative denaturation of the Hb molecule leads to Heinz body formation and is probably the mechanism for the precipitation of unstable Hb. Heinz bodies are usually removed by the spleen; after splenectomy, they increase in the peripheral blood and may appear in >50% of RBCs.


2,3-Diphosphoglycerate (2,3-DPG)

2,3-DPG assists in transporting oxygen in the RBC. 2,3-DPG increases in response to hypoxia or anemia and decreases in acidosis. Levels are lower in newborns and even lower in premature newborns. 


Iron tests

Iron (Fe), Total Iron-Binding Capacity (TIBC), and Transferrin Tests

Iron is necessary for the production of Hb. Iron is contained in several components. Transferrin (also called siderophilin), a transport protein largely synthesized by the liver, regulates iron absorption. High levels of transferrin relate to the ability of the body to deal with infections. Total iron-binding capacity (TIBC) correlates with serum transferrin, but the relation is not linear. A serum iron test without a TIBC and transferrin determination has very limited value except in cases of iron poisoning. Transferrin saturation is a better index of iron saturation; it is evaluated as follows:


Ferritin

Ferritin, a complex of ferric (Fe 2+) hydroxide and a protein, apoferritin, originates in the reticuloendothelial system. Ferritin reflects the body iron stores and is the most reliable indicator of total-body iron status. A bone marrow examination is the only better test. Bone marrow aspiration may be necessary in some cases, such as low-normal ferritin and low serum iron in the anemia of chronic disease.


Iron Stain (Stainable Iron in Bone Marrow; Prussian Blue Stain)

In the bone marrow, normoblasts containing iron granules (stainable) are known as sideroblasts. Erythrocytes (RBCs) that contain stainable iron are called siderocytes. Normally, about 33% of the normoblasts are sideroblasts. Other storage iron is readily identifiable in monophages in bone marrow particles on the marrow slides.


Tests for hemoglobin disorders

Hemoglobin Electrophoresis

Normal and abnormal Hb can be detected by electrophoresis, which matches hemolyzed RBC material against standard bands for the various Hb types known. The most common forms of normal adult Hb are Hb A 1, Hb A 2, and Hb F (fetal Hb). Of the various types of abnormal Hb (hemoglobinopathies), the best known are Hb S (responsible for sickle cell anemia) and Hb C (results in a mild hemolytic anemia). The most common abnormality is a significant increase in Hb A 2, which is diagnostic of the thalassemias, especially ß-thalassemia trait. 


Hemoglobin A 2 (Hb A 2)

Hb A 2 levels have special application to the diagnosis of ß-thalassemia trait, which may be present even though the peripheral blood smear is normal. The microcytosis and other morphologic changes of ß-thalassemia trait must be differentiated from iron deficiency. Low MCV may be present in most patients with ß-thalassemia trait, but it does not differentiate iron-deficient patients.


Hemoglobin S (Sickle Cell Test; Sickledex)

Sickle cell disease is a term for a group of hereditary blood disorders. Sickle cell anemia is caused by an abnormality of Hb, the red protein in red blood cells that carries oxygen from the lungs to the tissues. People with sickle cell disease make an abnormal Hb, hemoglobin S (Hb S). The red blood cells of a person with sickle cell disease do not last as long as "normal" red blood cells.

This result is chronic anemia. Also, these red blood cells lose their normal disk shape. They become rigid and deformed and take on a "sickle" or crescent shape. These oddly shaped cells are not flexible enough to squeeze through small blood vessels. This may result in blood vessels being blocked. The areas of the body served by those blood vessels will then be deprived of their blood circulation, damaging tissues and organs. This homozygous state of Hb S disease is associated with considerable morbidity and mortality. 

The heterozygous state presents little mortality.

This blood measurement is routinely done as a screening test for sickle cell anemia or trait and to confirm these disorders. This test detects Hb S, an inherited, recessive gene. An examination is made of erythrocytes for the sickle-shaped forms characteristic of sickle cell anemia or trait. This is done by removing oxygen from the erythrocyte. In erythrocytes with normal Hb, the shape is retained, but erythrocytes containing Hb S assume a sickle shape. However, the distinction between sickle cell trait and sickle cell disease is done by electrophoresis, which identifies an Hb pattern.


