Blood is a complex mixture of suspended cellular components (erythrocytes, leukocytes and thrombocytes) and dissolved substances (electrolytes, proteins, carbohydrates, amino acids, etc.). Clinical hematology is concerned primarily with the cellular components of blood.
If blood is withdrawn from a vein and placed in a plain, untreated test tube, the blood will clot. Blood specimens of this type are used to harvest blood serum for testing. After the blood has clotted (5-20 minutes) the tube is centrifuged to compact the clotted cellular mass which is more dense than the supernatant serum. The supernatant serum is then aspirated and used for chemical or immunological testing. If a fresh blood specimen is promptly mixed with an anticoagulant (there are several: oxalates, citrates, ethylene diamine tetraacetates, heparins), the whole blood will remain fluid, allowing the cells to remain homogenously suspended in the blood plasma. If the anticoagulated whole blood specimen is allowed to stand for a sufficient length of time or the specimen is centrifuged, the cells will sediment to the bottom of the test tube, leaving a supernatant fluid called blood plasma. The sedimented tube may be mixed to resuspend the cells to a state nearly identical to circulating whole blood. Anticoagulated whole blood specimens are required for most hematology testing, i.e., cell counts, etc. The principle difference between serum and plasma is that serum contains no fibrinogen. The fibrinogen is consumed in its conversion to an insoluble fibrin matrix which traps the cells and forms the clot.
| The photo at right shows a rack of tubed blood specimens. The red-stoppered tubes contain serum and a centrifuged clot. The green-stoppered tubes contain centrifuged, heparin-anticoagulated whole blood. Common measurements in hematology are quantitative and qualitative measurement of RBC's, hemoglobin content, total WBC count, differential WBC and platelet count. Erythrocytes are the hemoglobin-containing Red Blood Cells (or RBC corpuscles) which are necessary for the transport of oxygen from the lungs to the tissues and for the transport of carbon dioxide from the tissues to the lungs. Leukocytes (or |  |
 | Neutrophils phagocytize bacteria and increase in number in acute bacterial infections. Monocytes increase in number in chronic bacterial infections and neutropenia. Lymphocytes are immune leukocytes and increase in viral infections. Eosinophils are increased in alergic reactions and parasitic infections. Basophils are associated with inflammation, acute alergic reactions and chemotaxis. Moderately large increases in lymphocytes (leukemoid reaction) are associated with infectious lymphocytosis (infections by adenovirus, enterovirus or Coxsackie A virus). Very large increases of leukocytes are associated with specific leukemias, eg. acute lymphocytic or myelogenous leukemias. These are confirmed by bone marrow biopsy. |
| Adjacent is a typical CBC (Complete Blood Count or Hemogram) consisting of WBC, RBC, Hemoglobin, Platelet, RBC Indices and Differential WBC. Note that evaluation of the CBC components is based on comparison to established Normal or Reference Ranges (low, normal and high). Furthermore, many test's reference ranges are either sex or age dependent, or both. The clinical significance of laboratory findings is interpreted by medical practitioners (primarily physicians) to assist in the diagnosis and treatment of their patients. Today, the measurements of the cellular components of blood are very much automated by elaborate, computerized instruments. The instrument below is a Beckman-Coulter MAXM which is capable of performing an automated CBC. All of the tasks which would be required for manual "wet-bench" methods of performance of a CBC have been incorporated in this instrument; precise sampling, mixing with appropriate reagent volumes, cell counting and sizing, and hemoglobin measurement are performed on bar-coded samples, and the results are collated and printed locally and to remote printers and fax machines with little user intervention.  Wet-bench methods are still in use in small remote labs in this country and abroad. Every medical technology student must perform these manual methods prerequisite to an understanding of automated methods. Hemoglobin is converted to cyanmethemoglobin and measured photometrically. Blood is diluted into an isotonic saline solution, loaded into a ruled counting chamber, and the RBC's and platelets are counted microscopically. Blood is diluted into a dilute acid solution to rupture the RBC's, leaving the WBC's intact for counting microscopically. A thin blood smear on a microscope slide is stained with Wright's stain, and then a differential WBC count is performed microscopically. Click here for the details of a manually performed CBC. |  |
 | The reticulocyte (abbreviated "retic") count is another manual hematology test which has been automated. Reticulocytes are immature RBC's containing RNA prematurely released into the circulatory system from the bone marrow as a usual response to some anemias or blood loss. In the absence of anemia there is normally a small percentage of the RBC's which are reticulated. The manual version of this test consists of staining RBC's with New Methelene Blue (a supravital stain, i.e., a stain which stains living cells), and preparing a thin film of the stained RBC's on a microscope slide. The stained reticulocytes are counted and expressed as a percentage of the total RBC's. |
| Sickle cell screening tests for the presence of Hemoglobin-S, one of several hemoglobin variants. Hemoglobin S is formed as the result of a single-gene defect causing substitution of valine for glutamic acid in position 6 of the beta chain of adult hemoglobin. Persons homozygous for hemoglobin S (HbSS) have sickle cell anemia. Hemoglobin-S is found in individuals of African descent. "Sickle cells" obtain their name from the characteristic sickle shape that Hemoglobin-S-containing RBC's assume under decreased oxygen tension. The screening for Hemoglobin-S employs its decreased solubility in a buffered Sodium |  |
Blood is of prime importance in the normal physiologic function of our major organ systems. In order for it to be effective, blood must be in a liquid or non-coagulated state. Another important function of blood is to maintain an intact circulatory system following trauma. The process by which blood is maintained fluid within the vessel walls and the ability of the system to prevent excessive blood loss upon injury is termed hemostasis. The balance between the forces that cause blood to solidify or to remain fluid is very delicate and involves several interacting systems.