Methemoglobin (Hemoglobin M)

Methemoglobin is formed when the iron in the heme portion of deoxygenated Hb is oxidized to a ferric form rather than a ferrous form. In the ferric form, oxygen and iron cannot combine. The formation of methemoglobin is a normal process and is kept within bounds by the reduction of methemoglobin to Hb. Methemoglobin causes a shift to the left of the oxyhemoglobin dissociation curve. When a high concentration of methemoglobin is produced in the RBCs, it reduces their capacity to combine with oxygen; anoxia and cyanosis result.

This test is used to diagnose hereditary or acquired methemoglobinemia in patients with symptoms of anoxia or cyanosis and no evidence of cardiovascular or pulmonary disease. Hb M is an inherited disorder of the Hb that produces cyanosis.


Sulfhemoglobin

Sulfhemoglobin is an abnormal Hb pigment produced by the combination of inorganic sulfides with Hb. Sulfhemoglobinemia manifests as a cyanosis. Sulfhemoglobinemia often accompanies drug-induced methemoglobinemia. This test is indicated in persons with cyanosis. Sulfhemoglobinemia may occur in association with the administration of various drugs and toxins. The symptoms are few, but cyanosis is intense even though the concentration of sulfhemoglobin seldom exceeds 10%


Carboxyhemoglobin; Carbon Monoxide (CO)

Carboxyhemoglobin is formed when Hb is exposed to carbon monoxide (CO). The affinity of Hb for CO is 240 times greater than for oxygen. CO poisoning causes anoxia because the carboxyhemoglobin formed does not permit Hb to combine with oxygen. This test is done to detect CO poisoning. Because carboxyhemoglobin is not capable of transporting oxygen, hypoxia results, causing headache, nausea, vomiting, vertigo, collapse, or convulsions. Death may result from anoxia and irreversible tissue changes. Carboxyhemoglobin produces a cherry-red or violet color of the blood and skin, but this may not be present in chronic exposure. The most common causes of CO toxicity are automobile exhaust fumes, coal gas, water gas, and smoke inhalation from fires. Smoking is a minor cause.


Myoglobin (Mb)

Myoglobin (Mb) is the oxygen-binding protein of striated muscle. It resembles Hb but is unable to release oxygen except at extremely low tension. Injury to skeletal muscle results in release of myoglobin. It is not specific to myocardial muscle. Myoglobin is not tightly bound to protein and is rapidly excreted in the urine. The myoglobin test is used as an early marker of muscle damage in myocardial infarction and to detect injury damage or necrosis to skeletal muscle. Serum myoglobin is found earlier than creatine kinase (CK) enzymes in acute myocardial infarction.


Haptoglobin (Hp)

Haptoglobin (Hp) is a transport glycoprotein synthesized solely in the liver. It is a carrier for free Hb in plasma; its primary physiologic function is the preservation of iron. Haptoglobin binds Hb and carries it to the reticuloendothelial system.

A decrease in Hp (with normal liver function) is most likely to occur with increased consumption of Hp due to intravascular hemolysis. The concentration of Hp is inversely related to the degree of hemolysis and to the duration of hemolytic episode.


Bart's Hemoglobin

Bart's Hb is an unstable Hb with high oxygen affinity. When there is complete absence of production of the chain of Hb and deletion of all four globin genes, the disorder is known as Bart's hydrops fetalis. Both parents of the affected infant have heterozygous thalassemia; they are almost all Southeast Asians. Affected infants are either stillborn or die shortly after birth.

This test determines the percentage of the abnormal Bart's Hb in cord blood and identifies a-thalassemia hemoglobinopathies.


Paroxysmal Nocturnal Hemoglobinuria (PNH) Test; Acid Hemolysis Test; Ham's Test

PNH was first described by a patient who noted hemoglobinuria after sleep. In many patients, the hemolysis is irregular or occult. PNH is a hemolytic anemia in which there is also production of defective platelets and granulocytes. The diagnostic feature of PNH is an increased sensitivity of the erythrocytes to complement-mediated lysis. Although patients with PNH can present with hemoglobinuria or a hemolytic anemia, they may also present with iron deficiency (because of urinary loss of blood), bleeding secondary to thrombocytopenia, thrombosis, renal abnormalities, or neurologic abnormalities.


Other blood tests for anemia

Vitamin B 12 (VB 12)

Vitamin B 12 (VB 12), also known as the antipernicious anemia factor, is necessary for the production of RBCs. It is obtained only from ingestion of animal protein and requires an intrinsic factor for absorption. Both VB 12 and folic acid depend on a normally functioning intestinal mucosa for their absorption and are important for the production of red blood cells. Levels of VB 12 and folate are usually tested in conjunction with one another because the diagnosis of macrocytic anemia requires measurement of both.


Folic Acid (Folate)

Folic acid is needed for normal RBC and WBC function and for the production of cellular genes. Folic acid is a more potent growth promoter than VB 12, although both depend on the normal functioning of intestinal mucosa for their absorption. Folic acid, like VB 12, is required for DNA production. Folic acid is formed by bacteria in the intestines, is stored in the liver, and is present in eggs, milk, leafy vegetables, yeast, liver, fruits, and other elements of a well-balanced diet. This test is indicated for the differential diagnosis of megaloblastic anemia and in the investigation of folic acid deficiency, iron deficiency, and hypersegmental granulocytes.

Erythropoietin (Ep)

Erythropoietin (Ep) is a glycoprotein hormone that regulates erythropoiesis. The levels of Ep in anemia are primarily determined by the degree of anemia; Ep is inversely related to red blood cell volume and Hct. Ep is used to investigate obscure anemias. This test is useful in differentiating primary from secondary polycythemia and in detecting the recurrence of Ep-producing tumors. It is also used as an indicator of need for Ep therapy in patients with renal failure (end-stage renal disease).


Tests of hemostasis and coagulation

The prime functions of the coagulation mechanism are to protect the integrity of the blood vessels while maintaining the fluid state of blood. Serious medical problems or even death may occur with the inability to stem the loss of blood, or for the inability for a normal clot to form.

Two general forms of hypercoagulability exist: hyperreactivity of the platelet system, which results in arterial thrombosis, and accelerated activity of the clotting system, which results in venous thrombosis. Hypercoagulability refers to an unnatural tendency toward thrombosis. The thrombus is the actual insoluble mass (fibrin or platelets) present in the bloodstream or chambers of the heart.

Conditions and classifications associated with hypercoagulability include the following:

Platelet Abnormalities. These conditions are associated with arteriosclerosis, diabetes mellitus, increased blood lipids or cholesterol levels, increased platelet levels, and smoking. Arterial thrombosis may be related to blood flow disturbances, vessel wall changes, and increased platelet sensitivity to factors causing platelet adherence and aggregation.

Clotting System Abnormalities. These are associated with congestive heart failure, immobility, artificial surfaces (eg, artificial heart valves), damaged vasculature, use of oral contraceptives or estrogen, pregnancy and the postpartum state, and the postsurgical state. Other influences include malignancy, myeloproliferative (bone marrow) disorders, obesity, lupus disorders, and genetic predisposition.

Venous Thrombosis. This can be related to stasis of blood flow, to coagulation alterations, or to increases in procoagulation factors or decreases in anticoagulation factors

Disorders of Hemostasis

Congenital Vascular Abnormalities (Vessel Wall Structure Defects). Defects of the actual blood vessel are poorly defined and difficult to test. Hereditary telangiectasia is the most commonly recognized vascular abnormality. Laboratory studies are normal, so the diagnosis must be made from clinical signs and symptoms. Patients frequently report epistaxis and symptoms of anemia. Another abnormality is congenital hemangiomas (Kasabach-Merritt syndrome).

Acquired Abnormalities of the Vessel Wall Structure. Causes include Henoch-Schönlein purpura as an allergic response to infection or drugs, diabetes mellitus, rickettsial diseases, septicemia, and amyloidosis present with some degree of vascular abnormalities. Purpura can also be associated with steroid therapy and easy bruising in females (infectious purpura), or it can be a result of drug use.

Hereditary Connective Tissue Disorders. These include Ehlers-Danlos syndrome (hyperplastic skin and hyperflexible joints) and pseudoxanthoma elasticum (rare connective tissue disorder).