When you cut yourself, the process of coagulation begins by the formation of a blood clot. This is followed shortly after by digestion or breakdown of the clot. Patients clot and/or bleed because of a variety of identifiable hemostatic abnormalities. Logical and effective treatment depends upon the proper identification of the abnormality. The coagulation or hemostasis laboratory performs tests to determine the cause and to monitor the proper treatment of the defect.
Platelets, Vascular and Clotting Factors - a brief discussion of their function
Platelets -
small, anuclear cytoplasmic disks. In an unstimulated state, the shape
is discoid.
Hemostasis -
the process in circulation where the blood is maintained fluid in vessels and
without major loss in case of injury.
Coagulation factors -
Components that exist in the circulation and supply the necessary constituents
for clot formation.Hemostasis: The property of the circulation where the circulating fluid is maintained within the blood vessels is referred to as hemostasis. The process depends on a delicate and complex interplay of at least 4 systems: vascular, plasma coagulation factors, platelets and fibrinolytic system.
Vascular System: Blood normally flows smoothly through the vascular system without cellular adherence to the vessel wall. The thin layer of endothelial cells lining the inner surface of the various vessels helps to maintain a thrombo-resistant surface. When vascular injury occurs following trauma or in certain vessel diseases, the endothelial cells interact with platelets and clotting factors to form a blood clot at the site of injury.
Platelets and Hemostasis: The platelet has at least a fourfold function: (1) In response to vascular injury, platelets are stimulated to initiate the formation of a primary hemostatic plug, (2) the platelet contributes phospholipid (sometimes referred to as platelet factor 3 or PF3) to the coagulation cascade, (3) they help maintain vascular integrity through endothelial support and (4) platelets may have a role in inflammatory response, possibly by activating the fifth component of complement.
There is a sequence of events which occurs at the site of vascular injury. First, the platelet is attracted to the exposed sub-endothelial layer of collagen and adheres to it. To accomplish this, the platelet undergoes a shape change. Secondly, the platelets release intrinsic adenosine diphosphate (ADP), among other substances. The released ADP stimulates other platelets to stick together at the wound site, and, thirdly, aggregation occurs. In this process, platelets adhere to each other to form a beginning plug. Finally, coagulation occurs and fibrin forms around the platelet aggregate to initiate repair. (See figure 1)
Coagulation Factors: The coagulation factors circulate in the plasma as cofactors or as procoagulants, and, when activated supply some of the components needed for clot formation. According to the international nomenclature system, coagulation cofactors and procoagulants were assigned roman numerals in the order of their discovery and don't correspond to their location in the coagulation sequence of activation. (See figure 2) The coagulation factors are generated in the liver cells, except for Factor VIII (at least the Von Willebrand's portion), which is produced in multiple organs, possibly the endothelial cells and megakarocytes.
The model generally used to describe the mechanism of coagulation is the cascade system. The cascade is separated into three areas: the intrinsic system, commonly measured by the aPTT test, which is activated by surface contact; the extrinsic system, commonly measured by the PT test, which is activated by vascular injury, and, the common pathway, which is set into motion by activation from the intrinsic and/or the extrinsic pathway. Because of the variety of constituents involved with the common pathway, there are several different tests that could be used to monitor activity. The systems and tests are described later.
Primary Hemostasis: Following injury to a blood vessel, all of the systems are activated. For sake of ease, the hemostatic process is divided into 2 components; primary hemostasis and secondary hemostasis. Primary hemostasis depends upon the response of the platelet and blood vessel wall to the injury. When the small blood vessels are injured, blood platelets adhere and aggregate at the site of injury, reducing and finally arresting bleeding.
Secondary Hemostasis: Secondary Hemostasis starts when the cascade system of Coagulation is activated by substances released at the time of blood vessel injury.
These coagulation factors, which are proteins, with the exception of Calcium and Thromboplastin, can conveniently be divided into three families: the fibrinogen, prothrombin, and contact family. The fibrinogen family includes fibrinogen, Factors V, VIII, and XIII. The prothrombin family includes Factors II, VII, IX, X, Protein C and Protein S. The contact family of plasma coagulation proteins include: Factor XII or Hageman factor, Factor XI, Fletcher factor or Prekallikrein (PK), Fitzgerald factor or High Molecular Weight Kininogen (HMWK) and possibly the Passovoy factor. They are all involved in the mechanism that generates insoluble fibrin as a final product, by means of the coagulation cascade. Disorders of secondary hemostasis many times involve a change in the coagulation proteins. These changes can be a decreased level of a particular factor or a defect in the way the factor functions.
Extrinsic Pathway
Enzyme:
Organic compound, frequently a protein, capable of accelerating or producing by
catalytic action some change in a substrate for which it is often specific.
Extrinsic pathway:
Pathway in which fibrin is formed as the result of the release of tissue
thromboplastin into the circulation.
Prothrombin time: A laboratory coagulation test which measures the general level
of clottability of a plasma sample. It is sensitive to the factors of the
extrinsic clotting system.
INR:
International Normalized Ratio which provides a convenient method for
standardizing the monitoring of Warfarin therapy.Hemostasis is defined as a property of circulation whereby blood is maintained within a vessel and the ability of the system to prevent excessive blood loss when injured. One of the major components needed to provide hemostasis is the coagulation system which involves the clotting proteins or clotting factors. The coagulation factors, except for calcium and thromboplastin, are proteins and are involved in a sequential reaction or coagulation cascade. The last step of the cascade leads to insoluble fibrin as the end product. The reactions leading to fibrin formation can be divided into the extrinsic, intrinsic and common pathways. The extrinsic pathway is initiated by the release of tissue thromboplastin (Factor III) which is exposed to the blood when there is damage to the blood vessel. Factor VII which is a circulation coagulation factor, forms a complex with tissue thromboplastin and calcium. This complex rapidly converts Factor X to the enzyme form Factor Xa. Factor Xa catalyzes the prothrombin (Factor II) to thrombin (Factor IIa) reaction which is needed to convert fibrinogen (Factor I) to fibrin. See figure 3 for "coagulation cascade" diagram depicting the extrinsic, intrinsic and common pathways. The Prothrombin Time or PT is a laboratory screening test used to detect coagulation disorders. It measures the activity of the factors of the extrinsic pathway including factors II, V, VII, X, and fibrinogen. The extrinsic factors not measured in the PT test are Factors III (Thromboplastin), and IV (Calcium). The PT is also used to monitor oral anticoagulant therapy such as warfarin.
Warfarin is a drug used in patient therapy to prevent thrombosis. It inhibits the synthesis of the vitamin K dependent factors, factors II, VII, IX and X by blocking the regeneration of vitamin K and shows a dose dependent effect. As more warfarin is ingested orally, the greater the reduction in the functional levels of vitamin K dependent factors. See figure 4 for the effect of warfarin on the synthesis of clotting factors. Because 3 of the 4 factors affected by warfarin are evaluated by the PT test, it is commonly used to monitor therapy.
The PT test is performed by adding tissue thromboplastin and calcium to plasma and measuring the time for clot formation. It can be performed either manually by tilt tube method or mechanically by use of a fibrometer or a photo-optical instrument. The PT reagent used in the testing provides the tissue thromboplastin and calcium. The sources of thromboplastin can be human or rabbit brain, lung, placental, brain/lung combination, or produced by recombinant technology. The necessary calcium is added to the reagent either at the time of manufacture or prior to testing.
The PT can be done as either a one-stage or a two-stage assay, although the one-stage procedure is the most widely used and preferred. Thromboplastin reagent (0.2 ml) is warmed at 37C then forcibly added to plasma (0.1ml) which also has been heated to 37C and a timer is started. As soon as the clot forms indicating fibrin formation, the timing stops and the time is recorded to the nearest tenth of a second. The expected normal range for a PT is 10-14 seconds depending on the type of reagent used.
Variation in the composition and responsiveness of PT reagents have necessitated the use for standardization. The International Normalized Ratio or INR was developed for the purpose of standardizing the monitoring of warfarin therapy.