Acquired Connective Tissue Defects. These can be caused by scurvy (vitamin C deficiency) or senile purpura.

Quantitative Platelet Abnormalities. These are associated with Glanzmann's thrombasthenia, a hereditary autosomal-recessive disorder that can produce severe bleeding, especially with trauma and surgical procedures. Platelet factor 3 differences associated with aggregation, adhesion, or release defects may be manifested in storage-pool disease, May-Hegglin anomaly, Bernard-Soulier syndrome, and Wiskott-Aldrich syndrome. Dialysis and use of drugs such as aspirin, other antiinflammatory agents, dipyridamole, and prostaglandin E also can be tied to platelet abnormalities.

Congenital Coagulation Abnormalities. These include hemophilia A and B (deficiencies of factors VIII and IX, respectively), rare autosomal recessive traits (hemophilia C), and autosomal dominant traits (eg, von Willebrand's disease).

Acquired Coagulation Abnormalities. These are associated with several disease states and are much more common than inherited deficiencies.


Bleeding Time (Ivy Method; Template Bleeding Time)

Bleeding time measures the primary phase of hemostasis: the interaction of the platelet with the blood vessel wall and the formation of a hemostatic plug. Bleeding time is the best single screening test for platelet function disorders and is one of the primary screening tests for coagulation disorders.

This test is of value in detecting vascular abnormalities and platelet abnormalities or deficiencies. It is not recommended for routine presurgical workup.
A small stab wound is made in either the earlobe or the forearm; the bleeding time (the amount of time it takes to form a clot) is recorded. The duration of bleeding from a punctured capillary depends on the quantity and quality of platelets and the ability of the blood vessel wall to constrict.

The principal use of this test today is in the diagnosis of von Willebrand's disease, an inherited defective molecule of factor VIII and a type of pseudohemophilia. It has been established that aspirin may cause abnormal bleeding in some normal persons, but the bleeding time test has not proved consistently valuable in identifying such persons.


Platelet Count; Mean Platelet Volume (MPV)

Platelets (thrombocytes) are the smallest of the formed elements in the blood. These cells are nonnucleated, round or oval, flattened, disk-shaped structures. Platelet activity is necessary for blood clotting, vascular integrity and vasoconstriction, and the adhesion and aggregation activity that occurs during the formation of platelet plugs that occlude (plug) breaks in small vessels. Thrombocyte development takes place primarily in the bone marrow. The life span of a platelet is about 7.5 days. Normally, two thirds of all the body platelets are found in the circulating blood and one third in the spleen.

The platelet count is of value for assessing bleeding disorders that occur with thrombocytopenia, uremia, liver disease, or malignancies and for monitoring the course of disease associated with bone marrow failure. This test is indicated when the estimated platelet count (on a blood smear) appears abnormal. It is also part of a coagulation profile or workup. The mean platelet volume (MPV) is sometimes ordered in conjunction with a platelet count. The MPV indicates the uniformity of size of the platelet population. It is used for the differential diagnosis of thrombocytopenia.


Platelet Aggregation

Platelet aggregation is used to evaluate congenital qualitative functional disorders of adhesion, release, or aggregation. It is rarely used to evaluate acquired bleeding disorders.


Thrombin Time (TT); Thrombin Clotting Time (TCT)

Stage III fibrinogen defects can be detected by the TT test. It can detect DIC and hypofibrinogenemia and may also be used for monitoring streptokinase therapy. The test actually measures the time needed for plasma to clot when thrombin is added. Normally, a clot forms rapidly; if it does not, a stage III deficiency is present ( Fig. 2.1). A TT test is often included as part of a panel for coagulation defects.


Partial Thromboplastin Time (PTT); Activated Partial Thromboplastin Time (APTT)

The PTT, a one-stage clotting test, screens for coagulation disorders. Specifically, it can detect deficiencies of the intrinsic thromboplastin system and also reveals defects in the extrinsic coagulation mechanism pathway.


Activated Coagulation Time (ACT)

The ACT test evaluates coagulation status. The ACT responds linearly to heparin level changes and responds to wider ranges of heparin concentrations than does the APTT. The ACT however, assays overall coagulation activity. Therefore, prolonged values may not be exclusively the result of heparin.

The ACT can be a bedside procedure and requires only 0.4 mL of blood. Heparin infusion or reversal with protamine can then be titrated almost immediately according to the ACT results. 

ACT also is routinely used during dialysis, coronary artery bypass procedures, arteriograms, and percutaneous transluminal coronary arteriography. This test is hard to standardize, and no controls are available; therefore, it is used with caution mainly in cardiac surgery. The results are backed up by the APTT. 


Coagulant Factors (Factor Assay)

Assay of specific factors of coagulation is done in the investigation of inherited and acquired bleeding disorders. For example, tests of factor VIII–related antigen are used in the differential diagnosis of classic hemophilia and von Willebrand's disease in cases in which there is no family history of bleeding and bleeding times are borderline or abnormal. A test for ristocetin cofactor is done to help diagnose von Willebrand's disease by determining the degree or rate of platelet aggregation that is taking place.


Plasminogen (Plasmin; Fibrinolysin)

Plasminogen is a glycoprotein, synthesized in the liver, present in plasma. Under normal circumstances, plasminogen is a part of any clot because of the tendency of fibrin to absorb plasminogen from the plasma. When plasminogen activators perform their function, plasmin is formed within the clot; this gradually dissolves the clot while leaving time for tissue repair. Free plasmin also is released to the plasma; however, antiplasmins there immediately destroy any plasmin released from the clot (see Fig. 2.2).

This test is done to determine plasminogen activity in persons with thrombosis or DIC. When pathologic coagulation processes are involved, excessive free plasmin is released to the plasma. In these situations, the available antiplasmin is depleted, and plasmin begins destroying components other than fibrin, including fibrinogen, factors V and VIII, and other factors. Plasmin acts more quickly to destroy fibrinogen because of fibrinogen's instability.


Fibrinolysis (Euglobulin Lysis Time; Diluted Whole Blood Clot Lysis)

Primary fibrinolysis, without any sign of intravascular coagulation, is extremely rare. Secondary fibrinolysis is usually seen and follows or occurs simultaneously with intravascular coagulation. This secondary fibrinolysis is a protective mechanism against generalized clotting.

This test is done to evaluate a fibrinolytic activity. Shortened time indicates excessive fibrinolytic activity. Lysis is marked and rapid with primary fibrinolysis but can be minimal in secondary fibrinolysis. The diluted whole blood is used to monitor urokinase and streptokinase therapy.


Fibrin Split Products (FSPs); Fibrin Degradation Products (FDPs)

When fibrin is split by plasmin, positive tests for fibrin degradation (split) products, identified by the letters X, Y, D, and E, are produced. These products have an anticoagulant action and inhibit clotting when they are present in excess in the circulation. Increased levels of FDPs may occur with a variety of pathologic processes in which clot formation and lysis occur. This test is done to establish the diagnosis of DIC and other thromboembolic disorders.


D-Dimer

D-Dimers are produced by the action of plasmin on cross-linked fibrin. They are not produced by the action of plasmin on unclotted fibrinogen or FDPs and therefore are specific for fibrin. The presence of D-dimer confirms that both thrombin generation and plasmin generation have occurred.

This test is used in the diagnosis of DIC disease and to screen for venous thrombosis and acute myocardial infarction. The D-dimer test is more specific for DIC than are tests for FSPs. The test verifies in vivo fibrinolysis because D-dimers are produced only by the action of plasmin on cross-linked fibrin, not by the action of plasmin on unclotted fibrinogen. A positive D-dimer test is presumptive evidence for DIC but is not diagnostic.


Fibrinopeptide A (FPA)
Fibrinopeptides A and B are formed by the action of thrombin on fibrinogen; therefore, the presence of FPA indicates that thrombin has acted on fibrinogen.

The measurement is the most sensitive assay done to determine thrombin action. FPA reflects the amount of active intravascular blood clotting; this occurs in a subclinical DIC, which is common in patients with leukemia of various types and may be associated with tumor progression. FPA elevations can occur without intravascular thrombosis, decreasing the value of a positive test.