Several factors may contribute to the differing degrees of responsiveness observed for various thromboplastin reagents. Some of these include the species and tissue source, the relative concentrations of other components of the reagent formula etc. The responsiveness of the thromboplastin reagent needs to be considered to make the PT an effective way of monitoring warfarin treatment. The responsiveness of a thromboplastin reagent toward plasma samples from patients receiving warfarin is described by a value called the International Sensitivity Index (ISI).
The calculation of the INR is obtained by using the following calculation:
The lower the ISI, the more responsive the reagent. The differences in the responsiveness of thromboplastins to the reduction of clotting factors II, VII and X are responsible for the difference in dosage of oral anticoagulants.
In summary, defects in the normal hemostatic mechanism can be listed as two types. One is the failure of any of the processes that lead to the hemostatic plug formation which may lead to a bleeding disorder and inappropriate activation of the hemostatic mechanism which may cause thrombosis. Laboratory investigations and determinations are needed to identify the exact nature of the underlying bleeding disorder. Screening tests such as the PT are initially performed. Based on these results, further, more complex testing may be needed leading to follow-up corrective action and treatment.
Intrinsic Pathway
Activated partial thromboplastin time (APTT)
One of the tests used for screening patients for a bleeding tendency.
Specifically, adequate levels of the coagulation factors XII, XI, IX,
VIII, X, V and II must be present for the test to be normal. The test also
serves as the basis for other test procedures such as certain factor assay
tests.
Intrinsic
Originating from withinThe intrinsic pathway of Coagulation is activated when circulating Factor XII comes in contact with and is bound to a negatively charged surface. This causes a change in the molecular configuration of Factor XII and in concert with HMWK and prekallikrein it becomes an active enzyme, XIIa. This activated enzyme is then able to bring about a similar change in Factor XI. After activation, Factor XIa, in a calcium dependent reaction, converts Factor IX to its active form, Factor IXa. A phospholipid surface is also needed for Factor IXa conversion and is provided by activated platelets, as Platelet Factor Three (PF3). Factor IX can also be activated by the tissue factor, Factor VII complex; the initiating complex of the extrinsic pathway. Factor X can be activated to Factor Xa by either the Factor VIIa complex or by the complex of Factor IXa and Factor VIII. Factor Xa in the presence of Factor V, calcium and phospholipid surface converts Factor II (prothrombin) to Factor IIa (thrombin) which converts Factor I (fibrinogen) to fibrin (See figure 5).
Activated partial thromboplastin time (aPTT) is an assay used to screen for abnormalities of the intrinsic clotting system. It is also used to monitor the anticoagulant effect of circulating heparin.
An aPTT assay is performed by adding to platelet poor plasma a Factor XII activator, a phospholipid, and calcium ions. Factors I, II, V, VIII, IX, X, XI, XII, prekallikrein (Fletcher Factor) and high molecular weight kininogen (HMWK) are measured. An abnormal aPTT result might indicate the presence of an acquired inhibitor or a deficiency in any of the coagulation factors except Factors VII and XIII.
For in vitro analysis, some commonly used activators are glass, ellagic acid, kaolin, silica and celite. All of these except glass, are used in aPTT reagents and serve the same function of activating the clotting mechanism. Phospholipids are platelet substitutes and accelerate the reactions involved. Sources of phospholipids are rabbit brain, cephalin (dehydrated rabbit brain), bovine brain, and soy bean.
When adequate levels of all the coagulation factors are present in plasma, the aPTT test result is normal. Normal ranges of the factors vary from approximately 50-150% of normal. In general an aPTT reagent should be able to detect factor levels of 30% or less. If aPTT results are prolonged and there is no indication of a factor deficiency, an acquired inhibitor may be present.
Heparin will also cause a prolonged aPTT. This commercial product is prepared from beef lung or porcine intestinal mucosa and is administered via intravenous or subcutaneous injection. Heparin with its plasma co-factor Antithrombin III, inhibits coagulation immediately after being administered. It is the drug of choice for treating venous thrombosis by preventing fibrin formation.
The aPTT, although useful in monitoring high level heparin therapy, has had variable effectiveness in monitoring low dose heparin therapy and low molecular weight forms of heparin.
Natural Inhibitors
Antithrombin III
Natural inhibitor of the coagulation system
Protein C
Natural inhibitor of the coagulation system
Protein S
Protein C co-factor Once Coagulation is initiated, the body has mechanisms for avoiding massive thrombus formation. Physiologic balancing of the Hemostatic mechanism to limit uncontrolled bleeding and clotting is an important aspect in the Hemostatic response. There are a variety of biological control mechanisms which aid in the control of blood coagulation. These include the ability of the liver and the reticulo-endothelial system to clear activated clotting factors from the circulation, the prevention of the high concentrations of activated factors at a given location within the circulation by a constant blood flow, and natural inhibitors in the plasma such as Antithrombin III and the Protein C-S System.