Prothrombin Fragment (F1 + 2)

The prothrombin F1 + 2 fragment is liberated from the prothrombin molecule when it is activated by factor Xa to form thrombin. Thrombin may be rapidly inactivated by antithrombin III. The F1 + 2 fragment, however, has a half-life of about 1.5 hours, making it a useful marker for activated coagulation.

Prothrombin F1 + 2 is used to detect activation of the coagulation system before actual thrombosis occurs. It is used to identify patients with low-grade intravascular coagulation (DIC) and to judge the effectiveness of oral anticoagulant therapy. F1 + 2 levels may assist in the study of the hypercoagulable states and in the assessment of thrombotic risk.


Fibrin Monomers (Protamine Sulfate Test; Fibrin Split Products)

A positive test result reflects the presence of fibrin monomers, indicative of thrombin activity and consistent with a diagnosis of intravascular coagulation. A negative result does not mean that intravascular coagulation is not present. A positive result may also be seen in some cases of severe liver disease and in inflammatory disorders caused by accumulation of products of coagulation in the circulation.

The detection of fibrin monomers and early-stage FSPs in plasma is useful in the diagnosis of DIC. Heparin therapy does not interfere with this test.


Fibrinogen

Fibrinogen is a complex protein (polypeptide) that, with enzyme action, is converted to fibrin. The fibrin, along with platelets, forms the network for the common blood clot. Although it is of primary importance as a coagulation protein, fibrinogen is also an acute-phase protein reactant. It is increased in diseases involving tissue damage or inflammation. This test is done to investigate abnormal PT, APTT, and TT and to screen for DIC and fibrin-fibrinogenolysis. It is part of a coagulation panel.


Protein C (PC Antigen)

Protein C, a vitamin K–dependent protein that prevents thrombosis, is produced in the liver and circulated in the plasma. It functions as an anticoagulant by inactivating factors V and VIII. Protein C is also a profibrinolytic agent (ie, it enhances fibrinolysis). The protein C mechanism therefore functions to prevent extension of intravascular thrombi. This test is used for evaluation of patients suspected of having congenital protein C deficiency. 

Resistance to protein C is caused by an inherited defect in the factor V gene (factor V Leiden) and causes significant risk for thrombosis. It is the underlying defect in up to 60% of patients with unexplained thrombosis and is the most common cause of pathologic thrombosis. If functional protein C is abnormal, a protein C resistance test should be performed.

This test evaluates patients with severe thrombosis and those with an increased risk or predisposition to thrombosis. Patients with partial protein C or partial protein S deficiency (heterozygotes) may experience venous thrombotic episodes, usually in early adult years. There may be deep vein thromboses, episodes of thrombophlebitis or pulmonary emboli (or both), and manifestations of a hypercoagulable state. Patients who are heterozygous may have type I protein C deficiency, with decreased protein C antigen, or type II deficiency, with normal protein C antigen levels but decreased functional activity. 


Protein S

Both protein S and protein C are dependent on vitamin K for their production and function. A deficiency of either one is associated with a tendency toward thrombosis. Protein S serves as a cofactor to enhance the anticoagulant effects of activated protein C. Slightly more than half of protein S is complexed with C4 binding protein and is inactive. Activated protein C in the presence of protein S rapidly inactivates factors V and VIII.


Antithrombin III (AT-III; Heparin Cofactor Activity)

AT-III inhibits the activity of activated factors XII, XI, IX, and X as well as factor II. AT-III is the main physiologic inhibitor of activated factor X, on which it appears to exert its most critical effect. AT-III is a "heparin cofactor." Heparin interacts with AT-III and thrombin, increasing the rate of thrombin neutralization (inhibition) but decreasing the total quantity of thrombin inhibited.

This test detects a decreased level of antithrombin that is indicative of thrombotic tendency. Only the test of functional activity gives a direct clue to thrombotic tendency. In some families, several members may have a combination of recurrent thromboembolism and reduced plasma antithrombin (30%–60%).

A significant number of patients with mesenteric venous thrombosis have AT-III deficiency. It has been recommended that patients with such thrombotic disease be screened for AT-III levels to identify those patients who may benefit from coumarin anticoagulant prophylaxis rather than heparin therapy. 



------------------------------------------ Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123 Email:ehab10f@gmail.com,ehababoueladab@yahoo.com ------------------------------------------
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