Antithrombin III (AT-III) is the most important inhibitor of the coagulation enzymes. AT-III binds to activated factors rendering them inactive (Figure 6). The primary function is to inactivate thrombin. Inactive factors and cofactors are not neutralized by AT-III, since it only binds to the enzymatic factors. The process of binding the active forms of the clotting factors (XIIa, XIa, Xa, IXa) and thrombin to AT-III is greatly accelerated by heparin to an almost instant neutralization. AT-III inhibits not only coagulation enzymes but also plasmin and kallikrein.
Patients with decreased AT-III levels are subject to an increased risk of thromboembolism even in cases of slightly reduced AT-III levels, therefore the Antithrombin III assay is an important part of a prethrombotic workup.
Antithrombin III levels are affected by several other disease states. Individuals suffering from severe hepatic disorders such as cirrhosis or acute hepatitis have significantly depressed AT-III levels, while disease accompanied by inflammation may show elevations. Protein C is an inhibitor of the activated Factors Va and VIIIa. (See figure 6) This is its anticoagulant function. Protein C also inactivates tissue plasminogen activator inhibitor (PAI) which increases the activity of tissue plasminogen activator (tPA) which enhances fibrinolytic activity. Therefore, it can be said that Protein C has both anticoagulant and fibrinolytic functions. Just as Antithrombin III has a co-factor which is heparin, Protein C has a co-factor which is Protein S. Both Protein C and Protein S are vitamin K dependent factors. Enhancement of Protein C anticoagulant functions is achieved by Protein S. Patients with Protein C and/or Protein S deficiencies have a thrombotic tendency. Patients also may acquire deficiencies of Protein C and Protein S with liver disease and disseminated intravascular coagulation (DIC).
Fibrinolysis
Fibrinolysis:
Dissolution and localization of a fibrin clot.
Plasmin:
Active portion of fibrinolytic system: has the ability to dissolve formed fibrin
clots; also has similar effect on other plasma proteins and clotting factors.The last stage of coagulation is fibrinolysis, which is the dissolution and localization of a fibrin clot. These functions are carried out by enzymes and their inhibitors. A disruption or breach of the fine balance of this fibrinolytic system can result in bleeding or thrombosis.
The components of the fibrinolytic system are schematically shown in Figure 7. Fibrinolysis is mediated by activation of plasminogen to plasmin. This is accomplished by:
Intrinsic activation (plasma based) initiated through Factor XIIa and allikrien.
Thus, the contact system of coagulation serves as an intrinsic activator.
Extrinsic activation (cellular based) initiated by way of stimuli such as vascular
injury, ischemia, exercise, stress and pyrogens.
Exogenous (Therapeutic) activation (drug based) includes streptokinase, urokinase and
tPA tissue plasminogen activator).Activators of plasminogen convert it to the active enzyme plasmin. Plasmin, in turn, acts to split the fibrin clot into fibrin degradation products. To balance this activity there are inhibitors. The most important inhibitor of plasminogen activators is PAI-1, which is fast acting. Alpha2-antiplasmin, another principal inhibitor of fibrinolysis, inhibits plasmin (See figure 8).
Soluble fibrinogen is cleaved by thrombin to form fibrin monomers. The fibrin monomers aggregate to form fibrin polymers, unstable fibrin clots. Thrombin also activates factor XIII to an activated enzyme, factor XIIIa, which in the presence of calcium converts fibrin polymers to a stable fibrin clot. Plasmin can degrade or split both fibrinogen and fibrin into fragments, X, Y, D and E. Fibrinogen degradation products (FDP) are the products of fibrinogenolysis and are detected by the FDP assay. Fibrin degradation products (fdp) are the product of fibrinolysis. The only time D-dimers (cross linked D-domains) are present is after the degradation of a stable fibrin clot (See figure 9).
There are many conditions that can affect the fibrinolytic system resulting in an increased or decreased activity of fibrinolysis. Samples of such conditions are Disseminated Intravascular Coagulation (DIC), trauma from surgical procedures or accidents, deficiencies in or consumption of the various inhibitors and activators of the fibrinolytic system.
Continued study of the fibrinolytic system unlocks it's complexities . Always on the horizons are newer and more sensitive and specific methods of evaluating this system, thus providing better diagnostic tools.
References: Hemostasis Basics, Dade Behring, 2000